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+MIT License
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+[{"sourceid":"4806292","sourcedb":"","project":"","target":"","text":"Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi. Phosphatidylinositol 4-kinase beta (PI4KB, also known as PI4K IIIβ) is a soluble cytosolic protein yet its function is to phosphorylate membrane lipids. It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P). PI4P is an essential lipid found in various membrane compartments including the Golgi and trans-Golgi network (TGN), the plasma membrane and the endocytic compartments. In these locations, PI4P plays an important role in cell signaling and lipid transport, and serves as a precursor for higher phosphoinositides or as a docking site for clathrin adaptor or lipid transfer proteins. A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories. These structures are essential for effective viral replication. Recently, highly specific PI4KB inhibitors were developed as potential antivirals. PI4K kinases must be recruited to the correct membrane type to fulfill their enzymatic functions. Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins. In contrast, type III PI4Ks (PI4KA and PI4KB) are soluble cytosolic proteins that are recruited to appropriate membranes indirectly via protein-protein interactions. The recruitment of PI4KA to the plasma membrane by EFR3 and TTC7 is relatively well understood even at the structural level, but, the actual molecular mechanism of PI4KB recruitment to the Golgi is still poorly understood. Acyl-coenzyme A binding domain containing protein 3 (ACBD3, also known as GCP60 and PAP7) is a Golgi resident protein. Its membrane localization is mediated by the interaction with the Golgi integral protein golgin B1/giantin. ACBD3 functions as an adaptor protein and signaling hub across cellular signaling pathways. ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2. ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus. We present a biochemical and structural characterization of the molecular complex composed of the ACBD3 protein and the PI4KB enzyme. We show that ACBD3 can recruit PI4KB to model membranes as well as redirect PI4KB to cellular membranes where it is not naturally found. Our data also show that ACBD3 regulates the enzymatic activity of PI4KB kinase through membrane recruitment rather than allostery. Results ACBD3 and PI4KB interact with 1:1 stoichiometry with submicromolar affinity In order to verify the interactions between ACBD3 and PI4KB we expressed and purified both proteins. To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423–522) was removed (Fig. 1A). This internal deletion does not significantly affect the kinase activity(SI Fig. 1A) or interaction with ACBD3 (SI Fig. 1B,C). In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction. Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain. We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel). Because it has been reported that ACBD3 can dimerize in a mammalian two-hybrid assay, we were interested in determining the stoichiometry of the ACBD3:PI4KB protein complex. The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively. This result suggests that both proteins are monomeric and the stoichiometry of the ACBD3: PI4KB protein complex is 1:1 (Fig. 1C, left panel). Similar results were obtained for the complex of the Q domain of ACBD3 and the N-terminal region of PI4KB (Fig. 1C, right panel). We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR). SPR measurements revealed a strong interaction with a Kd value of 320 +/−130 nM (Fig. 1D, SI Fig. 1D). We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range. Structural analysis of the ACBD3:PI4KB complex Full length ACBD3 and PI4KB both contain large intrinsically disordered regions that impede crystallization. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of the complex to determine which parts of the complex are well folded (SI Fig. 2). However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB. For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization. As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules. Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously. Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44. Over 93% of non-exchangeable side-chain signals were assigned for the free ACBD3 Q domain. Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances. The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB). Assignments obtained for non-exchangeable side-chain signals were over 99% complete. The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation. Structural statistics for the final water-refined sets of structures are shown in SI Table 1. This structure revealed that the Q domain forms a two helix hairpin. The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A). Preceding the kinase helix are three ordered residues (Val42, Ile43, and Asp44) that also contribute to the interaction (Fig. 2B). The remaining part of the PI4KB N-termini, however, is disordered (SI Fig. 5). Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C). To corroborate the structural data, we introduced a number of point mutations and validated their effect on complex formation using an in vitro pull-down assay (Fig. 2D). Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D). ACBD3 efficiently recruits the PI4KB enzyme to membranes We next sought to determine if the ACBD3:PI4KB interaction drives membrane localization of the PI4KB enzyme. To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate – the PI lipid. We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A). We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel). We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B). Given this result, overexpression of the Q domain should also interfere with the PI4KB dependent Golgi functions. Ceramide transport and accumulation in Golgi is a well-known PI4KB dependent process. We have used fluorescently labeled ceramide and analyzed its trafficking in non-transfected cells and cell overexpressing the Q domain. As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi. We further analyzed the function of ACBD3:PI4KB interaction in membrane recruitment of PI4KB in living cells using fluorescently tagged proteins. We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system. We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP. The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D). We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin. As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase. In every case the ACDB3 Q domain was rapidly (within 5 minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2). Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel). However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel). ACBD3 increases PI4KB enzymatic activity by recruiting PI4KB to close vicinity of its substrate To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate. We observed no effect on the kinase activity of PI4KB (Fig. 4A) suggesting that ACBD3 does not directly affect the enzyme (e.g. induction of a conformation change). However, in vivo ACBD3 is located at the Golgi membranes, whereas in this experiment, ACBD3 was located in the solution and PI is provided as micelles. We therefore designed a more physiologically relevant experiment. For this, we again turned to the GUV system with ACBD3 localized to the GUV membrane. The GUVs contained 10% PI to serve as a substrate for PI4KB kinase. The buffer also contained CFP-SidC, which binds to PI4P with nanomolar affinity. This enabled visualization of the kinase reaction using a confocal microscope. We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3. Reaction was also performed in the absence of ATP as a negative control (Fig. 4B). These experiments showed that PI4KB enzymatic activity increases when ACBD3 is membrane localized (Fig. 4C, SI Fig. 6). We conclude that enzyme activation proceeds through a membrane recruitment mechanism. Discussion Membrane recruitment of PI4KB enzyme is crucial to ensure its proper function at the Golgi and TGN. However, the molecular mechanism and structural basis for PI4KB interaction with the membrane is poorly understood. In principle, any of the binding partners of PI4KB could play a role in membrane recruitment. To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins. The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase. Although Rab11 does not appear to be required for recruitment of PI4KB to the Golgi, PI4KB is required for Golgi recruitment of Rab11. Arf1, the other small GTP binding protein, is known to influence the activity and localization of PI4KB, but it does not appear to interact directly with PI4KB (our unpublished data). The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear. NCS-1 is an N-terminally myristoylated protein that participates in exocytosis. It is expressed only in certain cell types, suggesting that if it contributes to PI4KB membrane recruitment, it does so in a tissues specific manner. The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation. However, 14-3-3 proteins do not appear to interfere with membrane recruitment of this kinase. ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase. For these reasons we focused on the interaction of the PI4KB enzyme with the Golgi resident ACBD3 protein in this study. Here we present the mechanism for membrane recruitment of PI4KB by the Golgi resident ACBD3 protein. We show that these proteins interact directly with a Kd value in the submicromolar range. The interaction is sufficient to recruit PI4KB to model membranes in vitro as well as to the mitochondria where PI4KB is never naturally found. To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain. The Q domain adopts a helical hairpin fold that is further stabilized upon binding the kinase helix (Fig. 2A). Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase. Based on our and previously published structures we built a pseudoatomic model of PI4KB multi-protein assembly on the membrane (Fig. 5) that illustrates how the enzyme is recruited and positioned towards its lipidic substrate and how it in turn recruits Rab11. +RNA viruses replicate at specific PI4P-enriched membranous compartments. These are called replication factories (because they enhance viral replication) or membranous webs (because of their appearance under the electron microscope). To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid. Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3. Our data suggest that they could do this via 3A:ACBD3:PI4KB complex formation. The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication. This could eventually have implications for therapeutic intervention to combat picornaviruses-mediated diseases ranging from polio to the common cold. Materials and Methods Plasmid construction, protein expression, and purification All proteins used in this study were recombinant and were expressed in E. coli using previously developed protocols. Briefly, full-length human ACBD3 (UniProtKB entry Q9H3P7) and PI4KB (UniProtKB entry Q9UBF8, isoform 1) lipid kinase and their deletion mutants were cloned into a previously modified pRSFD vector (Novagen) that already contained an N-terminal 6xHis tag followed by a GB1 solubility tag and TEV protease cleavage site. Mutations were generated using the Phusion Site-Directed Mutagenesis Kit (Thermo Scientific). The plasmids used are listed in the SI (SI Table 2). The proteins were expressed in E. coli BL21 Star cells as previously described. Upon overnight expression in autoinduction media bacterial cells were harvested and lysed in lysis buffer (50 mM Tris pH 8, 300 mM NaCl, 3 mM β-mercaptoethanol, 20 mM imidazole, 10% glycerol). The lysate was incubated with the Ni-NTA resin (Macherey-Nagel) and then extensively washed with the lysis buffer. The protein was eluted with the lysis buffer supplemented with 300 mM imidazole. When appropriate, tags were removed with TEV protease, and the protein was further purified using the size exclusion chromatography on Superdex 75 or Superdex 200 columns (GE Healthcare) in SEC buffer (10 mM Tris pH 8, 200 mM NaCl, 3 mM β-ME). Proteins were concentrated to 1–5 mg/ml (measured spectroscopically) and stored at −80 °C until needed. In vitro pull-downs Ni-NTA sepharose beads (Macherey-Nagel) were mixed with both binding partners (one of which was tagged with an N-terminal 6xHis tag) at a final concentration of 1 μM in a final volume of 200 μL binding buffer (30 mM Tris pH 8, 200 mM NaCl, 10 mM imidazole, and 1 mM TCEP). After 30 min incubation at 4 °C the beads were washed twice with 200 μL of the binding buffer, and total protein was directly eluted with the Laemmli sample buffer and analyzed by SDS-PAGE. SPR (Surface plasmon resonance) and AUC (Analytical ultracentrifugation) PI4KB was chip-immobilized as detailed in the SI. Afterwards, the ACBD3 protein was injected in a series of concentrations for 3 min and then dissociation was monitored for another 5 min. The data were fit to a single-exponential model. Rate constants of association and dissociation were obtained by fitting the observed change in resonance signal using the following equations: where c is the protein concentration, t is time, kon is the association rate constant, koff is the dissociation rate constant, D1 and D2 are the linear drift terms, and Ras, Rdis, R0, R1, and Rmax are corresponding changes in the relative response signal. AUC was used to perform sedimentation velocity experiments using a ProteomeLab XL-I Beckman Coulter analytical ultracentrifuge equipped with an AN50Ti rotor. All measurements were performed in 10 mM Tris pH 8, 200 mM NaCl, and 3 mM β-mercaptoethanol at 20 °C and 48000 rpm. All data were collected using an absorbance (230 nm and 280 nm) optical system. Data analysis was performed with the SEDFIT package and data were analyzed using a sedimentation coefficient distribution model c(s). In vitro kinase assay In vitro kinase activity was measured using a bioluminescent ADP-Glo assay (Promega) as described previously. Briefly, reactions were carried out in a total volume of 5 μL in 384-well plates by diluting the indicated amounts of the PI4KB enzyme and/or ACBD3 protein into the kinase buffer (20 mM Tris pH 7.5, 5 mM MgCl2, 0.2% Triton-X100, 0.1 mg/mL BSA, 2 mM DTT, 50 μM phosphatidylinositol). Reaction was initiated by adding ATP to a final concentration of 100 μM. Samples were incubated for 60 min at 25 °C and the amount of hydrolyzed ATP was measured according to the manufacturer’s protocol using a TECAN infinite M 1000 plate reader. NMR spectroscopy NMR spectra were acquired at 25 °C on a 600 MHz and 850 MHz Bruker Avance spectrometers, both of which were equipped with a triple-resonance (15N/13C/1H) cryoprobe. The sample volume was 0.35 mL, with a 280 μM concentration for the free Q domain and a 470 μM concentration for the ACBD3:PI4KB complex in the NMR buffer (25 mM sodium phosphate pH 6.5, 100 mM NaCl, 1 mM TCEP, 0.01% NaN3), 5% D2O/95% H2O. A series of double- and triple-resonance spectra were recorded to determine essentially complete sequence-specific resonance backbone and side-chain assignments. Constraints for 1H-1H distance required to calculate the structure of free Q domain and ACBD3:PI4KB complex were derived from 3D 15N/1H NOESY-HSQC and 13C/1H NOESY-HMQC, which were acquired using a NOE mixing time of 100 ms. The families of converged structures for the ACBD3:PI4KB complex and free Q domain were calculated using standard software as detailed in the SI. The structures with the lowest total energy were selected and validated. The statistics for the resulting structures are summarized in SI Table 1. Protein labeling with fluorescent dyes PI4KB was labeled on native cysteine residues. Briefly, pure recombinant protein was incubated overnight at 4 °C with a 3x molar excess of Alexa 488 C5 maleimide (Life Technologies). The reaction was quenched by adding 10 mM β-mercaptoethanol (βME) and the protein was repurified by size exclusion chromatography. Giant Unilamellar Vesicle Preparation and Imaging Giant Unilamellar Vesicles (GUVs) composed of POPC (54.9 mol %), POPS (10 mol %), cholesterol (20 mol %), PI (10 mol %), DGS-NTA(Ni) [1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) ] (5 mol %) (Avanti Polar lipids), and ATTO647N-DOPE (0.1 mol %) (ATTO-TEC GmbH) were prepared by electroformation as described previously, please see SI. Live Cell Imaging COS-7 cells were plated onto 29-mm-diameter poly-L-Lysine coated glass bottom dishes (In Vitro Scientific) at 100,000 cells/well density and transfected using the Lipofectamine2000 reagent (Invitrogen) with plasmid DNAs (0.5–1 mg/well) according to manufacturer’s instructions. The plasmids are described in SI Table 2. 24 hr post transfection, COS-7 cells were washed with a modified Krebs-Ringer solution (10 mM Na-HEPES pH 7.4, 120 mM NaCl, 4.7 mM KCl, 2 mM CaCl2, 0.7 mM MgSO4, 10 mM glucose) in the same dish and were imaged using an LSM 710 confocal microscope (Carl Zeiss MicroImaging) with a 63 × 1.4-numerical-aperture planapochromatic objective. For ceramide uptake experiments, COS-7 cells were loaded with 0.05 μM BODIPY® FL C5-Ceramide (Molecular Probes, ThermoFisher Scientific) complexed with BSA in modified Krebs-Ringer solution at room temperature for 20 min. Cells were then washed three times and imaged using the above mentioned settings. Immunofluorescent imaging COS-7 cells were plated onto 25-mm-diameter poly-L-Lysine coated circular glass coverslips in six-well plates (100,000 cells/well), and transfected using the Lipofectamine2000 reagent (Invitrogen) with plasmid DNAs (0.5–1 mg/well) according to manufacturer’s instructions. Twenty four hours post transfection, cells were washed with PBS, fixed with 4% paraformaldehyde, stained with mouse anti-PI4KB primary antibody (BD Transduction Laboratories, 1:500 dilution) and then after washing with PBS stained with Alexa Fluor 647 conjugated donkey anti-mouse secondary antibody (Molecular Probes, ThermoFisher Scientific, 1:500 dilution). Cover slips were mounted and observed with the above mentioned microscopy settings. HD exchange Hydrogen/deuterium exchange was performed as previously described with the following modifications. The exchange was done in 10 mM Tris-HCl pD 8.0, 200 mM NaCl at 20 °C. Protein concentration during the exchange was 1 μM. Aliquots (50 μL) were removed after 10, 20, 60, 120, 600, 1800, and 3600 s and the exchange was quenched by the addition of 50 μL of 0.25 M glycine-HCl pH 2.3 and rapid freezing in liquid nitrogen. Prior to the analysis each sample was quickly thawed and injected onto an immobilized rhizopuspepsin column (bed volume 66 μL). Digestion was driven by a flow of 0.4% formic acid in water at a flow rate of 100 μL/min (LC-20AD pump, Shimadzu). The resulting peptides were trapped and desalted online on a peptide microtrap (Optimize Technologies). After a desalting step (3 min), peptides were separated using a linear gradient of 10–25% buffer B for 2 min, followed by a quick jump to 99% buffer B (buffer A = 0.4% formic acid/2% acetonitrile in water; buffer B = 95% acetonitrile/0.4% formic acid in water). The outlet of the LC system was interfaced to an electrospray ionization source of a Fourier transform ion cyclotron resonance mass spectrometer (12 T SolariX XR, Bruker Daltonics). Exchange was followed on 32 peptides from PI4KB (N) and 26 peptides from ACBD3(Q), covering in both cases 100% of the protein sequence. Peptides were identified by LC-MS/MS and MASCOT search against a database containing the sequences of the studied proteins. Data from H/D exchange were analyzed by program DeutEx written in the laboratory (unpublished). Additional Information Accession codes: The structures and assigned chemical shifts for the free Q domain and the ACBD3:PI4KB complex were deposited in PDB database under accession codes 2N72 and 2N73, and BMRB database under accession codes 25790 and 25791. How to cite this article: Klima, M. et al. Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein. Sci. Rep. 6, 23641; doi: 10.1038/srep23641 (2016). Supplementary Material Phosphatidylinositol 4-kinases: Function, structure, and inhibition Mammalian phosphatidylinositol 4-kinases as modulators of membrane trafficking and lipid signaling networks Phosphoinositides: Tiny Lipids with Giant Impact on Cell Regulation Phosphatidylinositol 4-kinases: hostages harnessed to build panviral replication platforms Highly Selective Phosphatidylinositol 4-Kinase IIIbeta Inhibitors and Structural Insight into Their Mode of Action 2-Alkyloxazoles as potent and selective PI4KIIIbeta inhibitors demonstrating inhibition of HCV replication The crystal structure of the phosphatidylinositol 4-kinase IIalpha Structural insights into assembly and regulation of the plasma membrane phosphatidylinositol 4-kinase complex Plasticity of PI4KIIIalpha interactions at the plasma membrane Acyl-coenzyme A binding domain containing 3 (ACBD3; PAP7; GCP60): an emerging signaling molecule Identification and characterization of a novel Golgi protein, GCP60, that interacts with the integral membrane protein giantin GCP60 preferentially interacts with a caspase-generated golgin-160 fragment The mammalian Golgi regulates numb signaling in asymmetric cell division by releasing ACBD3 during mitosis NMDA receptor-nitric oxide transmission mediates neuronal iron homeostasis via the GTPase Dexras1 The 3A protein from multiple picornaviruses utilizes the golgi adaptor protein ACBD3 to recruit PI4KIIIbeta ACBD3-mediated recruitment of PI4KB to picornavirus RNA replication sites Golgi protein ACBD3 mediates neurotoxicity associated with Huntington’s disease Structures of PI4KIIIbeta complexes show simultaneous recruitment of Rab11 and its effectors Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIbeta with Rab11 ACBD3 interaction with TBC1 domain 22 protein is differentially affected by enteroviral and kobuviral 3A protein binding Large, dynamic, multi-protein complexes: a challenge for structural biology Phosphatidylinositol 4-kinase IIIbeta regulates the transport of ceramide between the endoplasmic reticulum and Golgi PI4P and PI(4,5)P-2 Are Essential But Independent Lipid Determinants of Membrane Identity Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells A homogeneous and nonisotopic assay for phosphatidylinositol 4-kinases The Legionella longbeachae Icm/Dot substrate SidC selectively binds phosphatidylinositol 4-phosphate with nanomolar affinity and promotes pathogen vacuole-endoplasmic reticulum interactions Phosphatidylinositol 4-kinasebeta is critical for functional association of rab11 with the Golgi complex Structural insights into activation of phosphatidylinositol 4-kinase (Pik1) by yeast frequenin (Frq1) Neuronal calcium sensor 1 and phosphatidylinositol 4-OH kinase beta interact in neuronal cells and are translocated to membranes during nucleotide-evoked exocytosis Phospho-specific binding of 14-3-3 proteins to phosphatidylinositol 4-kinase III beta protects from dephosphorylation and stabilizes lipid kinase activity A complex comprising phosphatidylinositol 4-kinase III beta, ACBD3, and Aichi virus proteins enhances phosphatidylinositol 4-phosphate synthesis and is critical for formation of the viral replication complex 14-3-3 protein interacts with and affects the structure of RGS domain of regulator of G protein signaling 3 (RGS3) Structural basis for membrane targeting by the MVB12-associated beta-prism domain of the human ESCRT-I MVB12 subunit Structure of the human FOXO4-DBD-DNA complex at 1.9 A resolution reveals new details of FOXO binding to the DNA Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling The high-resolution crystal structure of phosphatidylinositol 4-kinase IIbeta and the crystal structure of phosphatidylinositol 4-kinase IIalpha containing a nucleoside analogue provide a structural basis for isoform-specific inhibitor design Sequence-specific assignment and secondary structure determination of the 195-residue complex formed by the Mycobacterium tuberculosis proteins CFP-10 and ESAT-6 NMR assignment of the mTOR domain responsible for rapamycin binding Endosomal sorting complex required for transport (ESCRT) complexes induce phase-separated microdomains in supported lipid bilayers Structural insight into the calcium ion modulated interdomain electron transfer in cellobiose dehydrogenase The Phyre2 web portal for protein modeling, prediction and analysis The authors declare no competing financial interests. Author Contributions M.K. and A.D. carried out DNA cloning, M.K., A.B., D.C. and E.B. carried out protein expression and purification, M.K. performed pull-down assays, L.R. carried out analytical ultracentrifugation, M.K. and J.T. performed S.P.R. experiments, R.H. and V.V. carried out NMR experiments, structure refinement, and deposition, A.B. and P.M. performed HDX/MS experiments, D.C. carried out in vitro kinase assay, E.B. performed protein labeling, E.B. and J.H. carried out GUV preparation and imaging, D.T. and N.S. performed some of the cloning and the cell-based experiments, E.B. supervised the project, E.B., M.K., M.N., V.V. and T.B. wrote the manuscript, all authors contributed to data analysis and commented on the manuscript. Biochemical characterization of the ACBD3:PI4KB complex. (A) Schematic representation of the ACBD3 and PI4KB constructs used for the experiments. ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD). PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes. (B) In vitro pull-down assay. Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3. The inputs and bound proteins were analyzed on SDS gels stained with Coomassie Blue. The asterisks mark the bands corresponding to specific interactions. Cropped gels ran the same experimental conditions are shown. Please, see SI Fig. 9 for original full-length gels. (C) Analytical Ultracentrifugation. AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance. SPR analysis of the PI4KB binding to immobilized ACBD3. Sensorgrams for four concentrations of PI4KB are shown. Structural analysis of the ACBD3:PI4KB complex. (A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region. Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex. The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. ACBD3 is shown in magenta and PI4KB in orange. (C) Top view of the kinase helix. The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta). Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein. Wild type proteins and selected point mutants of both PI4KB and ACBD3 were used. Inputs and bound proteins were analyzed on SDS gels and stained with Coomassie Blue. Cropped gels ran the same experimental conditions are shown. Please, see SI Fig. 9 for original full-length gels. ACBD3 is sufficient to recruit the PI4KB kinase to membranes. (A) GUVs recruitment assay. Top – Virtually no membrane bound kinase was observed when 600 nM PI4KB was added to the GUVs. Bottom – in the presence of 600 nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs. (B) Golgi displacement experiment. Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained. Middle panel: The same experiment performed with GFP alone. Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. Middle panel – The same experiment performed with mRFP-FKBP alone. Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment. – The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex. Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment. Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin. Right – The same experiment performed using the H264A Q domain mutant. ACBD3 indirectly increases the activity of PI4KB. (A) Micelles-based kinase assay – PI in TX100 micelles was used in a luminescent kinase assay and the production of PI4P was measured. Bar graph presents the mean values of PI4P generated in the presence of the proteins as indicated, normalized to the amount of PI4P generated by PI4KB alone. Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor. (C)–Quantification of the GUV phosphorylation assay – Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions. The mean membrane intensity value is relative to the background signal and the difference between the membrane and background signal in the reference system lacking ATP. The error bars stand for SEM based on three independent experiments (also SI Fig. 6). Pseudoatomic model of the PI4KB multiprotein complex assembly. PI4KB in orange, Rab11 in purple, ACBD3 in blue. The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server. The GOLD domain is tethered to the membrane by GolginB1 (also known as Giantin) which is not shown for clarity. Intrinsically disordered linkers are modeled in an arbitrary but physically plausible 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diff --git a/annotated_BioC_JSON/PMC4817029_ann.json b/annotated_BioC_JSON/PMC4817029_ann.json
new file mode 100644
index 0000000000000000000000000000000000000000..b516d39873896157ba5e40955a077b1bcdab78f2
--- /dev/null
+++ b/annotated_BioC_JSON/PMC4817029_ann.json
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+[{"sourceid":"4817029","sourcedb":"","project":"","target":"","text":"Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme. The production of biofuels from renewable sources is an important element of the global strategy for generating sustainable energy with reduced environmental impact. Current technologies for obtaining liquid biofuels and green chemicals rely on the enzymatic digestion of lignocellulosic biomass from a variety of feedstocks. Plant biomass-the most abundant source of carbohydrates on Earth-is primarily composed of cellulose microfibrils surrounded by a hydrated heteropolymeric matrix of hemicellulose and lignin. Plant biomass may be subjected to thermo-chemical pretreatments and enzymatic reactions to produce soluble fermentable sugars. The canonical model of hydrolytic degradation of cellulose requires at least three classes of enzymes. Cellobiohydrolases (CBHs) processively cleave the glycosidic bonds at the reducing and non-reducing ends of cellulose chains in crystalline regions to produce cellobiose. Endoglucanases (EGs) introduce random cuts in the amorphous regions of cellulose and create new chain extremities for CBH attack; thus, these enzymes act synergistically. The released cellobiose molecules are then enzymatically converted into glucose by β-glucosidases. The molecular architecture of glycoside hydrolases (GHs) frequently consists of a catalytic domain (CD), where hydrolysis occurs, and one or more ancillary modules (AMs), which are usually connected by less structured linkers. The most common type of AMs are carbohydrate-binding modules (CBMs), which are able to recognize and bind specific carbohydrate chains. Generally distinct and independent structural domains, the CBMs facilitate carbohydrate hydrolysis by increasing the local concentration of enzymes at the surface of insoluble substrates, thereby targeting the CD component to its cognate ligands. CBMs might also disrupt the crystalline structure of cellulose microfibrils, although the underlying mechanism remains poorly understood. Thus, CBMs enhance the accessibility of CDs to carbohydrate chains to improve enzymatic activity, making them important candidates for the development of effective biomass-degrading enzymes in industrial settings. Although there are examples of active GHs that lack AMs, the majority of the enzymes depend on AMs for activity. In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B). A pioneer work of Sakon et al. revealed that rigid structural extension of the GH9 CD by a type C CBM3 imprints a processive mode of action to this endoglucanase. Further publications showed that CBM-based structural extensions of the active site are important for substrate engagement and recognition. Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module. Removal of the CBM46 caused a ~60-fold reduction of the activity of the enzyme against β-glucans, but showed little or no effect against xyloglucan hydrolysis. Moreover, the CBM46 mediated a significant increase in the BhCel5B activity in plant cell wall settings. Modeling of cellotriose in the negative subsites of the active site of BhCel5B demonstrated the structural conservation of the -1 position, but provided little information about direct interactions between CBM46 and the substrate. It was speculated that β-1,3 kink of the β-glucan might allow the ligand to reach for the CBM46, whereas pure β-1,4 linkages in the backbone of xyloglucan chains would restrict binding to the CD, thus explaining the lack of influence of the CBM46 on the enzymatic activity of BhCel5B against xyloglucans in solution. It was also argued that the CBM46 could potentialize the activity by driving BhCel5B towards xyloglucan-rich regions in the context of the plant cell walls, but no large-scale conformational adjustments of the AMs have been shown to occur or suggested to take part in the enzymatic activity. The mechanisms of ligand binding mediated by large-scale conformational changes in proteins following the induced-fit or conformational selection models have recently attracted considerable attention. Although initially introduced as contradictory theories, these two limiting cases can be unified considering the flux description concept or the extended conformational selection model. While local ligand-induced conformational adjustments have been reported for carbohydrate-active enzymes, cognate ligands recognition and hydrolysis mediated by a large-scale conformational mobility of distinct domains in multidomain settings is uncommon for endoglucanases. Here, we report the crystal structure of a full-length GH5_4 enzyme from Bacillus licheniformis (BlCel5B) that exhibits two AMs (Ig-like module and CBM46) appended to the CD. We structurally and functionally characterize the enzyme using a combination of protein crystallography, small-angle X-ray scattering (SAXS), molecular dynamics computer simulations and site-directed mutagenesis, and show that the AMs and their conformational mobility are essential for the enzymatic activity of BlCel5B. We find that the large-scale conformational adjustments of the distal CBM46 mediated by the Ig-like hinge domain are crucial in active-site assembly for optimal substrate binding and hydrolysis. We propose that the BlCel5B conformational selection/induced-fit mechanism of hydrolysis represents a novel paradigm that applies to several GH5_4 members and, possibly, to a number of other multidomain GHs. Results BlCel5B Crystal Structure BlCel5B crystals in the substrate-free form and complexed with cellopentaose (C5) were obtained and diffracted to 1.7 Å and 1.75 Å resolutions, respectively (Supplementary Table 1). The substrate-free and complexed structures exhibited no substantial conformational differences (with the exception of the substrate). Because of minor variations in the loops located distal to the substrate-binding site, a root mean squared deviation (rmsd) of 0.33 Å between the complexed and substrate-free structures was observed. A single protein chain occupies the asymmetric unit, and most of the residues were built, with the exception of the first 17 residues and those in the loop between L398 and P405 due to weak electron density. The BlCel5B structure comprises three distinct domains: an N-terminal CD (residues 18 to 330), an Ig-like module (residues 335 to 428) and a family 46 CBM (residues 432 to 533) (Fig. 1A,B). Similarly to other members of the GH5 family, the CD of BlCel5B has a typical TIM barrel fold with eight inner β-strands and eight outer α helices that are interconnected by loops and three short α helices. Very short linkers, D429-D430-P431 and V331-P332-N333-A334, connect the CBM46 to the Ig-like module and the Ig-like module to the CD, respectively. Both Ig-like module and CBM46 have a β-sandwich fold composed of two β-sheets of four and three antiparallel β-strands interconnected by loops and a short α helix between strands β3 and β4 (Fig. 1C). A structural comparison between the Ig-like module and the CBM46 using the Dali server yielded an rmsd of 2.3 Å and a Z-score of 10.2. However, despite their structural resemblance, these modules share only 17% sequence identity. A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd = 1.3 Å and Z-score = 15.3. The CBM46 from BhCel5B is the most structurally similar to BlCel5B CBM46, with rmsd = 1.6 Å and Z-score = 12.4. The sequence identity relative to BhCel5B, however, is low (28% for Ig-like and 25% for CBM46). The Ig-like module, adjacent to the CD, contains only one tyrosine (Y367) exposed to solvent and no tryptophan residues. Because aromatic residues play a major role in glucose recognition, this observation suggests that substrate binding may not be the primary function of Ig-like module. In contrast, the CBM46 has three tryptophan residues, two of which face the CD substrate binding site (Fig. 1A), indicating that it may be actively engaged in the carbohydrate binding. Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule. The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end. The ability of BlCel5B to cleave C5 into glucose and C4 molecules in solution was demonstrated by enzymatic product profile mass spectrometry analysis (Fig. 2A). The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D). These residues belong to the CD and are conserved in the GH5 family. BlCel5B enzymatic activity BlCel5B exhibits optimum activity toward carboxymethylcellulose (CMC; 8.7 U/mg) at a pH of 4.0 and 55 °C and retains approximately half of its maximum activity at 80 °C, demonstrating considerable thermal stability (Fig. 2B,C). BlCel5B is also active on β-glucan (34 U/mg), lichenan (17.8 U/mg) and xyloglucan (15.7 U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-β-mannan or the insoluble substrate Azo-Avicel. Kinetic parameters were calculated assuming Michaelis-Menten behavior with CMC as substrate: KM = 1.78 g L−1 and Vmax = 1.41 × 10−4 g s−1 mg protein−1 (Fig. 2D). Although BlCel5B is not a highly active enzyme against one specific substrate as compared to others GH5_4, it has the advantage of being active against different substrates with β-1,3 and/or β-1,4 glycosidic linkages. To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A. These mutants were expressed and purified as described for the wild-type enzyme. Strikingly, neither of the deletion variants exhibited detectable activity toward any of the substrates tested using full-length BlCel5B (Table 1), demonstrating that the Ig-like module and the CBM46 are essential for BlCel5B activity. Thermal shift assays were conducted to confirm structural stability of the mutants (Supplementary Fig. 1). All of the constructs showed similar melting temperatures: 62 °C for BlCel5B, 58 °C for BlCel5BΔCBM46, 56 °C for BlCel5BΔIg-CBM46, 65 °C for BlCel5BW479A and 59 °C for BlCel5BW479A, thus confirming their proper overall fold. We also examined the function of the CBM46 inner surface residues W479 and W481 (Fig. 1A) in BlCel5B activity by performing enzymatic assays with W479A and W481A mutants. Both mutations reduced enzymatic activity toward all tested substrates (Table 1), with W481A having a stronger effect than W479A (~64% vs. 79% activity relative to wt BlCel5B using β-glucan and ~10% vs. 50% using CMC). This indicates that CBM46 must interact with the substrate via residues W479 and W481. However, since the BlCel5B crystal structure exhibits no close contact between these residues and the substrate, these results suggest the existence of large-amplitude interdomain motions that may enable direct interactions between CBM46 and the carbohydrate. BlCelB5 dynamics and binding-site architecture Molecular dynamics (MD) simulations were performed to investigate the conformational mobility of BlCel5B. In the simulations of the crystal structure for BlCel5B bound to C4, the substrate dissociates from the protein within the first 100 ns of the simulation time (Supplementary Fig. 2A). This observation suggests that cellotetraose does not exhibit detectable affinity for this specific BlCel5B conformation in solution, as one might otherwise expect for a reaction product. No changes beyond local fluctuations were observed in any of the three BlCel5B domains within the time scale of these runs (400 ns; Supplementary Fig. 2B). However, the CBM46 and Ig-like domains did exhibit rigid body-like motions relative to the CD, with rmsd values around 2.3 Å and 1.8 Å, respectively, suggesting that BlCel5B may execute large-amplitude interdomain motions over longer time scales (Supplementary Fig. 2B,C). Accordingly, simulations were then performed using accelerated molecular dynamics (aMD) techniques to probe BlCel5B interdomain motions. aMD enhances conformational sampling by raising the basins of the dihedral potential energy surface without affecting the general form of the atomistic potential, thereby increasing transition rates between different local minima. aMD trajectories corresponding to more than 1.0 μs of conventional MD runs were generated. During these simulations, we observed occlusive conformations between CBM46 and CD that resulted in a rearrangement of the enzyme’s architecture around the active site (Video S1). Figure 3A shows BlCel5B in the crystallographic conformation (red) and in a selected configuration obtained with aMD (blue) in the absence of the substrate. Interdomain motions were gauged by the time evolution of the distance between the α carbons of residues I120 and E477 (represented as spheres in Fig. 3A), belonging to the CD and CBM46, respectively. Figure 3C shows that the I120-E477 distance (red curve) gradually decreases from ~35 Å to ~7 Å within the first half of the 1.0 μs aMD trajectory, indicating a transition between the semi-open (crystallographic) and occluded (aMD sampled) configurations. During the second half of the aMD simulation, the full-length enzyme remained in the closed conformation, with the CBM46 covering the carbohydrate-binding site. These results suggest that BlCel5B undergoes large-scale interdomain movements that enable interactions between CBM46 and the substrate bound to the CD. To study the interactions of BlCel5B with a non-hydrolyzed glucan chain, we built a model structure with a cellooctaose (C8) chain spanning the entire positive (+1 to +4) and negative (−4 to −1) subsites of the enzyme. Starting from the crystallographic BlCel5B conformation, the C8 molecule deviated significantly from the active site and assumed a non-productive binding mode (Supplementary Fig. 2D). This observation suggests that the open conformation of BlCel5B is not able to hold the substrate in a position suitable for hydrolysis (Supplementary Fig. 2E). However, after subjecting the BlCel5B-C8 complex to a 0.5 μs aMD simulation with harmonic restraints on the C8 chain to prevent it from deviating from the productive binding mode, the CBM46 readily closed over the CD and trapped the C8 chain in position for hydrolysis (Fig. 3B). In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C). This BlCel5B-C8 configuration remains stable over an additional 500 ns of conventional MD simulation with no restraints (Fig. 3C cyan line, Supplementary Fig. 2E,F). A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D. Together, these results indicate that CBM46 is a key component of the catalytic active complex, providing an explanation as to why CBM46 is essential for the enzymatic activity of BlCel5B. To enable substantially longer time scales compared to atomistic simulations, we further explored the dynamics of BlCel5B using coarse-grained MD (CG-MD) simulations. We performed three independent ~120 μs CG-MD simulations, for a total of approximately 360 μs of sampling. The distance between the α carbons of two residues centrally positioned in the CD and CBM46 (Fig. 4A) was monitored, and the results shown in Fig. 4B indicate that the wide-amplitude events described above frequently appear in this time scale. The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure. BlCel5B conformers fit the SAXS envelope SAXS experiments were conducted to assess BlCel5B conformational states in solution, and the results revealed the enzyme in its monomeric form, with average values of Rg = 27.17 Å and Dmax = 87.59 Å (Supplementary Table 2). The ab initio dummy atom model (DAM) demonstrated that the SAXS-derived BlCel5B molecular envelope could not be single-handedly filled by any of the main conformational states encountered in the simulations (Fig. 4D). It is known that a Kratky plot exhibits a peak with an elevated baseline at high q for a monodisperse system composed of multi-domain particles with flexible extensions. Indeed, an elevation of the baseline toward a hyperbolic-like curve was observed for BlCel5B, indicating a considerable degree of molecular mobility in solution (Supplementary Fig. 3). Thus, the conformational heterogeneity of the enzyme can be decomposed in structural terms as a combination of conformational states identified in our crystallographic and MD studies. We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope. The resulting average scattering curve from this model fits the experimental protein scattering intensity, with χ = 1.89 (Supplementary Fig. 3). GH5_4 phylogenetic analysis To date, there are 427 sequences classified as subfamily 4 members in the CAZy database. After the exclusion of partial sequences and the suppression of highly identical members (higher than 90% identity), 144 sequences containing between 277 and 400 residues were aligned and used to construct a phylogenetic tree (Supplementary Fig. 4A). According to PFAM database conserved domain classification, 128 GH5 enzymes have an architecture consisting of an N-terminal catalytic module, a CBM_X2 module and an unknown module of approximately 100 residues at the C-terminus (Supplementary Fig. 4B). Of these, 12 enzymes have an additional CBM1, and 5 have a CBM2 at the N-terminal region. Based on this PFAM architecture and CAZy subfamily classification, all the 144 enzymes (including BlCel5B) belong to the GH5_4 subfamily and group together in the same branch of the phylogenetic tree, evidencing a common ancestor. These results support the hypothesis that the enzymes may employ the same mechanism by which ligand binding is mediated by an extensive conformational breathing of the enzyme that involves the large-scale movement of CBM46 around the Ig-like module (CBM_X2) as a structural hinge. Discussion Growing interest in biotechnological applications of enzymes exhibiting activity toward lignocellulosic biomass has sparked efforts in the discovery and development of novel enzymes, as well as the search for a deeper understanding of their mechanisms of action. Here, we elucidate the trimodular molecular architecture of the full-length BlCel5B, a member of the GH5_4 subfamily, for which large-scale conformational dynamics appears to play a central role in its enzymatic activity. Full-length BlCel5B is active on both cellulosic and hemicellulosic substrates and auxiliary modules are crucial for its activity. Most carbohydrate-active enzymes are modular and consist of a catalytic domain appended to one or more separate AMs. AMs, such as CBMs, typically recognize carbohydrates and target their cognate catalytic domains toward the substrate. Because the structural analysis of the protein is challenging if the linkers connecting the structural subunits of the enzyme are long and flexible, the standard approach is to study the domains separately. In this work, a combination of protein crystallography, computational molecular dynamics, and SAXS analyses enabled the identification of a new conformational selection-based molecular mechanism that involves GH5 catalytic domain and two AMs in full-length BlCel5B. We observed that the BlCel5B distal CBM46 is directly involved in shaping the local architecture of the substrate-binding site. Although the CD alone appears unable to bind the substrate for catalysis, the AMs exhibit open-close motions that allow the substrate to be captured in a suitable position for hydrolysis. Here, we advocate that large-amplitude motions of AMs are crucial for assembling the enzyme into its active conformation, highlighting a new function of CBMs. This mechanism of substrate binding closely resembles the extended conformational selection model, with the induced-fit mechanism of reaction as its limiting case. To the best of our knowledge, this enzymatic mechanism has not been proposed previously for any GH. The CD binding site of BlCel5B is open and relatively flat and is thus barely able to properly hold the substrate in position for catalysis without assistance from the CBM46. In contrast, other GH5s belonging to subfamily 4 listed in the Protein Data Bank exhibit a deep binding cleft or tunnel that can effectively entrap the substrate for catalysis (Fig. 5). Due to the marked interdomain conformational rearrangement observed in our simulations, the CBM46 generates a confined binding site in BlCel5B that resembles the binding site architecture of the other GH5 enzymes that lack AMs. Thus, BlCel5B appears to have adopted a strategy of CBM46-mediated interactions for proper functioning. Although the homologous BhCel5B has the same domain architecture of BlCel5B and belongs to the same subfamily (a comparison of the sequence and structure of BlCel5B and BhCel5B is presented in Supplementary Fig. 5), its binding site exhibits important differences that may impact the catalytic mechanism. The BhCel5B binding site is V-shaped and deeper than the BlCel5B binding site (Figs 5 and 6). This is due to the loop between residues F177 and R185 from BhCel5B (absent in the BlCel5B), which contains residue W181 that forms part of the binding cleft (Fig. 6). Consistently, although BhCel5B CBM46 is important for β-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates. A closer inspection of results of the phylogenetic analysis, more specifically of the clade composed by GH5_4 enzymes with trimodular architecture (Supplementary Fig. 4C), reveals subclades whose main characteristic is the varying length of the loop located between residues 161 and 163 (BlCel5B residue numbering). Therefore, our results show that BlCel5B represents a smaller group of enzymes that are completely dependent on its AMs for hydrolysis of plant cell wall polysaccharides, and that the underlying mechanism may rely on large-scale interdomain motions. The amino acid sequence of the BlCel5B Ig-like module is recognized by BLASTP as belonging to CBM_X2, a poorly described group that has been compared with CBM-like accessory modules without a defined function. Despite the similarity of BlCel5B Ig-like module to CBMs, it lacks an identifiable aromatic residue-rich carbohydrate-binding site. Nonetheless, according to our results, the Ig-like module seems to play an important function as a structural hinge, dynamically holding the CBM46 and CD in positions that are appropriate for enzymatic activity. Based on the results of our crystallographic, computer simulation, and SAXS structural analyses, as well as site-directed mutagenesis and activity assays, we propose a molecular mechanism for BlCel5B substrate binding, which might apply to other GH5_4 subfamily enzymes that share this tri-modular architecture. BlCel5B can be found in several different conformational states ranging from CBM46/CD closed (or occluded) to extended conformations (Fig. 7). In extended configurations, the substrate may dock at the shallow substrate binding site of CD in one of the semi-closed conformations of the enzyme; however, its binding is properly stabilized for hydrolysis only with the aid of induced-fit repositioning mediated by CBM46. After cleavage, the intrinsic dynamics of BlCel5B would eventually allow the opening of the active site for product release. The proposed mechanism is consistent with our mutagenesis and enzymatic activity assays, which show that the Ig-like module and CBM46 are indispensable for BlCel5B catalytic activity and, together with the CD, form the unique catalytic domain of the enzyme. These experiments reveal a novel function for CBMs in which they are intimately involved in the assembly of the active site and catalytic process. Computer simulations suggest that large-scale motions of the CBM46 and Ig-like domains mediate conformational selection and final induced-fit adjustments to trap the substrate at the active site and promote hydrolysis. SAXS data support the modeling results, providing compelling evidence for highly mobile domains in solution. Methods Cloning, Expression and Purification The gene encoding BlCel5B (GenBank: AAU23417.1) was amplified from Bacillus licheniformis genomic DNA (ATCC 14580) without the predicted signal peptide sequence (nucleotides 1 to 81) using the primers Blcel5B_Fw and Blcel5B_Rv (Supplementary Table 3). The fragment was cloned into the expression vector pETTRXA-1a/LIC by ligation-independent cloning (LIC), as described elsewhere. The same method was used for construction of domain deletions. For Ig-like + CBM46 deletion, Δ(Ig-CBM46), the fragment encoding the CD (nucleotides 82 to 1086) was amplified using the primers Blcel5B_Fw and Blcel5BΔ1087-1683_Rv. For CBM46 deletion, ΔCBM46, the fragment encoding the CD + Ig-like (nucleotides 82 to 1377) was amplified using the primers Blcel5B_Fw and Blcel5BΔ1378-1683_Rv (Supplementary Table 3). Both fragments were cloned into pETTRXA-1a/LIC. The wt protein BlCel5B, mutated proteins and AM deletions were expressed in E. coli Rosetta2 (DE3) strain. The cells were grown at 37 °C and 150 RPM in Luria Bertani Broth medium supplemented with 50 μg/mL kanamycin to an A600 of 1.5–2.0, after which the temperature was reduced to 20 °C and protein expression was induced with 1 mM IPTG for 6 h. The extract was then loaded onto a NiNTA resin (Qiagen) equilibrated with a washing buffer (5 mM imidazole, 100 mM NaCl, 50 mM Tris-HCl, pH 8.0). Non-absorbed material was washed with ten times column volume with washing buffer and the purified protein was eluted with 200 mM imidazole, 100 mM NaCl, 50 mM Tris-HCl at pH = 7.0. His6 tag was removed by overnight digestion with TEV (Tobacco Etch Virus) at 4 °C, and untagged protein was purified by gel filtration through a HiLoad 16/60 Superdex 200 column in buffer containing 50 mM NaCl, 25 mM Tris-HCl at pH 7.0. Site-directed Mutagenesis The BlCel5B point mutations W479A and W481A were obtained by the inverse PCR method of site-directed mutagenesis. Phusion® “High-Fidelity” DNA polymerase (NEB, USA) was used for amplifications with the plasmid pETTRXA-1a/LIC-Blcel5B as a template. Mutagenic primers Blcel5BW479A_Fw/Rv and Blcel5BW481A_Fw/Rv (Supplementary Table 3) were generated by HTP-OligoDesigner tool (http://www.ifsc.usp.br/htpoligo/). Activity Assays Enzymatic activity assays were performed by a colorimetric method using the 3,5-dinitrosalicylic acid (DNS), with glucose being a standard for the calibration curves. Assays of optimal temperature and pH were performed in triplicate with 1% medium-viscosity CMC as the substrate. For optimal temperature, the reaction mixture containing 10 μL of enzyme at 0.1 mg/mL, 50 μL of 1% (w/v) CMC and 40 μL of 50 mM sodium citrate buffer (pH 5.0) was incubated at 30 to 80 °C for 15 min and stopped by adding 100 μL of DNS solution. After this, the mixture was incubated again for 5 min at 100 °C and the absorbance was measured at 540 nm with a spectrophotometer. For optimal pH determination, the same amount of enzyme and substrate were diluted in 40 mM acetate/borate/phosphate buffer (ABF) with different pH values ranging from 2.0 to 10.0. The reactions were carried out under the predetermined optimal temperature. The substrate specificity of the enzyme was determind using rye arabinan, xyloglucan, β-glucan, galactomannan, lichenan, β-mannan, Azo-Avicel and CMC as substrates. The substrates were diluted in water to 1% (w/v), and the reaction mixture was composed of 10 mL of purified enzyme at a concentration of 0.1 mg/mL, 0.4 mL of 50 mM sodium citrate buffer at pH 5.0, and 0.5 mL of 1% (w/v) substrate aqueous solution. The reaction was incubated at 50 °C for 15 min, followed by treatment with DNS as mentioned above. Enzyme unit was defined as the amount of enzyme that produces 1.0 μM of glucose in one minute for each substrate. The kinetic parameters were determined by increasing concentrations of CMC. Reactions were performed in 50 mM sodium citrate buffer (pH = 4.0) at 50 °C, and measured by DNS method as well. Kinetic constants were determined by non-linear regression using OriginPro 8.0. Thermal Shift Assays The thermal denaturation assays were performed using a Real Time PCR Machine (Stratagene Mx3005P) as described by Dupeux and co-workers. Briefly, the enzymes were diluted to 10 μM in 50 mM sodium citrate buffer (pH = 4.0) containing 1x SYPRO orange dye (Thermo Fisher Scientific). The fluorescence emission of the probe was monitored (excitation and emission at 492 and 516 nm, respectively) varying the temperature between 25 and 75 °C with the rate of 1 °C/min. Cellopentaose Cleavage Experiment The full-length BlCel5B and AM deletion constructs were tested for product formation from cellopentaose. Cellopentaose (1.0 mM) was incubated with 25 μg of purified enzyme in 10 mM ammonium bicarbonate buffer (pH 7.0) in a total volume of 50 μL. The reaction was incubated for 90 min at 50 °C and then stopped by treatment at 100 °C for 5 min. After centrifugation for 10 min at 16,000 g the samples were subjected to MALDI/TOF-MS. Samples were supplemented with NaCl to a final concentration of 20 mM and 1 μL of the supernatant was co-crystallized with 1 μL 2,5-dihydroxybenzoic acid (10 mg/mL) in acetonitrile 30% and spotted on the target plate. The products were analyzed on Microflex LT MALDI-TOF (Bruker Daltonics) operating in positive ion mode. A single spectrum was obtained by averaging four independent spectra generated by 300 laser shots at 60% potency. Crystallization, Data Collection, and Structure Determination After purification, BlCel5B was concentrated to 10 mg/mL for crystallization trials. Crystallization screens were set up using the sitting-drop vapor-diffusion method on a Cartesian PixSys 4200 (Genomic Solutions, United Kingdom) in a 96-well plate with drops formed by 100 nL protein solution plus 100 nL reservoir solution. The commercial kits Crystal Screen and Index (Hampton) were used as initial conditions. Crystals were grown at 18 °C between 3 and 7 days, and screened for diffraction. Crystals were supplemented with cryoprotection solution, flash cooled in liquid nitrogen and diffraction data were collected at 100 K, at beamline ID23-1 (wavelength of 0.97 Å) from the European Synchrotron Radiation Facility (Grenoble, France). A crystal grown in condition containing 22.5% PEG 4000, 14% isopropanol and 0.1 M sodium citrate, pH 6.0, was selected to collect diffraction data to 1.7 Å resolution. The complex of the enzyme with substrate was obtained by crystal soaking with five times molar excess of cellopentaose for 24 hours. Diffraction data for the complexed enzyme were collected at 1.75 Å resolution. Data were integrated with iMosflm and scaled with Aimless. The structure was solved by molecular replacement with Phaser using an endoglucanase from Clostridium cellulovoran (PDB code: 3NDY) as the search model. Coot was used for density fitting, and refinement was performed with PHENIX. Atomistic simulations We took the BlCel5B structure complexed with cellotetraose as the starting configuration for the MD simulations. The missing residues were taken from the apo BlCel5B structure after structural alignment using the LovoAlign server. Hydrogen atoms were then added according to the protonation states determined at the optimum pH of 4.0 using the H + + server. The following residues were considered protonated: H55, H77, D89, E96, E103, H114, E129, E159, E197, D198, E202, H205, E208, D211, H220, E245, E248, E260, H278, H292, D306, E312, E371, E375, E476, H416, E477, E489, D497, and E524. The remaining protonatable residues were considered in the standard protonation state. The BlCel5B-cellotetraose complex was then immersed in a rectangular simulation box of dimensions such that a solvent layer at least 16 Å thick surrounded the protein. The simulation box, built with Packmol, also contained 0.10 M NaCl aqueous solution with excess counter ions to keep the system electrically neutral. The final system comprised approximately 85500 atoms. The simulations were performed using NAMD with the CHARMM force field and the TIP3P water model. Periodic boundary conditions were employed, using particle mesh Ewald to handle electrostatics and a 12-Å cutoff radius for truncating short-range potentials. Bonds involving hydrogen atoms were constrained at their equilibrium lengths and a time step of 2 fs was used to integrate the equations of motion. The simulations were carried out under constant pressure and temperature of 1 atm and 310 K, respectively, employing the Langevin barostat and thermostat. Accelerated Molecular Dynamics In accelerated molecular dynamics, the trajectory is propagated on a modified potential aimed to enhance conformational sampling. Whenever the potential energy drops below a given threshold E, a boost ΔV(r) is applied, so that the escaping rates of local minima increase. When the potential energy gets over the threshold E, the system evolves on the original energy surface. This method has the advantage of conserving the general shape of the potential energy surface and of requiring no prior definition of reaction coordinates, so the system is allowed to explore freely its conformational space. Here, we restricted the energy boost only to the dihedral potential energy, as changes in torsion angles are the main source of conformational changes in proteins. The energy boost assumes the form of equation (1) that depends on the energy threshold E and on the parameter α – which modulates the shape of the potential energy surface where the boost is applied. We set the parameters E and α according to previous studies, which recommend that E equals the average dihedral energy obtained from a conventional MD simulation plus 4 kcal/mol times the number of residues, and α equals 0.8 kcal/mol times the number of residues. The average dihedral energy was 2275.5 kcal/mol and the BlCel5B has 516 residues, so we set E = 2275.5 + 4 × 516 = 4339.5 kcal/mol and α = 0.8 × 516 = 418.8 kcal/mol. Simulation procedures BlCel5B-cellotetraose – Having built the system, we carried out the following steps for equilibration: (i) 1000 steps of energy minimization followed by 100 ps of MD simulation with all non-solvent heavy atoms fixed; (ii) same as (i), but with only the α carbons fixed; (iii) 5 ns of MD with all atoms free. After these preliminary steps, a trajectory lasting 400 ns was generated using conventional MD and then the aMD dihedral boost was applied for additional 1.0 μs. After 100 ns of conventional MD, the cellotetraose dissociated and the simulation began to represent the dynamics of unbound state of BlCel5B. BlCel5B-cellooctaose To build the cellooctaose chain, we extended the original cellotetraose chain in the crystal structure with 4 additional glucose residues spanning regions around the BlCel5B positive subsites. Then, we submitted the system to the following procedure: (i) 1000 steps of energy minimization followed by 1 ns of MD keeping all the non-solvent heavy atoms fixed, except the 4 modeled glucose residues of the cellooctaose chain; (ii) same as step (i), but with only the α carbons fixed; (iii) 1 ns of MD with only the non-modeled glucose residues fixed. We then performed a 200-ns-long MD with three harmonic potentials involving cellooctaose chain: first, between C3 atom (CHARMM atom names) of the second glucose residue from the cellooctaose non-reducing end and the CD2 atom of the W47 tryptophan residue; second, between the OH3 atom of the forth glucose residue from the cellooctaose non-reducing end and HE2 atom of the H113 histidine residue; and third, between the HE2 atom of the catalytic residue E159 and O4 glycosidic oxygen between the fourth and fifth glucose unit of the cellooctaose chain. After these preliminary relaxation steps, the harmonic potentials were removed and the trajectory was propagated by 400 ns using MD. To get a model of the BlCel5B-cellooctaose complex in the closed conformation, we took the configuration after 80 ns of the restrained 200-ns MD simulation as the starting point for a 500-ns-long restrained aMD simulation, in which the CBM46 moved towards the CD in the presence of the harmonically-restrained cellooctaose chain. After this procedure, we released the restraints and propagated the closed BlCel5B-cellooctaose complex for additional 500 ns of conventional, restraint-free MD simulation. Coarse-grained MD simulations The coarse-grained model was constructed from the minimized all-atom protein. We have used the domELNEDIN CG model for the protein. In this representation, an elastic network is used within each domain as a structural scaffold in order to maintain the overall shape of the protein, and a slightly modified version of MARTINI CG model describes the interactions involving beads not connected by harmonic springs. The delimitation of each domain was quite clear considering the short linkers connecting them and the recognition of their structural patterns in databases. We assumed CD, Ig-like module, and CBM46 as consisted of residues 18–331, 332–430, and 431–533, respectively. Therefore, there were elastic network bonds only within these domains (domELNEDIN CG model in Supplementary Fig. 6A). The protonation state of each residue bead in the protein was the same adopted in the atomistic simulations. The system was then solvated by 10000 standard MARTINI CG water beads, including 10% of antifreeze particles. Also, 58 chloride and 48 sodium ions were added for charge neutrality. The size of final system was 109 Å × 109 Å × 109 Å. Preliminary simulations were performed to test the elastic network (EN) parameters. We have tested six different ENs in 100 ns of simulations, using combinations of cut-off distance (Rc) of 8 Å and 9 Å with spring force constant (ks) of 500, 800 and 1000 kJ mol−1 nm−2. The time evolution of root mean square deviation relative to the crystal structure as well as the mobility profile of the protein in these simulations were compared to the correspondent data from a 100 ns atomistic simulation. From this procedure, the parameters Rc = 9 Å and ks = 500 kJ mol−1 nm−2 resulted in the best match between atomistic and coarse-grained simulations (Supplementary Fig. 6B). The coarse-grained simulations were carried out using GROMACS. Periodic boundary conditions were employed. Van der Waals interactions were shifted to zero in the range 0.9–1.2 nm, and the electrostatic interactions, in the range 0.0–1.2 nm. The simulations were performed in the isothermal-isobaric ensemble (NpT), employing the Berendsen thermostat and barostat for temperature and pressure control, respectively, with time constants τT = 0.5 ps and τp = 1.2 ps. The CG simulations were carried out using the following protocol: the system was first minimized for 1000 steps using the steepest descent method. Then, it was submitted to a relaxation procedure comprising gradual increasing in time step or temperature. In the first stage of relaxation, the protein beads were restrained with a 1000 kJ mol−1 nm−2 force constant and a 50 ps simulation was carried out at 50 K, using the short time step of 1 fs. In the second stage, the time step was increased up to 5 ps lasting 1000 ps of simulation time. In the last stage of relaxation, all the system is released to move and it underwent a gradual increase in temperature, consisting on five segments of 100 ps at 50, 100, 150, 200 and 310 K. After achieving the desired temperature of 310 K, we performed three production simulations using 20-fs timestep. We have used a random number generator for assigning velocities to generate three independent simulations. In general, smoothing of the energy surface in CG model makes the time scales faster. A speed up factor of 4 is typically employed to rescale the time scale of MARTINI CG systems. Therefore, all CG simulations times described here and in the main text are effective times, i.e., 4× simulation time. Small Angle X-ray Scattering SAXS data were collected at the SAXS2 beamline of the Brazilian Synchrotron Light Laboratory-LNLS (Campinas, Brazil) on a bi-dimensional position sensitive CCD detector (MarResearch, USA) using the radiation wavelength 1.54 Å. The sample-detector distance of 1000 mm allowed covering the momentum transfer range 0.01 Å−1 \u003c q \u003c 0.35 Å−1 (q = 4πsin θ/λ, where 2θ is the scattering angle). The protein samples were prepared in McIlvaine’s buffer at 50 mM, pH 5 and 20 °C. In each measurement, two successive frames of 300s were recorded for each sample at 1 and 2 mg/mL to monitor radiation damage. The patterns were integrated using the FIT2D program. The comparative analysis for each scattering curve at 1 and 2 mg/mL of BlCel5B (data not shown), as well as the radius of gyration values (Rg), indicated that concentration and aggregation effects did not exist. The linearity of the Guinier plot indicated that the preparation was monodisperse. The radius of gyration of the molecules (Rg) was estimated by two methods, using the Guinier equation-I(q) = I(0).exp[(−q2.Rg2)/3], q.Rg \u003c 1.3- and also with the inverse Fourier transform in GNOM. The same program was used to obtain the distance distribution function P(r) and the maximum diameter Dmax. Ten independent dummy atom models (DAMs) were restored by the ab initio proceeding implemented in DAMMIN package. The best model, selected using normalized spatial discrepancy parameter computed by DAMAVER program, was superimposed on the crystallographic model with the SUPCOMB. Then, based on the enzyme conformations reported by MD and protein crystallography, the computed X-ray scattering profile was fitted to a given experimental SAXS data by minimizing the χ function in the FOXS program. To assess the inter-domain information, the contribution of individual conformer and the flexibility of BlCel5B, we proceed in two approaches. First, the theoretical profiles and experimental data comparison was performed to infer the best-fit conformation of the ensemble-based analysis by the ensemble optimization method – EOM, which assumes coexistence of a range of conformations in solution for which an average scattering intensity fits the experimental SAXS data; all models were generated with the three individual domains (Ig-like, CBM46, and CD) free to randomly move in order to cover the entire conformational space. The second approach was based on a fractional volume calculation from three conformation members extracted from the MD simulations, each with a distinct scatter curve. OLIGOMER provided solution of a system of linear equations between the experimental and generated conformations by MD. The simulated scattering curves from the MD and crystallographic models were obtained using the CRYSOL. Phylogenetic assignment Sequences for all GH5 members, in which only the catalytic domain were considered, were downloaded from PFAM database and their classification into subfamilies were obtained within the CAZy database. The sequences belonging to subfamily 4 were selected and those that had over 90% identity or represented partial coverage were rejected. Based on their multiple sequence alignment, the phylogenetic tree was constructed using the maximum likelihood method implemented in the MEGA program version 6.06. One hundred Bootstrap replications were performed to examine the reliability of the phylogenetic tree. Additional Information How to cite this article: Liberato, M. V. et al. Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism. Sci. Rep. 6, 23473; doi: 10.1038/srep23473 (2016). Supplementary Material Deconstruction of lignocellulosic biomass to fuels and chemicals Carbohydrate-binding modules: fine-tuning polysaccharide recognition Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance X-ray structure and molecular dynamics simulations of endoglucanase 3 from Trichoderma harzianum: structural organization and substrate recognition by endoglucanases that lack cellulose binding module Structure and mechanism of endo/exocellulase E4 from Thermomonospora fusca Structural insights into the catalytic mechanism of a family 18 exo-chitinase Phosphoglucan-bound structure of starch phosphatase Starch Excess4 reveals the mechanism for C6 specificity Family 46 Carbohydrate-binding Modules Contribute to the Enzymatic Hydrolysis of Xyloglucan and β-1,3-1,4-Glucans through Distinct Mechanisms The conformation and function of a multimodular glycogen-degrading pneumococcal virulence factor Application of a Theory of Enzyme Specificity to Protein Synthesis Molecular recognition by induced fit: how fit is the concept? Enzymes with lid-gated active sites must operate by an induced fit mechanism instead of conformational selection Conformational selection or induced fit: a flux description of reaction mechanism Induced fit, conformational selection and independent dynamic segments: an extended view of binding events The crystal structure of a family 5 endoglucanase mutant in complexed and uncomplexed forms reveals an induced fit activation mechanism Insights into ligand-induced conformational change in Cel5A from Bacillus agaradhaerens revealed by a catalytically active crystal form Complex structures of Thermoactinomyces vulgaris R-47 alpha-amylase 2 with acarbose and cyclodextrins demonstrate the multiple substrate recognition mechanism Dali server: conservation mapping in 3D Structural Features of a Bacteroidetes-Affiliated Cellulase Linked with a Polysaccharide Utilization Locus Accelerated molecular dynamics: a promising and efficient simulation method for biomolecules Structural characterization of flexible proteins using small-angle X-ray scattering Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law The carbohydrate-active enzymes database (CAZy) in 2013 Pfam: the protein families database Crystal structure of the catalytic domain of a bacterial cellulase belonging to family 5 A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH12 Substrate binding of a GH5 endoglucanase from the ruminal fungus Piromyces rhizinflata Solution structure of the module X2 1 of unknown function of the cellulosomal scaffolding protein CipC of Clostridium cellulolyticum High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC) A novel simple and rapid PCR-based site-directed mutagenesis method Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar A thermal stability assay can help to estimate the crystallization likelihood of biological samples iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM An introduction to data reduction: space-group determination, scaling and intensity statistics Phaser crystallographic software Features and development of Coot PHENIX: a comprehensive Python-based system for macromolecular structure solution Convergent algorithms for protein structural alignment H++: a server for estimating pKas and adding missing hydrogens to macromolecules PACKMOL: a package for building initial configurations for molecular dynamics simulations Scalable molecular dynamics with NAMD All-atom empirical potential for molecular modeling and dynamics studies of proteins CHARMM Additive All-Atom Force Field for Glycosidic Linkages between Hexopyranoses Comparison of simple potential functions for simulating liquid water Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems Implementation of Accelerated Molecular Dynamics in NAMD Routine Access to Millisecond Time Scale Events with Accelerated Molecular Dynamics The MARTINI Coarse-Grained Force Field: Extension to Proteins Combining an Elastic Network With a Coarse-Grained Molecular Force Field: Structure, Dynamics, and Intermolecular Recognition Conformational flexibility of the leucine binding protein examined by protein domain coarse-grained molecular dynamics GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation A buffer solution for colorimetric comparison  Determination of the regularization parameter in indirect-transform methods using perceptual criteria Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing FoXS: a web server for rapid computation and fitting of SAXS profiles PRIMUS : a Windows PC-based system for small-angle scattering data analysis CRYSOL – a Program to Evaluate X-ray Solution Scattering of Biological Macromolecules from Atomic Coordinates MEGA6: Molecular Evolutionary Genetics Analysis version 6.0 Author Contributions M.V.L. and I.P. designed the experiments; C.M.C. performed the gene mutations and cloning; M.V.L. and V.O.A.P. expressed and purified the enzymes and characterized the enzymatic activities; M.V.L. crystallized and determined the crystal structures, with data collection supervised by A.P.; M.A.K. performed mass spectrometry experiment; E.A.A. and M.O.N. collected and treated SAXS data. R.L.S., E.T.P. and M.S.S. designed the computer simulations; R.L.S. performed Accelerated Molecular Dynamics; E.T.P. performed Coarse-grained MD Simulations; M.V.L., R.L.S., M.S.S. and I.P. wrote the manuscript with the input from all the other authors; M.S.S. and I.P. supervised the project. Crystal models of BlCel5B. Complete structure is shown as a cartoon illustration in (a) and a van der Waals surface in (b). The CD module (red) has a typical TIM-barrel fold, and its substrate-binding site is adjacent to CBM46 (blue). Despite the proximity of the binding site in the crystallographic model, the CBM46 residues W479 and W481 are distant from the substrate cellotetraose (yellow). The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray. BlCel5B enzymatic activity characterization. (a) MALDI/TOF-MS spectra of the products released after incubation of BlCel5B and its two deletion constructs (ΔCBM46 and ΔIg-CBM46) with the substrate cellopentaose (C5). The first three spectra show the substrate, enzyme and buffer controls. The forth spectrum reveals that full length BlCel5B is capable of enzymatic hydrolysis of C5 into smaller oligosaccharides such as C4, C3 and C2. The last two spectra show that the C-terminal deletions eliminate the enzyme activity. BlCel5B activities on CMC as functions of pH and temperature are shown in (b) and (c), respectively. The enzyme exhibits optimal pH of 4.0 and optimal temperature of 55 °C, retaining about 50% of its activity at 80 °C. (d) Michaelis-Menten curve using CMC as a substrate. Open-close transitions of BlCel5B. (a) BlCel5B in the absence of substrate and (b) in the presence of cellooctaose, as observed in our aMD simulations. The distance between the α carbon of residues I120 (CD) and E477 (CBM46), illustrated as spheres in (a), is plotted in (c), revealing a transition by the decrease in the distance from 40 Å to 7 Å (substrate-free) or 20 Å (in presence of cellooctaose). For the substrate-free enzyme, the red line refers to a 1 μs-long aMD; for the BlCel5B-cellooctaose complex, the first 500 ns refers to aMD (in blue) and the second 500 ns to conventional MD (in turquoise). (d) A snapshot of the BlCel5B-cellooctaose complex, highlighting the tryptophan residues that interact with the glucan chain in subsites −4 to +4. Residues W479 and W481 belong to CBM46 and only become available for substrate interactions in the closed configuration of BlCel5B. Large-scale movements of BlCel5B modules and superposition of their representative conformations with the SAXS envelope. (a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations. Different colors separated by vertical lines correspond to independent simulations of approximately 120 μs. (c) The distance distribution indicates three major peaks: closed or occluded CBM46/CD conformations (I); semi-open (II), which is similar to the crystallographic structure; and extended conformers (III). (d) Superimposition of the three representative molecular conformations of BlCel5B with the SAXS model. (e) Average structures obtained from the simulation segments corresponding to population groups I-III, which are individually superposed on the SAXS envelope. Comparison of the binding site shape of GH5_4 enzymes available on the Protein Data Bank. (a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE). Comparison of the binding cleft of the BlCel5B and BhCel5B. The main difference between BlCel5B and BhCel5B is that the latter exhibits a deeper cleft due to the presence of residue W181 in the loop between F177 and R185. We conjecture that this difference in the binding site architecture relates to the importance that the CBM46 plays in the BlCel5B enzymatic mechanism. Proposed molecular mechanism of BlCel5B conformational selection. As suggested by the simulations and SAXS data, BlCel5B spans multiple conformations ranging from closed to extended CBM46/CD states. In a given open state, the substrate may reach the active site and become entrapped by the capping of CBM46 onto CD and induced-fit conformational adjustments. After hydrolysis, the reaction product is released to yield apo-BlCel5B, which becomes ready for a new cycle. Activity of BlCel5B constructs against tested substrates. Substrate (1%)\tRelative Activity (%)\t \tWT*\tW479A\tW481A\tΔCBM46\tΔIg-CBM46\t \tβ-glucan\t100\t79.1\t63.6\tnd\tnd\t \tCMC\t25.5\t12.2\t2.4\tnd\tnd\t \tLichenan\t52.4\t41\t28.6\tnd\tnd\t \tXyloglucan\t45.2\t41.2\t30.8\tnd\tnd\t \tAzo-Avicel\tnd**\tnd\tnd\tnd\tnd\t \tArabinoxylan\tnd\tnd\tnd\tnd\tnd\t \tGalactomannan\tnd\tnd\tnd\tnd\tnd\t \t1,4-β-mannan\tnd\tnd\tnd\tnd\tnd\t \t *WT = wild type. **nd = not 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diff --git a/annotated_BioC_JSON/PMC4980666_ann.json b/annotated_BioC_JSON/PMC4980666_ann.json
new file mode 100644
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--- /dev/null
+++ b/annotated_BioC_JSON/PMC4980666_ann.json
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+[{"sourceid":"4980666","sourcedb":"","project":"","target":"","text":"N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes Influenza virus PA endonuclease has recently emerged as an attractive target for the development of novel antiviral therapeutics. This is an enzyme with divalent metal ion(s) (Mg2+ or Mn2+) in its catalytic site: chelation of these metal cofactors is an attractive strategy to inhibit enzymatic activity. Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays. Several N-acylhydrazones were found to have promising anti-influenza activity in the low micromolar concentration range and good selectivity. Computational docking studies are carried on to investigate the key features that determine inhibition of the endonuclease enzyme by N-acylhydrazones. Moreover, we here describe the crystal structure of PA-Nter in complex with one of the most active inhibitors, revealing its interactions within the protein’s active site. Influenza virus is an enveloped virus with a segmented negative-oriented single-stranded RNA genome, belonging to the Orthomyxoviridae. Seasonal influenza A and B viruses affect each year approximately 5–10% of the adult and 20–30% of the paediatric population, and there is a permanent risk of sudden influenza pandemics, such as the notorious ‘Spanish flu’ in 1918 and the swine-origin H1N1 pandemic in 2009. Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir). The M2 inhibitors have limited clinical utility due to their central nervous system side effects and widespread resistance, as in the case of the 2009 pandemic H1N1 virus; resistance is also a growing concern for oseltamivir. Therefore, there is an urgent need for new antiviral drugs with an entirely different mode of action. The influenza virus polymerase complex is composed of three subunits: PB1, PB2 and PA. The PA subunit performs the ‘cap-snatching’ endonuclease reaction, the PB2 subunit is responsible for initial binding of the capped RNAs, while the actual RNA synthesis is performed by the PB1 protein. Given its crucial role in the viral life cycle, the influenza virus polymerase is widely recognized as a superior target for antiviral drug development and, in particular, inhibition of the PA endonuclease has deserved much attention in recent years. The endonuclease catalytic site resides in the N-terminal domain of PA (PA-Nter; residues 1~195). It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions. The two-metal-ion model is consistent with numerous biochemical findings. Since the intracellular concentration of Mg2+ is at least 1000-fold higher than that of Mn2+, magnesium may be more biologically relevant. A controversy about number and type of metal ions exists also for the active site of HIV-1 integrase. HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1). In recent years, we focused our research on chemical scaffolds that are able to chelate metal ions of PA-Nter, resulting in inhibition of influenza virus replication. N-acylhydrazones represent an appealing class of chelating ligands with a broad spectrum of biological activities, such as activity against HIV, hepatitis A, vaccinia and influenza virus. In the present work, we report the biological activity of a series of N-acylhydrazones (Fig. 2), as determined in an enzymatic assay with PA-Nter endonuclease as well as in cell-based influenza viral ribonucleoprotein (vRNP) reconstitution and virus yield assays. Several N-acylhydrazones were found to have promising anti-influenza activity with 50% effective concentration values (EC50) in the range of 3–20 μM and good selectivity (Table 1 and Fig. 3). Computational docking studies of two candidate ligands in the PA-Nter active site gave information about the features that could determine inhibition of endonuclease activity. Moreover, we describe the X-ray crystal structure of PA-Nter in complex with one of the most active inhibitors. Results and Discussion Chemistry N-acylhydrazones 1–27 (Fig. 2) were prepared in high yields by following literature methods (Fig. 2A); they were characterized by spectroscopic tools, mass spectrometry and elemental analysis. Even if isomerism around the C = N bond is possible, 1–27 are present in the E form in solution, as evidenced by the chemical shift values of the HC = N and NH protons in the 1H-NMR spectrum. Exceptions are represented by the alkyl-derivatives 3 and 4 (2:1 and 5:3 E:Z ratio, respectively). If R’ (Fig. 2A) is a 2-hydroxy substituted phenyl ring, the corresponding acylhydrazones can coordinate one or, depending on denticity, two metal centers (modes A and B in Fig. 4). Starting from N’-(2,3-dihydroxybenzylidene)-semicarbazide (1) and its methoxy-analogue (2), we modified the acylhydrazonic substituent R” (3–8, 18, 19, Fig. 2A). In 18 and 19, also the gallic moiety can be involved in the chelation of the metal cofactors (mode C, Fig. 4). In order to investigate the role of hydroxyl substituents 9–11, 13–17, 20–23 and 27 were also synthesized. Compound 12 was synthesized in order to confirm the crucial influence of the gallic moiety. Finally, 26 was here considered, because it is an inhibitor of HIV RNase H, another enzyme with two magnesium ions in its active site. Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+. Different reaction conditions were used (1:1 and 1:2 metal to ligand ratio, up to 4 equivalents of triethylamine), but in any case the same chemical species Mg(HL)2∙4H2O was recovered and conveniently characterized. The use of a coordinating solvent as d6-DMSO causes partial decoordination of the ligand, but the 1H-NMR spectrum in MeOD, instead, shows only the signals attributable to the complex. In the 13C-NMR spectrum, the signal of the C = O quaternary carbon is practically unaffected by complexation, suggesting that the C = O group is weakly involved in the coordination to the metal ion. This is confirmed, in the IR spectrum, by the shift of about 20 cm−1 of the C = O absorption, while a shift of 30–50 cm−1 is expected when the carbonylic oxygen is tightly bound to the metal ion. ESI-mass spectra and elemental analysis confirmed the formula Mg(HL)2∙4H2O. The interaction between the N-acylhydrazone ligands and the magnesium cation was investigated also by means of UV-visible spectroscopy (UV-visible titrations of 23 and 19 with increasing amount of Mg(CH3COO)2 are shown in Figure S1). The spectrum of 19 includes a band at 313 nm assignable to n-π* transitions of the C = N and C = O groups. By adding increasing equivalents of Mg(CH3COO)2, the absorption around 400 nm increases, and a new band appears with a maximum at 397 nm. The opposite trend is observed in the range 300–350 nm, where an isosbestic point is present close to 335 nm. When the same experiment was performed with 23, a different behavior was observed. Increasing concentration of Mg2+, in fact, caused a diminution in the maximum absorption, an isosbestic point is visible at about 345 nm, but a new band at 400 nm does not appear. Ligands 19 and 23 coordinate the Mg2+ ions in different ways: 19 chelates the metal ion by using the deprotonated salicyl oxygen and the iminic nitrogen, while for 23, the gallic moiety is supposed to be involved (Fig. 4A,B versus C), leading to different, less extensive, modifications of the UV spectrum. These results will be revisited during the discussion of the biological activity. Inhibition of the PA-Nter enzyme All the compounds were tested for their ability to inhibit the influenza endonuclease in an enzymatic plasmid-based assay with recombinant PA-Nter, as well as in cell-based influenza methods (i.e. virus yield and vRNP reconstitution assays). The results are shown in Table 1 and summarized in Fig. 3 to visualize the structure-activity relationships; Figure S2 shows the dose-response curves for three representative compounds (i.e. 10, 13 and 23) in either the PA-enzyme or vRNP reconstitution assay. The moderate activity (IC50 = 24 μM) of N’-2,3-dihydroxybenzylidene semicarbazide (1) was completely lost when the NH2 moiety was replaced by a hydrophobic heptyl chain (3), but it is less affected when a phenyl or a 2-hydroxyphenyl is present (5 and 7, IC50 = 84 and 54 μM, respectively). When the hydroxyl in position 3 on R1 (2,3-dihydroxybenzylidene) was replaced by a methoxy group (2-hydroxy-3-methoxybenzylidene), the activity disappeared (compounds 2, 4, 6 and 8). The activity is unaffected (IC50 values ranging from 45 to 75 μM) when going from two hydroxyls in R1 (7) to compounds with three hydroxyls (i.e. 9, 10 and 11). Similarly, 11 (R1 = 3,4,5-trihydroxyphenyl, R2 = 2-hydroxyphenyl) had comparable activity as 27 (R1 = 3,4,5-trihydroxyphenyl, R2 = NH2). Within the series carrying a 2-hydroxyphenyl R2 group, the activity of 11 is particularly intriguing. 11 does not have the possibility to chelate in a tridentate ONO fashion (mode A in Fig. 4), but it can coordinate two cations by means of its three OH groups in R1 (mode C, Fig. 4). Note that a similar chelating mode was observed in a crystal structure, solved by Cusack and coworkers, of PA-Nter endonuclease in complex with the inhibitor EGCG. The PA-Nter inhibitory activity strongly depends on the number and position of hydroxyl substituents in R1 and R2: this is clearly highlighted by the data obtained with compounds 13–23, in which R2 is a 3,4,5-trihydroxyphenyl (gallic) group, the most active scaffold in our series. The analogue carrying an unsubstituted aromatic ring as R1 (compound 13) had moderate activity (IC50 = 69 μM). When one OH was added at position 2 of the R1 ring (14), the activity was lost. Adding a second OH substituent at position 5 resulted in strong activity (compound 15, IC50 = 9 μM); medium activity for a 3-OH (18; IC50 = 83 μM), and marginal activity when the second OH is at position 4 (17, IC50 ≥ 370 μM). The addition of a 3-methoxy group (19) abolished all inhibitory activity. This cannot be related to variations in the chelating features displayed by the R1 moiety, since compounds 14–19 all have, in theory, the capacity to chelate one metal ion through the ortho-OH and iminic nitrogen (mode A in Fig. 4). Moreover, compound 18 can, in principle, chelate the two M2+ ions in the active site according to mode B (Fig. 4), yet it (IC50 = 83 μM) has nine-fold lower activity than 15, that does not possess this two-metal chelating feature. Therefore, we hypothesized that the inhibitory activity of the series containing the gallic moiety is determined by: (i) the capacity of the moiety R2 to chelate two metal ions in the active site of the enzyme, according to mode C (Fig. 4); and (ii) the presence and position of one or more hydroxyl substituents in R1, which may possibly result in ligand-protein interactions (e.g. through hydrogen bonds). This assumption was supported by molecular docking calculations and X-ray analysis of inhibitor 23 in complex with PA-Nter (vide infra). At this point, change of the substituents in R1 represents the next logical step. Substitution of the 5-hydroxyl in 15 by a methoxy group (16) causes a dramatic drop in activity (IC50 = 9 and 454 μM for 15 and 16, respectively). When two or three OH groups are present in R1, their spatial disposition greatly affects the activity. In particular, all the compounds with a trihydroxylated phenyl group as R1 (i.e. 20, 21, 22 and 23) were able to inhibit PA-Nter quite potently. The lowest IC50 values were obtained for 21 and 23 (IC50 = 13 and 7 μM, respectively), which both have one of their three hydroxyl groups at position 5. The most active compound in this series was 23, which lacks the hydroxyl group at position 2 of R1, further confirming that this function is undesirable or even detrimental for inhibitory activity against PA-Nter, as already noticed above for 14. Consistent with a crucial role of the R2 gallic moiety in metal chelation, the strong activity of 15 was completely lost in its 3,4,5-trimethoxy analogue 12. On the other hand, the R2 gallic containing compounds displayed moderate activity (IC50 values around 40 μM) when R1 was absent (i.e. the 3,4,5-trihydroxybenzohydrazide 28, Fig. 2), or composed of an extended ring system (26) or a pyrrole ring (25). Still lower activity was seen with the pyridine analogue 24. Evidently, the 3,4,5-trihydroxybenzyl moiety at R2 is fundamental but not sufficient to ensure potent PA-Nter endonuclease inhibition, since the interactions of R1 with the amino acid side chains of the protein appear crucial in modulating activity. Inhibition of vRNP activity or virus replication in cells To determine the anti-influenza virus activity of compounds 1–28 in cell culture, we performed an influenza vRNP reconstitution assay in human embryonic kidney 293 T (HEK293T) cells, then subjected the active compounds (i.e. EC50 \u003c 100 μM) to a virus yield assay in influenza virus-infected Madin-Darby canine kidney (MDCK) cells (Table 1 and Fig. 3). For some N-acylhydrazone compounds, we observed quite potent and selective activity in the vRNP reconstitution assay. This indicates that they are able to inhibit viral RNA synthesis and suggests that they could be classified as original PA inhibitors. Values for EC50 (vRNP) or EC90 (virus yield) in the range of 0.4–18 μM were obtained for compounds 15 and 20–23, which all carry a 3,4,5-trihydroxyphenyl as R2, and possess either two (15) or three (20–23) hydroxyl substituents in the R1 moiety. As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells. The hydrazide 28 displayed weak (virus yield) to moderate (vRNP reconstitution) activity, albeit less than the most active molecules in the 3,4,5-trihydroxyphenyl series (i.e. 18 and 21–23). Even if there are no data indicating that the compounds reported in the paper are subject to hydrolysis, the activity of 28 could raise the concern that for some N-acylhydrazones the antiviral activity in cell culture may be related to their intracellular hydrolysis. However, this is unlikely, since the antiviral potency showed large differences (i.e. EC50 values between 0.42 and 29 μM) for compounds with the same R2 but different R1 groups, meaning that R1 does play a role in modulating the antiviral effect. Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8. Two notable exceptions are 18 and 19 (containing a 2,3-dihydroxybenzylidene or 2-hydroxy-3-methoxybenzylidene R1, respectively) which were not cytotoxic at 200 μM and displayed favorable antiviral selectivity. Some N-acylhydrazone compounds were devoid of activity in the enzymatic assay, yet showed good to moderate efficacy in cell culture (e.g. 14 and 19, having EC50 values of 2.2 and 7.1 μM, respectively). For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay. On the other hand, this difference was 8-fold or more for 7, 10, 14, 22, 25 and 28. Some N-acylhydrazone compounds showed good to moderate efficacy in the vRNP assay (e.g. 14 and 19, having EC50 values of 2.3 and 5.7 μM, respectively), yet were devoid of activity in the enzymatic assay. This observation suggests that they may inhibit the viral polymerase in an endonuclease-independent manner. To achieve a clear insight into the antiviral profile of the N-acylhydrazones, specific mechanistic experiments are currently ongoing in our laboratory, in which we are analyzing in full depth their effects on virus entry, polymerase-dependent RNA synthesis or the late stage (maturation and release) of the virus replication cycle. Docking studies In order to explore the possible binding mode of the synthesized compounds, docking simulations by GOLD program were performed by using the structural coordinates (PDB code 4AWM) for the PA-Nter endonuclease in complex with EGCG. Considering that the position of the side-chains of some residues changes depending on which pocket the ligand is occupying, we superimposed some X-ray structures of complexes between PA-Nter endonuclease and known active ligands. It was observed that the side-chain of amino acid Tyr24 shows greater movement than the other residues and for this reason we considered it as a flexible residue during the docking procedure. First, test docking calculations, using EGCG, L-742,001 and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one (Fig. 1), were carried out to compare experimental and predicted binding modes and validate docking procedure. Their best docking poses agreed well with the experimental binding modes (rmsd values of 0.8, 1.2 and 0.7, respectively). Next, docking of several N-acylhydrazones was performed and this generated a number of possible binding conformations, highlighting that the active site cavity of the PA endonuclease is quite spacious, as already demonstrated by crystallographic studies, and confirming the ability of this scaffold to chelate the two M2+ ions in different ways (Mode A-C in Fig. 4). Figure 5 displays the first (panel A) and second (panel B) GOLD cluster docked solutions for compound 23. These two complex structures represent the largest clusters with similar fitness values (59.20 and 58.65, respectively). In both cases, 23 appears able to coordinate the two M2+ ions in the active site through the three contiguous OH groups (Fig. 5). In addition, 23 was predicted to form two hydrogen bonding interactions, i.e. with the catalytic Lys134 on the one side and Glu26 on the other side. Furthermore, in these two different binding modes, 23 forms π–π interactions with the aromatic ring of Tyr24, in a fashion similar to that described for other endonuclease inhibitors, i.e. EGCG and L-742,001. The best docked conformation for compound 15 (Fig. 6, fitness value 68.56), which has an activity slightly lower than 23, reveals a different role for the gallic moiety. The ligand seems to form two hydrogen bonding interactions with Tyr130 as well as a cation–π interaction with Lys134. Tyr130 lies in a pocket that also contains Arg124, a residue that was proposed to have a crucial role in binding of the RNA substrate. Compound 15 appears further stabilized by hydrogen bonding interactions between two hydroxyl groups and Arg82 and Asp108. In this case, chelation of the two M2+ ions is carried out by involving the imine group (mode A in Fig. 4). It is important to highlight that compounds 23 and 15, although in different ways, both are able to chelate the metal cofactors and to establish interactions with highly conserved aminoacids (Tyr24, Glu26, Arg124, Tyr130 and Lys134) that are very important for both endonuclease activity and transcription in vitro. The crucial role of such interactions is underlined by the differences in activity between 15 (IC50 = 9.0 μM) and 19 (\u003e500 μM): their coordinating features are similar, since both coordinate to the divalent metal ion through the phenolic oxygen, the iminic nitrogen and the carbonylic oxygen (mode A in Fig. 4), but the biological activity could be related to their different ability to engage interactions with the protein environment. Crystallographic Studies Attempts were made to co-crystallize PA-Nter with 15, 20, 21 and 23 in one to four molar excess. While crystals appeared and diffracted well, upon data processing, no or very little electron density for the inhibitors was observed. Attempts to soak apo crystals in crystallization solution containing 5 mM inhibitor overnight also did not result in substantial electron density for the inhibitor. As a last resort, dry powder of the inhibitor was sprinkled over the crystallization drop containing apo crystals and left over night. This experiment was successful for compound 23, the crystals diffracted to 2.15 Å and diffraction data were collected (PDB ID 5EGA). The refined structure shows unambiguous electron density for the inhibitor (Table S1 and Fig. 7). The complex structure confirms one of the two binding modes predicted by the docking simulations (Fig. 5, panel B). The galloyl moiety chelates the manganese ions, while the trihydroxyphenyl group stacks against the Tyr24 side chain. It is interesting to note that two of these hydroxyl groups are in position to form hydrogen bonds with the side chain of Glu26 and Lys34 (Fig. 7). These interactions suggest that other functional groups, e.g. halogens, could be used in place of the hydroxyl groups for better interactions with Glu26 and Lys34 side chains, and the inhibitory potency of these compounds could be further improved. Conclusions The development of new agents for the treatment of influenza infection that exert their action by inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is a strategy that recently is gaining a lot of interest. The results here presented add the N-acylhydrazone scaffold to the library of the chelating molecules with potent antiviral activity (EC90 \u003c 5 μM, virus yield assay in influenza virus-infected MDCK cells). The structure of the N-acylhydrazone 23 co-crystallized with PA-Nter is important not only because confirms that the polyhydroxypheyl group efficiently coordinates two metal ions in the active site of the enzyme, but also because highlights the importance of the (flexible) inhibitor backbone in order to engage effective interactions with crucial aminoacids of the protein. Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase could represent another example, after carbonic anhydrase, histone deacetylase, and HIV-1 integrase, of metal binding as a successful strategy in drug design. Experimental Section Materials and methods. Chemistry All reagents of commercial quality were purchased from Sigma-Aldrich and used without further purification. The purity of the compounds was determined by elemental analysis and verified to be ≥95% for all synthesized molecules. NMR spectra were recorded at 25 °C on a Bruker Avance 400 FT spectrophotometer. The attenuate total reflectance IR spectra were recorded by means of a Nicolet-Nexus (Thermo Fisher) spectrophotometer by using a diamond crystal plate in the range of 4000–400 cm−1. Elemental analyses were performed by using a FlashEA 1112 series CHNS/O analyzer (Thermo Fisher) with gas-chromatographic separation. Electrospray mass spectral analyses (ESI-MS) were performed with an electrospray ionization (ESI) time-of-flight Micromass 4LCZ spectrometer. MS spectra were acquired in positive EI mode by means of a direct exposure probe mounting on the tip of a Re-filament with a DSQII Thermo Fisher apparatus, equipped with a single quadrupole analyzer. UV–Vis spectra were recorded on an Evolution 260 Bio Thermo spectrophotometer by using cells of 1 cm path length. UV-vis absorption spectra of 19 and 23 were registered using a ca. 10−5 M solution in methanol. Each metal/ligand system was studied by titrating a 2.8 ml sample of the ligand solution with a methanolic solution of Mg(CH3COO)2; 8–12 spectra of samples with M:L molar ratio ranging from 0 to 6 were measured. Synthesis of the ligands (general procedure) All the N-acylhydrazones were prepared in a manner similar to reported procedures. Briefly, to a solution of the aldehyde in absolute ethanol or toluene, an equimolar amount of the hydrazide dissolved in the same solvent was added. The mixture was refluxed for 6 hours, cooled at room temperature and concentrated in vacuum. The resulting precipitate was filtered off, washed with cold ethanol and dried in vacuum. 3,4,5-trihydroxybenzohydrazide (28) and 3,4,5-trimethoxybenzohydrazide (29) were obtained by reaction of the corresponding methyl esters with hydrazine monohydrate. Hydrazine was added to an ethanol suspension of the ester and stirred at room temperature until the solute completely dissolved. Reaction mixture was then refluxed overnight. On concentrating the solution, a precipitate was observed, which was filtered and washed with cold ethanol. Chemical characterization of 1–29 and of Mg(HL)2 4H2O is collected in the Supplementary Information. Computational Studies The crystal structure of PA-Nter endonuclease in complex with EGCG was retrieved from the RCSB Protein Data Bank (entry code 4AWM). The ligand and water molecules were discarded and the hydrogens were added to the protein by Discovery Studio 2.5. The charge on the metal ions was set as +2. EGCG, L-742,001, and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one structures were extracted from their X-ray complexes (PDB IDs 4AWM, 4W9S and 4E5H respectively). The other ligand structures were constructed using Discovery Studio 2.5.5 (Accelrys, Discovery Studio) and energy minimized using the Smart Minimizer protocol (1000 steps) which combines the Steepest Descent and the Conjugate Gradient methods. The minimized ligands were docked in their corresponding proteins by means of GOLD Suite 5.0.1. The region of interest used by the GOLD program was defined in order to contain the residues within 15 Å from the original position of the ligand in the X-ray structure. The side-chain of residue Tyr24 was allowed to rotate according to the internal rotamer libraries in GOLD Suite 5.0.1. GoldScore was chosen as fitness function. The standard default settings were used in all calculations and the ligands were submitted to 100 genetic algorithm runs. The “allow early termination” command was deactivated. Results differing by less than 0.75 Å in ligand-all atom rmsd, were clustered together. The best GOLD-calculated conformation was used both for analysis and representation. Plasmid-based endonuclease assay This enzymatic assay was performed according to a previously published method. One microgram of recombinant PA-Nter (residues 1–217 from the PA protein of influenza virus strain A/X-31) was incubated with 1 μg (16.7 nM) of single-stranded circular DNA plasmid M13mp18 (Bayou Biolabs, Metairie, Louisiana) in the presence of the test compounds and at a final volume of 25 μL. The assay buffer contained 50 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM β-mercaptoethanol and 1 mM MnCl2. The reaction was incubated at 37 °C for 2 h and then stopped by heat inactivation (80 °C, 20 min), followed by visualization of the endonucleolytic digestion of the plasmid by gel electrophoresis on a 1% agarose gel with ethidium bromide staining. The amount of remaining intact plasmid was quantified by ImageQuant TL software (GE Healthcare, Diegem, Belgium). The percentage inhibition of PA endonuclease activity was plotted against the compound concentration on a semi-logarithmic plot, using GraphPad Prism software (GraphPad Software, La Jolla, CA). The 50% inhibitory concentrations (IC50) were obtained by nonlinear least-squares regression analysis of the results from three independent experiments. 2,4-Dioxo-4-phenylbutanoic acid (DPBA; Interchim, Montluçon, France) was included as the reference compound. Cells and media MDCK cells (a kind gift from M. Matrosovich, Marburg, Germany) and HEK293T cells (purchased from Thermo Fisher Scientific, Waltham, MA) were cultivated in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, and 0.075% sodium bicarbonate. During virus experiments, the MDCK cells were maintained in MDCK infection medium, consisting of Ultra MDCK medium (Lonza, Basel, Switzerland) supplemented with 0.0225% sodium bicarbonate, 2 mM L-glutamine, and 2 μg/ml tosyl phenylalanyl chloromethyl ketone-treated trypsin (Sigma-Aldrich, St. Louis, MO). The cells were incubated in a humidified atmosphere containing 5% CO2. vRNP reconstitution assay The procedure to determine the inhibitory effect of the compounds on influenza virus vRNPs reconstituted in HEK293T cells, is described in full detail elsewhere. Briefly, the four relevant plasmids (i.e. the expression plasmids for PB1, PB2, PA and NP) were combined with the fluc reporter plasmid, and co-transfected into HEK293T cells using Lipofectamin 2000 (Invitrogen, Life Technologies, Gent, Belgium). After incubation at 37 °C for 24 h in the presence of serial dilutions of the test compounds, the ONE-Glo luciferase assay system (Promega, Madison, WI) was used to determine luciferase activity. EC50 was defined as the compound concentration causing 50% reduction in the vRNP-driven firefly luciferase signal, as compared to cells receiving medium instead of compound. These EC50 values were calculated by interpolation assuming a semi-log dose-response effect using GraphPad Prism software. In parallel, compound cytotoxic activity was determined in untransfected HEK293T cells which had been incubated with serial dilutions of the compounds for 24 h, using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay; Promega). These spectrophotometric data were used to calculate the 50% cytotoxic concentration (CC50), i.e. the concentration reducing cell viability by 50%, as compared to wells receiving medium instead of compound. Ribavirin (Virazole; ICN Pharmaceuticals, Costa Mesa, CA) was included as the reference compound. Virus yield assay We previously published in full detail the virus yield assay to determine the anti-influenza virus activity in MDCK cell cultures. Briefly, one day prior to infection, MDCK cells were seeded into 96-well plates at 25,000 cells per well. At day 0, serial dilutions of the test compounds were added, immediately followed by infection with influenza A/PR/8/34 virus. After 24 h incubation at 35 °C, the virus amount in the supernatants was estimated by determining the viral genome copy number in a one-step quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay (CellsDirect One-Step qRT-PCR kit; Invitrogen), with influenza virus M1-specific primers and probe. The compound concentration values causing a 2-log10 (EC99) and a 1-log10 (EC90) reduction in viral RNA (vRNA) copy number at 24 h p.i., as compared to the virus control receiving no compound, were calculated by interpolation from data of at least three experiments. In parallel, the CC50 values after 24 h incubation with compounds were determined in uninfected MDCK cells, using the spectrophotometric MTS cell viability assay described above, respectively. Ribavirin was included as the reference compound. Crystallographic analysis A PAN construct (PANΔLoop) with a loop (residues 51–72) deleted and replaced with GGS from A/California/04/2009 H1N1 strain was used for the crystallographic studies. The details of cloning, over-expression and purification are described elsewhere. Briefly, the gene was cloned into pET52b vector and transformed into BL21 (DE3) cells, and the protein was expressed in LB medium overnight at 18 °C after induction at an OD600 ~0.8 with 0.2 mM isopropyl-β-thiogalactopyranoside (IPTG). The protein was purified from cell lysates by HisTrap affinity chromatography and the 10xHis tag was removed by digestion with thrombin. The protein was further purified by gel filtration using a Superdex 75 size-exclusion chromatography column in 20 mM Tris pH 8.0, 150 mM NaCl and 1 mM TCEP. The protein was concentrated to 10–12 mg/ml for crystallization. Crystals were grown in 0.2 M MgCl2, 2 mM MnCl2, 0.1 M Tris pH 8.5, 30% (w/v) PEG 4000 using the hanging drop method. For determination of the protein-inhibitor complex structure, the powder of the inhibitor was sprinkled on a 2 μl drop of a 1:1 ratio mixture of protein solution and well solution, on a cover slide hanging over 500 μl well solution, and left overnight. Next day, the crystals were cryo-protected using well solution supplemented with 25% ethylene glycol and flash frozen in liquid nitrogen. The data were collected at the 22-ID beam line maintained by Southeast Regional Collaborative Access Team (SERCAT) at the Advanced Photon Source, Argonne National Laboratory. The data were indexed, integrated and scaled using the HKL2000 suite of programs. Phase determination, structure refinement and model building were completed using Phaser, Refmac and Coot (part of the CCP4 package). The apo structure of PANΔLoop (PDB ID: 5DES) was used as starting model for molecular replacement. The details of the data collection and refinement statistics are given in Table S1. Additional Information How to cite this article: Carcelli, M. et al. N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes. Sci. Rep. 6, 31500; doi: 10.1038/srep31500 (2016). Supplementary Material Orthomyxoviridae: the viruses and their replication World Health Organization. Influenza (seasonal) - Fact sheet N°211. http://www.who.int/mediacentre/factsheets/fs211/en/ (accessed 19-1-2015). 1918 Influenza: the mother of all pandemics Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans Amantadine and rimantadine for influenza A in adults. Cochrane Database Surveillance of resistance to adamantanes among influenza A(H3N2) and A(H1N1) viruses isolated worldwide Global transmission of oseltamivir-resistant influenza Multidrug-resistant 2009 pandemic influenza A(H1N1) viruses maintain fitness and transmissibility in ferrets Influenza virus resistance to antiviral therapy Emerging antiviral strategies to interfere with influenza virus entry Structure of influenza A polymerase bound to the viral RNA promoter Influenza A virus polymerase: structural insights into replication and host adaptation mechanisms  Viral enzymes containing magnesium: metal binding as a successful strategy in drug design The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site The N-terminal domain of PA from bat-derived influenza-like virus H17N10 has endonuclease activity Nucleoside monophosphate complex structures of the endonuclease domain from the influenza virus polymerase PA subunit reveal the substrate binding site inside the catalytic center Structural and biochemical basis for development of influenza virus inhibitors targeting the PA endonuclease Crystallographic fragment screening and structure-based optimization yields a new class of influenza endonuclease inhibitors Metal ion catalysis of RNA cleavage by the influenza virus endonuclease Metalloproteomics, metalloproteomes, and the annotation of metalloproteins Magnesium basics Discovery of raltegravir, a potent, selective orally bioavailable HIV-integrase inhibitor for the treatment of HIV-AIDS infection Carbamoyl pyridone HIV-1 integrase inhibitors 3. A diastereomeric approach to chiral nonracemic tricyclic ring systems and the discovery of dolutegravir (S/GSK1349572) and (S/GSK1265744) Anti-influenza virus activities of 4-substituted 2,4-dioxobutanoic acid inhibitors An integrated biological approach to guide the development of metal-chelating inhibitors of influenza virus PA endonuclease A novel antiviral agent which inhibits the endonuclease of influenza viruses Metal-chelating 2-hydroxyphenyl amide pharmacophore for inhibition of influenza virus endonuclease Use of a pharmacophore model to discover a new class of influenza endonuclease inhibitors Phenyl substituted 4-hydroxypyridazin-3(2H)-ones and 5-hydroxypyrimidin-4(3H)-ones: inhibitors of influenza A endonuclease Anti-influenza activity of marchantins, macrocyclic bisbibenzyls contained in liverworts Green tea catechins inhibit the endonuclease activity of influenza A virus RNA polymerase Antiviral effect of catechins in green tea on influenza virus Investigation of the salicylaldehyde thiosemicarbazone scaffold for inhibition of influenza virus PA endonuclease Biological activities of hydrazone derivatives A review exploring biological activities of hydrazones Interaction of HIV-1 reverse transcriptase ribonuclease H with an acylhydrazone inhibitor Discovery of novel inhibitors of LEDGF/p75-IN protein–protein interactions A broad HIV-1 inhibitor blocks envelope glycoprotein transitions critical for entry Synthesis and antiviral evaluation of some sugar arylglycinoylhydrazones and their oxadiazoline derivatives A versatile salicyl hydrazonic ligand and its metal complexes as antiviral agents Computation-guided discovery of influenza endonuclease inhibitors Conformational behaviour and E/Z isomerization of N-acyl and N-aroylhydrazones Mutational analysis of the binding pockets of the diketo acid inhibitor L-742,001 in the influenza virus PA endonuclease Structural analysis of specific metal chelating inhibitor binding to the endonuclease domain of influenza pH1N1 (2009) polymerase Development and validation of a genetic algorithm for flexible docking. Mutational and metal binding analysis of the endonuclease domain of the influenza virus polymerase PA subunit Magnesium-dependent RNA binding to the PA endonuclease domain of the avian influenza polymerase Amino Acid Residues in the N-Terminal Region of the PA Subunit of Influenza A Virus RNA Polymerase Play a Critical Role in Protein Stability, Endonuclease Activity, Cap Binding, and Virion RNA Promoter Binding A novel small-molecule inhibitor of influenza A virus acts by suppressing PA endonuclease activity of the viral polymerase Virtual Screening and Biological Validation of Novel Influenza Virus PA Endonuclease Inhibitors Synthesis, characterization, X-ray crystallography, acetyl cholinesterase inhibition and antioxidant activities of some novel ketone derivatives of gallic hydrazide-derived Schiff bases Development and validation of a genetic algorithm for flexible docking Novel inhibitors of influenza virus fusion: structure-activity relationship and interaction with the viral hemagglutinin Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor Processing of X-ray diffraction data collected in oscillation mode Overview of the CCP4 suite and current developments Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. M.C., D.R., A.G. and M.S. drug design and chemical synthesis; L.D.L. docking studies; G.K. and S.W.W. crystallographic studies; A.S. and L.N. biological studies. Chemical structures of some prototype inhibitors of influenza virus endonuclease. Inhibitor activity in enzymatic assays (IC50, μM) as reported in: aref., bref., cref., dref.. General synthesis for N-acylhydrazones 1–27 and hydrazides 28 and 29 (A). Chemical structures of compounds 1–27 (B). Overview of the structure-activity relationship for compounds 1–27. Scheme of possible binding modes of the studied N-acylhydrazones. First (A) and second (B) GOLD cluster docked solutions of compound 23 (orange and cyan, respectively) in complex with PA endonuclease. Key residues of the pocket are presented using PyMOL [ http://www.pymol.org] and LIGPLUS [Laskowski, R. A.; Swindells, M. B. Journal of chemical information and modeling\n2011,\n51, 2778]. Hydrogen bonds are illustrated by dotted lines, while the divalent metal ions are shown as purple spheres. Schematic drawings of the interactions of the first (C) and second (D) GOLD cluster docked solutions generated using LIGPLUS. Dashed lines are hydrogen bonds and ‘eyelashes’ show residues involved in hydrophobic interactions. (A) Binding mode of compound 15 (orange) in complex with PA endonuclease. Key residues of the pocket are presented using PyMOL [ http://www.pymol.org] and LIGPLUS [Laskowski, R. A.; Swindells, M. B. Journal of chemical information and modeling\n2011,\n51, 2778]. Hydrogen bonds are illustrated by dotted lines while the divalent metal ions are shown as purple spheres. (B) Schematic drawing of the interactions of compound 15 generated using LIGPLUS. Dashed lines are hydrogen bonds and ‘eyelashes’ show residues involved in hydrophobic interactions. Crystal structure of PANΔLoop in complex with compound 23. Active site residues are shown in sticks with green carbons, manganese atoms are shown as purple spheres and water molecules as red spheres. Compound 23 is shown in sticks with yellow carbons. Protein secondary structure is shown as ribbons in salmon color. 2Fo-Fc electron density map contoured at 1σ is shown as blue mesh. Hydrogen bonds and metal coordination are shown with dotted lines. The H-bond distances from the side chain carboxyl group of Glu26 to p-OH and m-OH of the trihydroxyphenyl group of the inhibitor are 2.7 Å and 3.0 Å, respectively. The H-bond distance from the side chain of Lys34 to p-OH of the trihydroxyphenyl group is 3.6 Å. The H-bond distance to the water molecule from m-OH of the galloyl moiety is 3.0 Å, which in turn is H-bonded to the side chain of Tyr130 with a distance of 2.7 Å. Crystal structure has been deposited in the RCSB Protein Data Bank with PDB ID: 5EGA. Inhibitory activity of the N-acylhydrazones 1–27 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays. Compound\tEnzyme assay with PA-Ntera\tVirus yield assay in influenza virus-infected MDCK cellsb\tvRNP reconstitution assay in HEK293T cellsc\t \tAntiviral activity\tCytotoxicity\tSId\tActivity\tCytotoxicity\t \tIC50\tEC99\tEC90\tCC50\tEC50\tCC50\t \t(1)\t24\tNDf\tND\tND\t \t107\t\u003e200\t \t(2)\t\u003e500\tND\tND\tND\t \t\u003e100\t\u003e200\t \t(3)\t\u003e500\tND\tND\t\u003e200\t \t5.9\t48\t \t(4)\t\u003e500\tND\tND\t\u003e200\t \t6.3\t33\t \t(5)\t67\t\u003e25\t\u003e25\t≥146\t \t2.6\t10\t \t(6)\t\u003e500\t\u003e50\t\u003e50\t\u003e200\t \t15\t14\t \t(7)\t54\t172\t100\t\u003e200\t\u003e2.0\t3.2\t8.9\t \t(8)\t\u003e500\t\u003e12.5\t\u003e12.5\t\u003e200\t \t1.9\t15\t \t(9)\t34\t16\t5.3\t\u003e200\t\u003e38\t5.5\t\u003e200\t \t(10)\t68\t14\t8.5\t111\t\u003e13\t0.40\t132\t \t(11)\t45\t30\t12\t\u003e200\t\u003e17\t5.6\t\u003e200\t \t(12)\t\u003e500\t\u003e12.5\t\u003e12.5\t\u003e200\t \t20\t39\t \t(13)\t69\t71\t34\t\u003e200\t\u003e5.9\t6.3\t\u003e200\t \t(14)\t\u003e500\t63\t37\t\u003e200\t\u003e5.4\t2.3\t\u003e200\t \t(15)\t8.9\t18\t7.5\t≥172\t≥23\t14\t\u003e200\t \t(16)\t454\t67\t28\t\u003e200\t\u003e7.1\t5.2\t\u003e200\t \t(17)\t482\t21\t8.1\t\u003e200\t\u003e25\t7.1\t\u003e200\t \t(18)\t83\t6.2\t2.2\t\u003e200\t\u003e91\t3.3\t\u003e200\t \t(19)\t\u003e500\t53\t26\t\u003e200\t\u003e7.7\t5.7\t\u003e200\t \t(20)\t18\t35\t11\t\u003e200\t\u003e18\t2.2\t\u003e200\t \t(21)\t13\t8.3\t3.6\t\u003e200\t\u003e56\t2.5\t\u003e200\t \t(22)\t75\t7.4\t3.4\t\u003e200\t\u003e59\t0.42\t\u003e200\t \t(23)\t8.7\t11\t3.5\t\u003e200\t\u003e57\t3.1\t\u003e200\t \t(24)\t131\t58\t26\t\u003e200\t\u003e7.7\t25\t\u003e200\t \t(25)\t40\t132\t70\t\u003e200\t\u003e2.9\t4.1\t\u003e200\t \t(26)\t30\t36\t13\t\u003e200\t\u003e15\t5.5\t\u003e200\t \t(27)\t36\tND\tND\tND\t \t21\t\u003e200\t \t(28)\t40\t158\t85\t\u003e200\t\u003e2.4\t7.2\t\u003e200\t \tDPBAe\t5.3\tND\tND\tND\t \tND\tND\t \tRibavirin\tND\t13\t8.5\t\u003e200\t\u003e24\t9.4\t\u003e200\t \t aRecombinant PA-Nter was incubated with the ssDNA plasmid substrate, a Mn2+-containing buffer and test compounds. Cleavage of the substrate was assessed after 2 hr incubation. The IC50 represents the compound concentration (in μM) required to obtain 50% inhibition of cleavage, calculated by nonlinear least-squares regression analysis (using GraphPad Prism software) of the results from 2–4 independent experiments. bMDCK cells were infected with influenza A virus (strain A/PR/8/34) and incubated with the compounds during 24 h. The virus yield in the supernatant was assessed by real-time qPCR. The EC99 and EC90 values represent the compound concentrations (in μM) producing a 2-log10 or 1-log10 reduction in virus titer, respectively, determined in 2–3 independent experiments. The cytotoxicity, assessed in uninfected MDCK cells, was expressed as the CC50 value (50% cytotoxic concentration, determined with the MTS cell viability assay, in μM). cHEK293T cells were co-transfected with the four vRNP-reconstituting plasmids and the luciferase reporter plasmid in the presence of the test compounds. The EC50 represents the compound concentration (in μM) producing 50% reduction in vRNP-driven firefly reporter signal, estimated at 24 h after transfection. The EC50 value was derived from data from 2–4 independent experiments, by nonlinear least-squares regression analysis (using GraphPad Prism software). The CC50 (in μM), i.e. the 50% cytotoxic concentration, was determined in untransfected HEK293T cells by MTS cell viability assay. dSI, selectivity index, defined as the ratio between the CC50 and EC90. eDPBA, 2,4-dioxo-4-phenylbutanoic acid. fND, not 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+[{"sourceid":"4981400","sourcedb":"","project":"","target":"","text":"Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA The Arabidopsis protein FPA controls flowering time by regulating the alternative 3′-end processing of the FLOWERING LOCUS (FLC) antisense RNA. FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain. The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower. We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution. The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them. Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs. Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain. Introduction Eukaryotic messenger RNAs (mRNAs) are made as precursors through transcription by RNA polymerase II (Pol II), and these primary transcripts undergo extensive processing, including 3′-end cleavage and polyadenylation. In addition, alternative 3′-end cleavage and polyadenylation is an essential and ubiquitous process in eukaryotes. Misregulation of (alternative) 3′-end processing can lead to various genetic defects, cancer and other diseases. There is currently great interest in understanding the molecular mechanisms and functional impacts of alternative 3′-end processing. Recently, the split ends (SPEN) family of proteins was identified as RNA binding proteins that regulate alternative 3′-end cleavage and polyadenylation. They are characterized by possessing N-terminal RNA recognition motifs (RRMs) and a conserved SPEN paralog and ortholog C-terminal (SPOC) domain (Fig 1A). The SPOC domain is believed to mediate protein-protein interactions and has diverse functions among SPEN family proteins, but the molecular mechanism of these functions is not well understood. Sequence conservation of SPOC domains. (A). Domain organization of A. thaliana FPA. (B). Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP. Residues in surface patch 1 are indicated with the orange dots, and those in surface patch 2 with the green dots. The secondary structure elements in the structure of FPA SPOC are labeled. Residues that are strictly conserved among the five proteins are shown in white with a red background, and those that are mostly conserved in red. FPA, a SPEN family protein in Arabidopsis thaliana and other plants, was found to regulate the 3′-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene. FPA promotes the 3′-end processing of class I FLC antisense RNAs, which includes the proximal polyadenylation site. This is associated with histone demethylase activity and down-regulation of FLC transcription. However, the functional mechanism of this complex is still not clear. Although a SPOC domain is found in all the SPEN family proteins, its sequence conservation is rather low. For example, the sequence identity between the SPOC domains of A. thaliana FPA and human SMRT/HDAC1 Associated Repressor Protein (SHARP) is only 19% (Fig 1B). Currently, the SHARP SPOC domain is the only one with structural information. As a first step toward understanding the molecular basis for the regulation of alternative 3′-end processing and flowering by FPA, we have determined the crystal structure of the SPOC domain of A. thaliana FPA at 2.7 Å resolution. The overall structure is similar to that of the SHARP SPOC domain, although there are also substantial conformational differences between them. The structure reveals a surface patch that is conserved among FPA homologs. Results and Discussion Structure of FPA SPOC domain The crystal structure of the SPOC domain of A. thaliana FPA has been determined at 2.7 Å resolution using the selenomethionyl single-wavelength anomalous dispersion method. The expression construct contained residues 433–565 of FPA, but only residues 439–460 and 465–565 are ordered in the crystal. The atomic model has good agreement with the X-ray diffraction data and the expected bond lengths, bond angles and other geometric parameters (Table 1). All the residues are located in the favored regions of the Ramachandran plot (data not shown). The structure has been deposited in the Protein Data Bank, with accession code 5KXF. Summary of crystallographic information. Resolution range (Å)1\t50–2.7 (2.8–2.7)\t \tNumber of observations\t78,008\t \tRmerge (%)\t10.5 (45.3)\t \tI/σI\t24.1 (6.3)\t \tRedundancy\t\t \tCompleteness (%)\t100 (100)\t \tR factor (%)\t19.2 (25.0)\t \tFree R factor (%)\t25.4 (35.4)\t \tRms deviation in bond lengths (Å)\t0.017\t \tRms deviation in bond angles (°)\t1.9\t \t 1The numbers in parentheses are for the highest resolution shell. The crystal structure of the FPA SPOC domain contains a seven-stranded, mostly anti-parallel β-barrel (β1-β7) and three helices (αA-αC) (Fig 2A). Only two of the neighboring strands, β1 and β3, are parallel to each other. Helix αB covers one end of the barrel, while helices αA and αC are located next to each other at one side of the barrel (Fig 2B). The other end of the β-barrel is covered by the loop connecting strands β2 and β3, which contains the disordered 461–464 segment. The center of the barrel is filled with hydrophobic side chains and is not accessible to the solvent. Crystal structure of the SPOC domain of A. thaliana FPA. (A). Schematic drawing of the structure of FPA SPOC domain, colored from blue at the N terminus to red at the C terminus. The view is from the side of the β-barrel. The disordered segment (residues 460–465) is indicated with the dotted line. (B). Structure of the FPA SPOC domain, viewed from the end of the β-barrel, after 90° rotation around the horizontal axis from panel A. All structure figures were produced with PyMOL (www.pymol.org). Comparisons to structural homologs of the SPOC domain Only five structural homologs of the FPA SPOC domain were found in the Protein Data Bank with the DaliLite server, suggesting that the SPOC domain structure is relatively unique. The top hit is the SPOC domain of human SHARP (Fig 3A), with a Z score of 12.3. The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D). The next structural homolog has a Z score of 3.0. Structural homologs of the FPA SPOC domain. (A). Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray). The bound position of a doubly-phosphorylated peptide from SMRT is shown in magenta. (B). Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 β-barrel domain (gray). Ku80 contains a homologous domain (green), which forms a hetero-dimer with that in Ku70. The two domains, and inserted segments on them, mediate the binding of dsDNA (orange). The red rectangle highlights the region of contact between the two β-barrel domains. (C). Overlay of the structures of the FPA SPOC domain (cyan) and the homologous domain in Chp1 (gray). The binding partner of Chp1, Tas3, is shown in green. The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (D). Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray). SHARP is a transcriptional co-repressor in the nuclear receptor and Notch/RBP-Jκ signaling pathways. The SPOC domain of SHARP interacts directly with silencing mediator for retinoid and thyroid receptor (SMRT), nuclear receptor co-repressor (N-CoR), HDAC, and other components to represses transcription. While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the β-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A). In addition, the SHARP SPOC domain has three extra helices. One of them covers the other end of the β-barrel, and the other two shield an additional surface of the side of the β-barrel from solvent. A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands β1 and β3 (Fig 3A). Such a binding mode probably would not be possible in FPA, as the peptide would clash with the β1-β2 loop. The Ku70-Ku80 hetero-dimer is involved in DNA double-strand break repair and the β-barrel domain contributes to DNA binding. In fact, the β-barrel domains of Ku70 and Ku80 form a hetero-dimer, primarily through interactions between the loops connecting the third and fourth strands of the barrel (Fig 3B). The open ends of the two β-barrels face the DNA binding sites, and contact the phosphodiester backbone of the dsDNA. In addition, a long insert connecting strands β2 and β3 in the two domains form an arch-like structure, encircling the dsDNA. Chp1 is a subunit of the RNA-induced initiation of transcriptional gene silencing (RITS) complex. The partner of Chp1, Tas3, is bound between the barrel domain and the second domain of Chp1, and the linker between the two domains is also crucial for this interaction (Fig 3C). It is probably unlikely that the β-barrel itself is sufficient to bind Tas3. Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A). Mediator is a coactivator complex that promotes transcription by Pol II. The Med25 subunit ACID is the target of the potent activator VP16 of the herpes simplex virus. The structure of ACID contains a helix at the C-terminus as well as an extended β1-β2 loop. Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands β1 and β3, that is used by the SHARP and Tas3 SPOC domains for binding their partners. A conserved surface patch in the FPA SPOC domain An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A). This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B). The first surface patch is electropositive in nature (Fig 4C), and residues Arg477 and Tyr515 are also conserved in the SHARP SPOC domain (Fig 1B). In fact, one of the phosphorylated residues of the SMRT peptide interacts with this surface patch (Fig 3A), suggesting that the FPA SPOC domain might also interact with a phosphorylated segment here. In comparison, the second surface patch is more hydrophobic in nature (Fig 4C). A conserved surface patch of FPA SPOC domain. (A). Two views of the molecular surface of FPA SPOC domain colored based on sequence conservation among plant FPA homologs. Purple: most conserved; cyan: least conserved. (B). Residues in the conserved surface patch of FPA SPOC domain. The side chains of the residues are shown in stick models, colored orange in the first sub-patch and green in the second. (C). Molecular surface of FPA SPOC domain colored based on electrostatic potential. Blue: positively charged; red: negatively charged. Testing the requirement of specific conserved amino acids for FPA functions We next examined the potential impact of the conserved surface patch on FPA function in vivo. We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain (Fig 1B) and were found to be functionally important. The mutations were introduced into a transgene designed to express FPA from its native control elements (promoter, introns and 3′ UTR). The resulting transgenes were then stably transformed into an fpa-8 mutant background so that the impact of the mutations on FPA function could be assessed. Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig). We examined independent transgenic lines expressing each R477A and Y515A mutation. In each case, we confirmed that detectable levels of FPA protein expression were restored close to wild-type levels in protein blot analyses using antibodies that specifically recognize FPA (S2 Fig). We then examined the impact of the surface patch mutations on FPA’s function in controlling RNA 3′-end formation by determining whether the mutant proteins functioned in FPA autoregulation and the repression of FLC expression. FPA autoregulates its expression by promoting cleavage and polyadenylation within intron 1 of its own pre-mRNA, resulting in a truncated transcript that does not encode functional protein. We used RNA gel blot analyses to reveal that in each of three independent transgenic lines for each single mutant, rescue of proximally polyadenylated FPA pre-mRNA can be detected (Fig 5A and 5B). We therefore conclude that neither of these mutations disrupted the ability of FPA to promote RNA 3′-end formation in its own transcript. Impact of individual FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA. RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A\n(A), or FPA::FPA Y515A\n(B) using poly(A)+ purified mRNAs. A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs. RNA size (kb) marker (Ambion). TUBULIN was detected as an internal control. Proximally and distally polyadenylated FPA transcripts are marked with arrows. The ratio of distal:proximal polyadenylated forms is given under each lane. (C,D) Impact of individual FPA SPOC domain mutations on FLC transcript levels. qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A\n(C), FPA::FPA Y515A\n(D) plants. Transcript levels were normalized to the control UBC. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates. We next examined whether the corresponding mutations disrupted the ability of FPA to control FLC expression. We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D). Since each surface patch mutation appeared to be insufficient to disrupt FPA functions on its own, we combined both mutations into the same transgene. We could again confirm that near wild-type levels of FPA protein were expressed from three independent transgenic lines expressing the FPA R477A;Y515A doubly mutated protein in an fpa-8 mutant background (S3 Fig). We found that FPA R477A;Y515A protein functioned like wild-type FPA to restore FPA pre-mRNA proximal polyadenylation (Fig 6A) and FLC expression to wild-type levels (Fig 6B). Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and FLC expression. (A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs. Black arrows indicate the proximally and distally polyadenylated FPA mRNAs. A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs. RNA size (kb) marker (Ambion). TUBULIN was detected as an internal control. The ratio of distal:proximal polyadenylated forms is given under each lane. (B). qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, and FPA::FPA R477A;Y515A plants. Transcript levels were normalized to the control UBC. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates. Together our findings suggest that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation, or that this combination of mutations is not sufficient to critically disrupt the function of the FPA SPOC domain. Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain. Materials and Methods Protein expression and purification The SPOC domain (residue 433–565) of A. thaliana FPA was sub-cloned into the pET28a vector (Novagen). The recombinant protein, with an N-terminal hexa-histidine tag, was over-expressed in E. coli BL21 Star (DE3) cells (Novagen), which were induced with 0.4 mM IPTG and allowed to grow at 20°C for 14–18 h. The soluble protein was purified by nickel-charged immobilized-metal affinity chromatography and gel filtration chromatography. The purified protein was concentrated and stored at –80°C in a buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 10 mM DTT and 5% (v/v) glycerol. The His-tag was not removed for crystallization. The selenomethionine labeled SPOC domain was expressed in E. coli B834(DE3) strain using LeMaster media and purified with the same protocol as the native protein. Protein crystallization Crystals of the native SPOC domain of FPA were grown at 20°C with the sitting-drop vapor diffusion method. The protein solution was at 30 mg/ml concentration, and the reservoir solution contained 0.2 M MgSO4, and 20% (v/v) PEG 3350. Fully-grown crystals were obtained two days after set-up. Crystals of the selenomethionine labeled SPOC domain were grown using the same condition as the native protein. The crystals were cryo-protected in the crystallization solution supplemented with 20% (v/v) glycerol and flash-frozen in liquid nitrogen for data collection at 100K. Data collection and processing A single-wavelength anomalous dispersion (SAD) X-ray diffraction data set on a selenomethionine labeled SPOC domain crystal was collected at the National Synchrotron Light Source (NSLS) beamline X29A using an ADSC Q315r CCD. The diffraction images were processed and scaled with the HKL package. The crystal belongs to space group P65, with unit cell parameters of a = b = 108.2 Å, and c = 34.2 Å. Structure determination and refinement The structure of the SPOC domain was solved by the selenomethionyl SAD method with the program SHELX. The phases were used by program PHENIX for automatic model building. Manual model rebuilding was carried out with Coot. The structure refinement was performed with the program PHENIX, with translation, libration, and screw-rotation (TLS) parameters. The data processing and refinement statistics are summarized in Table 1. The Ramachandran plot showed that 95.8% of the residues are located in the most favored regions, and 4.2% are in additional allowed regions. Generation of constructs with mutated genomic FPA sequence A series of constructs containing a mutated FPA genomic sequence was prepared based on pGreen I 0029 vector. pGreen I 0029 vector with inserted FPA genomic sequence was prepared. In this vector FPA genomic sequence is flanked by 2620bp of the native sequence upstream to the start codon and 1178bp downstream to the stop codon. The vector contains kanamycin resistance genes for both the bacteria and plant hosts. In order to obtain a series of constructs with mutated FPA genomic sequence, FPA sequence in this construct was modified using site-directed mutagenesis. Primers used to prepare required constructs are listed in S1 Table. After the mutagenesis reaction the presence of only the desired mutations was confirmed by sequencing of the whole FPA genomic sequence and flanking regions. Generation of Arabidopsis thaliana transgenic plants All transgenic plants were prepared in fpa-8 mutant background, which is in Col-0 accession. The prepared vectors for Arabidopsis transformations were introduced into electro-competent Agrobacterium tumefaciens cells (C58 CV3101 strain harbouring pSoup vector). The floral dip method was used for plant transformation. Transgenic plants were selected using kanamycin as a selection marker. Presence of the desired mutations in plants was confirmed with specific dCaps markers. Plant growth conditions Wild type Col-0 plants used in this study were obtained from the Nottingham Arabidopsis Stock Centre. Seed of fpa-8 and 35S::FPA:YFP were obtained from Professor Caroline Dean. Plants were grown in pots containing Universal Extra general purpose soil. The glasshouse temperature was maintained at 20°C and the 16 hour daylight was provided by high pressure sodium vapour lamps (Philips Powertone SON-T AGRO 400). In order to grow plants in sterile conditions, seeds were first surface sterilized by a 5 min treatment with sterilizing solution (3% v/v sodium hypochlorite, 0.02% v/v Triton X-100), followed by three washes with 0.02% v/v Triton X-100 and one wash with sterile water. The sterile seeds were sown on MS10 media supplemented with 0.8% w/v agar. MS10 medium was also supplemented with specific antibiotics if required. After sowing, the seeds were stratified at 4°C for two days in order to synchronize their germination. Plants were grown in the tissue culture room at the following conditions: temperature 22°C, 16 hours daylight provided by the Master TL-D 36W/840 (Philips) lamps. Plant protein analysis Total protein samples were prepared using extraction buffer containing: 40 mM Tris-HCl, pH 6.8; 0.1 mM EDTA, pH 8.0; 8 M urea; 1.43 M β-mercaptoethanol, 7% v/v Complete Protease Inhibitors (Roche) and 5 mM PMSF. Equal volumes of samples were separated on 8% SDS-PAGE. Proteins were transferred onto Protran nitrocellulose transfer membrane (Whatman) using wet Criterion blotter system (BioRad). The transfer was performed at room temperature for two hours at a stable voltage of 70 V. Membrane was blocked in 3% (w/v) Milk in TBS for 1h at room temperature followed by overnight incubation with anti-FPA antibody (dilution 1:100 in 3% (w/v) Milk in TBS). After washes the membrane was incubated for 75 min with goat anti-rabbit antibody (Thermo Scientific) (1:3000 dilution in 3% (w/v) Milk in TBS). Protein was detected using SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific). Blots were re-probed following treatment with low pH solution (25mM glycine-HCl, pH 2, 1% (w/v) SDS) followed by blocking for 1h at room temperature in 3% (w/v) Milk in TBS. The membrane was incubated overnight with anti-TUBB2A, tubulin, beta 2A antibody (ARP40177_P050 Aviva systems biology; (dilution 1:1000 in 3% (w/v) Milk in TBS). After washes the membrane was incubated for 75 min with goat anti-rabbit antibody (Thermo Scientific) [1:3000 dilution in 3% (w/v) Milk in TBS]. Signal was detected using SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific). RNA gel blot analysis and RT-qPCR RNA gel blot analysis and RT-qPCR method performed as previously described. Supporting Information References Pre-mRNA processing reaches back to transcription and ahead to translation Molecular mechanisms of eukaryotic pre-mRNA 3' end processing regulation RNA processing and its regulation: global insights into biological networks Structure and function of pre-mRNA 5'-end capping quality control and 3'-end processing Alternative mRNA polyadenylation in eukaryotes: an effective regulator of gene expression Alternative cleavage and polyadenylation: the long and short of it 3' end mRNA processing: molecular mechanisms and implications for health and disease Implications of polyadenylation in health and disease Split end family RNA binding proteins: novel tumor suppressors coupling transcriptional regulation with RNA processing SPOC: a widely distributed domain associated with cancer, apoptosis and transcription The crystal structure of the Split End protein SHARP adds a new layer of complexity to proteins containing RNA recognition motifs FPA, a gene involved in floral induction in Arabidopsis, encodes a protein containing RNA-recognition motifs The spen family protein FPA controls alternative cleavage and polyadenylation of RNA Alternative polyadenylation of antisense RNAs and flowering time control A conserved structural motif reveals the essential transcriptional repression function of Spen proteins and their role in developmental signaling Structural insights into the recruitment of SMRT by the corepressor SHARP under phosphorylative regulation Searching protein structure databases with DaliLite v.3 Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair The Chp1-Tas3 core is a multifunctional platform critical for gene silencing by RITS Structure and VP16 binding of the Mediator Med25 activator interaction domain Structure of the VP16 transactivator target in the Mediator Solution NMR structure of MED25(391–543) comprising the activator-interacting domain (ACID) of human mediator subunit 25 NMR structure of the human Mediator MED25 ACID domain Sharp, an inducible cofactor that integrates nuclear receptor repression and activation SHARP is a novel component of the Notch/RBP-Jkappa signalling pathway ConSurf: an algorithmic tool for the identification of functional regions in proteins by surface mapping of phylogenetic information Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure Processing of X-ray diffraction data collected in oscillation mode Determination of macromolecular structures from anomalous diffraction of synchrotron radiation Substructure solution with SHELXD PHENIX: building a new software for automated crystallographic structure determination Coot: model-building tools for molecular graphics pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation Widespread role for the flowering-time regulators FCA and FPA in RNA-mediated chromatin silencing Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis 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+[{"sourceid":"4993997","sourcedb":"","project":"","target":"","text":"Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity. Acetylation is one of the most ubiquitous modifications that plays a vital role in many biological processes, such as transcriptional regulation, protein-protein interaction, enzyme activity, protein stability, antibiotic resistance, biological rhythm and so on. Protein acetylation can be grouped into lysine Nε-acetylation and peptide N-terminal acetylation (Nt-acetylation). Generally, Nε-acetylation refers to the transfer of an acetyl group from an acetyl coenzyme A (Ac-CoA) to the ε-amino group of lysine. This kind of modification is catalyzed by lysine acetyltransferases (KATs), some of which are named histone acetyltransferases (HATs) because early studies focused mostly on the post-transcriptional acetylation of histones. Despite the prominent accomplishments in the field regarding Nε-acetylation by KATs for over 50 years, the significance of the more evolutionarily conserved Nt-acetylation is still inconclusive. Nt-acetylation is an abundant and evolutionarily conserved modification occurring in bacteria, archaea and eukaryotes. It is estimated that about 80–90% of soluble human proteins and 50–70% of yeast proteins are subjected to Nt-acetylation, where an acetyl moiety is transferred from Ac-CoA to the α-amino group of the first residue. Recently Nt-acetylome expands the Nt-acetylation to transmembrane proteins. Unlike Nε-acetylation that can be eliminated by deacetylases, Nt-acetylation is considered irreversible since no corresponding deacetylase is found to date. Although Nt-acetylation has been regarded as a co-translational modification traditionally, there is evidence that post-translational Nt-acetylation exists. During the past decades, a large number of Nt-acetylome researches have shed light on the functional roles of Nt-acetylation, including protein degradation, subcellular localization, protein-protein interaction, protein-membrane interaction, plant development, stress-response and protein stability. The Nt-acetylation is carried out by N-terminal acetyltransferases (NATs) that belong to the GNAT superfamily. To date, six NATs (NatA/B/C/D/E/F) have been identified in eukaryotes. About 40 percent of Nt-acetylation of soluble proteins in cells is catalyzed by NatA complex which is composed of the catalytic subunit Naa10p and the auxiliary subunit Naa15p. NatE was found to physically interact with the NatA complex without any observation of impact on NatA-activity. Two other multimeric complexes of NATs are NatB and NatC which contain the catalytic subunits Naa20 and Naa30 and the auxiliary subunits Naa25 and Naa35/Naa38, respectively. Furthermore, only the catalytic subunits Naa40 and Naa60 were found for NatD and NatF, respectively. Besides Nt-acetylation, accumulating reports have proposed Nε-acetylation carried out by NATs. There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family. Unlike other NATs that are highly conserved among lower and higher eukaryotes, NatF only exists in higher eukaryotes. Subsequent researches indicated that NatF displays its catalytic ability with both Nt-acetylation and lysine Nε-acetylation. As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91. Another important feature of NatF is that this protein is anchored on the Golgi apparatus through its C-terminal membrane-integrating region and takes part in the maintaining of Golgi integrity. With its unique intracellular organellar localization and substrate selectivity, NatF appears to provide more evolutionary information among the NAT family members. It was recently found that NatF facilitates nucleosomes assembly and that NAA60 knockdown in MCF7-cell inhibits cell proliferation, sensitizes cells to DNA damage and induces cell apoptosis. In Drosophila cells, NAA60 knockdown induces chromosomal segregation defects during anaphase including lagging chromosomes and chromosomal bridges. Much recent attention has also been focused on the requirement of NatF for regulation of organellar structure. In HeLa cells, NAA60 knockdown causes Golgi apparatus fragmentation which can be rescued by overexpression Naa60. The systematic investigation of publicly available microarray data showed that NATs share distinct tissue-specific expression patterns in Drosophila and NatF shows a higher expression level in central nervous system of Drosophila. In this study, we solved the structures of human Naa60 (NatF) in complex with coenzyme. The hNaa60 protein contains a unique amphipathic α-helix (α5) following its GNAT domain that might account for the Golgi localization of this protein. Crystal structures showed that the β7-β8 hairpin rotated about 50 degrees upon removing the C-terminal region of the protein and this movement substantially changed the geometry of the substrate-binding pocket. Remarkably, we find that Phe 34 may participate in the proper positioning of the coenzyme for the transfer reaction to occur. Further structure comparison and biochemical studies also identified other key structural elements essential for the enzyme activity of Naa60. Results Overall structure of hNaa60 In the effort to prepare the protein for structural studies, we tried a large number of hNaa60 constructs but all failed due to heavy precipitation or aggregation. Sequence alignment of Naa60 from different species revealed a Glu-Glu-Arg (EER) versus Val-Val-Pro (VVP) sequence difference near the N-terminus of the protein in Xenopus Laevis versus Homo sapiens (Fig. 1A). Considering that terminal residues may lack higher-order structure and hydrophobic residues in this region may expose to solvent and hence cause protein aggregation, we mutated residues 4–6 from VVP to EER for the purpose of improving solubility of this protein. According to previous studies, this N-terminal region should not interfere with hNaa60’s Golgi localization. We tried many hNaa60 constructs with the three-residues mutation but only the truncated variant 1-199 and the full-length protein behaved well. We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C). Hereafter, all deletions or point mutants of hNaa60 we describe here are with the EER mutation. The crystal structures of hNaa60(1-242)/Ac-CoA and hNaa60(1-199)/CoA were determined by molecular replacement and refined to 1.38 Å and 1.60 Å resolution, respectively (Table 1). The electron density maps were of sufficient quality to trace residues 1-211 of hNaa60(1-242) and residues 5-199 of hNaa60(1-199). The structure of hNaa60 protein contains a central domain exhibiting a classic GCN5-related N-acetyltransferase (GNAT) folding, along with the extended N- and C-terminal regions (Fig. 1B,C). The central domain includes nine β strands (β1-β9) and four α-helixes (α1-α4) and is highly similar to the known hNaa50p and other reported NATs (Fig. 1D). However, in hNaa60, there is an extra 20-residue loop between β3 and β4 that forms a small subdomain with well-defined 3D structure (Fig. 1B–D). Furthermore, the β7-β8 strands form an approximately antiparallel β-hairpin structure remarkably different from that in hNaa50p (Fig. 1D). The N- and C-terminal regions form helical structures (α0 and α5) stretching out from the central GCN5-domain (Fig. 1C). Interestingly, we found that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) (Figure S1), indicating that residues 200–242 may have some auto-inhibitory effect on the activity of the enzyme. However, since this region was not visible in the hNaa60(1-242) crystal structure, we do not yet understand how this happens. Another possibility is that since hNaa60 is localized on Golgi apparatus, the observed low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies. For the convenience of studying the kinetics of mutants, the mutagenesis studies described hereafter were all based on hNaa60 (1-199). An amphipathic α-helix in the C-terminal region may contribute to Golgi localization of hNaa60 There is one hNaa60 molecule in the asymmetric unit in the hNaa60(1-242)/Ac-CoA structure. The C-terminal region extended from the GCN5-domain forms an amphipathic helix (α5) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between α5-helix and a hydrophobic groove between the N-terminal β1 and β3 strands of the neighbor molecule (Fig. 2A). The C-terminal extension following α5-helix forms a β-turn that wraps around and interacts with the neighbor protein molecule through hydrophobic interactions, too. In the hNaa60(1-199)/CoA structure, a part of the α5-helix is deleted due to truncation of the C-terminal region (Fig. 1B). Interestingly, the remaining residues in α5-helix still form an amphipathic helix although the hydrophobic interaction with the N-terminal hydrophobic groove of a neighbor molecule is abolished and the helix is largely exposed in solvent due to different crystal packing (Fig. 2B). A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc. The β7-β8 hairpin showed alternative conformations in the hNaa60 crystal structures Superposition of hNaa60(1-242)/Ac-CoA, hNaa60(1-199)/CoA and hNaa50/CoA/peptide (PDB 3TFY) revealed considerable difference in the β7-β8 hairpin region despite the overall stability and similarity of the GNAT domain (Fig. 1D). In hNaa60(1-242), the β7-β8 hairpin is located in close proximity to the α1-α2 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (β6-β7 loop). Upon removing the C-terminal region of hNaa60, we observed that hNaa60 (1-199) molecules pack in a different way involving the β7-β8 hairpin in the crystal, leading to about 50 degree rotation of the hairpin which moves away from the α1-α2 loop (Figs 1D and 2C). This conformational change substantially altered the geometry of the substrate binding site, which could potentially change the way in which the substrate accesses the active site of the enzyme. In hNaa60(1-242), the β7-β8 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only. KAT activity of hNaa60 toward histone H4 has been noted in previous study, and our enzyme kinetic data also indicated that hNaa60 can acetylate H3-H4 tetramer in vitro (Figure S3). Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4). We also conducted liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis on a synthetic peptide (NH2-MKGKEEKEGGAR-COOH) after treatment with hNaa60(1-199), and the data confirmed that both the N-terminal α-amine and lysine side-chain ε-amine were robustly acetylated after the treatment (Table S1). Despite these observations, the mechanism for this alternative activity remains unknown. Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site. Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D). Interestingly, in the hNaa60(1-199) crystal structure, the displaced β7-β8 hairpin opened a second way for the substrate to access the active center that would readily accommodate the binding of the H4 peptide (Fig. 2E), thus implied a potential explanation for KAT activity of this enzyme from a structural biological view. However, since hNaa60(1-242) and hNaa60(1-199) were crystallized in different crystal forms, the observed conformational change of the β7-β8 hairpin may simply be an artifact related to the different crystal packing. Whether the KAT substrates bind to the β7-β8 hairpin displaced conformation of the enzyme needs to be verified by further structural and functional studies. Phe 34 facilitates proper positioning of the cofactor for acetyl-transfer The electron density of Phe 34 side-chain is well defined in the hNaa60(1-242)/Ac-CoA structure, but becomes invisible in the hNaa60(1-199)/CoA structure, indicating displacement of the Phe 34 side-chain in the latter (Fig. 3A,B). A solvent-derived malonate molecule is found beside Phe 34 and the ethanethioate moiety of Ac-CoA in the high-resolution hNaa60(1-242)/Ac-CoA structure (Fig. 3A). Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A). Interestingly, in the structure of hNaa60(1-199)/CoA, the terminal thiol of CoA adopts alternative conformations. One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the α1-α2 loop and away from the substrate amine (Fig. 3B). To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C). Phe 27 in hNaa50p (equivalent to Phe 34 in hNaa60) has been implicated to facilitate the binding of N-terminal methionine of the substrate peptide through hydrophobic interaction. However, in the hNaa60/Ac-CoA structure, a hydrophilic malonate molecule is found at the same location where the N-terminal methionine should bind as is indicated by the superposition (Fig. 3A), suggesting that Phe 34 may accommodate binding of hydrophilic substrate, too. Moreover, orientation of Phe 34 side-chain seems to be co-related to positioning of the terminus of the co-enzyme and important for placing it at a location in close proximity to the substrate amine. We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme. Indeed, our enzyme kinetic data showed that hNaa60(1-199) F34A mutant showed no detectable activity (Fig. 5A). In order to rule out the possibility that the observed loss of activity may be related to bad folding of the mutant protein, we studied the circular dichroism (CD) spectrum of the protein (Fig. 5B) and determined its crystal structure (Fig. 3C). Both studies proved that the F34A mutant protein is well-folded. Many studies have addressed the crucial effect of α1-α2 loop on catalysis, showing that some residues located in this area are involved in the binding of substrates. We propose that Phe 34 may play a dual role both in interacting with the peptide substrate (recognition) and in positioning of the ethanethioate moiety of Ac-CoA to the right location to facilitate acetyl-transfer. Structural basis for hNaa60 substrate binding Several studies have demonstrated that the substrate specificities of hNaa60 and hNaa50 are highly overlapped. The structure of hNaa50p/CoA/peptide provides detailed information about the position of substrate N-terminal residues in the active site of hNaa50. Comparing the active site of hNaa60(1-242)/Ac-CoA with hNaa50p/CoA/peptide revealed that key catalytic and substrate binding residues are highly conserved in both proteins (Fig. 4A). With respect to catalysis, hNaa50p has been shown to employ residues Tyr 73 and His 112 to abstract proton from the α-amino group from the substrate’s first residue through a well-ordered water. A well-ordered water was also found between Tyr 97 and His 138 in hNaa60 (1-199)/CoA and hNaa60 (1-242)/Ac-CoA (Fig. 4B). To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B). Purity of all proteins were also analyzed by SDS-PAGE (Figure S5). As show in Fig. 5A, the mutants Y97A, Y97F, H138A and H138F abolished the activity of hNaa60. In contrast, to mutate the nearby solvent exposed residue Glu 37 to Ala (E37A) has little impact on the activity of hNaa60 (Figs 4B and 5A). In conclusion, the structural and functional studies indicate that hNaa60 applies the same two base mechanism through Tyr 97, His 138 and a well-ordered water as was described for hNaa50. The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A). Residues Tyr 38, Asn 143 and Tyr 165 are located around the malonate and interact with it through direct hydrogen bonds or water bridge (Fig. 4C). Although malonate is negatively charged, which is different from that of lysine ε-amine or peptide N-terminal amine, similar hydrophilic interactions may take place when substrate amine presents in the same position, since Tyr 38, Asn 143 and Tyr 165 are not positively or negatively charged. In agreement with this hypothesis, it was found that the Y38A, N143A and Y165A mutants all showed remarkably reduced activities as compared to WT, implying that these residues may be critical for substrate binding (Figs 4C and 5A). The β3-β4 loop participates in the regulation of hNaa60-activity Residues between β3 and β4 of hNaa60 form a unique 20-residue long loop (residues 73–92) that is a short turn in many other NAT members (Fig. 1D). Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain. We therefore used mass spectrometry to analyze if Lys 79 was acetylated in our bacterially purified proteins, and observed no modification on this residue (Figure S6). To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively. Interestingly, both K79R and K79Q mutants led to an increase in the catalytic activity of hNaa60, while K79A mutant led to modest decrease of the activity (Fig. 5A). These data indicate that the acetylation of Lys 79 is not required for optimal catalytic activity of hNaa60 in vitro. It is noted that the β3-β4 loop of hNaa60 acts like a door leaf to partly cover the substrate-binding pathway. We hence hypothesize that the β3-β4 loop may interfere with the access of the peptide substrates and that the solvent-exposing Lys 79 may play a potential role to remove the door leaf when it hovers in solvent (Fig. 4D). Acidic residues Glu 80, Asp 81 and Asp 83 interact with His 138, His 159 and His 158 to maintain the conformation of the β3-β4 loop, thus contribute to control the substrate binding (Fig. 4D). To verify this hypothesis, we mutated Glu 80, Asp 81 and Asp 83 to Ala respectively. In line with our hypothesis, E80A, D81A and D83A mutants exhibit at least 2-fold increase in hNaa60-activity (Fig. 5A). Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity. Discussion Nt-acetylation, which is carried out by the NAT family acetyltransferases, is an ancient and essential modification of proteins. Although many NATs are highly conserved from lower to higher eukaryotes and the substrate bias of them appears to be partially overlapped, there is a significant increase in the overall level of N-terminal acetylation from lower to higher eukaryotes. In this study we provide structural insights into Naa60 found only in multicellular eukaryotes. The N-terminus of hNaa60 harbors three hydrophobic residues (VVP) that makes it very difficult to express and purify the protein. This problem was solved by replacing residues 4–6 from VVP to EER that are found in Naa60 from Xenopus Laevis. Since Naa60 from human and from Xenopus Laevis are highly homologous (Fig. 1A), we speculate that these two proteins should have the same biological function. Therefore it is deduced that the VVP to EER replacement on the N-terminus of hNaa60 may not interfere with its function. However, in the hNaa60(1-242) structure the N-terminus adopts an α-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing. Hence it is not clear if the N-terminal end of wild-type hNaa60 is an α-helix, and what roles the hydrophobic residues 4–6 play in structure and function of wild-type hNaa60. In addition to the three-residue mutation (VVP to EER), we also tried many other hNaa60 constructs, but only the full-length protein and the truncated variant 1-199 behaved well. The finding that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) is intriguing. We speculate that low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies or there remains some undiscovered auto-inhibitory regulation in the full-length protein. The hNaa60 protein was proven to be localized on Golgi apparatus. Aksnes and colleagues predicted putative transmembrane domains and two putative sites of S-palmitoylation, by bioinformatics means, to account for Golgi localization of the protein. They then mutated all five cysteine residues of hNaa60’s to serine, including the two putative S-palmitoylation sites. However, these mutations did not abolish Naa60 membrane localization, indicating that S-palmitoylation is unlikely to (solely) account for targeting hNaa60 on Golgi. Furthermore, adding residues 217–242 of hNaa60 (containing residues 217–236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182–216 are important for Golgi localization of hNaa60. We found that residues 190–202 formed an amphipathic helix with an array of hydrophobic residues located on one side. This observation is reminiscent of the protein/membrane interaction through amphipathic helices in the cases of KalSec14, Atg3, PB1-F2 etc. In this model an amphipathic helix can immerse its hydrophobic side into the lipid bilayer through hydrophobic interactions. Therefore we propose that the amphipathic helix α5 may contribute to Golgi localization of hNaa60. This model, though may need further studies, is supported by the Aksnes studies. Previous studies indicated that members of NAT family are bi-functional NAT and KAT enzymes. However, known structures of NATs do not well support this hypothesis, since the β6-β7 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the α1-α2 loop, which would be good for the NAT but not KAT activity of the enzyme. Kinetic studies have been conducted to compare the NAT and KAT activity of hNaa50 in vitro, and indicate that the NAT activity of Naa50 is much higher than KAT activity. However, the substrate used in this study for assessing KAT activity was a small peptide which could not really mimic the 3D structure of a folded protein substrate in vivo. Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro. Conformational change of the β7-β8 hairpin (corresponding to the β6-β7 loop of other NATs) is noted in our structures (Figs 1D and 2C), which might provide an explanation to the NAT/KAT dual-activity in a structural biological view, but we were unable to rule out the possibility that the observed conformational change of this hairpin might be an artifact related to crystal packing or truncation of the C-terminal end of the protein. Further studies are therefore needed to reveal the mechanism for the KAT activity of this enzyme. The relationship between enzyme, co-enzyme and substrates has been documented for several years. In early years, researchers found adjustment of GCN5 histone acetyltransferase structure when it binds CoA molecule. The complexed form of NatA is more suitable for catalytic activation, since the α1-α2 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA). In the structure of hNaa50/CoA/peptide, Phe 27 in the α1-α2 loop appears to make hydrophobic interaction with the N-terminal Met of substrate peptide. However, the hNaa60(1-242)/Ac-CoA crystal structure indicated that its counterpart in hNaa60, Phe 34, could also accommodate the binding of a hydrophilic malonate that occupied the substrate binding site although it maintained the same conformation as that observed in hNaa50. Interestingly, the terminal thiol of CoA adopted alternative conformations in the structure of hNaa60(1-199)/CoA. One was to approach the substrate amine; the other was to approach the α1-α2 loop and away from the substrate amine. Same alternative conformations of CoA were observed in the hNaa60(1-199)(F34A) crystal structure, and our kinetic data showed that the F34A mutation abolished the activity of the enzyme. Taken together, our data indicated that Phe 34 in hNaa60 may play a role in placing co-enzyme at the right location to facilitate the acetyl-transfer. However, these data did not rule out that possibility that Phe 34 may coordinate the binding of the N-terminal Met through hydrophobic interaction as was proposed by previous studies. Furthermore, we showed that hNaa60 adopts the classical two base mechanism to catalyze acetyl-transfer. Although sequence identity between hNaa60 and hNaa50 is low, key residues in the active site of both enzymes are highly conserved. This can reasonably explain the high overlapping substrates specificities between hNaa60 and hNaa50. Another structural feature of hNaa60 that distinguishes it from other NATs is the β3-β4 long loop which appears to inhibit the catalytic activity of hNaa60. However, this loop also seems to stabilize the whole hNaa60 structure, because deletion mutations of this region led to protein precipitation and aggregation (Figure S7). A previous study suggested that the auto-acetylation of Lys 79 was important for hNaa60-activity, whereas the point mutation K79R did not decrease the activity of hNaa60 in our study. Meanwhile, no electron density of acetyl group was found on Lys 79 in our structures and mass spectrometry analysis. Hence, it appears that the auto-acetylation of hNaa60 is not an essential modification for its activity for the protein we used here. As for the reason why K79R in Yang’s previous studies reduced the activity of the enzyme, but in our studies it didn’t, we suspect that the stability of this mutant may play some role. K79R is less stable than the wild-type enzyme as was judged by its poorer gel-filtration behavior and tendency to precipitate. In our studies we have paid special attention and carefully handled this protein to ensure that we did get enough of the protein in good condition for kinetic assays. The intracellular environment is more complicated than our in vitro assay and the substrate specificity of hNaa60 most focuses on transmembrane proteins. The interaction between hNaa60 and its substrates may involve the protein-membrane interaction which would further increase the complexity. It is not clear if the structure of hNaa60 is different in vivo or if other potential partner proteins may help to regulate its activity. Nevertheless, our study may be an inspiration for further studies on the functions and regulation of this youngest member of the NAT family. Methods Cloning, expression and purification of Homo sapiens Naa60 (hNaa60) The cDNA encoding hNaa60 residues 1–242 (full-length) or residues 1–199 were amplified by PCR and inserted into the pET23a vector, which had been modified to provide an N-terminal 6xHis-tag followed by a tobacco etch virus (TEV) protease cleavage site. The VVP to EER (residues 4–6) mutation and other mutations for functional studies were introduced using the quick change method. The protein was expressed in Escherichia coli BL21 (DE3) or Escherichia coli BL21 (DE3) pLysS at 16 °C for 15 h in the presence of 0.1 mM IPTG. Cells were harvested at 4 °C by centrifugation (4,000 g for 10 min) and resuspended in buffer A containing 20 mM Tris, pH 8.0, 500 mM NaCl, 50 mM imidazole, 10% glycerol, 1 mM protease inhibitor PMSF (Phenylmethylsulfonyl fluoride) and 1 mM Tris (2-carboxyethyl)phosphine (TCEP) hydrochloride. Cells were lysed by sonication and the lysate was cleared by centrifugation (18,000 g at 4 °C for 20 min). Then the supernatant was loaded onto a 5-mL Chelating Sepharose column (GE Healthcare) charged with Ni2+ and washed with buffer B (20 mM Tris, pH 8.0, 500 mM NaCl, 50 mM imidazole, 1% glycerol and 1 mM TCEP). The protein was eluted with buffer C (20 mM Tris, pH 8.0, 500 mM NaCl, 300 mM imidazole, 1% glycerol and 1 mM TCEP). The eluent was digested by His-tagged TEV protease and concentrated by ultrafiltration at the same time. After 3 hours, the concentrated eluent was diluted 10 times with buffer D (20 mM Tris, pH 8.0, 500 mM NaCl, 1% glycerol and 1 mM TCEP) and the diluent was passed through the nickel column once again to remove the His-tagged TEV protease and the un-cleaved His-hNaa60 protein. The flow-through was concentrated to 500 μl and loaded onto a Superose 6 or Superdex 200 10/300 gel-filtration column (GE Healthcare) equilibrated with buffer E (20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP). Fractions containing the protein were collected and concentrated to a final concentration of 10 mg/ml for crystallization or acetyltransferases assays. Circular Dichroism (CD) Spectroscopy CD spectra of the proteins were obtained using a Jasco J-810 circular dichroism spectropolarimeter scanning from 190 to 250 nm with a 1 mm quartz cuvette. The wild-type and mutant proteins were examined at 4.5 μM concentration in 20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP at room temperature. All samples were centrifuged at 10,000 g for 5 min before analysis. Crystallization, data collection and structure determination The purified hNaa60(1-242), hNaa60(1-199) or F34A(1-199) protein was mixed with acetyl coenzyme A (Ac-CoA) or coenzyme A (CoA) (Sigma), respectively, at a 1:5 molar ratio before crystallization. All crystals were made by the hanging-drop vapor diffusion method. The crystallization reservoir solution for hNaa60(1-242) was 10 mM Tris pH 8.0, 75 mM NaCl, 0.5% glycerol, 3% v/v Tacsimate pH 4.0 (Hampton Research) and 7.5% w/v polyethylene glycol 3350 (PEG 3350), and for hNaa60(1-199) was 0.2 M L-Proline, 0.1 M HEPES pH 7.5, 10% w/v PEG 3350. Crystals of F34A mutation were obtained in 0.2 M Lithium Sulfate monohydrate, 0.1 M Tris pH 8.5, 20% w/v PEG 3350. The crystals were flash-frozen in liquid nitrogen in a cryo-protectant made of the reservoir solution supplemented with 25% glycerol. The diffraction data were collected at the Shanghai SSRF BL18U1 beamline or at the Argonne National Laboratory APS ID19 beamline at 100 K. The data were processed with HKL3000. The hNaa60(1-199) structure was determined by molecular replacement with Phaser using a previously reported GNAT family acetyltransferase structure (PDB 2AE6) as the search model. The hNaa60(1-242) structure was solved by molecular replacement using hNaa60(1-199) structure as the search model. To improve the model quality, the programs ARP/wARP in CCP4 or simulated-annealing in CNS were used. Iterative cycles of manual refitting and crystallographic refinement were performed using COOT and Phenix. Ac-CoA/CoA and malonate were modeled into the closely fitting positive Fo-Fc electron density and then included in following refinement cycles. Topology and parameter files for Ac-CoA/CoA and malonate were generated using PRODRG. All figures for the molecular models were prepared using the PyMOL program. Statistics of diffraction data processing and structure refinement are shown in Table 1. Acetyltransferase assay Acetyltransferase assay of hNaa60 was conducted as described previously. Briefly, a reaction cocktail containing 100 mM Tris-HCl buffer, pH 8.5, 0.07% alkylated BSA, 0.01% NP-40, 1 mM EDTA, 150 μM Ac-CoA (Sigma) was prepared and varied concentrations of the substrate peptide (0–400 μM) (NH2-MKGKEEKEGGAR-COOH) was added in a 1.5-mL microfuge tube, and then the respective enzyme was added to initiate the reaction with a final assay volume of 100 μL. The reaction was carried out for 20 minutes at 37 °C. Aliquots (40 μL) of the reaction were then removed and quenched with 40 μL of ice-cold isopropanol in individual wells of a 96-well black microplate (Corning), and then mixed with 80 μl of 25 μM 7-diethylamino-3-(49 maleimidylphenyl)-4-methylcoumarin (CPM) (Sigma) in 100 mM Tris-HCl (pH 8.5) and 1% Triton X-100 and allowed to react in darkness for 10 minutes prior to reading. The fluorescence signal was monitored using a Varioskan Flash plate reader (Thermo Scientific) at Exmax = 385 nm and Emmax = 465 nm. Substrate inhibition appeared at high concentrations of substrate peptide prevented our kinetics assays from reaching saturation of the enzyme. Therefore, we determined the value of kcat/Km by fitting our data to the equation: v = (kcat/Km)[ET][S] when the substrate concentration was far less than Km. The assays were done in triplicate. The slope of the line indicates the kcat/Km value of the enzyme (Figure S1). Additional Information How to cite this article: Chen, J.-Y. et al. Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase. Sci. Rep. 6, 31425; doi: 10.1038/srep31425 (2016). Supplementary Material Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation Structure and ligand of a histone acetyltransferase bromodomain Regulation of the p300 HAT domain via a novel activation loop N-terminal acetylation of cellular proteins creates specific degradation signals Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes Melatonin production: proteasomal proteolysis in serotonin N-acetyltransferase regulation GCN5-related N-acetyltransferases: a structural overview Histone acetyltransferase complexes: one size doesn’t fit all The biological functions of Naa10 - From amino-terminal acetylation to human disease Protein N-terminal acetyltransferases in cancer An organellar nalpha-acetyltransferase, naa60, acetylates cytosolic N termini of transmembrane proteins and maintains Golgi integrity N-terminal modifications of cellular proteins: The enzymes involved, their substrate specificities and biological effects Proteome-derived peptide libraries allow detailed analysis of the substrate specificities of N(alpha)-acetyltransferases and point to hNaa10p as the post-translational actin N(alpha)-acetyltransferase Protein N-terminal acetyltransferases: when the start matters Emerging Functions for N-Terminal Protein Acetylation in Plants Human Naa50p (Nat5/San) displays both protein N alpha- and N epsilon-acetyltransferase activity HAT4, a Golgi apparatus-anchored B-type histone acetyltransferase, acetylates free histone H4 and facilitates chromatin assembly NatF contributes to an evolutionary shift in protein N-terminal acetylation and is important for normal chromosome segregation Developmental roles of protein N-terminal acetylation An N-terminal Amphipathic Helix Binds Phosphoinositides and Enhances Kalirin Sec14 Domain-mediated Membrane Interactions Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3 The Influenza A Virus PB1-F2 Protein Targets the Inner Mitochondrial Membrane via a Predicted Basic Amphipathic Helix That Disrupts Mitochondrial Function Amphipathic helices and membrane curvature Structure of a ternary Naa50p (NAT5/SAN) N-terminal acetyltransferase complex reveals the molecular basis for substrate-specific acetylation Molecular basis for N-terminal acetylation by the heterodimeric NatA complex The molecular basis for histone H4- and H2A-specific amino-terminal acetylation by NatD Hat2p recognizes the histone H3 tail to specify the acetylation of the newly synthesized H3/H4 heterodimer by the Hat1p/Hat2p complex Implications for the evolution of eukaryotic amino-terminal acetyltransferase (NAT) enzymes from the structure of an archaeal ortholog Structure of Tetrahymena GCN5 bound to coenzyme A and a histone H3 peptide HKL-3000: the integration of data reduction and structure solution–from diffraction images to an initial model in minutes Phaser crystallographic software ARP/wARP and automatic interpretation of protein electron density maps Crystallography \u0026 NMR system: A new software suite for macromolecular structure determination Coot: model-building tools for molecular graphics PHENIX: a comprehensive Python-based system for macromolecular structure solution PRODRG, a program for generating molecular topologies and unique molecular descriptors from coordinates of small molecules Application of a fluorescent histone acetyltransferase assay to probe the substrate specificity of the human p300/CBP-associated factor Scaling and assessment of data quality Author Contributions J.-Y.C., L.L., C.-L.C., M.-J.L. and X.Y. designed and performed the experiments. K.T. collected diffraction data at APS. C.-H.Y. conceived and instructed the project. All authors are involved in data analysis. J.-Y.C. and C.-H.Y. wrote the manuscript. Overall structure of Naa60. (A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus). Alignment was generated using NPS@ and ESPript.3.0 (http://espript.ibcp.fr/ESPript/ESPript/). Residues 4–6 are highlighted in red box. (B) The structure of hNaa60(1-199)/CoA complex is shown as a yellow cartoon model. The CoA molecule is shown as sticks. (C) The structure of hNaa60(1-242)/Ac-CoA complex is presented as a cartoon model in cyan. The Ac-CoA and malonate molecules are shown as cyan and purple sticks, respectively. The secondary structures are labeled starting with α0. (D) Superposition of hNaa60(1-242) (cyan), hNaa60(1-199) (yellow) and hNaa50 (pink, PDB 3TFY). The Ac-CoA of hNaa60(1-242)/Ac-CoA complex is represented as cyan sticks. Amphipathicity of the α5 helix and alternative conformations of the β7-β8 hairpin. (A) The α5 helix of hNaa60(1-242) in one asymmetric unit (slate) interacts with another hNaa60 molecule in a neighboring asymmetric unit (cyan). A close view of the interaction is shown in red box. Side-chains of hydrophobic residues on α5 helix and the neighboring molecule participating in the interaction are shown as yellow and green sticks, respectively. (B) The α5 helix of hNaa60(1-199) in one asymmetric unit (yellow) interacts with another hNaa60 molecule in the neighboring asymmetric units (green). A close view of the interaction is shown in the red box. Side-chains of hydrophobic residues on α5 helix and the neighboring molecule (green) participating in the interaction are shown as yellow and green sticks, respectively. The third molecule (pink) does not directly interact with the α5 helix. (C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E). The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D). The α-amine of the NAT substrate and ε-amine of the KAT substrate (along with the lysine side-chain) subject to acetylation are shown as sticks. Electron density map of the active site. The 2Fo-Fc maps contoured at 1.0σ are shown for hNaa60(1-242)/Ac-CoA (A), hNaa60(1-199)/CoA (B) and hNaa60(1-199) F34A/CoA (C). The putative substrate peptide binding site is indicated by the peptide (shown as pink sticks) from the hNaa50/CoA/peptide complex structure after superimposing hNaa50 on the hNaa60 structures determined in this study. The black arrow indicates the α-amine of the first Met (M1) (all panels). The purple arrow indicates the acetyl moiety of Ac-CoA (A). The red arrow indicates the alternative conformation of the thiol moiety of the co-enzyme when Phe 34 side-chain is displaced (B) or mutated to Ala (C). Structural basis for hNaa60 catalytic activity. (A) Superposition of hNaa60 active site (cyan) on that of hNaa50 (pink, PDB 3TFY). Side-chains of key catalytic and substrate-binding residues are highlighted as sticks. The malonate molecule in the hNaa60(1-242)/Ac-CoA structure and the peptide in the hNaa50/CoA/peptide structure are shown as purple and yellow sticks respectively. (B) A close view of the active site of hNaa60. Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks. The water molecules participating in catalysis in the hNaa60 and hNaa50 structures are showed as green and red spheres, separately. (C) The interaction between the malonate molecule and surrounding residues observed in the hNaa60(1-242)/Ac-CoA structure. The yellow dotted lines indicate the hydrogen bonds. (D) A zoomed view of β3-β4 loop of hNaa60. Key residues discussed in the text (cyan), the malonate (purple) and Ac-CoA (gray) are shown as sticks. The yellow dotted lines indicate the salt bridges. Catalytic activity of hNaa60 and mutant proteins. (A) Catalytic efficiency (shown as kcat/Km values) of hNaa60 (1-199) WT and mutants. Error bars indicate the Standard Error (SE). (B) CD spectra of wild-type and mutant proteins from 250 nm to 190 nm. The sample concentration was 4.5 μM in 20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP at room temperature. Data collection and refinement statistics. Structure and PDB ID\thNaa60(1-242)/Ac-CoA 5HGZ\thNaa60(1-199)/CoA 5HH0\thNaa60(1-199)F34A/CoA 5HH1\t \tData collection*\t \t Space group\tP212121\tP21212\tP21212\t \tCell dimensions\t \t a, b, c (Å)\t53.3, 57.4, 68.8\t67.8, 73.8, 43.2\t66.7, 74.0, 43.5\t \t α,β,γ (°)\t90.0, 90.0, 90.0\t90.0, 90.0, 90.0\t90.0, 90.0, 90.0\t \tResolution (Å)\t50–1.38 (1.42–1.38)\t50–1.60 (1.66–1.60)\t50–1.80 (1.86–1.80)\t \tRp.i.m.(%)**\t3.0 (34.4)\t2.1 (32.5)\t2.6 (47.8)\t \tI/σ\t21.5 (2.0)\t31.8 (2.0)\t28.0 (2.4)\t \tCompleteness (%)\t99.8 (99.1)\t99.6 (98.5)\t99.9 (99.7)\t \tRedundancy\t6.9 (5.0)\t6.9 (6.2)\t6.3 (5.9)\t \tRefinement\t \t Resolution (Å)\t25.81–1.38\t33.55–1.60\t43.52–1.80\t \t No. reflections\t43660\t28588\t20490\t \t Rwork/Rfree\t0.182/0.192\t0.181/0.184\t0.189/0.209\t \tNo. atoms\t \t Protein\t1717\t1576\t1566\t \t Ligand/ion\t116\t96\t96\t \t Water\t289\t258\t168\t \tB-factors\t \t Protein\t23.8\t32.0\t37.4\t \t Ligand/ion\t22.2\t34.6\t43.7\t \t Water\t35.1\t46.4\t49.1\t \tR.m.s. deviations\t \t Bond lengths (Å)\t0.018\t0.017\t0.015\t \t Bond angles (°)\t1.529\t1.651\t1.581\t \tRamachandran Plot\t \t Favoured region\t98.98%\t98.93%\t98.96%\t \t Allowed region\t1.02%\t1.07%\t1.04%\t \t Outliers\t0.00%\t0.00%\t0.00%\t \t *Values in parentheses are for highest-resolution shell. One crystal was used for each data set. **Rp.i.m., a redundancy-independent R factor was used to evaluate the diffraction data quality as was proposed by 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+[{"sourceid":"5012862","sourcedb":"","project":"","target":"","text":"Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage. We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid. We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface. EncFtn adopts an open decameric structure that is topologically distinct from other ferritins. While EncFtn acts as a ferroxidase, it cannot mineralize iron. Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage. This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins. DOI: http://dx.doi.org/10.7554/eLife.18972.001 eLife digest Iron is essential for life as it is a key component of many different enzymes that participate in processes such as energy production and metabolism. However, iron can also be highly toxic to cells because it readily reacts with oxygen. This reaction can damage DNA, proteins and the membranes that surround cells. To balance the cell’s need for iron against its potential damaging effects, organisms have evolved iron storage proteins known as ferritins that form cage-like structures. The ferritins convert iron into a less reactive form that is mineralised and safely stored in the central cavity of the ferritin cage and is available for cells when they need it. Recently, a new family of ferritins known as encapsulated ferritins have been found in some microorganisms. These ferritins are found in bacterial genomes with a gene that codes for a protein cage called an encapsulin. Although the structure of the encapsulin cage is known to look like the shell of a virus, the structure that the encapsulated ferritin itself forms is not known. It is also not clear how encapsulin and the encapsulated ferritin work together to store iron. He et al. have now used the techniques of X-ray crystallography and mass spectrometry to determine the structure of the encapsulated ferritin found in some bacteria. The encapsulated ferritin forms a ring-shaped doughnut in which ten subunits of ferritin are arranged in a ring; this is totally different from the enclosed cages that other ferritins form. Biochemical studies revealed that the encapsulated ferritin is able to convert iron into a less reactive form, but it cannot store iron on its own since it does not form a cage. Thus, the encapsulated ferritin needs to be housed within the encapsulin cage to store iron. Further work is needed to investigate how iron moves into the encapsulin cage to reach the ferritin proteins. Some organisms have both standard ferritin cages and encapsulated ferritins; why this is the case also remains to be discovered. DOI: http://dx.doi.org/10.7554/eLife.18972.002 Introduction Encapsulin nanocompartments are a family of proteinaceous metabolic compartments that are widely distributed in bacteria and archaea. They share a common architecture, comprising an icosahedral shell formed by the oligomeric assembly of a protein, encapsulin, that is structurally related to the HK97 bacteriophage capsid protein gp5. Gp5 is known to assemble as a 66 nm diameter icosahedral shell of 420 subunits. In contrast, both the Pyrococcus furiosus and Myxococcus xanthus encapsulin shell-proteins form 32 nm icosahedra with 180 subunits; while the Thermotoga maritima encapsulin is smaller still with a 25 nm, 60-subunit icosahedron. The high structural similarity of the encapsulin shell-proteins to gp5 suggests a common evolutionary origin for these proteins. The genes encoding encapsulin proteins are found downstream of genes for dye-dependent peroxidase (DyP) family enzymes, or encapsulin-associated ferritins (EncFtn). Enzymes in the DyP family are active against polyphenolic compounds such as azo dyes and lignin breakdown products; although their physiological function and natural substrates are not known. Ferritin family proteins are found in all kingdoms and have a wide range of activities, including ribonucleotide reductase, protecting DNA from oxidative damage, and iron storage. The classical iron storage ferritin nanocages are found in all kingdoms and are essential in eukaryotes; they play a central role in iron homeostasis, where they protect the cell from toxic free Fe2+ by oxidizing it and storing the resulting Fe3+ as ferrihydrite minerals within their central cavity. The encapsulin-associated enzymes are sequestered within the icosahedral shell through interactions between the shell’s inner surface and a short localization sequence (Gly-Ser-Leu-Lys) appended to their C-termini. This motif is well-conserved, and the addition of this sequence to heterologous proteins is sufficient to direct them to the interior of encapsulins. A recent study of the Myxococcus xanthus encapsulin showed that it sequesters a number of different EncFtn proteins and acts as an ‘iron-megastore’ to protect these bacteria from oxidative stress. At 32 nm in diameter, it is much larger than other members of the ferritin superfamily, such as the 12 nm 24-subunit classical ferritin nanocage and the 8 nm 12-subunit Dps (DNA-binding protein from starved cells) complex; and is thus capable of sequestering up to ten times more iron than these ferritins. The primary sequences of EncFtn proteins have Glu-X-X-His metal coordination sites, which are shared features of the ferritin family proteins. Secondary structure prediction identifies two major α-helical regions in these proteins; this is in contrast to other members of the ferritin superfamily, which have four major α-helices (Supplementary file 1). The ‘half-ferritin’ primary sequence of the EncFtn family and their association with encapsulin nanocompartments suggests a distinct biochemical and structural organization to other ferritin family proteins. The Rhodospirillum rubrum EncFtn protein (Rru_A0973) shares 33% protein sequence identity with the M. xanthus (MXAN_4464), 53% with the T. maritima (Tmari_0787), and 29% with the P. furiosus (PF1192) homologues. The GXXH motifs are strictly conserved in each of these species (Supplementary file 1). Here we investigate the structure and biochemistry of EncFtn in order to understand iron storage within the encapsulin nanocompartment. We have produced recombinant encapsulin (Enc) and EncFtn from the aquatic purple-sulfur bacterium R. rubrum, which serves as a model organism for the study of the control of the bacterial nitrogen fixation machinery, in Escherichia coli. Analysis by transmission electron microscopy (TEM) indicates that their co-expression leads to the production of an icosahedral nanocompartment with encapsulated EncFtn. The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins. We identify a symmetrical iron bound ferroxidase center (FOC) formed between subunits in the decamer and additional metal-binding sites close to the center of the ring and on the outer surface. We also demonstrate the metal-dependent assembly of EncFtn decamers using native PAGE, analytical gel-filtration, and native mass spectrometry. Biochemical assays show that EncFtn is active as a ferroxidase enzyme. Through site-directed mutagenesis we show that the conserved glutamic acid and histidine residues in the FOC influence protein assembly and activity. We use our combined structural and biochemical data to propose a model for the EncFtn-catalyzed sequestration of iron within the encapsulin shell. Results Assembly of R. rubrum EncFtn encapsulin nanocompartments in E. coli Full-frame transmission electron micrographs of R. rubrum nanocompartments. (A/B) Negative stain TEM image of recombinant R. rubrum encapsulin and EncFtn-Enc nanocompartments. All samples were imaged at 143,000 x magnification; the scale bar length corresponds to 50 nm. (C) Histogram showing the distribution of nanocompartment diameters. A model Gaussian nonlinear least square function was fitted to the data to obtain a mean diameter of 24.6 nm with a standard deviation of 2.0 nm for encapsulin (grey) and a mean value of 23.9 nm with a standard deviation of 2.2 nm for co-expressed EncFtn and encapsulin (EncFtn-Enc, black). DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.004 Purification of recombinant R. rubrum encapsulin nanocompartments. (A) Recombinantly expressed encapsulin (Enc) and co-expressed EncFtn-Enc were purified by sucrose gradient ultracentrifugation from E. coli B834(DE3) grown in SeMet medium. Samples were resolved by 18% acrylamide SDS-PAGE; the position of the proteins found in the complexes as resolved on the gel are shown with arrows. (B/C) Negative stain TEM image of recombinant encapsulin and EncFtn-Enc nanocompartments. Samples were imaged at 143,000 x magnification, with scale bar shown as 25 nm. Representative encapsulin and EncFtn-Enc complexes are indicated with red arrows. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.003 We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A). TEM imaging of uranyl acetate-stained samples revealed that, when expressed in isolation, the encapsulin protein forms empty compartments with an average diameter of 24 nm (Figure 1B and Figure 1—figure supplement 1A/C), consistent with the appearance and size of the T. maritima encapsulin. We were not able to resolve any higher-order structures of EncFtn by TEM. Protein purified from co-expression of the encapsulin and EncFtn resulted in 24 nm compartments with regions in the center that exclude stain, consistent with the presence of the EncFtn within the encapsulin shell (Figure 1C and Figure 1—figure supplement 1B/C). R. rubrum EncFtn forms a metal-ion stabilized decamer in solution Purification of recombinant R. rubrum EncFtnsH. (A) Recombinant SeMet-labeled EncFtnsH produced with 1 mM Fe(NH4)2(SO4)2 in the growth medium was purified by nickel affinity chromatography and size-exclusion chromatography using a Superdex 200 16/60 column (GE Healthcare). Chromatogram traces measured at 280 nm and 315 nm are shown with the results from ICP-MS analysis of the iron content of the fractions collected during the experiment. The peak around 73 ml corresponds to a molecular weight of around 130 kDa when compared to calibration standards; this is consistent with a decamer of EncFtnsH. The small peak at 85 ml corresponds to the 13 kDa monomer compared to the standards. Only the decamer peak contains significant amounts of iron as indicated by the ICP-MS analysis. (B) Peak fractions from the gel filtration run were resolved by 15% acrylamide SDS-PAGE and stained with Coomassie blue stain. The bands around 13 kDa and 26 kDa correspond to EncFtnsH, as identified by MALDI peptide mass fingerprinting. The band at 13 kDa is consistent with the monomer mass, while the band at 26 kDa is consistent with a dimer of EncFtnsH. The dimer species only appears in the decamer fractions. (C) SEC-MALLS analysis of EncFtnsH from decamer fractions and monomer fractions allows assignment of an average mass of 132 kDa to decamer fractions and 13 kDa to monomer fractions, consistent with decamer and monomer species (Table 2). DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.005 Determination of the Fe/EncFtnsH protein ratio by ICP-MS. EncFtnsH was purified as a SeMet derivative from E. coli B834(DE3) cells grown in SeMet medium with 1 mM Fe(NH4)2(SO4)2. Fractions from SEC were collected, acidified and analysed by ICP-MS. EncFtnsH concentration was calculated based on the presence of two SeMet per mature monomer. Samples where the element was undetectable are labelled with n.d. These data were collected from EncFtnsH fractions from a single gel-filtration run. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.006 Peak\tEncFtnsHretention volume (ml)\tElement concentration (µM)\tDerived EncFtnsHconcentration (µM)\tDerived Fe/ EncFtnsH monomer\t \tCa\tFe\tZn\tSe\t \tDecamer\t66.5\tn.d.\t6.7\tn.d.\t24.6\t12.3\t0.5\t \t68.3\tn.d.\t28.4\tn.d\t124.5\t62.3\t0.5\t \t70.1\t2.9\t93.7\t2.4\t301.7\t150.9\t0.6\t \t71.9\t6.9\t120.6\t3.7\t379.8\t189.9\t0.6\t \t73.7\t1.9\t64.4\t0.8\t240.6\t120.3\t0.5\t \t75.5\t0.9\t21.1\tn.d.\t101.7\t50.8\t0.4\t \t77.3\tn.d.\t6.2\tn.d.\t42.6\t21.3\t0.3\t \t79.1\t0.1\t2.4\tn.d.\t26.5\t13.3\t0.2\t \t\t80.9\t1.0\t1.5\tn.d.\t22.3\t11.2\t0.1\t \t\t82.7\tn.d.\t0.2\tn.d.\t29.2\t14.6\tn.d\t \tMonomer\t84.5\tn.d.\t0.1\tn.d.\t34.9\t17.5\tn.d\t \t86.3\tn.d.\tn.d\tn.d.\t28.9\t14.4\tn.d\t \t88.1\tn.d.\tn.d.\tn.d.\t17.4\t8.7\tn.d.\t \t89.9\tn.d.\tn.d.\tn.d.\t5.5\t2.8\tn.d.\t \t91.7\tn.d.\tn.d.\tn.d.\t0.1\t0.07\t0.2\t \t Estimates of EncFtnsH molecular weight from SEC-MALLS analysis. EncFtnsH was purified from E. coli BL21(DE3) grown in minimal medium (MM) by nickel affinity chromatography and size-exclusion chromatography. Fractions from two peaks (decamer and monomer) were pooled separately (Figure 1C) and analysed by SEC-MALLS using a Superdex 200 10/300 GL column (GE Healthcare) and Viscotek SEC-MALLS instruments (Malvern Instruments) (Figure 2C). The decamer and monomer peaks were both symmetric and monodisperse, allowing the estimation of the molecular weight of the species in these fractions. The molecular weights are quoted to the nearest kDa due to the resolution limit of the instrument. The proteins analyzed by SEC-MALLS came from single protein preparation. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.007 Molecular Weight (kDa)\tDecamer peak\tMonomer peak\t \tTheoretical\t133\t13\t \tEncFtnsH-decamer fractions\t132\t15\t \tEncFtnsH-monomer fractions\t126\t13\t \t We purified recombinant R. rubrum EncFtn as both the full-length sequence (140 amino acids) and a truncated C-terminal hexahistidine-tagged variant (amino acids 1–96 plus the tag; herein EncFtnsH). In both cases the elution profile from size-exclusion chromatography (SEC) displayed two peaks (Figure 2A). SDS-PAGE analysis of fractions from these peaks showed that the high molecular weight peak was partially resistant to SDS and heat-induced denaturation; in contrast, the low molecular weight peak was consistent with monomeric mass of 13 kDa (Figure 2B). MALDI peptide mass fingerprinting of these bands confirmed the identity of both as EncFtn. Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the SEC fractions showed 100 times more iron in the oligomeric fraction than the monomer (Figure 2A, blue scatter points; Table 1), suggesting that EncFtn oligomerization is associated with iron binding. In order to determine the iron-loading stoichiometry in the EncFtn complex, further ICP-MS experiments were performed using selenomethionine (SeMet)-labelled protein EncFtn (Table 1). In these experiments, we observed sub-stoichiometric metal binding, which is in contrast to the classical ferritins. Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) analysis of samples taken from each peak gave calculated molecular weights consistent with a decamer for the high molecular weight peak and a monomer for the low molecular weight peak (Figure 2C, Table 2). Effect of metal ions on the oligomeric state of EncFtnsH in solution. (A/B) EncFtnsH-monomer was incubated with one mole equivalent of various metal salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column. Co2+ and Zn2+ induced the formation of the decameric form of EncFtnsH; while Mn2+, Mg2+ and Fe3+ did not significantly alter the oligomeric state of EncFtnsH. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.009 PAGE analysis of the effect of metal ions on the oligomeric state of EncFtnsH. 50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE. (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.010 Effect of Fe2+ and protein concentration on the oligomeric state of EncFtnsH in solution. (A) Recombinant EncFtnsH was purified by Gel filtration Superdex 200 chromatography from E. coli BL21(DE3) grown in MM or in MM supplemented with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+). A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH\nin vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column (GE Healthcare). Both Fe2+ salts tested induced the formation of decamer indicated by the peak between 1.2 and 1.6 ml. Monomeric and decameric samples of EncFtnsH are shown as controls. Peaks around 0.8 ml were seen as protein aggregation. (C) Analytical gel filtration of EncFtn monomer at different concentrations to illustrate the effect of protein concentration on multimerization. The major peak shows a shift towards a dimer species at high concentration of protein, but the ratio of this peak (1.5–1.8 ml) to the decamer peak (1.2–1.5 ml) does not change when compared to the low concentration sample. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.008 Gel-filtration peak area ratios for EncFtnsH decamer and monomer on addition of different metal ions. EncFtnsH was produced in E. coli BL21(DE3) cultured in MM and MM with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+) and purified by gel-filtration chromatography using an Superdex 200 16/60 column (GE Healthcare). Monomer fractions of EncFtnsH purified from MM were pooled and run in subsequent analytical gel-filtration runs over the course of three days. Samples of EncFtnsH monomer were incubated with one molar equivalent of metal ion salts at room temperature for two hours before analysis by analytical gel filtration chromatography (AGF) using a Superdex 200 10/300 GL column. The area for resulting protein peaks were calculated using the Unicorn software (GE Healthcare); peak ratios were calculated to quantify the propensity of EncFtnsH to multimerize in the presence of the different metal ions. The change in the ratios of monomer to decamer over the three days of experiments may be a consequence of experimental variability, or the propensity of this protein to equilibrate towards decamer over time. The increased decamer: monomer ratio seen in the presence of Fe2+, Co2+, and Zn2+ indicates that these metal ions facilitate multimerization of the EncFtnsH protein, while the other metal ions tested do not appear to induce multimerization. The analytical gel filtration experiment was repeated twice using two independent preparations of protein, of which values calculated from one sample are presented here. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.011 Method\tSample\tMonomer area\tDecamer area\tDecamer/Monomer\t \tGel filtration Superdex 200 chromatography\tEncFtnsH-MM\t64.3\t583.6\t0.1\t \tEncFtnsH-MM+Fe2+\t1938.4\t426.4\t4.5\t \tAnalytical Gel filtration Day1\tEncFtnsH-decamer fractions\t20.2\t1.8\t11.2\t \tEncFtnsH-monomer fractions\t2.9\t21.9\t0.1\t \tFe(NH4)2(SO4)2/EncFtnsH-monomer\t11.0\t13.0\t0.8\t \tFeSO4-HCl/EncFtnsH-monomer\t11.3\t11.4\t1.0\t \tAnalytical Gel filtration Day2\tEncFtnsH-monomer fractions\t8.3\t22.8\t0.4\t \tCoCl2/EncFtnsH-monomer\t17.7\t14.5\t1.2\t \tMnCl2/EncFtnsH-monomer\t3.1\t30.5\t0.1\t \tZnSO4/EncFtnsH-monomer\t20.4\t9.0\t2.3\t \tFeCl3/EncFtnsH-monomer\t3.9\t28.6\t0.1\t \tAnalytical Gel filtration Day3\tEncFtnsH-monomer fractions\t6.3\t23.4\t0.3\t \tMgSO4/EncFtnsH-monomer\t5.8\t30.2\t0.2\t \tCa acetate/EncFtnsH-monomer\t5.6\t25.2\t0.2\t \t We purified EncFtnsH from E. coli grown in MM with or without the addition of 1 mM Fe(NH4)2(SO4)2. The decamer to monomer ratio in the sample purified from cells grown in iron-supplemented media was 4.5, while that from the iron-free media was 0.11, suggesting that iron induces the oligomerization of EncFtnsH in vivo (Figure 3A, Table 3). To test the metal-dependent oligomerization of EncFtnsH in vitro, we incubated the protein with various metal cations and subjected samples to analytical SEC and non-denaturing PAGE. Of the metals tested, only Fe2+, Zn2+ and Co2+ induced the formation of significant amounts of the decamer (Figure 3B, Figure 3—figure supplement 1/2). While Fe2+ induces the multimerization of EncFtnsH, Fe3+ in the form of FeCl3 does not have this effect on the protein, highlighting the apparent preference this protein has for the ferrous form of iron. To determine if the oligomerization of EncFtnsH was concentration dependent we performed analytical SEC at 90 and 700 µM protein concentration (Figure 3C). At the higher concentration, no increase in the decameric form of EncFtn was observed; however, the shift in the major peak from the position of the monomer species indicated a tendency to dimerize at high concentration. Crystal structure of EncFtnsH Electrostatic surface of EncFtnsH. The solvent accessible surface of EncFtnsH is shown, colored by electrostatic potential as calculated using the APBS plugin in PyMOL. Negatively charged regions are colored red and positive regions in blue, neutral regions in grey. (A) View of the surface of the EncFtnsH decamer looking down the central axis. (B) Orthogonal view of (A). (C) Cutaway view of (B) showing the charge distribution within the central cavity. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.013 Crystal structure of EncFtnsH. (A) Overall architecture of EncFtnsH. Transparent solvent accessible surface view with α-helices shown as tubes and bound metal ions as spheres. Alternating subunits are colored blue and green for clarity. The doughnut-like decamer is 7 nm in diameter and 4.5 nm thick. (B) Monomer of EncFtnsH shown as a secondary structure cartoon. (C/D) Dimer interfaces formed in the decameric ring of EncFtnsH. Subunits are shown as secondary structure cartoons and colored blue and green for clarity. Bound metal ions are shown as orange spheres for Fe3+ and grey and white spheres for Ca2+. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.012 We determined the crystal structure of EncFtnsH by molecular replacement to 2.0 Å resolution (see Table 1 for X-ray data collection and refinement statistics). The crystallographic asymmetric unit contained thirty monomers of EncFtn with visible electron density for residues 7 – 96 in each chain. The protein chains were arranged as three identical annular decamers, each with D5 symmetry. The decamer has a diameter of 7 nm and thickness of 4 nm (Figure 4A). The monomer of EncFtn has an N-terminal 310-helix that precedes two 4 nm long antiparallel α-helices arranged with their long axes at 25° to each other; these helices are followed by a shorter 1.4 nm helix projecting at 70° from α2 (Figure 4B). The C-terminal region of the crystallized construct extends from the outer circumference of the ring, indicating that the encapsulin localization sequence in the full-length protein is on the exterior of the ring and is thus free to interact with its binding site on the encapsulin shell protein. The monomer of EncFtnsH forms two distinct dimer interfaces within the decamer (Figure 4 C/D). The first dimer is formed from two monomers arranged antiparallel to each other, with α1 from each monomer interacting along their lengths and α3 interdigitating with α2 and α3 of the partner chain. This interface buries one third of the surface area from each partner and is stabilized by thirty hydrogen bonds and fourteen salt bridges (Figure 4C). The second dimer interface forms an antiparallel four-helix bundle between helices 1 and 2 from each monomer (Figure 4D). This interface is less extensive than the first and is stabilized by twenty-one hydrogen bonds, six salt bridges, and a number of metal ions. The arrangement of ten monomers in alternating orientation forms the decamer of EncFtn, which assembles as a pentamer of dimers (Figure 4A). Each monomer lies at 45° relative to the vertical central-axis of the ring, with the N-termini of alternating subunits capping the center of the ring at each end, while the C-termini are arranged around the circumference. The central hole in the ring is 2.5 nm at its widest in the center of the complex, and 1.5 nm at its narrowest point near the outer surface, although it should be noted that a number of residues at the N-terminus are not visible in the crystallographic electron density and these may occupy the central channel. The surface of the decamer has distinct negatively charged patches, both within the central hole and on the outer circumference, which form spokes through the radius of the complex (Figure 4—figure supplement 1). EncFtn ferroxidase center Putative ligand-binding site in EncFtnsH. (A) Wall-eyed stereo view of the dimer interface of EncFtn. Protein chains are shown as sticks, with 2mFo-DFc electron density shown in blue mesh and contoured at 1.5 σ and mFo-DFc shown in green mesh and contoured at 3 σ. (B) Wall-eyed stereo view of putative metal binding site at the external surface of EncFtnsH. Protein chains and electron density maps are shown as in (A). DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.015 EncFtnsH metal binding sites. (A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH. Protein residues are shown as sticks with blue and green carbons for the different subunits, iron ions are shown as orange spheres and calcium as grey spheres, and the glycolic acid ligand is shown with yellow carbon atoms coordinated above the di-iron center. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ and the NCS-averaged anomalous difference map is shown as an orange mesh and contoured at 10 σ. (B) Iron coordination within the FOC including residues Glu32, Glu62, His65 and Tyr39 from two chains. Protein and metal ions are shown as in A. Coordination between the protein and iron ions is shown as yellow dashed lines with distances indicated. (C) Coordination of calcium within the dimer interface by four glutamic acid residues (E31 and E34 from two chains). The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.014 The electron density maps of the initial EncFtnsH model displayed significant positive peaks in the mFo-DFc map at the center of the 4-helix bundle dimer (Figure 5—figure supplement 1). Informed by the ICP-MS data indicating the presence of iron in the protein we collected diffraction data at the experimentally determined iron absorption edge (1.74 Å) and calculated an anomalous difference Fourier map using this data. Inspection of this map showed two 10-sigma peaks between residues Glu32, Glu62 and His65 of two adjacent chains, and a statistically smaller 5-sigma peak between residues Glu31 and Glu34 of the two chains. Modeling metal ions into these peaks and refinement of the anomalous scattering parameters allowed us to identify these as two iron ions and a calcium ion respectively (Figure 5A). An additional region of asymmetric electron density near the di-iron binding site in the mFo-DFc map was modeled as glycolic acid, presumably a breakdown product of the PEG 3350 used for crystallization. This di-iron center has an Fe-Fe distance of 3.5 Å, Fe-Glu-O distances between 2.3 and 2.5 Å, and Fe-His-N distances of 2.5 Å (Figure 5B). This coordination geometry is consistent with the di-nuclear ferroxidase center (FOC) found in ferritin. It is interesting to note that although we did not add any additional iron to the crystallization trials, the FOC was fully occupied with iron in the final structure, implying that this site has a very high affinity for iron. The calcium ion coordinated by Glu31 and Glu34 adopts heptacoordinate geometry, with coordination distances of 2.5 Å between the metal ion and carboxylate oxygens of Glu31 and Glu34 (E31/34-site). A number of ordered solvent molecules are also coordinated to this metal ion at a distance of 2.5 Å. This heptacoordinate geometry is common in crystal structures with calcium ions (Figure 5C). While ICP-MS indicated that there were negligible amounts of calcium in the purified protein, the presence of 140 mM calcium acetate in the crystallization mother liquor favors the coordination of calcium at this site. The fact that the protein does not multimerize in solution in the presence of Fe3+ may indicate that these metal binding sites have a lower affinity for the ferric form of iron, which is the product of the ferroxidase reaction. A number of additional metal-ions were present at the outer circumference of at least one decamer in the asymmetric unit (Figure 5D). These ions are coordinated by His57, Glu61 and Glu64 from both chains in the FOC dimer and are 4.5 Å apart; Fe-Glu-O distances are between 2.5 and 3.5 Å and the Fe-His-N distances are 4 and 4.5 Å. Comparison of quaternary structure of EncFtnsH and ferritin. (A) Aligned FOC of EncFtnsH and Pseudo-nitzschia multiseries ferritin (PmFtn). The metal binding site residues from two EncFtnsH chains are shown in green and blue, while the PmFtn is shown in orange. Fe2+ in the FOC is shown as orange spheres and Ca2+ in EncFtnsH is shown as a grey sphere. The two-fold symmetry axis of the EncFtn FOC is shown with a grey arrow (B) Cross-section surface view of quaternary structure of EncFtnsH and PmFtn as aligned in (A) (dashed black box). The central channel of EncFtnsH is spatially equivalent to the outer surface of ferritin and its outer surface corresponds to the mineralization surface within ferritin. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.017 Comparison of the symmetric metal ion binding site of EncFtnsH and the ferritin FOC. (A) Structural alignment of the FOC residues in a dimer of EncFtnsH (green/blue) with a monomer of Pseudo-nitzschia multiseries ferritin (PmFtn) (PDBID: 4ITW) (orange). Iron ions are shown as orange spheres and a single calcium ion as a grey sphere. Residues within the FOC are conserved between EncFtn and ferritin PmFtn, with the exception of residues in the position equivalent to H65’ in the second subunit in the dimer (blue). The site in EncFtn with bound calcium is not present in other family members. (B) Secondary structure of aligned dimeric EncFtnsH and monomeric ferritin highlighting the conserved four-helix bundle. EncFtnsH monomers are shown in green and blue and aligned PmFtn monomer in orange as in A. (C) Cartoon of secondary structure elements in EncFtn dimer and ferritin. In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between α2 and α3 in ferritin PmFtn. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.016 Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins  (Figure 6A). The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65’) (Figure 6A). Structural superimposition of the FOCs of ferritin and EncFtn brings the four-helix bundle of the ferritin fold into close alignment with the EncFtn dimer, showing that the two families of proteins have essentially the same architecture around the di-iron center (Figure 6B). The linker connecting helices 2 and 3 of ferritin is congruent with the start of the C-terminal helix of one EncFtn monomer and the N-terminal 310 helix of the second monomer (Figure 6C). Mass spectrometry of the EncFtn assembly Native IM-MS analysis of the apo-EncFtnsH monomer. (A) Mass spectrum of apo-EncFtnsH acquired from 100 mM ammonium acetate pH 8.0 under native MS conditions. The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1. The +8 to +15 protein charge states have observed CCS between 20–26 nm2, which is significantly higher than the calculated CCS for an EncFtnsH monomer taken from the decameric assembly crystal structure (15.8 nm2). The mobility of the +7 charge state displays broad drift-time distribution with maxima consistent with CCS of 15.9 and 17.9 nm2. Finally, the 6+ charge state of EncFtnsH has mobility consistent with a CCS of 12.3 nm2, indicating a more compact/collapsed structure. It is clear from this data that apo-EncFtnsH exists in several gas phase conformations. The range of charge states occupied by the protein (6+ to 15+) and the range of CCS in which the protein is observed (12.3 nm2 – 26 nm2) are both large. In addition, many of the charge states observed have higher charge than the theoretical maximal charge on spherical globular protein, as determined by the De La Mora relationship (ZR = 0.0778m; for the EncFtnsH monomer ZR = 8.9) Fernandez. As described by Beveridge et al., all these factors are indicative of a disordered protein. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.019 Gas-phase disassembly of the holo-EncFtnsH decameric assembly. The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles). The mass of the ejected-monomer is consistent with apo- EncFtnsH (13.2 kDa), suggesting unfolding of the monomer (and loss of Fe) occurs during ejection from the complex. This observation of asymmetric charge partitioning of the sub-complexes with respect to the mass of the complex is consistent with the 'typical' pathway of dissociation of protein assemblies by CID, as described by. In addition, a third, lower abundance, charge state distribution is observed which overlaps the EncFtn ejected monomer charge state distribution; this region of the spectrum is highlighted in (B). This distribution is consistent with an ejected EncFtnsH dimer (orange circles). Interestingly, closer analysis of the individual charge state of this dimeric CID product shows that this sub-complex exists in three forms – displaying mass consistent with an EncFtnsH dimer binding 0, 1, and 2 Fe ions. This is highlighted in (C), where the 15+ charge state of the EncFtnsH dimer is shown; 3 peaks are observed with m/z 1760.5, 1763.8, and 1767.0 Th – the lowest peak corresponds to neutral masses of 26392.5 Da [predicted EncFtnsH dimer, (C572H884N172O185S2)2; 26388.6 Da]. The two further peaks have a delta-mass of ~+50 Da, consistent with Fe binding. We interpret these observations as partial ‘atypical’ CID fragmentation of the decameric complex – i.e. fragmentation of the initial complex with retention of subunit and ligand interactions. A schematic summary of these results is displayed in (D). We postulate the high stability of this iron-bound dimer sub-complex is due to the metal coordination at the dimer interface, increasing the strength of the dimer interface. Taken together, these observations support our findings that the topology of the decameric EncFtnsH assembly is arranged as a pentamer of dimers, with two Fe ions at each dimer interface. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.020 Native mass spectrometry and ion mobility analysis of iron loading in EncFtnsH. All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Native nanoelectrospray ionization (nESI) mass spectrometry of EncFtnsH at varying iron concentrations. A1, nESI spectrum of iron-free EncFtnsH displays a charge state distribution consistent with EncFtnsH monomer (blue circles, 13,194 Da). Addition of 100 µM (A2) and 300 µM (A3) Fe2+ results in the appearance of a second higher molecular weight charge state distribution consistent with a decameric assembly of EncFtnsH (green circles, 132.6 kDa). (B) Ion mobility (IM)-MS of the iron-bound holo-EncFtnsH decamer. Top, Peaks corresponding to the 22+ to 26+ charge states of a homo-decameric assembly of EncFtnsH are observed (132.6 kDa). Top Insert, Analysis of the 24+ charge state of the assembly at m/z 5528.2 Th. The theoretical average m/z of the 24+ charge state with no additional metals bound is marked by a red line (5498.7 Th); the observed m/z of the 24+ charge state indicates that the EncFtnsH assembly binds between 10 (green line, 5521.1 Th) and 15 Fe ions (blue line, 5532.4 Th) per decamer. Bottom, The arrival time distributions (ion mobility data) of all ions in the EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). Bottom right, The arrival time distribution of the 24+ charge state (dashed blue box) has been extracted and plotted. The drift time for this ion is shown (ms), along with the calibrated collision cross section (CCS), Ω (nm2). DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.018 In order to confirm the assignment of the oligomeric state of EncFtnsH and investigate further the Fe2+-dependent assembly, we used native nano-electrospray ionization (nESI) and ion-mobility mass spectrometry (IM-MS). As described above, by recombinant production of EncFtnsH in minimal media we were able to limit the bioavailability of iron. Native MS analysis of EncFtnsH produced in this way displayed a charge state distribution consistent with an EncFtnsH monomer (blue circles, Figure 7A1) with an average neutral mass of 13,194 Da, in agreement with the predicted mass of the EncFtnsH protein (13,194.53 Da). Under these conditions, no significant higher order assembly was observed and the protein did not have any coordinated metal ions. Titration with Fe2+ directly before native MS analysis resulted in the appearance of a new charge state distribution, consistent with an EncFtnsH decameric assembly (+22 to +26; 132.65 kDa) (Figure 7A2/3). After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa). MS analysis of EncFtnsH after addition of further Fe2+ did not result in iron loading above this stoichiometry. Therefore, the extent of iron binding seen is limited to the FOC and Glu31/34 secondary metal binding site. These data suggest that the decameric assembly of EncFtnsH does not accrue iron in the same manner as classical ferritin, which is able to sequester around 4500 iron ions within its nanocage. Ion mobility analysis of the EncFtnsH decameric assembly, collected with minimal collisional activation, suggested that it consists of a single conformation with a collision cross section (CCS) of 58.2 nm2 (Figure 7B). This observation is in agreement with the calculated CCS of 58.7 nm2derived from our crystal structure of the EncFtnsH decamer. By contrast, IM-MS measurements of the monomeric EncFtnsH at pH 8.0 under the same instrumental conditions revealed that the metal-free protein monomer exists in a wide range of charge states (+6 to +16) and adopts many conformations in the gas phase with collision cross sections ranging from 12 nm2 to 26 nm2 (Figure 7—figure supplement 1). These observations are indicative of an unstructured protein with little secondary or tertiary structure. Thus, IM-MS studies highlight that higher order structure in EncFtnsH is mediated/stabilized by metal binding, an observation that is in agreement with our solution studies. Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure. We subsequently performed gas phase disassembly of the decameric EncFtnsH using collision-induced dissociation (CID) tandem mass spectrometry. Under the correct CID conditions, protein assemblies can dissociate with retention of subunit and ligand interactions, and thus provide structurally-informative evidence as to the topology of the original assembly; this has been termed ‘atypical’ dissociation. For EncFtnsH, this atypical dissociation pathway was clearly evident; CID of the EncFtnsH decamer resulted in the appearance of a dimeric EncFtnsH subcomplex containing 0, 1, or 2 iron ions (Figure 7—figure supplement 2). In light of the crystal structure, this observation can be rationalized as dissociation of the EncFtnsH decamer by disruption of the non-FOC interface with at least partial retention of the FOC interface and the FOC-Fe. Thus, this observation supports our crystallographic assignment of the overall topology of the EncFtnsH assembly as a pentameric assembly of dimers with two iron ions located at the FOC dimer interface. In addition, this analysis provides evidence that the overall architecture of the complex is consistent in the crystal, solution and gas phases. Ferroxidase activity TEM visualization of iron-loaded bacterial nanocompartments and ferritin. Decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin, at 8.5 µM, were mixed with 147 µM, 1 mM, 1 mM and 215 µM acidic Fe(NH4)2(SO4)2, respectively. Protein mixtures were incubated at room temperature for 1 hr prior to TEM analysis with or without uranyl acetate stain. (A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.022 Spectroscopic evidence for the ferroxidase activity and comparison of iron loading capacity of apoferritin, EncFtnsH, encapsulin, and EncFtn-Enc. (A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer. The background oxidation of iron at 20 and 100 μM in enzyme-free controls are shown for reference. (B) Encapsulin and EncFtn-Enc complexes at 10 μM asymmetric unit concentration were incubated with Fe2+ at 20 and 100 μM and progress curves for iron oxidation at A315 were measured in a UV/visible spectrophotometer. Enzyme free controls for background oxidation of Fe2+ are shown for reference. (C) Histogram of the iron loading capacity per biological assembly of EncFtnsH, encapsulin, EncFtn-Enc and apoferritin. The results shown are for three technical replicates and represent the optimal iron loading by the complexes after three hours when incubated with Fe2+. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.021 In light of the identification of an iron-loaded FOC in the crystal structure of EncFtn and our native mass spectrometry data, we performed ferroxidase and peroxidase assays to demonstrate the catalytic activity of this protein. In addition, we also assayed equine apoferritin, an example of a classical ferritin enzyme, as a positive control. Unlike the Dps family of ferritin-like proteins, EncFtn showed no peroxidase activity when assayed with the substrate ortho-phenylenediamine. The ferroxidase activity of EncFtnsH was measured by recording the progress curve of Fe2+ oxidation to Fe3+ at 315 nm after addition of 20 and 100 µM Fe2+ (2 and 10 times molar ratio Fe2+/FOC). In both experiments the rate of oxidation was faster than background oxidation of Fe2+ by molecular oxygen, and was highest for 100 µM Fe2+ (Figure 8A). These data show that recombinant EncFtnsH acts as an active ferroxidase enzyme. When compared to apoferritin, EncFtnsH oxidized Fe2+ at a slower rate and the reaction did not run to completion over the 1800 s of the experiment. Addition of higher quantities of iron resulted in the formation of a yellow/red precipitate at the end of the reaction. We also performed these assays on purified recombinant encapsulin; which, when assayed alone, did not display ferroxidase activity above background Fe2+ oxidation (Figure 8B). In contrast, complexes of the full EncFtn encapsulin nanocompartment (i.e. the EncFtn-Enc protein complex) displayed ferroxidase activity comparable to apoferritin without the formation of precipitates (Figure 8B). We attributed the precipitates observed in the EncFtnsH ferroxidase assay to the production of insoluble Fe3+ complexes, which led us to propose that EncFtn does not directly store Fe3+ in a mineral form. This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures. To analyze the products of these reactions and determine whether the EncFtn and encapsulin were able to store iron in a mineral form, we performed TEM on the reaction mixtures from the ferroxidase assay. The EncFtnsH reaction mixture showed the formation of large, irregular electron-dense precipitates (Figure 8—figure supplement 1A). A similar distribution of particles was observed after addition of Fe2+ to the encapsulin protein (Figure 8—figure supplement 1B). In contrast, addition of Fe2+ to the EncFtn-Enc nanocompartment resulted in small, highly regular, electron dense particles of approximately 5 nm in diameter (Figure 8—figure supplement 1C); we interpret these observations as controlled mineralization of iron within the nanocompartment. Addition of Fe2+ to apoferritin resulted in a mixture of large particles and small (~2 nm) particles consistent with partial mineralization by the ferritin and some background oxidation of the iron (Figure 8—figure supplement 1D). Negative stain TEM of these samples revealed that upon addition of iron, the EncFtnsH protein showed significant aggregation (Figure 8—figure supplement 1F); while the encapsulin, EncFtn-Enc system, and apoferritin are present as distinct nanocompartments without significant protein aggregation (Figure 8—figure supplement 1G–I). Iron storage in encapsulin nanocompartments The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage. To investigate this further, we added Fe2+ at various concentrations to samples of apo-ferritin, EncFtn, isolated encapsulin, and the EncFtn-Enc protein complex, and subjected these samples to a ferrozine assay to quantify the amount of iron associated with the proteins after three hours of incubation. The maximum iron loading capacity of these systems was calculated as the quantity of iron per biological assembly (Figure 8C). In this assay, the EncFtnsH decamer binds a maximum of around 48 iron ions before excess iron induces protein precipitation. The encapsulin shell protein can sequester about 2200 iron ions before significant protein loss occurs, and the reconstituted EncFtn-Enc nanocompartment sequestered about 4150 iron ions. This latter result is significantly more than the apoferritin used in our assay, which sequesters approximately 570 iron ions in this assay (Figure 8C, Table 5). Consideration of the functional oligomeric states of these proteins, where EncFtn is a decamer and encapsulin forms an icosahedral cage, and estimation of the iron loading capacity of these complexes gives insight into the role of the two proteins in iron storage and mineralization. EncFtn decamers bind up to 48 iron ions (Figure 8C), which is significantly higher than the stoichiometry of fifteen metal ions visible in the FOC and E31/34-site of the crystal structure of the EncFtnsH decamer and our MS analysis. The discrepancy between these solution measurements and our MS analysis may indicate that there are additional metal-binding sites on the interior channel and exterior faces of the protein; this is consistent with our identification of a number of weak metal-binding sites at the surface of the protein in the crystal structure (Figure 5D). These observations are consistent with hydrated Fe2+ ions being channeled to the active site from the E31/34-site and the subsequent exit of Fe3+ products on the outer surface, as is seen in other ferritin family proteins. While the isolated encapsulin shell does not display any ferroxidase activity, it binds around 2200 iron ions in our assay (Table 5). This implies that the shell can bind a significant amount of iron on its outer and inner surfaces. While the maximum reported loading capacity of classical ferritins is approximately 4500 iron ions, in our assay system we were only able to load apoferritin with around 570 iron ions. However, the recombinant EncFtn-Enc nanocompartment was able to bind over 4100 iron ions in the same time period, over seven times the amount seen for the apoferritin. We note we do not reach the experimental maximum iron loading for apoferritin and therefore the total iron-loading capacity of our system may be significantly higher than in this experimental system. Taken together, our data show that EncFtn can catalytically oxidize Fe2+ to Fe3+; however, iron binding in EncFtn is limited to the FOC and several surface metal binding sites. In contrast, the encapsulin protein displays no catalytic activity, but has the ability to bind a considerable amount of iron. Finally, the EncFtn-Enc nanocompartment complex retains the catalytic activity of EncFtn, and sequesters iron within the encapsulin shell at a higher level than the isolated components of the system, and at a significantly higher level than the classical ferritins. Furthermore, our recombinant nanocompartments may not have the physiological subunit stoichiometry, and the iron-loading capacity of native nanocompartments is potentially much higher than the level we have observed. Mutagenesis of the EncFtnsHferroxidase center Purification of recombinant R. rubrum EncFtnsH FOC mutants. Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex 200 size-exclusion chromatography. (A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.023 To investigate the structural and biochemical role played by the metal binding residues in the di-iron FOC of EncFtnsH we produced alanine mutations in each of these residues: Glu32, Glu62, and His65. These EncFtnsH mutants were produced in E. coli cells grown in MM, both in the absence and presence of additional iron. The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9). For all of the mutants studied, no change in oligomerization state was apparent upon addition of Fe2+ in vitro. Native mass spectrometry of EncFtnsH mutants. All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles). (C) E62A EncFtnsH displays charge states consistent with a decamer (green circles). (D) H65A EncFtnsH displays charge states consistent with both monomer (blue circles) and dimer (purple circles). DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.024 In addition to SEC studies, native mass spectrometry of the apo-EncFtnsH mutants was performed and compared with the wild-type apo-EncFtnsH protein (Figure 10). As described above, the apo-EncFtnsH has a charge state distribution consistent with an unstructured monomer, and decamer formation is only initiated upon addition of ferrous iron. Both the E32A mutant and E62A mutant displayed charge state distributions consistent with decamers, even in the absence of Fe2+. This gas-phase observation is consistent with SEC measurements, which indicate both of these variants were also decamers in solution. Thus it seems that these mutations allow the decamer to form in the absence of iron in the FOC. In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed. We propose that the observed differences in the oligomerization state of the E32A and E62A mutants compared to wild-type are due to the changes in the electrostatic environment within the FOC. At neutral pH the glutamic acid residues are negatively charged, while the histidine residues are predominantly in their uncharged state. In the wild-type (WT) EncFtnsH this leads to electrostatic repulsion between subunits in the absence of iron. Coordination of Fe2+ in this site stabilizes the dimer and reconstitutes the active FOC. The geometric arrangement of Glu32 and Glu62 in the FOC explains their behavior in solution and the gas phase, where they both favor the formation of decamers due to the loss of a repulsive negative charge. The FOC in the H65A mutant is destabilized through the loss of this metal coordinating residue and potential positive charge carrier, thus favoring the monomer in solution and the gas phase. Data collection and refinement statistics. Statistics for the highest-resolution shell are shown in parentheses. Friedel mates were averaged when calculating reflection numbers and statistics. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.025 \tWT\tE32A\tE62A\tH65A\t \tData collection\t\t\t\t\t \tWavelength (Å)\t1.74\t1.73\t1.73\t1.74\t \tResolution range (Å)\t49.63 - 2.06 (2.10 - 2.06)\t48.84 - 2.59 (2.683 - 2.59)\t48.87 - 2.21 (2.29 - 2.21)\t48.86 - 2.97 (3.08 - 2.97)\t \tSpace group\tP 1 21 1\tP 1 21 1\tP 1 21 1\tP 1 21 1\t \tUnit cell (Å) a b  c β (°)\t98.18 120.53 140.30 95.36\t97.78 120.28 140.53 95.41\t98.09 120.23 140.36 95.50\t98.03 120.29 140.43 95.39\t \tTotal reflections\t1,264,922 (41,360)\t405,488 (36,186)\t1,069,345 (95,716)\t323,853 (32,120)\t \tUnique reflections\t197,873 (8,766)\t100,067 (9,735)\t162,379 (15,817)\t66,658 (6,553)\t \tMultiplicity\t6.4 (4.7)\t4.1 (3.7)\t6.6 (6.1)\t4.9 (4.9)\t \tAnomalous multiplicity\t3.2 (2.6)\tN/A\tN/A\tN/A\t \tCompleteness (%)\t99.2 (88.6)\t99.0 (97.0)\t100 (97.0)\t100 (99.0)\t \tAnomalous completeness (%)\t96.7 (77.2)\tN/A\tN/A\tN/A\t \tMean I/sigma(I)\t10.6 (1.60)\t8.46 (1.79)\t13.74 (1.80)\t8.09 (1.74)\t \tWilson B-factor\t26.98\t40.10\t33.97\t52.20\t \tRmerge\t0.123 (0.790)\t0.171 (0.792)\t0.0979 (1.009)\t0.177 (0.863)\t \tRmeas\t0.147 (0.973)\t0.196 (0.923)\t0.1064 (1.107)\t0.199 (0.966)\t \tCC1/2\t0.995 (0.469)\t0.985 (0.557)\t0.998 (0.642)\t0.989 (0.627)\t \tCC*\t0.999 (0.846)\t0.996 (0.846)\t0.999 (0.884)\t0.997 (0.878)\t \tImage DOI\t10.7488/ds/1342\t10.7488/ds/1419\t10.7488/ds/1420\t10.7488/ds/1421\t \tRefinement\t\t\t\t\t \tRwork\t0.171 (0.318)\t0.183 (0.288)\t0.165 (0.299)\t0.186 (0.273)\t \tRfree\t0.206 (0.345)\t0.225 (0351)\t0.216 (0.364)\t0.237 (0.325)\t \tNumber of non-hydrogen atoms\t23,222\t22,366\t22,691\t22,145\t \tmacromolecules\t22,276\t22,019\t21,965\t22,066\t \tligands\t138\t8\t24\t74\t \twater\t808\t339\t702\t5\t \tProtein residues\t2,703\t2,686\t2,675\t2,700\t \tRMS(bonds) (Å)\t0.012\t0.005\t0.011\t0.002\t \tRMS(angles) (°)\t1.26\t0.58\t1.02\t0.40\t \tRamachandran favored (%)\t100\t99\t100\t99\t \tRamachandran allowed (%)\t0\t1\t0\t1\t \tRamachandran outliers (%)\t0\t0\t0\t0\t \tClash score\t1.42\t1.42\t1.79\t0.97\t \tAverage B-factor (Å2)\t33.90\t42.31\t41.34\t47.68\t \tmacromolecules\t33.80\t42.35\t41.31\t47.60\t \tligands\t40.40\t72.80\t65.55\t72.34\t \tsolvent\t36.20\t38.95\t41.46\t33.85\t \tPDB ID\t5DA5\t5L89\t5L8B\t5L8G\t \t Iron loading capacity of EncFtn, encapsulin and ferritin. Protein samples (at 8.5 µM) including decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were mixed with Fe(NH4)2(SO4) (in 0.1% (v/v) HCl) of different concentrations in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl buffer at room temperature for 3 hrs in the air. Protein-Fe mixtures were centrifuged at 13,000 x g to remove precipitated material and desalted prior to the Fe and protein content analysis by ferrozine assay and BCA microplate assay, respectively. Fe to protein ratio was calculated to indicate the Fe binding capacity of the protein. Protein stability was compromised at high iron concentrations; therefore, the highest iron loading with the least protein precipitation was used to derive the maximum iron loading capacity per biological assembly (underlined and highlighted in bold). The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin. Errors are quoted as the standard deviation of three technical repeats in both the ferrozine and BCA microplate assays. The proteins used in Fe loading experiment came from a single preparation. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.026 Protein sample\tFe(NH4)2(SO4)2 loading (µM)\tFe detected by ferrozine assay (µM)\tProtein detected by BCA microplate assay (µM)\tFe / monomeric protein\tMaximum Fe loading per biological assembly unit\t \t8.46 µM EncFtnsH-10mer\t0\t4.73 ± 2.32\t5.26 ± 0.64\t0.90 ± 0.44\t\t \t39.9\t9.93 ± 1.20\t5.36 ± 0.69\t1.85 ± 0.22\t\t \t84\t17.99 ± 2.01\t4.96 ± 0.04\t3.63 ± 0.41\t\t \t147\t21.09 ± 1.94\t4.44 ± 0.21\t4.75 ± 0.44\t48 ± 4\t \t224\t28.68 ± 0.30\t3.73 ± 0.53\t7.68 ± 0.08\t\t \t301\t11.27 ± 1.10\t2.50 ± 0.05\t4.51 ± 0.44\t\t \t8.50 µM Encapsulin\t0\t-1.02 ± 0.54\t8.63 ± 0.17\t-0.12 ± 0.06\t\t \t224\t62.24 ± 2.49\t10.01 ± 0.58\t6.22 ± 0.35\t\t \t301\t67.94 ± 3.15\t8.69 ± 0.42\t7.81 ± 0.36\t\t \t450\t107.96 ± 8.88\t8.50 ± 0.69\t12.71 ± 1.05\t\t \t700\t97.51 ± 3.19\t7.26 ± 0.20\t13.44 ± 0.44\t\t \t1000\t308.63 ± 2.06\t8.42 ± 0.34\t36.66 ± 0.24\t2199 ± 15\t \t1500\t57.09 ± 0.90\t1.44 ± 0.21\t39.77 ± 0.62\t\t \t2000\t9.2 ± 1.16\t0.21 ± 0.14\t44.73 ± 5.63\t\t \t8.70 µM EncFtn-Enc\t0\t3.31 ± 1.57\t6.85 ± 0.07\t0.48 ± 0.23\t\t \t224\t116.27 ± 3.74\t7.63 ± 0.12\t15.25 ± 0.49\t\t \t301\t132.86 ± 4.03\t6.66 ± 0.31\t19.96 ± 0.61\t\t \t450\t220.57 ± 27.33\t6.12 ± 1.07\t36.06 ± 4.47\t\t \t700\t344.03 ± 40.38\t6.94 ± 0.17\t49.58 ± 5.82\t\t \t1000\t496.00 ± 38.48\t7.19 ± 0.08\t68.94 ± 5.35\t4137 ± 321\t \t1500\t569.98 ± 73.63\t5.73 ± 0.03\t99.44 ± 12.84\t\t \t2000\t584.30 ± 28.33\t4.88 ± 0.22\t119.62 ± 5.80\t\t \t8.50 µM Apoferritin\t0\t3.95 ± 2.26\t9.37 ± 0.24\t0.42 ± 0.25\t\t \t42.5\t10.27 ± 1.12\t8.27 ± 0.30\t1.24 ± 0.18\t\t \t212.5\t44.48 ± 2.76\t7.85 ± 0.77\t5.67 ± 0.83\t\t \t637.5\t160.93 ± 4.27\t6.76 ± 0.81\t23.79 ± 3.12\t571 ± 75\t \t1275\t114.92 ± 3.17\t3.84 ± 0.30\t29.91 ± 2.95\t\t \t1700\t91.40 ± 3.37\t3.14 ± 0.35\t29.13 ± 3.86\t\t \t To understand the impact of the mutants on the organization and metal binding of the FOC, we determined the X-ray crystal structures of each of the EncFtnsH mutants (See Table 4 for data collection and refinement statistics). The crystal packing of all of the mutants in this study is essentially isomorphous to the EncFtnsH structure. All of the mutants display the same decameric arrangement in the crystals as the EncFtnsH structure, and the monomers superimpose with an average RMSDCα of less than 0.2 Å. FOC dimer interface of EncFtnsH-E32A mutant. (A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-E32A. Protein residues are shown as sticks with blue and green carbons for the different subunits. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ. (B) Views of the FOC of the EncFtnsH-E32Amutant. Protein atoms shown as in (A). DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.028 FOC dimer interface of EncFtnsH-E62A mutant. (A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-E62A. Protein residues are shown as sticks with blue and green carbons for the different subunits. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ. The single coordinated calcium ion is shown as a grey sphere. (B) Views of the FOC of the EncFtnsH-E62A mutant. Protein atoms shown as in (A). DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.029 FOC dimer interface of EncFtnsH-H65A mutant. (A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-H65A. Protein residues are shown as sticks with blue and green carbons for the different subunits. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ. The coordinated calcium ions are shown as a grey spheres with coordination distances in the FOC highlighted with yellow dashed lines. (B) Views of the FOC of the EncFtnsH-H65A mutant. Protein atoms and metal ions shown as in (A). DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.030 Comparison of the EncFtnsH FOC mutants vs wild type. The structures of the three EncFtnsH mutants were all determined by X-ray crystallography. The E32A, E62A and H65A mutants were crystallized in identical conditions to the wild type. EncFtnsH structure and were essentially isomorphous in terms of their unit cell dimensions. The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue. Of the mutants, only H65A has any coordinated metal ions, which appear to be calcium ions from the crystallization condition. The overall organization of FOC residues is retained in the mutants, with almost no backbone movements. Significant differences center around Tyr39, which moves to coordinate the bound calcium ions in the H65A mutant; and Glu32, which moves away from the metal ions in this structure. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.027 Close inspection of the region of the protein around the FOC in each of the mutants highlights their effect on metal binding (Figure 11 and Figure 11—figure supplement 1–3). In the E32A mutant the position of the side chains of the remaining iron coordinating residues in the FOC is essentially unchanged, but the absence of the axial-metal coordinating ligand provided by the Glu32 side chain abrogates metal binding in this site. The Glu31/34-site also lacks metal, with the side chain of Glu31 rotated by 180° at the Cβ in the absence of metal (Figure 11—figure supplement 1). The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11—figure supplement 2). The H65A mutant diverges significantly from the wild type in the position of the residues Glu32 and Tyr39 in the FOC. E32 appears in either the original orientation as the wild type and coordinates Ca2+ in this position, or it is flipped by 180° at the Cβ, moving away from the coordinated calcium ion in the FOC. Tyr39 moves closer to Ca2+ compared to the wild-type and coordinates the calcium ion (Figure 11—figure supplement 3). A single calcium ion is present in the entry site of this mutant; however, Glu31 of one chain is rotated away from the metal ion and is not involved in coordination. Taken together the results of our data show that these changes to the FOC of EncFtn still permit the formation of the decameric form of the protein. While the proteins all appear decameric in crystals, their solution and gas-phase behavior differs considerably and the mutants no longer show metal-dependent oligomerization. These results highlight the importance of metal coordination in the FOC for the stability and assembly of the EncFtn protein. Progress curves recording ferroxidase activity of EncFtnsH mutants. 20 µM wild-type EncFtnsH, E32A, E62A and H65A mutants were mixed with 20 µM or 100 µM acidic Fe(NH4)2(SO4)2, respectively. Absorbance at 315 nm was recorded for 1800 s at 25°C as an indication of Fe3+ formation. Protein free samples (dashed and dotted lines) were measured for Fe2+ background oxidation as controls. Assays were performed with three technical repeats. Error bars were showed in shadows behind each curves. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.032 Relative ferroxidase activity of EncFtnsH mutants. EncFtnsH, and the mutant forms E32A, E62A and H65A, each at 20 µM, were mixed with 100 µM acidic Fe(NH4)2(SO4)2. Ferroxidase activity of the mutant forms is determined by measuring the absorbance at 315 nm for 1800 s at 25 °C as an indication of Fe3+ formation. The relative ferroxidase activity of mutants is plotted as a proportion of the activity of the wild-type protein using the endpoint measurement of A315. Three technical repeats were performed and the plotted error bars represent the calculated standard deviations. The FOC mutants showed reduced ferroxidase activity to varied extents, among which E62A significantly abrogated the ferroxidase activity. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.031 To address the question of how mutagenesis of the iron coordinating residues affects the enzymatic activity of the EncFtnsH protein we recorded progress curves for the oxidation of Fe2+ to Fe3+ by the different mutants as before. Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%; the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12). Collectively, the effect of mutating these residues in the FOC confirms the importance of the iron coordinating residues for the ferroxidase activity of the EncFtnsH protein. Discussion Phylogenetic tree of ferritin family proteins. The tree was built using the Neighbor-Joining method based on step-wise amino acid sequence alignment of the four-helical bundle portions of ferritin family proteins (Supplementary file 1). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree; the likely root of the tree is indicated by a red arrow. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 2.5). The analysis involved 104 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 262 positions in the final dataset. Evolutionary analyses were conducted in MEGA7  DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.033 Our study reports on a new class of ferritin-like proteins (EncFtn), which are associated with bacterial encapsulin nanocompartments (Enc). By studying the EncFtn from R. rubrum we demonstrate that iron binding results in assembly of EncFtn decamers, which display a unique annular architecture. Despite a radically different quaternary structure to the classical ferritins, the four-helical bundle scaffold and FOC of EncFtnsH are strikingly similar to ferritin (Figure 6A). A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn). The analysis revealed that the EncFtn and Non-EncFtn proteins form groups distinct from the other clearly delineated groups of ferritins, and represent outliers in the tree (Figure 13). While it is difficult to infer ancestral lineages in protein families, the similarity seen in the active site scaffold of these proteins highlights a shared evolutionary relationship between EncFtn proteins and other members of the ferritin superfamily that has been noted in previous studies (; ). From this analysis, we propose that the four-helical fold of the classical ferritins may have arisen through gene duplication of an ancestor of EncFtn. This gene duplication would result in the C-terminal region of one EncFtn monomer being linked to the N-terminus of another and thus stabilizing the four-helix bundle fold within a single polypeptide chain (Figure 6B). Linking the protein together in this way relaxes the requirement for the maintenance of a symmetrical FOC and thus provides a path to the diversity in active-site residues seen across the ferritin family (Figure 6A, residues Glu95, Gln128 and Glu131 in PmFtn, Supplementary file 1). Relationship between ferritin structure and activity The quaternary arrangement of classical ferritins into an octahedral nanocage and Dps into a dodecamer is absolutely required for their function as iron storage compartments. The oxidation and mineralization of iron must be spatially separated from the host cytosol to prevent the formation of damaging hydroxyl radicals in the Fenton and Haber-Weiss reactions. This is achieved in all ferritins by confining the oxidation of iron to the interior of the protein complex, thus achieving sequestration of the Fe3+ mineralization product. A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A). This overlay highlights the fact that the ferroxidase center of EncFtn faces in the opposite direction relative to the classical ferritins and is essentially inside out regarding iron storage space (Figure 6—figure supplement 1B, boxed region). Analysis of each of the single mutations (E32A, E62A and H65A) made in the FOC highlights the importance of the iron-coordinating residues in the catalytic activity of EncFtn. Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC. The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site. The absolute requirement for the spatial separation of oxidation and mineralization in ferritins suggests that the EncFtn family proteins are not capable of storing iron minerals due to the absence of an enclosed compartment in their structure (Figure 6—figure supplement 1B). Our biochemical characterization of EncFtn supports this hypothesis, indicating that while this protein is capable of oxidizing iron, it does not accrue mineralized iron in an analogous manner to classical ferritins. While EncFtn does not store iron itself, its association with the encapsulin nanocage suggests that mineralization occurs within the cavity of the encapsulin shell. Our ferroxidase assay data on the recombinant EncFtn-Enc nanocompartments, which accrue over 4100 iron ions per complex and form regular nanoparticles, are consistent with the encapsulin protein acting as the store for iron oxidized by the EncFtn enzyme. TEM analysis of the reaction products shows the production of homogeneous iron nanoparticles only in the EncFtn-Enc nanocompartment (Figure 8—figure supplement 1). Model of iron oxidation in encapsulin nanocompartments. (A) Model of EncFtnsH docking to the encapsulin shell. A single pentamer of the icosahedral T. maritima encapsulin structure (PDBID: 3DKT) is shown as a blue surface with the encapsulin localization sequence of EncFtn shown as a purple surface. The C-terminal regions of the EncFtn subunits correspond to the position of the localization sequences seen in 3DKT. Alignment of EncFtnsH with 3DKT positions the central channel directly above the pore in the 3DKT pentamer axis (shown as a grey pentagon). (B) Surface view of EncFtn within the encapsulin nanocompartment (grey and blue respectively). The lumen of the encapsulin nanocompartment is considerably larger than the interior of ferritin (shown in orange behind the encapsulin for reference) and thus allows the storage of significantly more iron. The proposed pathway for iron movement through the encapsulin shell and EncFtn FOC is shown with arrows. (C) Model ofiron oxidation within an encapsulin nanocompartment. As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.034 Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A). Thus, it appears that the EncFtn decamer is the physiological state of this protein. This arrangement positions the central ring of EncFtn directly above the pore at the five-fold symmetry axis of the encapsulin shell and highlights a potential route for the entry of iron into the encapsulin and towards the active site of EncFtn. A comparison of the encapsulin nanocompartment and the ferritin nanocage highlights the size differential between the two complexes (Figure 14B) that allows the encapsulin to store significantly more iron. The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment. This arrangement of multiple reaction centers in a single protein assembly is reminiscent of classical ferritins, which has 24 FOCs distributed around the nanocage. Our structural data, coupled with biochemical and ICP-MS analysis, suggest a model for the activity of the encapsulin iron-megastore (Figure 14C). The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4—figure supplement 1). The five metal-binding sites on the interior of the ring (Glu31/34-sites) may select for the Fe2+ ion and direct it towards their cognate FOCs. We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by  in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14). Here we describe for the first time the structure and biochemistry of a new class of encapsulin-associated ferritin-like protein and demonstrate that it has an absolute requirement for compartmentalization within an encapsulin nanocage to act as an iron store. Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment. Materials and methods Cloning Genes of interest were amplified by PCR using R. rubrum ATCC 11,170 genomic DNA (DSMZ) as the template and KOD Hot Start DNA Polymerase (Novagen). Primers used in this study are listed in Supplementary file 2. PCR products were visualized in 0.8% agarose gel stained with SYBR Safe (Life Technologies, UK). Fragments of interest were purified by gel extraction (Qiagen, UK) before digestion by endonuclease restriction enzymes (Thermo Fisher Scientific, UK) at 37°C for 1 hr, followed by ligation with similarly digested vector pET-28a(+) or pACYCDuet-1 at room temperature for 1 hr. Ligation product was transformed into chemically competent Escherichia coli Top10 cells and screened against 50 ng/μl kanamycin for pET-28a(+) based constructs or 34 ng/μl chloramphenicol for pACYCDuet-1 based constructs. DNA insertion was confirmed through Sanger sequencing (Edinburgh Genomics, The University of Edinburgh, UK). Sequence verified constructs were transformed into E. coli BL21(DE3) or Tuner(DE3) for protein production. Alternatively, plasmids transformed into E. coli B834(DE3) cells were cultured in selenomethionine medium. Protein production and purification A single colony of E. coli BL21(DE3) or Tuner(DE3) cells, transformed with protein expression plasmid, was transferred into 10 ml LB medium, or M9 minimal medium (MM), supplemented with appropriate antibiotic, and incubated overnight at 37 °C with 200 rpm shaking. The overnight pre-culture was then inoculated into 1 liter of LB medium and incubated at 37 °C with 200 rpm shaking. Recombinant protein production was induced at OD600= 0.6 by the addition of 1 mM IPTG and the incubation temperature was reduced to 18°C for overnight incubation. Cells were pelleted by centrifugation at 4000 g for 20 min at 4 °C, and resuspended 10-fold (volume per gram of cell pellet) in PBS to wash cells before a second centrifugation step. Cells were resuspended in 10-times (v/w) of appropriate lysis buffer for the purification method used (see details of buffers below) and lysed by sonication on ice, with ten cycles of 30-second burst of sonication at 10 µm amplitude and 30 s of cooling. Cell lysate was clarified by centrifugation at 20,000 x g, 30 min, 4 °C; followed by filtration using a 0.22 µM syringe filter (Millipore, UK). Selenomethionine labelled protein was produced by growing a single colony of E. coli B834 (DE3) cells transformed with protein expression plasmids in 100 ml LB medium supplemented with appropriate antibiotic overnight at 37 °C with shaking at 200 rpm. The overnight pre-culture was pelleted by centrifugation 3,000 x g, 4 °C, 15 min and washed twice with M9 minimal medium. The washed cells were transferred to 1 liter of SeMet medium, which contains M9 minimal medium, 40 mg/L of each L-amino acid (without methionine), 40 mg/L selenomethionine, 2 mM MgSO4, 0.4% (w/v) glucose and 1 mM Fe(NH4)2(SO4)2. Cells were incubated at 37 °C with 200 rpm shaking and recombinant protein production was induced at OD600= 0.6 by the addition of 1 mM IPTG and the incubation temperature was reduced to 18 °C for overnight incubation. Cells were harvested and lysed as above. His-tagged protein purification Clarified cell lysate was loaded onto a 5 ml HisTrap column (GE Healthcare, UK) pre-equilibrated with HisA buffer (50 mM Tris-HCl, 500 mM NaCl and 50 mM imidazole, pH 8.0). Unbound proteins were washed from the column with HisA buffer. His-tagged proteins were then eluted by a step gradient of 50% HisA buffer and 50% HisB buffer (50 mM Tris-HCl, 500 mM NaCl and 500 mM imidazole, pH 8.0). Fractions containing the protein of interest, as determined by 15% (w/v) acrylamide SDS-PAGE, were pooled before loading onto a gel-filtration column (HiLoad 16/600 Superdex 200, GE Healthcare) equilibrated with GF buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl). Fractions were subjected to 15% SDS-PAGE and those containing the protein of interest were pooled for further analysis. Sucrose gradient ultracentrifugation purification Co-expressed encapsulin and EncFtn (EncFtn-Enc) and encapsulin protein were both purified according to the protocol used by M. Sutter. Briefly, EncFtn-Enc or encapsulin was expressed based on pACYCDuet-1 vector. The E. coli cells were grown, induced, harvested and sonicated in a similar way as described above. GF buffer used in this purification contains 50 mM Tris-HCl, pH 8.0, and 150 mM NaCl. To remove RNA contamination, the lysate was supplemented with 50 μg/ml RNase A and rotated at 10 rpm and room temperature for 2 hrs, followed by centrifugation at 34,000 x g and 4 °C for 20 min and filtering through 0.22 µM syringe filter. Proteins were pelleted through 38% (w/v) sucrose cushion by ultracentrifugation at 100,000 x g and 4 °C for 21 hrs. 10% - 50% (w/v) sucrose gradient ultracentrifugation was applied to further separate the proteins at 100,000 x g and 4 °C for 17 hrs. Protein was dialyzed against GF buffer to remove sucrose before being used in chemical assays or TEM. Transmission electron microscopy TEM imaging was performed on purified encapsulin, EncFtn, and EncFtn-Enc and apoferritin. Purified protein at 0.1 mg/ml concentration was spotted on glow-discharged 300 mesh carbon-coated copper grids and excess liquid wicked off with filter paper (Whatman, UK). The grids were washed with distilled water and blotted with filter paper three times before staining with 0.2% uranyl acetate, blotting and air-drying. Grids were imaged using a JEM1400 transmission electron microscope and images were collected with a Gatan CCD camera. Images were analyzed using ImageJ (NIH, Bethesda, MD) and size-distribution histograms were plotted using Prism 6 (GraphPad software). To observe iron mineral formation by TEM, protein samples at 8.5 µM concentration including EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were supplemented with acidic Fe(NH4)2(SO4)2 at their maximum iron loading ratio in room temperature for 1 hr. The mixtures were subjected to TEM analysis with or without uranyl acetate staining. TEM experiments without Fe loading were repeated three times, a representative set of images are presented here. Proteins loaded with Fe and imaged by TEM were from single preparation. Protein crystallization and X-ray data collection EncFtnsH was purified by anion exchange and Superdex 200 size- exclusion chromatography and concentrated to 10 mg/ml (based on extinction coefficient calculation). Crystallization drops were set up using the hanging drop vapor diffusion method at 292 K. Glass coverslips were set up with 1–2 μl protein mixed with 1 μl well solution (0.14 M calcium acetate and 15% (w/v) PEG 3350) and sealed over 1 ml of well solution. Crystals appeared after 5 days and were harvested from the well using a LithoLoop (Molecular Dimensions Limited, UK), transferred briefly to a cryoprotection solution containing well solution supplemented with 1 mM FeSO4 (in 0.1% (v/v) HCl), 20% (v/v) PEG 200, and subsequently flash cooled in liquid nitrogen. Crystals of the EncFtnsHsingle mutations were produced in the same manner as for the EncFtnsH wild-type protein. All crystallographic datasets were collected on the macromolecular crystallography beamlines at Diamond Light Source (Didcot, UK) at 100 K using Pilatus 6M detectors. Diffraction data were integrated and scaled using XDS and symmetry related reflections were merged with Aimless . Data collection statistics are shown in Table 4. The resolution cut-off used for structure determination and refinement was determined based on the CC1/2 criterion proposed by. The structure of EncFtnsH was determined by molecular replacement using PDB ID: 3K6C as the search model, modified to match the sequence of the target protein using Chainsaw. A single solution comprising three decamers in the asymmetric unit was found by molecular replacement using Phaser. The initial model was rebuilt using Phenix.autobuild followed by cycles of refinement with Phenix.refine, with manual rebuilding and model inspection in Coot . The final model was refined with isotropic B-factors, torsional NCS restraints, and with anomalous group refinement. The model was validated using MolProbity. Structural superimpositions were calculated using Coot. Crystallographic figures were generated with PyMOL. Multiple sequence alignment of EncFtn and ferritin family proteins was performed using Clustal Omega Sievers and Higgins, 2014 and displayed with Espript 3.0. Model refinement statistics are shown in Table 4. The final models and experimental data are deposited in the PDB and diffraction image files are available at the Edinburgh DataShare repository. Horse spleen apoferritin preparation Horse spleen apoferritin purchased from Sigma Aldrich (UK) was dissolved in deaerated MOPS buffer (100 mM MOPS, 100 mM NaCl, 3 g/100 ml Na2S2O4 and 0.5 M EDTA, pH 6.5). Protein was dialyzed against 1 liter MOPS buffer in room temperature for two days before buffer exchanging to GF buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl) in a vivaspin column with 5 kDa cut-off (Sartorius, UK) for several times. Fe content of apoferritin was detected using ferrozine assay. Protein concentration was determined using Pierce Microplate BCA Protein Assay Kit. Apoferritin containing less than 0.5 Fe per 24-mer was used in the ferroxidase assay. Apoferritin used in the Fe loading capacity experiment was prepared in the same way with 5–15 Fe per 24-mer. Ferroxidase assay 1 mM and 200 µM Fe(NH4)2(SO4)2 stock solutions were prepared in 0.1% (v/v) HCl anaerobically. Protein solutions with 20 µM FOC were diluted from ~10 mg/ml frozen stock in GF buffer (50 mM Tris-HCl, pH 8.0 and 150 mM NaCl) anaerobically. Ferroxidase activity was initiated by adding 450 μl protein to 50 μl of acidic Fe(NH4)2(SO4)2 at the final concentration of 100 µM and 20 µM in the air, respectively. The ferroxidase activity was measured by monitoring the Fe3+ formation which gives rise to the change of the absorbance at 315 nm. Absorbance at 315 nm was recorded every second over 1800 s using a quartz cuvette in a JASCO V-730 UV/VIS spectrophotometer (JASCO Inc., Easton, MD). In recombinantly coexpressed nanocompartments the ratio of EncFtn to Enc was assumed as 2 to 1, assuming each of the twelve pentameric vertices of the icosahedral encapsulin were occupied with decameric EncFtn. The data are presented as the mean of three technical replicates with error bars indicating one standard deviation from the mean. Proteins used here were from a single preparation. Iron loading capacity of ferritins In order to determine the maximum iron loading capacity, around 8.5 µM proteins including decameric EncFtnsH, Encapsulin, EncFtn-Enc and apoferritin were loaded with various amount of acidic Fe(NH4)2(SO4)2 ranging from 0 to 1700 µM. Protein mixtures were incubated in room temperature for 3 hrs before desalting in Zebra spin desalting columns (7 kDa cut-off, Thermo Fisher Scientific, UK) to remove free iron ions. The protein concentration was determined using PierceMicroplate BCA assay kit (Thermo Fisher Scientific). The protein standard curve was plotted according to the manufacturer. The Fe content in the samples was determined using modified ferrozine assay. Briefly speaking, 100 μl protein sample was mixed with 100 μl mixture of equal volume of 1.4 M HCl and 4.5% (w/v) KMnO4 and incubated at 60 °C for 2 hrs. 20 μl of the iron-detection reagent (6.5 mM ferrozine, 6.5 mM neocuproine, 2.5 M ammonium acetate, and 1 M ascorbic acid dissolved in H2O) was added to the cooled tubes. 30 min later, 200 μl of the solution was transferred into a well of 96-well plate and the absorbance at 562 nm was measured on the plate reader Spectramax M5 (Molecular Devices, UK). The standard curve was plotted using various concentrations of FeCl3 (in 10 mM HCl) diluted in the gel-filtration buffer. Three technical repeats were performed for both the ferrozine and microplate BCA assays. Samples analyzed by ICP-MS were prepared in the same way by mixing protein and ferrous ions and desalting. The proteins used in the Fe loading experiment came from a single preparation. Peroxidase assay The peroxidase activity of EncFtnsH was determined by measuring the oxidation of ortho-phenylenediamine (OP) by H2O2 . EncFtnsH decameric and monomeric fractions purified from MM were both used in the assay. Ortho-phenylenediamine was prepared as a 92.5 mM stock solution in 50 mM Tris-HCl (pH 8.0). 80, 70, 60, 50, 40, 30, 20 and 10 mM of OP were prepared by diluting the stock solution in the 50 mM Tris-HCl (pH 8.0). 100 μl of each diluted OP was added to a 96-well plate in 3 repeats. 1 μl of 32 µM protein was supplemented into each well to a final concentration of 160 nM, followed by the addition of 2 μl of 30% H2O2. After 15 min shaking in the dark, the reaction was stopped by adding 100 μl of 0.5 M H2SO4. The peroxidase activity was measured by monitoring the absorbance at 490 nm in the SpectraMax M5 Microplate Reader (Molecular Devices). ICP-MS analysis Protein samples were diluted 50-fold into a solution of 2.5% HNO3 (Suprapur, Merck, UK) containing 20 µg/L Pt as internal standard. Matrix-matched elemental standards (containing analyte metal concentrations 0 – 1000 µg/L) were prepared by serial dilution from individual metal standard stocks (VWR) with identical solution compositions, including the internal standard. All standards and samples were analyzed by ICP-MS using a Thermo x-series instrument (Thermo Fisher Scientific) operating in collision cell mode (using 3.0 ml min-1 flow of 8% H2 in He as the collision gas). Isotopes 44Ca, 56Fe, 66Zn, 78Se, and 195Pt were monitored using the peak-jump method (100 sweeps, 25–30 ms dwell time on 5 channels per isotope, separated by 0.02 atomic mass units) in triplicate. The protein samples used in ICP-MS came from a single protein preparation. Mass spectrometry analysis For native MS analysis, all protein samples were buffer exchanged into 100 mM ammonium acetate (pH 8.0; adjusted with dropwise addition of 1% ammonia solution) using Micro Biospin Chromatography Columns (Bio-Rad, UK) prior to analysis and the resulting protein samples were analyzed at a final concentration of ~5 µM (oligomer concentration). In order to obtain Fe-bound EncFtn, 100 µM or 300 µM of freshly prepared FeCl2 was added to apo-EncFtnsH (monomer peak) immediately prior to buffer exchange into 100 mM ammonium acetate (pH 8.0). Samples were analyzed on a quadrupole ion-mobility time of flight instrument (Synapt G2, Waters Corp., Manchester, UK), equipped with a nanomate nanoelectrospray infusion robot (Advion Biosciences, Ithaca, NY). Instrument parameters were tuned to preserve non-covalent protein complexes. After optimization, typical parameters were: nanoelectrospray voltage 1.54 kV; sample cone 50 V; extractor cone 0 V; trap collision voltage 4 V; source temperature 80°C; and source backing pressure 5.5 mbar. For improved mass resolution the sample cone was raised to 155 V. Ion mobility mass spectrometry (IM-MS) was performed using the travelling-wave mobility cell in the Synapt G2, employing nitrogen as the drift gas. Typically, the IMS wave velocity was set to 300 m/s; wave height to 15 V; and the IMS pressure was 1.8 mbar. All native MS experiments were performed on samples from two independent protein preparations. For collision cross section determination, IM-MS data was calibrated using denatured equine myoglobin and data was analyzed using Driftscope v2.5 and MassLynx v4.1 (Waters Corp., UK). Theoretical collision cross sections (CCS) were calculated from pdb files using IMPACT software v. 0.9.1. In order to obtain information on the topology of the EncFtnsH assembly, gas-phase dissociation of the Fe-associated EncFtnsH complex was achieved by increasing the sample cone and/or trap collision voltage prior to MS analysis. SEC-MALLS Size-exclusion chromatography (ÄKTA-Micro; GE Healthcare) coupled to UV, static light scattering and refractive index detection (Viscotec SEC-MALS 20 and Viscotec RI Detector:VE3580; Malvern Instruments, UK) were used to determine the molecular mass of fractions decamer and monomer of EncFtnsH in solution individually. Protein concentration was determined by measurement of absorbance at 280 nm and calculated using the extinction coefficient ε0.1%= 1.462 mg−1 ml-1 cm−1. 100 μl of 1.43 mgml-1 fractions of EncFtnsH decamer and 4.03 mg ml -1 fractions of EncFtnsH monomer were run individually on a Superdex 200 10/300 GL size-exclusion column pre-equilibrated in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl at 22°C with a flow rate of 0.5 ml/min. Light scattering, refractive index (RI) and A280nm were analyzed by a homo-polymer model (OmniSEC software, v 5.1; Malvern Instruments) using the following parameters for fractions of decamer and monomer: the extinction coefficient (dA/dc) at 280 nm was 1.46 AU mg ml−1 and specific refractive index increment (dn/dc) was 0.185 ml g−1. The proteins analyzed by SEC-MALLS came from single protein preparation. Metal binding analysis by PAGE Recombinant EncFtnsH fractions at 50 µM concentration were incubated with one molar equivalent of metal ions at room temperature for 2 hrs. Half of each sample was mixed with 5 x native loading buffer (65 mM Tris-HCl, pH 8.5, 20% glycerol and 0.01% bromophenol blue) and run on non-denaturing PAGE gels (10% acrylamide) and run in Tris/glycine buffer, 200 V, 4 °C for 50 min. The remaining samples were left for an additional three hours prior to SDS-PAGE (15% acrylamide) analysis. SDS-PAGE gels were run at room temperature at 200 V, room temperature for 50 min. Gels were stained with Coomassie Brilliant Blue R250 and scanned after de-staining in water. The proteins used in this experiment came from single protein preparation. Analytical size-exclusion chromatography For analysis of the multimeric state of EncFtn proteins by analytical size-exclusion gel-filtration chromatography (AGF) 25 μl of 90 µM protein was loaded into Superdex 200 PC 3.2/30 column (GE Healthcare) at 15 °C with GF buffer running at 0.05 ml/min and pressure limit 0.45 MPa. In order to use AGF to determine how metal ions influence the assembly of EncFtnsH, 90 µM EncFtnsH monomer fractions were mixed with equal molar concentrations of metal ion solutions including FeSO4 in 0.1% (v/v) HCl, Fe(NH4)2(SO4)2, FeCl3, CoCl2, calcium acetate (CaAc), ZnSO4 and MnCl2 at room temperature for 2 hrs prior to AGF analysis. Protein samples without metal titration were also analyzed as a control group. Both monomer and decamer fractions of EncFtnsH left at room temperature for 2 hrs, or overnight, were also analysed as controls to show the stability of the protein samples in the absence of additional metal ions. The AGF results have been repeated twice using two independent preparations of protein, of which only one representative trace is presented in the paper. Accession codes and datasets Coordinates and structure factors for the structures presented in this paper have been deposited in the PDB under the following accession codes: EncFtnsH, 5DA5; EncFtnsH-E32A, 5L89; EncFtnsH-E62A, 5L8B; EncFtnsH-H65A, 5L8G (DOIs for X-ray diffraction image data are shown in Table 4). All MS datasets presented in this paper can be found, in the raw format at http://dx.doi.org/10.7488/ds/1449. Funding Information This paper was supported by the following grants:  to Didi He.  to Sam Hughes, Kirsten Altenbach, David J Clarke.   to Emma Tarrant, Kevin J Waldron.   to David J Clarke, Jon Marles-Wright.   to Jon Marles-Wright. Additional information Competing interests The authors declare that no competing interests exist. Author contributions DH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. SH, Acquisition of data, Analysis and interpretation of data. SV-H, Acquisition of data, Analysis and interpretation of data. AG, Acquisition of data, Drafting or revising the article. KA, Acquisition of data, Contributed unpublished essential data or reagents. ET, Acquisition of data, Analysis and interpretation of data. CLM, Acquisition of data, Contributed unpublished essential data or reagents. KJW, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. DJC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. JM-W, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article. Additional files Major datasets The following datasets were generated: References Unusual clustering of carboxyl side chains in the core of iron-free ribonucleotide reductase PHENIX: a comprehensive Python-based system for macromolecular structure solution Towards automated crystallographic structure refinement with phenix.refine The crystal structure of a virus-like particle from the hyperthermophilic archaeon Pyrococcus furiosus provides insight into the evolution of viruses The Ferritin-like superfamily: Evolution of the biological iron storeman from a rubrerythrin-like ancestor Mössbauer spectroscopic investigation of structure-function relations in ferritins Moving Fe2+ from ferritin ion channels to catalytic OH centers depends on conserved protein cage carboxylates Structural insights into the ferroxidase site of ferritins from higher eukaryotes A Mass-Spectrometry-Based Framework To Define the Extent of Disorder in Proteins Ferroxidase activity of recombinant Desulfovibrio vulgaris rubrerythrin The iron redox and hydrolysis chemistry of the ferritins Mechanisms of iron mineralization in ferritins: one size does not fit all Identification of a Minimal Peptide Tag for in Vivo and in Vitro Loading of Encapsulin Mineralization in ferritin: an efficient means of iron storage MolProbity: all-atom structure validation for macromolecular crystallography Characterization of a Mycobacterium tuberculosis nanocompartment and its potential cargo proteins Features and development of Coot An introduction to data reduction: space-group determination, scaling and intensity statistics Electrospray ionization of large multiply charged species proceeds via Dole’s charged residue mechanism Oligomeric states of proteins determined by size-exclusion chromatography coupled with light scattering, absorbance, and refractive index detectors ESPript/ENDscript: Extracting and rendering sequence and 3D information from atomic structures of proteins The crystal structure of Dps, a ferritin homolog that binds and protects DNA Moving Iron through ferritin protein nanocages depends on residues throughout each four α-helix bundle subunit The Role of Salt Bridges, Charge Density, and Subunit Flexibility in Determining Disassembly Routes of Protein Complexes Ferritin family proteins and their use in bionanotechnology The refined structure of a protein catenane: the HK97 bacteriophage capsid at 3.44 A resolution The catalytic center of ferritin regulates iron storage via Fe(II)-Fe(III) displacement Integration, scaling, space-group assignment and post-refinement Linking crystallographic model and data quality Calcium Ion Coordination: A Comparison with That of Beryllium, Magnesium, and Zinc MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets Solving the structure of human H ferritin by genetically engineering intermolecular crystal contacts Iron core mineralisation in prokaryotic ferritins Use of structural phylogenetic networks for classification of the ferritin-like superfamily Structure and composition of ferritin cores isolated from human spleen, limpet (Patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells Collision cross sections for structural collision cross sections for structural proteomics Phaser crystallographic software A virus capsid-like nanocompartment that stores iron and protects bacteria from oxidative stress  Structure and mechanism of iron translocation by a Dps protein from Microbacterium arborescens Mechanism of ferrous iron binding and oxidation by ferritin from a pennate diatom Covalent modification of the iron protein of nitrogenase from Rhodospirillum rubrum by adenosine diphosphoribosylation of a specific arginine residue Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells Characterization of dye-decolorizing peroxidases from Rhodococcus jostii RHA1 Recombinant expression and purification of \"virus-like\" bacterial encapsulin protein cages Self-sorting of foreign proteins in a bacterial nanocompartment The neighbor-joining method: a new method for reconstructing phylogenetic trees Clustal Omega, accurate alignment of very large numbers of sequences CHAINSAW : a program for mutating pdb files used as templates in molecular replacement Structural basis of enzyme encapsulation into a bacterial nanocompartment 10.7554/eLife.18972.048 Decision letter Losick Richard In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. [Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The previous decision letter after peer review is shown below.] Thank you for submitting your work entitled \"Structural characterisation of an encapsulated ferritin provides insight into iron storage in bacterial nanocompartments\" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Losick as the Senior Editor. Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife. In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently, and thus represent an interesting frontier in microbial cell biology. While this study significantly advances our understanding of the structural and biochemical relationship between encapsulins and EncFer, it requires significant revision prior to publication. We do, however, encourage the authors to resubmit when and if they are address the issues raised below. Reviewer #1:  1) Methods: What procedures and analyses did the author use to assess whether the iron added to the various ferritin derivatives was protein coated or was simply balls of rust attached to protein fragments? If the latter, it could easily generate reactive oxygen species in air under physiological conditions. 2) Results: A) Critical data, such as the comparison of maximum amount of iron bound by a monomer in the dodocamer is in the Supplementary information. B) The data in Supplementary file 2, shows that the amount of iron bound by an ENCFTN decamer monomer is sub -stoichiometric, ranging from 0.18 to 0.64. In a bona fide ferritin, with ~ 2000 iron atoms/ protein cage (24 subunits), the same parameter is much, much higher. Even an experimental situation: 24 subunit (monomer) ferritin with a biomineral prepared experimentally from apoferritin and containing, on average, only 1000 iron atoms/24 subunit cage, the equivalent parameter appears to be 1000/24 = 42. This Fe/protein ratio is 66 times more iron than in the test system described. Moreover, in nature, some ferritin protein cages contain as much as 4500 Fe atoms, several hundred times higher than the test system. Thus the significance of the experimental results in the paper are unclear. 3) Table 4: Data are shown for three proteins, Encapsulin, Enc-Ftn-10mer, and EncFTN-Enc. Missing are data for the starting material, 24 subunit ferritin or apoferritin (ferritin with the iron removed, by reduction and chelation, as a control.) Reviewer #2:  In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently. While the structure of the encapsulin shell has been determined, that of its cargo, the ferritin-like protein (EncFer), has remained elusive. Here, the authors provide the structure of one such cargo and show that it assembles in a manner that is topologically distinct from ferritin. Additionally, the authors provide evidence that metal binding promotes the assembly of the EncFer and that it does act as a ferroxidase. Altogether, there is a substantial amount of work here that will likely be viewed as a major step forward in understanding these unique bacterial organelles. I have a few suggestions and questions that are listed below: 1) The authors grow E. coli in minimal media with and without added iron to show that assembly is iron dependent. The output of these experiments is the ratio of decamer vs. monomer. However, we don't have information on whether the growth conditions altered either the total amount of protein produced or the total amount soluble complex/monomer. Perhaps, lower protein concentrations lead to less efficient assembly (a critical concentration is needed). 2) There is no information regarding the reason for the use of R. rubrum encapsulins. As far as I can tell, these have not been a model for either in vivo or in vitro work. Is there even evidence that they are produced by R. rubrum? What is their size/appearance in that organism? Do they have a physiological role? 3) Also, how similar are the Enc and EncFer to those of M. xanthus? Are the putative iron-binding sites conserved? 4) I would have liked to see some mutagenesis experiments to test the models of assembly, iron binding and ferroxidase activity. These do not have to be in vivo and can be performed in vitro with the available system. 5) I would like some more phylogenetic data for the model that ferritin evolved from EncFer. Perhaps, EncFer evolved from ferritin? Do any of the existing phylogenetic analyses support one model over another. 10.7554/eLife.18972.049 Author response [Editors’ note: the author responses to the first round of peer review follow.] In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently, and thus represent an interesting frontier in microbial cell biology. While this study significantly advances our understanding of the structural and biochemical relationship between encapsulins and EncFer, it requires significant revision prior to publication. We do, however, encourage the authors to resubmit when and if they are address the issues raised below.  Reviewer #1:  1) Methods: What procedures and analyses did the author use to assess whether the iron added to the various ferritin derivatives was protein coated or was simply balls of rust attached to protein fragments? If the latter, it could easily generate reactive oxygen species in air under physiological conditions.  The reviewer makes an excellent point here. To ascertain whether the iron in the assays forms ‘balls of rust’ we performed transmission electron microscopy on the ferroxidase reaction mixtures after completion of the reaction to assess the formation of free, or encapsulated iron minerals. We provide an additional supplemental figure (Figure 8—figure supplement 1) and discuss the observation of iron mineral crystals and nanoparticles in the main text, subsection “Ferroxidase activity”, last paragraph. We also attempted to use a commercial luminescence-based ROS detection kit on the reactions to address the possibility that H2O2 is produced as a reaction intermediate by the EncFtn protein. We found that the results from this particular kit were inconsistent between repeats, but for the benefit of the reviewer we provide a graph of the results obtained (see Author response image 1). These results show the production of ROS by apoferritin, which is consistent with the published data on the reaction mechanism of certain ferritins; however, no significant ROS were detected for the EncFtn or encapsulin proteins. DOI:\nhttp://dx.doi.org/10.7554/eLife.18972.037 We acknowledge that the reaction mechanism of the EncFtn merits further investigation in a follow up study. 2) Results: A) Critical data, such as the comparison of maximum amount of iron bound by a monomer in the dodocamer is in the Supplementary information.  We acknowledge that the data for iron loading merits inclusion in the main text, we have now moved this data and other supplementary data tables to the main text. B) The data in Supplementary file 2, shows that the amount of iron bound by an ENCFTN decamer monomer is sub -stoichiometric, ranging from 0.18 to 0.64. In a bona fide ferritin, with ~ 2000 iron atoms/ protein cage (24 subunits), the same parameter is much, much higher.  One of the central arguments of our paper is the fact that the EncFtnsH monomer must dimerize to produce a functional ferroxidase active site and that its iron binding properties are highly divergent from those of the classical ferritin nanocages. We have added additional text to the manuscript to highlight these differences (Introduction, last paragraph, and Mass spectrometry section) and discuss the functional consequences at length. Even an experimental situation: 24 subunit (monomer) ferritin with a biomineral prepared experimentally from apoferritin and containing, on average, only 1000 iron atoms/24 subunit cage, the equivalent parameter appears to be 1000/24 = 42. This Fe/protein ratio is 66 times more iron than in the test system described. Moreover, in nature, some ferritin protein cages contain as much as 4500 Fe atoms, several hundred times higher than the test system! Thus the significance of the experimental results in the paper are unclear.  We have clarified this key difference in the discussion of the iron storage function of the encapsulin nanocompartment (subsection “Iron storage in encapsulin nanocompartments”, second paragraph). The key conclusion of the paper is that the iron storage and iron oxidation functions that are combined in classical ferritins are split between the encapsulin nanocompartment and the EncFtn protein. 3) Table 4: Data are shown for three proteins, Encapsulin, Enc-Ftn-10mer, and EncFTN-Enc. Missing are data for the starting material, 24 subunit ferritin or apoferritin (ferritin with the iron removed, by reduction and chelation, as a control.)  The data for the starting material are shown in Table 5. Control data for apoferritin have been added to this table and are illustrated in Figure 8. We note that we do not reach the experimental maximum loading capacity for apoferritin; however, we also note that the EncFtn-encapsulin nanocompartment sequesters five times more iron than the ferritin under the same reaction conditions, supporting the published observations that these nanocompartments can store more iron than classical ferritin nanocages. Reviewer #2:  In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently. While the structure of the encapsulin shell has been determined, that of its cargo, the ferritin-like protein (EncFer), has remained elusive. Here, the authors provide the structure of one such cargo and show that it assembles in a manner that is topologically distinct from ferritin. Additionally, the authors provide evidence that metal binding promotes the assembly of the EncFer and that it does act as a ferroxidase. Altogether, there is a substantial amount of work here that will likely be viewed as a major step forward in understanding these unique bacterial organelles. I have a few suggestions and questions that are listed below:  1) The authors grow E. coli in minimal media with and without added iron to show that assembly is iron dependent. The output of these experiments is the ratio of decamer vs. monomer. However, we don't have information on whether the growth conditions altered either the total amount of protein produced or the total amount soluble complex/monomer. Perhaps, lower protein concentrations lead to less efficient assembly (a critical concentration is needed).  The reviewer makes an interesting point about growth conditions and we acknowledge that production of the protein in LB medium leads to varying protein yields and monomer/decamer proportions. We therefore adopted the use of M9 minimal medium throughout the study to give better reproducibility, which also enables better control of metal ion availability than the complex LB medium. Given the fact that the protein is produced recombinantly in E. coli it is not particularly instructive to prove the in vivoproduction of the EncFtn multimer in this host. We have added a panel to Figure 3 to show the effect of protein concentration on multimerization in vitro(Figure 3C). Our mass spectrometry results show that the protein spontaneously multimerized in the presence of iron in vitroto form decameric species and that this is metal ion concentration dependent (Figure 7). 2) There is no information regarding the reason for the use of R. rubrum encapsulins. As far as I can tell, these have not been a model for either in vivo or in vitro work. Is there even evidence that they are produced by R. rubrum? What is their size/appearance in that organism? Do they have a physiological role?  We have put a comment in the Introduction to introduce R. rubrum (last paragraph). A preliminary study in the laboratory identified encapsulins in a preparation of lipid vesicles from R. rubrum containing chromatophores. We chose to follow up on these structures in this study. We do not feel this particular information is key to the central argument of the paper. 3) Also, how similar are the Enc and EncFer to those of M. xanthus? Are the putative iron-binding sites conserved?  We have noted this in the Introduction of the manuscript. 4) I would have liked to see some mutagenesis experiments to test the models of assembly, iron binding and ferroxidase activity. These do not have to be in vivo and can be performed in vitro with the available system.  To address this question we have produced three FOC mutants of the EncFtn protein and characterized these in solution, by mass spectrometry, and crystallographically (section: Mutagenesis of the EncFtnsH Ferroxidase center). We thank the reviewer for this suggestion as it highlighted the importance of the FOC residues for assembly and activity, and our new data has provided interesting insights into the EncFtn protein. 5) I would like some more phylogenetic data for the model that ferritin evolved from EncFer. Perhaps, EncFer evolved from ferritin? Do any of the existing phylogenetic analyses support one model over another.  We now include a phylogenetic tree (Figure 13) and consider the question of ferritin evolution in the Discussion (first paragraph). None of the authors of this study are evolutionary biologists but we appreciate the difficulty inherent in tracing the history of protein folds, especially in bacterial lineages. We refer to previous studies in this section and make a suggestion that can be followed up in subsequent 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+[{"sourceid":"5014086","sourcedb":"","project":"","target":"","text":"Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf Summary Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling. We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains. The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor. We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD. DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC. Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2. Graphical Abstract Highlights The structure of the KREMEN 1 ectodomain is solved from three crystal forms Kringle, WSC, and CUB subdomains interact tightly to form a single structural unit The interface to DKKs is formed from the Kringle and WSC domains The CUB domain is found to interact directly with LRP6PE1PE2 Zebisch et al. describe the ectodomain structure of KREMEN 1, a receptor for Wnt antagonists of the DKK family. Apo structures and a complex with functional fragments of DKK1 and LRP6 shed light on the function of this dual-mode regulator of Wnt signaling. Introduction Signaling by Wnt morphogens is renowned for its fundamental roles in embryonic development, tissue homeostasis, and stem cell maintenance. Due to these functions, generation, delivery, and interpretation of Wnt signals are all heavily regulated in the animal body. Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6. Kremen proteins (Krm1 and Krm2) have been identified as additional high-affinity transmembrane receptors for Dkk. Krm and Dkk synergize in Wnt inhibition during Xenopus embryogenesis to regulate anterior-posterior patterning. Mechanistically it is thought that, in the presence of Dkk, Krm forms a ternary complex with Lrp6, which is then rapidly endocytosed. This amplifies the intrinsic Wnt antagonistic activity of Dkk by efficiently depleting the cell surface of the Wnt co-receptor. In accordance with this, Krm1−/− and Krm2−/− double knockout mice show a high bone mass phenotype typical of increased Wnt signaling, as well as growth of ectopic forelimb digits. Growth of ectopic digits is further enhanced upon additional loss of dkk expression. The Wnt antagonistic activity of Krm1 is also linked to its importance for correct thymus epithelium formation in mice. The importance of intact KRM1 for normal human development and health is highlighted by the recent finding that a homozygous mutation in the ectodomain of KRM1 leads to severe ectodermal dysplasia including oligodontia. Interestingly, the Wnt antagonistic activity of Krm is context dependent, and Krm proteins are actually dual-mode Wnt regulators. In the absence of Dkk, Krm1 and 2 change their function from inhibition to enhancement of Lrp6-mediated signaling. By direct binding to Lrp6 via the ectodomains, Krm proteins promote Lrp6 cell-surface localization and hence increase receptor availability. Further increasing the complexity of Krm functionality, it was recently found that Krm1 (but not Krm2) can also act independently of LRP5/6 and Wnt as a dependence receptor, triggering apoptosis unless bound to Dkk. Structurally, Krm1 and 2 are type I transmembrane proteins with a 40 kDa ectodomain and a flexible cytoplasmic tail consisting of 60–75 residues. The ectodomain consists of three similarly sized structural domains of around 10 kDa each: the N-terminal Kringle domain (KR) is followed by a WSC domain of unknown fold. The third structural domain is a CUB domain. An approximately 70-residue linker connects the CUB domain to the transmembrane span. An intact KR-WSC-CUB domain triplet and membrane attachment is required for Wnt antagonism. The transmembrane span and cytoplasmic tail can be replaced with a GPI linker without impact on Wnt antagonism. We sought to provide structural insights into the multi-functionality of this cell-surface receptor. The structures presented here reveal the unknown fold of the WSC domain and the tight interactions of all three domains. We further succeeded in determination of a low-resolution LRP6PE3PE4-DKK1CRD2-KRM1ECD complex, defining the architecture of the Wnt inhibitory complex that leads to Lrp6 cell-surface depletion. Results The recombinant production of the extracellular domain of Krm for structural studies proved challenging (see Experimental Procedures). We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown). Several crystal forms were obtained from these complexes, however, crystals always contained only KRM1 protein. We solved the structure of KRM1ECD in three crystal forms at 1.9, 2.8, and 3.2 Å resolution (Table 1). The high-resolution structure is a near full-length model (Figure 1). The small, flexible, and charged 98AEHED102 loop could only be modeled in a slightly lower resolution structure and in crystal form III. The KR, WSC, and CUB are arranged in a roughly triangular fashion with tight interactions between all three domains. The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements. The structurally most similar Kringle domain is that of human plasminogen (PDB: 1PKR) with an root-mean-square deviation (RMSD) of 1.7 Å for 73 aligned Cα (Figure 1B). The KRM1 structure reveals the fold of the WSC domain for the first time. The structure is best described as a sandwich of a β1-β5-β3-β4-β2 antiparallel β sheet and a single α helix. The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198). Using the PDBeFold server, we detected a surprising yet significant homology to PAN module domains. The closest structural relative is hepatocyte growth factor (HGF, PDB: 1GP9), which superposes with an RMSD of 2.3 Å for 58 aligned Cα (Figure 1B). The CUB domain bears two glycosylation sites. Although present, the quality of the electron density around N217 did not allow modeling of the sugar moiety. In crystal form I, a calcium ion is present at the canonical position coordinated by the carboxylates of D263, D266 (bidentate), and D306, as well as the carbonyl of N309 and a water molecule. The coordination sphere deviates significantly from perfectly octahedral (not shown). This might result in the site having a low affinity and may explain why calcium is not present in the two low-resolution crystal forms. Loss of calcium has led to loop rearrangements and partial disorder in these crystal forms. The closest structural relative is the CUB_C domain of Tsg-6 (PDB: 2WNO), which superposes with KRMCUB with an RMSD of 1.6 Å for 104 Cα (Figure 1B). A superposition of the three KRM1 structures reveals no major structural differences (Figure 1C) as anticipated from the plethora of interactions between the three domains. Minor differences are caused by the collapse of the Ca2+ binding site in crystal forms II and III and loop flexibility in the KR domain. The F207S mutation recently found to cause ectodermal dysplasia in Palestinian families maps to the hydrophobic core of the protein at the interface of the three subdomains (Figure 1A). Such a mutation is bound to severely destabilize the protein structure of KRM1, leading to disturbance of its Wnt antagonistic, Wnt stimulatory, and Wnt independent activity. Low-Resolution Insight into Ternary Complex Formation Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands. One face of the rather flat DKK1CRD2 fragment binds to the third β propeller of LRP6. Mutational analyses further implied that the LRP6PE3-averted face of DKK1CRD2 bears the Krm binding site, hence suggesting how Dkk can recruit both receptors into a ternary complex. To obtain direct insight into ternary complex formation by Lrp5/6, Dkk, and Krm, we subjected an LRP6PE3PE4-DKK1fl-KRM1ECD complex to crystallization trials. Diffraction data collected from the resulting crystals were highly anisotropic with diffraction extending in the best directions to 3.5 Å and 3.7 Å but only to 6.4 Å in the third direction. Despite the lack of high-resolution diffraction, the general architecture of the ternary complex is revealed (Figure 2A). DKK1CRD2 binds to the top face of the LRP6 PE3 β propeller as described earlier for the binary complex. KRM1ECD does indeed bind on the opposite side of DKK1CRD2 with only its KR and WSC domains engaged in binding (Figure 2A). Although present in the complex subjected to crystallization, we observe no density that could correspond to CRD1 or the domain linker (L). We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure 2B). A SUMO fusion of DKK1L-CRD2 displayed a similar (slightly higher) affinity. In contrast, a SUMO fusion of DKK1CRD1-L did not display binding for concentrations tested up to 325 μM (Figure 2B). Overall, the DKK1-KRM1 interface is characterized by a large number of polar interactions but only few hydrophobic contacts (Figure 2C). The crystal structure gives an explanation for DKK1 loss-of-binding mutations identified previously: R191 of DKK1 forms a double salt bridge to D125 and E162 of KRM1 (Figure 2C). A charge reversal as in the mouse Dkk1 (mDkk1) R197E variant would severely disrupt the binding. Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90. Again, a charge reversal as shown before for mDkk1 K232E would be incompatible with binding. The side chain of DKK1 S192 was also predicted to be involved in Krm binding. Indeed, we found (Figure 2C) that the side chain of D201 of KRM1 forms two hydrogen bonds to the side-chain hydroxyl and the backbone amide of S192 (mouse, S198). Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively. The carbonyl of DKK1 R224 is hydrogen bonded to Y105 and W106 of KRM1. We suspect that the Dkk charge reversal mutations performed in the murine background and shown to diminish Krm binding K211E and R203E (mouse K217E and R209E) do so likely indirectly by disruption of the Dkk fold. We further validated the DKK1 binding site on KRM1 by introducing glycosylation sites at the KR (90DVS92→NVS) and WSC (189VCF191→NCS) domains pointing toward DKK (Figures 2A and 2D). Introduction of N-linked glycans in protein-protein-binding sites is an established way of disrupting protein-binding interfaces. Both ectodomain mutants were secreted comparably with the wild-type, indicating correct folding, but failed to achieve any detectable binding in SPR using full-length DKK1 as analyte. In contrast, a mutant carrying an additional N-glycan outside the interface at the CUB domain (309NQA311→NQS), was wild-type-like in DKK1 binding (Figure 2D). Identification of a Direct LRP6-KRM1 Binding Site The LRP6PE3PE4-DKK1CRD2-KRM1ECD complex structure reveals no direct interactions between KRM1 and LRP6. We constructed in silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data. An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A). For this crude approximation of a true ternary complex, we noted very close proximity between the Ca2+-binding region of KRM1 and the top face of the PE2 β propeller of LRP6. The solvent-exposed residues R307, I308, and N309 of the central Ca2+-binding β connection loop of KRM1 would be almost ideally positioned for binding to this face, which is commonly used as a binding site on β propellers. Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure 3B). To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment. Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2 (Figure 3C). In contrast, no saturable binding was observed between KRM1 and LRP6PE3PE4. Introduction of an N-glycosylation site at N309 in KRM1ECD abolished LRP6PE1PE2 binding (Figure 3C), while binding to DKK1 was unaffected (Figure 2D). We conclude that the predicted binding site between KRM1CUB and LRP6PE2 is a strong candidate for mediating the direct Lrp6-Krm interaction, which is thought to increase Wnt responsiveness by stabilizing Lrp6 at the cell surface. Further experiments are required to pinpoint the exact binding site. Although LRP6PE1 appears somewhat out of reach in the modeled ternary complex, it cannot be excluded as the Krm binding site in the ternary complex and LRP6-Krm binary complex. The presence of DKK may govern which propeller (PE1 versus PE2) of LRP6 is available for Krm binding. Apparent binding across the proposed KRM1CUB-LRP6PE2 interface is expected to be higher once Krm is also cross-linked to LRP6PE3 via DKK1CRD2 (Figure 3D). Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3. It is therefore possible that the interplay of Krm and Dkk binding can promote changes in LRP6 ectodomain conformation with functional consequences; however, such ideas await investigation. Taken together, the structural and biophysical studies we report here extend our mechanistic understanding of Wnt signal regulation. We describe the ectodomain structure of the dual Wnt regulator Krm1, providing an explanation for the detrimental effect on health and development of a homozygous KRM1 mutation. We also reveal the interaction mode of Krm-Dkk and the architecture of the ternary complex formed by Lrp5/6, Dkk, and Krm. Furthermore, the ternary crystal structure has guided in silico and biophysical analyses to suggest a direct LRP6-KRM1 interaction site. Our findings provide a solid foundation for additional studies to probe how ternary complex formation triggers internalization, whereas Krm binding in the absence of Dkk stabilizes the Wnt co-receptor at the cell surface. Experimental Procedures Large-Scale Mammalian Expression and Protein Purification KrmECD fragments were cloned into pHLsec or variants thereof. Full ectodomain variants (e.g., KRM1 isoform 3, P30-T377) were well secreted into the conditioned medium (CM) of HEK293T cells, but exhibited extensive O-glycosylation (as judged from smeary bands in western blot), which would be detrimental to crystallization. Fragments truncated to the KR-WSC-CUB core gave sharp bands but were barely secreted. We therefore engineered an A23-G373 (isoform 1 numbering used throughout the article) full ectodomain construct (KRM1ECD-TEV) with a C-terminal His10 tag that contained a TEV protease cleavage site after E324. The expected sequence of the secreted protein is ETG-23APSPGLGPGPE31 … 320AVKEE324-GSENLYFQGGS-325LPQ … VPG373-THHHHHHHHHH (the isoform-2-specific PG insertion and the TEV site are underlined). This construct was well secreted and could be processed using TEV protease. However, 80%–90% of the protein eluted as aggregates from a size-exclusion column even before TEV treatment. The same applied to analog constructs for Krm1 from zebrafish, frog, and mouse. No monomeric protein at all could be obtained for several Krm2 constructs from multiple species. A KRM1ECD-TEV expressing stable GntI-deficient HEK293S cell line was generated by excision of an EcoRI-XhoI fragment, sub-cloning into pNeo-Sec-1, and selection of neomycin-resistant cells. The stable cell line showed expression levels superior to transiently transfected cells (not shown). Human LRP6PE1PE2, LRP6PE3PE4, and full-length DKK1 were produced in a similar way as described. Shorter constructs of DKK1 lacking the N-terminal flexible region and CRD1 were not secreted from HEK cells. However, using the approach of an N-terminal fusion to a modified SUMO protein as described earlier, we succeeded in secretory expression of a SUMO-DKK1Linker-CRD2 construct encompassing residues S141-H266. A variant of this containing a TEV cleavage site just before T181, SUMO-DKK1Linker-TEV-CRD2, was also well expressed and allowed removal of the flexible linker region. To obtain complexes of KRM1ECD-TEV, we (co-)transfected the stable cell line with DKK and LRP6PE3PE4 constructs described earlier. Binary and ternary KRM1ECD-DKK1fl and KRM1ECD-DKK1fl-LRP6PE3PE4 complexes were stable in gel-filtration eluting as distinct monodisperse peaks. Crystallization and Data Collection All samples subjected to crystallization were purified from CM by affinity and size-exclusion chromatography. After treatment with TEV protease and endoglycosidase F1 overnight using mass equivalents of 1%, samples were subjected to size-exclusion chromatography in 10 mM HEPES/NaOH (pH 7.5), 150 mM NaCl. The crystals giving rise to the 1.9 Å dataset for KRM1 in crystal form I were obtained from a KRM1ECD-DKK1Linker-CRD2 complex concentrated to 12 mg/mL. Out of this complex, KRM1ECD crystallized alone in 2.0 M ammonium sulfate, 5% (v/v) iso-propanol. For cryoprotection, crystals were transferred to mother liquor mixed 1:1 with 3.4 M sodium malonate (pH 7.0). The slightly less well-ordered crystal of crystal form I and crystals of form II were obtained from a KRM1ECD-DKK1CRD2 complex using the SUMO-DKK1Linker-TEV-CRD2 construct and releasing SUMO and the DKK linker region by TEV and 3C protease treatment. Crystals of form I (2.1 Å) appeared from protein at 12 mg/mL in 1.0 M (NH4)H2PO4, 0.100 M sodium citrate (pH 5.6) and were cryoprotected by transfer to 2.9 M sodium malonate (pH 5.0). Crystals of form II grew from protein concentrated to 17 mg/mL in 1.0 M MgSO4, 0.1 M trisodium citrate (final pH 5.6). For cryoprotection, crystals were transferred to mother liquor mixed 1:3 with 3.0 M ammonium sulfate, 18% glycerol. Crystal form III appeared after 11 months in a dried-out drop of condition H5 of the Morpheus screen. The protein concentration had been 9 mg/mL. For cryoprotection, fresh liquid from Morpheus/H5 was added. The ternary complex structure was obtained from an LRP6PE3PE4-DKK1fl-KRM1ECD complex at 9 mg/mL that grew in condition E10 of the PACTpremier screen (pH approximately 6.8) over the course of 2–11 months. For cryoprotection, 10% PEG200 was added. By mistake, the crystals were incubated for 1 hr with 1 mM platinum compound in this cryosolution before cryocooling. Structure Determination Diffraction data were collected at DIAMOND synchrotron light source at the beamlines detailed in Table 1. The structure was initially solved from crystal form III by molecular replacement (MR) with PHASER, placing models for the CUB domain (PDB: 2WNO, CUB_C domain of Tsg-6, 37% sequence identity), and the KR domain (PDB: 1PKR, Kringle 1 of plasminogen; 39% sequence identity). Traceable density for the WSC domain became immediately evident. The KRM1 structure was then built and refined by cycling between the various crystal forms. For the ternary complex, we obtained only a low-resolution, highly anisotropic dataset extending to Bragg spacings of 3.5 Å, 6.4 Å, and 3.7 Å along the three principle directions (\u003cI/σI\u003e = 2). All data to 3.5 Å were used during structure determination by MR. LRP6PE3PE4 (PDB: 4A0P) and KRM1ECD (both stripped of glycosylation sites) could be placed independently by PHASER, giving Z scores of \u003e10 and log likelihood gains (LLG) of \u003e200. The combined LLG was 673, increasing to 901 after rigid-body refinement. Strong electron density became apparent at glycosylation sites and close to methionines (see platinum soak above), further supporting the MR solution. Additional strong density was evident between LRP6 and KRM1, suggesting the presence of DKK1. A model of the DKK1CRD2 (PDB: 3S2K and 3S8V) could then be placed with PHASER by testing all rotation function peaks. This increased the LLG from 901 to 973 indicating a correct solution. The individually placed LRP6 and DKK models were then replaced with chains B and C from the LRP6-DKK complex in PDB: 3S2K. The structure was subjected to rigid-body refinement using single structural domains as individually positioned bodies. We then performed restrained refinement of the coordinates against the ellipsoidally truncated and anisotropically scaled diffraction data as obtained from the diffraction anisotropy server at UCLA. The resolution cutoffs were 3.5 Å, 6.4 Å, and 3.7 Å. Strong geometric restraints generated by PROSMART from the available high-resolution reference structures were used during refinement. No manual model building was attempted. Restrained refinement was followed by ten cycles of structure idealization. The final model had Rwork/Rfree errors of 32.5%/36.1% against the anisotropy-corrected data and 32.1%/35.5% against the unmodified but ellipsoidally truncated diffraction data. Surface Plasmon Resonance Equilibrium experiments were performed as described before with the addition of 2 mM CaCl2 for experiments investigating the direct LRP6PE1PE2-KRM1ECD interaction. Author Contributions M.Z. and V.A.J. performed experiments with support from Y.Z., who generated the stable cell line. M.Z. and E.Y.J. designed the research. M.Z. wrote the paper with input from all other authors. References Structural basis of Wnt signaling inhibition by Dickkopf binding to LRP5/6 Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow The structural basis of DKK-mediated inhibition of Wnt/LRP signaling Wnt antagonists bind through a short peptide to the first beta-propeller domain of LRP5/6 Metal ion-dependent heavy chain transfer activity of Tsg-6 mediates assembly of the cumulus-oocyte matrix Kremen1 and Dickkopf1 control cell survival in a Wnt-independent manner Structure and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and proteoglycan Structural insight into the mechanisms of Wnt signaling antagonism by Dkk Structural and functional studies of LRP6 ectodomain reveal a platform for Wnt signaling Crystal structures of the extracellular domain of LRP6 and its complex with DKK1 Wnt/beta-catenin signaling and disease Context-dependent activation or inhibition of Wnt-beta-catenin signaling by Kremen Kremen proteins interact with Dickkopf1 to regulate anteroposterior CNS patterning Targeted disruption of the Wnt regulator Kremen induces limb defects and high bone density Structure and properties of the Ca(2+)-binding CUB domain, a widespread ligand-recognition unit involved in major biological functions Kremen is required for neural crest induction in Xenopus and promotes LRP6-mediated Wnt signaling The evolution of the Wnt pathway Mutation of KREMEN1, a modulator of Wnt signaling, is responsible for ectodermal dysplasia including oligodontia in Palestinian families Notum deacylates Wnt proteins to suppress signalling activity Extracellular modulators of Wnt signalling Kremen2 modulates Dickkopf2 activity during Wnt/LRP6 signaling LDL-receptor-related protein 6 is a receptor for Dickkopf proteins Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling Phaser crystallographic software Molecular cloning and characterization of Kremen, a novel kringle-containing transmembrane protein The functions and possible significance of Kremen as the gatekeeper of Wnt signalling in development and pathology The complex world of WNT receptor signalling The Wnt signaling antagonist Kremen1 is required for development of thymic architecture Enhanced protein expression in mammalian cells using engineered SUMO fusions: secreted phospholipase A2 The crystal structures of two spermadhesins reveal the CUB domain fold Negative regulation of bone formation by the transmembrane Wnt antagonist Kremen-2 Production of cell surface and secreted glycoproteins in mammalian cells Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6 Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis The PAN module: the N-terminal domains of plasminogen and hepatocyte growth factor are homologous with the apple domains of the prekallikrein family and with a novel domain found in numerous nematode proteins A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae Characterization of the Kremen-binding site on Dkk1 and elucidation of the role of Kremen in dkk-mediated Wnt antagonism A new crystal form of the NK1 splice variant of HGF/SF demonstrates extensive hinge movement and suggests that the NK1 dimer originates by domain swapping The structure of recombinant plasminogen kringle 1 and the fibrin binding site Crystallographic evidence for a domain motion in rat nucleoside triphosphate diphosphohydrolase (NTPDase) 1 Structural and molecular basis of ZNRF3/RNF43 transmembrane ubiquitin ligase inhibition by the Wnt agonist R-spondin Accession Numbers Coordinates and structure factors have been deposited in the PDB with succession numbers PDB: 5FWS, 5FWT, 5FWU, 5FWV, and 5FWW. Supplemental Information Supplemental Information includes one figure and can be found with this article online at http://dx.doi.org/10.1016/j.str.2016.06.020. Structure of Unliganded KRM1ECD (A) The KRM1ECD fold (crystal form I) colored blue to red from the N to C terminus. Cysteines as ball and sticks, glycosylation sites as sticks. The bound calcium is shown as a gray sphere. The site of the F207S mutation associated with ectodermal dysplasia in humans is shown as mesh. (B) Superposition of the three KRM1ECD subdomains (solid) with their next structurally characterized homologs (half transparent). (C) Superposition of KRM1ECD from the three crystal forms. Alignment scores for each pairing are indicated on the dashed triangle. Insight into Ternary Complex Formation (A) The structure of the ternary LRP6PE3PE4-DKK1CRD2-KRM1ECD complex. DKK1 (orange) is sandwiched between the PE3 module of LRP6 (blue) and the KR-WSC domain pair of KRM1 (green). Colored symbols indicate introduced N-glycan attachment sites (see D). (B) SPR data comparing binding of full-length DKK1 and SUMO fusions of DKK1 truncations for binding to immobilized wild-type KRM1ECD. (C) Close-up view of the DKK1CRD2-KRM1ECD interface. Residues involved in interface formation are shown as sticks; those mentioned in the text are labeled. Salt bridges are in pink and hydrogen bonds in black. Model bias cannot be excluded as single atoms and bonds are not resolved at 6.4–3.5 Å. See also Figure S1. (D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange). See also symbols in (A). LRP6-KRM1 Direct Interaction and Summary (A) In a construction of a ternary complex with all four β propellers of LRP6 intact, the CUB domain points via its Ca2+-binding region toward the top face of the second β propeller. (B) Close-up view of the potential interaction site. In addition, LRP6PE2 has been superimposed with DKK1 (yellow) and SOST (pink) peptide complexes of LRP6PE1. (C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309. (D) Schematic representation of structural and biophysical findings and their implications for Wnt-dependent (left, middle) and independent (right) signaling. Conformational differences in the depictions of LRP6 are included purely for ease of representation. Diffraction and Refinement Statistics \tKRM1ECD\tKRM1ECD\tKRM1ECD\tKRM1ECD\tLRP6PE3PE4-DKKCRD2-KRM1ECD\t \tCrystal form\tI\tI\tII\tIII\tI\t \tX-ray source\tDiamond i04\tDiamond i03\tDiamond i03\tDiamond i04\tDiamond i04\t \tWavelength (Å)\t0.9793\t0.9700\t0.9700\t0.9795\t0.9795\t \tSpace group\tP3121\tP3121\tP43\tP41212\tC2221\t \tUnit cell a/α (Å/°)\t50.9/90\t50.5/90\t65.8/90\t67.8/90\t86.9/90\t \tb/β (Å/°)\t50.9/90\t50.5/90\t65.8/90\t67.8/90\t100.1/90\t \tc/γ (Å/°)\t188.4/120\t187.4/120\t75.0/90\t198.2/90\t270.7/90\t \tWilson B factor (Å2)\t31\t41\t76\t77\tNA\t \tResolution range (Å)\t47.10–1.90 (1.95–1.90)\t62.47–2.10 (2.16–2.10)\t75.00–2.80 (2.99–2.80)\t67.80–3.20 (3.42–3.20)\t67.68–3.50 (7.16–6.40, 3.92–3.50)\t \tUnique reflections\t23,300 (1,524)\t17,089 (1,428)\t7,964 (1,448)\t8,171 (1,343)\t8,070 (723, 645)\t \tAverage multiplicity\t9.1 (9.2)\t5.2 (5.3)\t3.7 (3.7)\t22.7 (12.6)\t3.8 (3.5, 4.4)\t \tCompleteness (%)\t99.8 (98.5)\t100 (100)\t99.8 (100)\t98.8 (93.4)\t51.6 (98.5, 14.1)\t \t\u003cI/σI\u003e\t11.4 (1.7)\t12.0 (1.7)\t14.9 (1.5)\t13.1 (1.9)\t4.6 (4.1, 2.2)\t \tRmerge (%)\t14.8 (158.3)\t9.3 (98.0)\t6.2 (98.9)\t29.8 (142.2)\t44.9 (40.5, 114.2)\t \tRpim (%)\t15.7 (55.3)\t10.3 (109.0)\t3.7 (53.8)\t6.3 (40.0)\t24.7 (23.9, 59.9)\t \t\t \tRefinement\t \t\t \tRwork (%)\t17.9\t18.4\t21.6\t20.2\t32.1\t \tRfree (%)\t22.7\t23.2\t30.7\t27.1\t35.5\t \t\t \tNo. of Non-Hydrogen Atoms\t \t\t \tProtein\t2,260\t2,301\t2,102\t2,305\t7,730\t \tN-glycans\t42\t42\t28\t28\t0\t \tWater\t79\t54\t0\t2\t0\t \tLigands\t6\t6\t2\t5\t0\t \t\t \tAverage B factor (Å2)\t \t\t \tProtein\t63\t65\t108\t84\t–\t \tN-glycans\t35\t46\t102\t18\t–\t \tWater\t68\t85\t–\t75\t–\t \tLigands\t36\t47\t91\t75\t66\t \t\t \tRMSD from Ideality\t \t\t \tBond lengths (Å)\t0.020\t0.016\t0.019\t0.016\t0.004\t \tBond angles (°)\t2.050\t1.748\t1.952\t1.796\t0.770\t \t\t \tRamachandran Plot\t \t\t \tFavored (%)\t96.8\t95.5\t96.9\t94.9\t92.3\t \tAllowed (%)\t99.7\t100.0\t100.0\t99.7\t99.8\t \tNumber of outliers\t1\t0\t0\t1\t2\t \tPDB code\t5FWS\t5FWT\t5FWU\t5FWV\t5FWW\t \t Values in parentheses refer to the highest-resolution shell. An additional shell given for the ternary complex corresponds to the last shell with near-complete diffraction data. NA, not 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diff --git a/annotated_BioC_JSON/PMC5063996_ann.json b/annotated_BioC_JSON/PMC5063996_ann.json
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+[{"sourceid":"5063996","sourcedb":"","project":"","target":"","text":"The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans* The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance. Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains. A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans. The mechanism of substrate recognition displayed by the enzyme, however, remains unclear. Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands. The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain. The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility. The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center. The −2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants. Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity. Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed. This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack. Introduction The plant cell wall is an important biological substrate. This complex composite structure is depolymerized by microorganisms that occupy important highly competitive ecological niches, whereas the process makes an important contribution to the carbon cycle. Lignocellulosic degradation is also of continued interest to environmentally sensitive industries such as the biofuels and biorefinery sectors, where the use of sustainable or renewable substrates is of increasing importance. Given that the plant cell wall is the most abundant source of renewable organic carbon on the planet, this macromolecular substrate has substantial industrial potential. An example of the chemical complexity of the plant cell wall is provided by xylan, which is the major hemicellulosic component. This polysaccharide comprises a backbone of β-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with α-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated. In addition, the Araf side chain decorations can also be esterified to ferulic acid that, in some species, provide a chemical link between hemicellulose and lignin. The precise structure of xylans varies between plant species, in particular in different tissues and during cellular differentiation. In specialized plant tissues, such as the outer layer of cereal grains, xylans are extremely complex, and side chains may comprise a range of other sugars including l- and d-galactose and β- and α-Xylp units. Indeed, in these cereal brans, xylans have very few backbone Xylp units that are undecorated, and the side chains can contain up to six sugars. Reflecting the chemical and physical complexity of the plant cell wall, microorganisms that utilize these composite structures express a large number of polysaccharide-degrading enzymes, primarily glycoside hydrolases, but also polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. These carbohydrate active enzymes are grouped into sequence-based families in the CAZy database. With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8. The extensive decoration of the xylan backbone generally restricts the capacity of these enzymes to attack the polysaccharide prior to removal of the side chains by a range of α-glucuronidases, α-arabinofuranosidases, and esterases. Two xylanases, however, utilize the side chains as essential specificity determinants and thus target decorated forms of the hemicellulose. The GH30 glucuronoxylanases require the Xylp bound at the −2 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites −1 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively). The GH5 arabinoxylanase (CtXyl5A) derived from Clostridium thermocellum displays an absolute requirement for xylans that contain Araf side chains. In this enzyme, the key specificity determinant is the Araf appended to O3 of the Xylp bound in the active site (−1 subsite). The reaction products generated from arabinoxylans, however, suggest that Araf can be accommodated at subsites distal to the active site. CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1. Previous studies of Fn3 domains have indicated that they might function as ligand-binding modules, as a compact form of peptide linkers or spacers between other domains, as cellulose-disrupting modules, or as proteins that help large enzyme complexes remain soluble. The dockerin domain recruits the enzyme into the cellulosome, a multienzyme plant cell wall degrading complex presented on the surface of C. thermocellum. CtCBM6 stabilizes CtGH5, and CtCBM62 binds to d-galactopyranose and l-arabinopyranose. The function of the CtCBM13 and Fn3 modules remains unclear. Similarly, the mechanism of substrate recognition and its impact on specificity are key unresolved issues. This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity. The data show that the plasticity in substrate recognition enables the enzyme to hydrolyze highly complex xylans that are not accessible to classical GH10 and GH11 endo-xylanases. Molecular architecture of GH5_34 enzymes. Modules prefaced by GH, CBM, or CE are modules in the indicated glycoside hydrolase, carbohydrate binding module, or carbohydrate esterase families, respectively. Laminin_3_G domain belongs to the concanavalin A lectin superfamily, and FN3 denotes a fibronectin type 3 domain. Segments labeled D are dockerin domains. Results Substrate Specificity of CtXyl5A Previous studies showed that CtXyl5A is an arabinoxylan-specific xylanase that generates xylooligosaccharides with an arabinose linked O3 to the reducing end xylose. The enzyme is active against both wheat and rye arabinoxylans (abbreviated as WAX and RAX, respectively). It was proposed that arabinose decorations make productive interactions with a pocket (−2*) that is abutted onto the active site or −1 subsite. Arabinose side chains of the other backbone xylose units in the oligosaccharides generated by CtXyl5A were essentially random. These data suggest that O3, and possibly O2, on the xylose residues at subsites distal to the active site and −2* pocket are solvent-exposed, implying that the enzyme can access highly decorated xylans. To test this hypothesis, the activity of CtXyl5A against xylans from cereal brans was assessed. CtXyl5a was incubated with a range of xylans for 16 h at 60 °C, and the limit products were visualized by TLC. These xylans are highly decorated not only with Araf and GlcA units but also with l-Gal, d-Gal, and d-Xyl. Indeed, very few xylose units in the backbone of bran xylans lack side chains. The data presented in Table 1 showed that CtXyl5A was active against corn bran xylan (CX). In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues). The limit products generated by CtXyl5A from CX consisted of an extensive range of oligosaccharides. These data support the view that in subsites out with the active site the O2 and O3 groups of the bound xylose units are solvent-exposed and will thus tolerate decoration. Kinetics of GH5_34 arabinoxylanases ND, not determined; NA, no activity. Enzyme\tVariant\tkcat/Km\t \tWAX\tRAX\tCX\t \t\t\tmin−1mg−1ml\t \tCtXyl5A\tCtGH5-CBM6-CBM13-Fn3-CBM62\t800\tND\t460\t \tCtXyl5A\tCtGH5-CBM6-CBM13-Fn3\t1,232\tND\t659\t \tCtXyl5A\tCtGH5-CBM6-CBM13\t1,307\tND\t620\t \tCtXyl5A\tCtGH5-CBM6\t488\tND\t102\t \tCtXyl5A\tCtGH5-CBM6: E68A\tNA\tNA\tNA\t \tCtXyl5A\tCtGH5-CBM6: Y92A\tNA\tNA\tNA\t \tCtXyl5A\tCtGH5-CBM6: N135A\t260\tND\tND\t \tCtXyl5A\tCtGH5-CBM6: N139A\tNA\tNA\tNA\t \tAcGH5\tWild type\t628\t1,641\t289\t \tGpGH5\tWild type\t2,600\t9,986\t314\t \tVbGH5\tWild type\tND\tND\tND\t \tVbGH5\tD45A\t102\t203\t23\t \t To explore whether substrate bound only at −2* and −1 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study. HPAEC analysis of the smallest oligosaccharide fraction (pool 4) contained two species with retention times of 14.0 min (oligosaccharide 1) and 20.8 min (oligosaccharide 2) (Fig. 2). Positive mode electrospray mass spectrometry showed that pool 4 contained exclusively molecular ions with a m/z = 305 [M + Na]+, which corresponds to a pentose-pentose disaccharide (molecular mass = 282 Da) as a sodium ion adduct, whereas a dimer of the disaccharide with a sodium adduct (m/z = 587 [2M+Na]+) was also evident. The monosaccharide composition of pool 4 determined by TFA hydrolysis contained xylose and arabinose in a 3:1 ratio. This suggests that the two oligosaccharides consist of two disaccharides: one consisting of two xylose residues and the other consisting of an arabinose linked to a xylose. Treatment of pool 4 with the nonspecific arabinofuranosidase, CjAbf51A, resulted in the loss of oligosaccharide 2 and the production of both xylose and arabinose, indicative of a disaccharide of xylose and arabinose. Incubation of pool 4 with a β-1,3-xylosidase (XynB) converted oligosaccharide 1 into xylose, demonstrating that this molecule is the disaccharide β-1,3-xylobiose. This view is supported by the inability of a β-1,4-specific xylosidase to hydrolyze oligosaccharide 1 or oligosaccharide 2 (data not shown). The crucial importance of occupancy of the −2* pocket for catalytic competence is illustrated by the inability of the enzyme to hydrolyze linear β-1,4-xylooligosaccharides. The generation of Araf-Xylp and Xyl-β-1,3-Xyl as reaction products demonstrates that occupancy of the −2 subsite is not essential for catalytic activity, which is in contrast to all endo-acting xylanases where this subsite plays a critical role in enzyme activity. Indeed, the data demonstrate that −2* plays a more important role in productive substrate binding than the −2 subsite. Unfortunately, the inability to generate highly purified (Xyl-β-1,4)n-[β-1,3-Xyl/Ara]-Xyl oligosaccharides from arabinoxylans prevented the precise binding energies at the negative subsites to be determined. Identification of the disaccharide reaction products generated from CX. The smallest reaction products were purified by size exclusion chromatography and analyzed by HPAEC (A) and positive mode ESI-MS (B), respectively. The samples were treated with a nonspecific arabinofuranosidase (CjAbf51A) and a GH3 xylosidase (XynB) that targeted β-1,3-xylosidic bonds. X, xylose; A, arabinose. The m/z = 305 species denotes a pentose disaccharide as a sodium adduct [M + Na]+, whereas the m/z = 587 signal corresponds to an ESI-MS dimer of the pentose disaccharide also as a sodium adduct [2M + Na]+. Crystal Structure of the Catalytic Module of CtXyl5A in Complex with Ligands To understand the structural basis for the biochemical properties of CtXyl5A, the crystal structure of the enzyme with ligands that occupy the substrate binding cleft and the critical −2* subsite were sought. The data presented in Fig. 3A show the structure of the CtXyl5A derivative CtGH5-CtCBM6 in complex with arabinose bound in the −2* pocket. Interestingly, the bound arabinose was in the pyranose conformation rather than in its furanose form found in arabinoxylans. O1 was facing toward the active site −1 subsite, indicative of the bound arabinose being in the right orientation to be linked to the xylan backbone via an α-1,3 linkage. As discussed on below, the axial O4 of the Arap did not interact with the −2* subsite, suggesting that the pocket might be capable of binding a xylose molecule. Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the −2* subsite in its pyranose conformation (Fig. 3B). These crystal structures support the biochemical data presented above showing that the enzyme generated β-1,3-xylobiose from CX, which would require the disaccharide to bind at the −1 and −2* subsites. A third product complex was generated by co-crystallizing the nucleophile inactive mutant CtGH5E279S-CtCBM6 with a WAX-derived oligosaccharide (Fig. 3C). The data revealed a pentasaccharide bound to the enzyme, comprising β-1,4-xylotetraose with an Araf linked α-1,3 to the reducing end xylose. The xylotetraose was positioned in subsites −1 to −4 and the Araf in the −2* pocket. Analysis of the three structures showed that O1, O2, O3, and the endocyclic oxygen occupied identical positions in the Arap, Araf, and Xylp ligands bound in the −2* subsite and thus made identical interactions with the pocket. O1 makes a polar contact with Nδ2 of Asn139, O2 is within hydrogen bonding distance with Oδ1 of Asn139 and the backbone N of Asn135, and O3 interacts with the N of Gly136 and Oϵ2 of Glu68. Although O4 of Arap does not make a direct interaction with the enzyme, O4 and O5 of Xylp and Araf, respectively, form hydrogen bonds with Oϵ1 of Glu68. Finally Tyr92 makes apolar parallel interactions with the pyranose or furanose rings of the three sugars. Representation of the residues involved in the ligands recognition at the −2* subsite. The protein backbone is represented as a cartoon in gray. Interacting residues are represented as stick in blue, and the catalytic residues and the mutated glutamate (into a serine) are in magenta. A, CtGH5-CBM6 in complex with an arabinopyranose. B, CtGH5-CBM6 in complex with a xylopyranose. C, CtGH5E279S-CBM6 in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose). The xylan backbone is shown transparently for more clarity. Densities shown in blue are RefMac maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ. The figure and all other structural figures were made with PyMOL unless otherwise stated. The importance of the interactions between the ligands and the side chains of the residues in the −2* pocket were evaluated by alanine substitution of these amino acids. The mutants E68A, Y92A, and N139A were all inactive (Table 1), demonstrating the importance of the interactions of these residues with the substrate and reinforcing the critical role the −2* subsite plays in the activity of the enzyme. N135A retained wild type activity because the O2 of the sugars interacts with the backbone N of Asn135 and not with the side chain. Because the hydroxyls of Xylp or Araf in the −2* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration. This may explain why the kcat/Km for CtXyl5A against WAX was 2-fold higher than against CX (Table 1). WAX is likely to have a higher concentration of single Araf decorations compared with CX and thus contain more substrate available to the arabinoxylanase. In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92. O3 (O1 of the Araf at the −2* subsite) makes a polar contact with Nδ2 of Asn139; the endocyclic oxygen hydrogens bonds with the OH of Tyr255. The Xylp in the active site makes strong parallel apolar interactions with Phe310. Substrate recognition in the active site is conserved between CtXyl5A and the closest GH5 structural homolog, the endoglucanase BaCel5A (PDB code 1qi2) as noted previously. Comparison of the ligand recognition at the distal negative subsites between CtGH5E279S-CBM6, the cellulase BaCel5A, and the xylanase GH10.\nA–C show CtGH5E279S-CBM6 is in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose). A, Poseview representation highlighting the hydrogen bonding and the hydrophobic interactions that occur in the negative subsites. C, density of the ligand shown in blue is RefMac maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ. D and E display BaCel5A in complex with deoxy-2-fluoro-β-d-cellotrioside (PDB code 1qi2), and F and G show CmXyn10B in complex with a xylotriose (PDB code 1uqy). The ligand are represented as sticks. B, D, and F are surface representations (CtGH5E279S-CBM6 in gray, BaCel5A in cyan, and the xylanase GH10 in light brown). C, E, and G show the protein backbone as a cartoon representation with the interacting residues represented as sticks. The black dashes represent the hydrogen bonds. The capacity of CtXyl5A to act on the highly decorated xylan CX indicates that O3 and possibly O2 of the backbone Xylp units are solvent-exposed. This is consistent with the interaction of the xylotetraose backbone with the enzyme distal to the active site. A surface representation of the enzyme (Fig. 4B) shows that O3 and O2 of xylose units at subsites −2 to −4 are solvent-exposed and are thus available for decoration. Indeed, these pyranose sugars make very weak apolar interactions with the arabinoxylanase. At −2, Xylp makes planar apolar interactions with the Araf bound to the −2* subsite (Fig. 4C). Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69. Comparison of the distal negative subsites of CtXyl5A with BaCel5A and a typical GH10 xylanase (CmXyn10B, PDB code 1uqy) highlights the paucity of interactions between the arabinoxylanase and its substrate out with the active site (Fig. 4). Thus, the cellulase contains three negative subsites and the sugars bound in the −2 and −3 subsites make a total of 9 polar interactions with the enzyme (Fig. 4, D and E). The GH10 xylanase also contains a −2 subsite that, similar to the cellulase, makes numerous interactions with the substrate (Fig. 4, F and G). The Influence of the Modular Architecture of CtXyl5A on Catalytic Activity CtXyl5A, in addition to its catalytic module, contains three CBMs (CtCBM6, CtCBM13, and CtCBM62) and a fibronectin domain (CtFn3). A previous study showed that although the CBM6 bound in an exo-mode to xylo- and cellulooligosaccharides, the primary role of this module was to stabilize the structure of the GH5 catalytic module. To explore the contribution of the other non-catalytic modules to CtXyl5A function, the activity of a series of truncated derivatives of the arabinoxylanase were assessed. The data in Table 1 show that removal of CtCBM62 caused a modest increase in activity against both WAX and CX, whereas deletion of the Fn3 domain had no further impact on catalytic performance. Truncation of CtCBM13, however, caused a 4–5-fold reduction in activity against both substrates. Members of CBM13 have been shown to bind to xylans, mannose, and galactose residues in complex glycans, hinting that the function of CtCBM13 is to increase the proximity of substrate to the catalytic module of CtXyl5A. Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown). It would appear, therefore, that CtCBM13 makes a structural contribution to the function of CtXyl5A. Crystal Structure of CtXyl5A-D To explore further the role of the non-catalytic modules in CtXyl5A the crystal structure of CtXyl5A extending from CtGH5 to CtCBM62 was sought. To obtain a construct that could potentially be crystallized, the protein was generated without the C-terminal dockerin domain because it is known to be unstable and prone to cleavage. Using this construct (CtXyl5A-D) the crystal structure of the arabinoxylanase was determined by molecular replacement to a resolution of 2.64 Å with Rwork and Rfree at 23.7% and 27.8%, respectively. The structure comprises a continuous polypeptide extending from Ala36 to Trp742 displaying four modules GH5-CBM6-CBM13-Fn3. Although there was some electron density for CtCBM62, it was not sufficient to confidently build the module (Fig. 5). Further investigation of the crystal packing revealed a large solvent channel adjacent to the area the CBM62 occupies. We postulate that the reason for the poor electron density is due to the CtCBM62 being mobile compared with the rest of the protein. The structures of CtGH5 and CtCBM6 have been described previously. Surface representation of the tetra-modular arabinoxylanase and zoom view on the CtGH5 loop. The blue module is the CtGH5 catalytic domain, the green module corresponds to the CtCBM6, the yellow module is the CtCBM13, and the salmon module is the fibronectin domain. Surfaces are semitransparent with the protein backbone represented as a cartoon. The CtGH5 loop is stabilized between the CtCBM6 and the CtCBM13 modules. The black dashes represent the hydrogen bonds. The protein backbone is represented as cartoon, and interacting residues are shown as sticks. CtCBM13 extends from Gly567 to Pro648. Typical of CBM13 proteins CtCBM13 displays a β-trefoil fold comprising the canonical pseudo 3-fold symmetry with a 3-fold repeating unit of 40–50 amino acid residues characteristic of the Ricin superfamily. Each repeat contains two pairs of antiparallel β-strands. A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v). The Fn3 module displays a typical β-sandwich fold with the two sheets comprising, primarily, three antiparallel strands in the order β1-β2-β5 in β-sheet 1 and β4-β3-β6 in β-sheet 2. Although β-sheet 2 presents a cleft-like topology, typical of endo-binding CBMs, the surface lacks aromatic residues that play a key role in ligand recognition, and in the context of the full-length enzyme, the cleft abuts into CtCBM13 and thus would not be able to accommodate an extended polysaccharide chain (see below). In the structure of CtXyl5A-D, the four modules form a three-leaf clover-like structure (Fig. 5). Between the interfaces of CtGH5-CBM6-CBM13 there are a number of interactions that maintain the modules in a fixed position relative to each other. The interaction of CtGH5 and CtCBM6, which buries a substantial apolar solvent-exposed surface of the two modules, has been described previously. The polar interactions between these two modules comprise 14 hydrogen bonds and 5 salt bridges. The apolar and polar interactions between these two modules likely explaining why they do not fold independently compared with other glycoside hydrolases that contain CBMs. CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of ∼450, 350, and 500 Å2, respectively, to form a compact heterotetramer. With respect to the CtCBM6-CBM13 interface, the linker (SPISTGTIP) between the two modules, extending from Ser514 to Pro522, adopts a fixed conformation. Such sequences are normally extremely flexible; however, the two Ile residues make extensive apolar contacts within the linker and with the two CBMs, leading to conformational stabilization. The interactions between CtGH5 and the two CBMs, which are mediated by the tip of the loop between β-7 and α-7 (loop 7) of CtGH5, not only stabilize the trimodular clover-like structure but also make a contribution to catalytic function. Central to the interactions between the three modules is Trp285, which is intercalated between the two CBMs. The Nϵ of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5). Indeed, loop 7 is completely disordered in the truncated derivative of CtXyl5A comprising CtGH5 and CtCBM6, demonstrating that the interactions with CtCBM13 stabilize the conformation of this loop. Although the tip of loop 7 does not directly contribute to the topology of the active site, it is only ∼12 Å from the catalytic nucleophile Glu279. Thus, any perturbation of the loop (through the removal of CtCBM13) is likely to influence the electrostatic and apolar environment of the catalytic apparatus, which could explain the reduction in activity associated with the deletion of CtCBM13. Similar to the interactions between CtCBM6 and CtCBM13, there are extensive hydrophobic interactions between CtCBM13 and CtFn3, resulting in very little flexibility between these modules. As stated above, the absence of CtCBM62 in the structure suggests that the module can adopt multiple positions with respect to the rest of the protein. The CtCBM62, by binding to its ligands (d-Galp and l-Arap) in plant cell walls, may be able to recruit the enzyme onto its target substrate. Xylans are not generally thought to contain such sugars. d-Galp, however, has been detected in xylans in the outer layer of cereal grains and in eucalyptus trees, which are substrates used by CtXyl5A. Thus, CtCBM62 may direct the enzyme to particularly complex xylans containing d-Galp at the non-reducing termini of the side chains, consistent with the open substrate binding cleft of the arabinoxylanase that is optimized to bind highly decorated forms of the hemicellulose. In general CBMs have little influence on enzyme activity against soluble substrates but have a significant impact on glycans within plant cell walls. Thus, the role of CBM62 will likely only be evident against insoluble composite substrates. Exploring GH5 Subfamily 34 CtXyl5A is a member of a seven-protein subfamily of GH5, GH5_34. Four of these proteins are distinct, whereas the other three members are essentially identical (derived from different strains of C. thermocellum). To investigate further the substrate specificity within this subfamily, recombinant forms of three members of GH5_34 that were distinct from CtXyl5A were generated. AcGH5 has a similar molecular architecture to CtXyl5A with the exception of an additional carbohydrate esterase family 6 module at the C terminus (Fig. 1). The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function. The Verrucomicobiae enzyme also has an N-terminal GH43 subfamily 10 (GH43_10) catalytic module. The fungal GH5_34, GpGH5, unlike the two bacterial homologs, comprises a single GH5 catalytic module lacking all of the other accessory modules (Fig. 1). GpGh5 is particularly interesting as Gonapodya prolifera is the only fungus of the several hundred fungal genomes that encodes a GH5_34 enzyme. In fact there are four potential GH5_34 sequences in the G. prolifera genome, all of which show high sequence homology to Clostridium GH5_34 sequences. G. prolifera and Clostridium occupy similar environments, suggesting that the GpGH5_34 gene was acquired from a Clostridium species, which was followed by duplication of the gene in the fungal genome. The sequence identity of the GH5_34 catalytic modules with CtXyl5A ranged from 55 to 80% (supplemental Fig. S1). All the GH5_34 enzymes were active on the arabinoxylans RAX, WAX, and CX but displayed no activity on BX (Table 1 and Fig. 6) and are thus defined as arabinoxylanases. The limit products generated by CtXyl5A, AcGH5, and GpGH5 comprised a range of oligosaccharides with some high molecular weight material. The oligosaccharides with low degrees of polymerization were absent in the VbGH5 reaction products. However, the enzyme generated a large amount of arabinose, which was not produced by the other arabinoxylanases. Given that GH43_10 is predominantly an arabinofuranosidase subfamily of GH43, the arabinose generated by VbGH5 is likely mediated by the N-terminal catalytic module (see below). Kinetic analysis showed that AcGH5 displayed similar activity to CtXyl5A against both WAX and RAX and was 2-fold less active against CX. When initially measuring the activity of wild type VbGH5 against the different substrates, no clear data could be obtained, regardless of the concentration of enzyme used the reaction appeared to cease after a few minutes. We hypothesized that the N-terminal GH43_10 rapidly removed single arabinose decorations from the arabinoxylans depleting the substrate available to the arabinoxylanase, explaining why this activity was short lived. To test this hypothesis, the conserved catalytic base (Asp45) of the GH43_10 module of VbGH5 was substituted with alanine, which is predicted to inactivate this catalytic module. The D45A mutant did not produce arabinose consistent with the arabinofuranosidase activity displayed by the GH43_10 module in the wild type enzyme (Fig. 6). The kinetics of the GH5_34 arabinoxylanase catalytic module was now measurable, and activities were determined to be between ∼6- and 10-fold lower than that of CtXyl5A. Interestingly, the fungal arabinoxylanase displays the highest activities against WAX and RAX, ∼4- and 6-fold higher, respectively, than CtXyl5A; however, there is very little difference in the activity between the eukaryotic and prokaryotic enzymes against CX. Attempts to express individual modules of a variety of truncations of AcGH5 and VbGH5 were unsuccessful. This may indicate that the individual modules can only fold correctly when incorporated into the full-length enzyme, demonstrating the importance of intermodule interactions to maintain the structural integrity of these enzymes. Products profile generated of GH5_34 enzymes. The enzymes at 1 μm were incubated with the four different xylans at 1% in 50 mm sodium phosphate buffer for 16 h at 37 °C (GpGH5, VbGH5, and AcGH5) or 60 °C. The limit products were separated by TLC. The xylooligosaccharide standards (X) are indicated by their degrees of polymerization. Discussion A characteristic feature of enzymes that attack the plant cell wall is their complex molecular architecture. The CBMs in these enzymes generally play a role in substrate targeting and are appended to the catalytic modules through flexible linker sequences. CtXyl5A provides a rare visualization of the structure of multiple modules within a single enzyme. The central feature of these data is the structural role played by two of the CBMs, CtCBM6 and CtCBM13, in maintaining the active conformation of the catalytic module, CtGH5. The crystallographic data described here are supported by biochemical data showing either that these two modules do not bind to glycans (CtCBM13) or that the recognition of the non-reducing end of xylan or cellulose chains (CtCBM6) is unlikely to be biologically significant. It should be emphasized, however, that glycan binding and substrate targeting may only be evident in the full-length enzyme acting on highly complex structures such as the plant cell wall, as observed recently by a CBM46 module in the Bacillus xyloglucanase/mixed linked glucanase BhCel5B. CtXyl5A is a member of GH5 that contains 6644 members. These proteins have been subdivided into 51 subfamilies based on sequence similarity. CtXyl5A is a member of subfamily GH5_34. Here we have explored the substrate specificity of the other members of this subfamily. Despite differences in sequence identity all of the homologs were shown to be arabinoxylanases. Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the −2* subsite, which are 1,3-linked to the xylose positioned in the active site. The presence of a CBM62 in CtXyl5A and AcGH5 suggests that these enzymes target highly complex xylans that contain d-galactose in their side chains. The absence of a “non-structural” CBM in GpGH5 may indicate that this arabinoxylanase is designed to target simpler arabinoxylans present in the endosperm of cereals. Although the characterization of all members of GH5_34 suggests that this subfamily is monospecific, differences in specificity are observed in other subfamilies of GHs including GH43 and GH5. Thus, as new members of GH5_34 are identified from genomic sequence data and subsequently characterized, the specificity of this family may require reinterpretation. An intriguing feature of VbGH5 is that the limited products generated by this enzymes are much larger than those produced by the other arabinoxylanases. This suggests that although arabinose decorations contribute to enzyme specificity (VbGH5 is not active on xylans lacking arabinose side chains), the enzyme requires other specificity determinants that occur less frequently in arabinoxylans. This has some resonance with a recently described GH98 xylanase that also exploits specificity determinants that occur infrequently and are only evident in highly complex xylans (e.g. CX). To conclude, this study provides the molecular basis for the specificity displayed by arabinoxylanases. Substrate specificity is dominated by the pocket that binds single arabinose or xylose side chains. The open xylan binding cleft explains why the enzyme is able to attack highly decorated forms of the hemicellulose. It is also evident that appending additional catalytic modules and CBMs onto the core components of these enzymes generates bespoke arabinoxylanases with activities optimized for specific functions. The specificities of the arabinoxylanases described here are distinct from the classical endo-xylanases and thus have the potential to contribute to the toolbox of biocatalysts required by industries that exploit the plant cell wall as a sustainable substrate. Experimental Procedures Cloning, Expression, and Purification of Components of CtXyl5A All recombinant forms of CtXyl5A used in this study were expressed in the cytoplasm of Escherichia coli because they lacked a signal peptide. DNA encoding CtGH5-CtCBM6 and CtXyl5A-D (CtXyl5A lacking the C-terminal dockerin domain (CtGH5-CtCBM6-CtCBM13-Fn3-CtCBM62)) were described previously. DNA encoding CtGH5-CtCBM6-CtCBM13-Fn3 and CtGH5-CtCBM6-CtCBM13 and mature Acetivibrio cellulolyticus GH5 (AcGH5) were amplified by PCR using plasmid encoding the full-length C. thermocellum arabinoxylanase or A. cellulolyticus genomic DNA as the respective templates. DNA encoding the G. prolifera GH5 (GpGH5) and V. bacterium GH5 (VbGH5) were initially generated by GeneArt® gene synthesis (Thermo Fisher Scientific). DNA encoding VbGH5 lacking the C-terminal cell surface anchoring residues was also amplified by PCR using the synthesized nucleic acid as the template. All the primers used in the PCRs required restriction sites and plasmids used are listed inj supplemental Table S1. All constructs were cloned such that the encoded proteins contain a C-terminal His6 tag. Site-directed mutagenesis was carried out using the PCR-based QuikChange method (Stratagene) deploying the primers listed in supplemental Table S1. To express the recombinant proteins, E. coli strain BL21(DE3), harboring appropriate recombinant plasmids, was cultured to mid-exponential phase in Luria broth at 37 °C. Isopropyl β-d-galactopyranoside at 1 mm was then added to induce recombinant gene expression, and the culture incubated for a further 18 h at 16 °C. The recombinant proteins were purified to \u003e90% electrophoretic purity by immobilized metal ion affinity chromatography using TalonTM (Clontech), cobalt-based matrix, and elution with 100 mm imidazole, as described previously. When preparing the selenomethionine derivative of CtXyl5A-D for crystallography, the proteins were expressed in E. coli B834 (DE3), a methionine auxotroph, cultured in medium comprising 1 liter of SelenoMet Medium BaseTM, 50 ml of SelenoMetTM nutrient mix (Molecular Dimensions), and 4 ml of a 10 mg/ml solution of l-selenomethionine. Recombinant gene expression and protein purification were as described above except that all purification buffers were supplemented with 10 mm β-mercaptoethanol. Enzyme Assays CtXyl5A-D and its derivatives were assayed for enzyme activity using the method of Miller to detect the release of reducing sugar. The standard assay was carried out in 50 mm sodium phosphate buffer, pH 7.0, containing 0.1 mg/ml BSA and at substrate concentrations ranging from 1 to 6 mg/ml. The pH and temperature optima were previously determined to be 7 and 60 °C, respectively, for the CtXyl5A-D and its derivatives. The optimum temperature for the other enzymes was found to be 37 °C, and pH optima of 5, 7, and 4 were determined for AcGH5, GpGH5 and VbGH5, respectively. All enzymes were assayed for activity at their individual temperature and pH optimum. A FLUOstar Omega microplate reader (BMG Labtech) was used to measure activity in 96-well plates. Overnight assays to assess end point products were carried out with 6 mg/ml substrate and 1 μm enzyme concentrations. The identification of potential reaction products was also assessed by HPAEC or TLC using methodology described previously. Oligosaccharide Analysis Approximately 5 g of CX or WAX were digested to completion (no further increase in reducing sugar and change in the HPAEC product profile) with 3 μm of CtXyl5A-D at 60 °C for 48 h. The oligosaccharide products were purified by size exclusion chromatography using a Bio-Gel P2 column as described previously. The structures of the oligosaccharides were analyzed by positive ion-mode infusion/offline electrospray ionization (ESI)-MS following either dilution with 30% acetonitrile or via desalting as described previously  Crystallography Purified SeMet CtXyl5A-D was concentrated and stored in 5 mm DTT, 2 mm CaCl2. Crystals of seleno-l-methionine-containing protein were obtained by hanging drop vapor diffusion in 40% (v/v) 2-methyl-2,4-pentandiol. The data were collected on Beamlines ID14-1 and ID14-4 at the European Synchrotron Radiation Facility (Grenoble, France) to a resolution of 2.64 Å. The data were processed using the programs iMOSFLM and SCALA from the CCP4 suite (Collaborative Computational Project, Number 4, 1994). The crystal belongs to the orthorhombic space group (P21212). The structure was solved by molecular replacement using independently solved structures of some of the modules of the CtXyl5A: CtGH5-CBM6 (PDB code 2y8k), Fn3 (PDB code 3mpc), and CtCBM62 (PDB codes 2y8m, 2yfz, and 2y9s) using PHASER. The CtCBM13 domain was built de novo. BUCCANEER and PHENIX were initially used for auto building. The structure was completed by iterative cycles of manual rebuilding in COOT in tandem with refinement with RefMac5. The final values for Rwork and Rfree) were 23.73 and 27.80%) using TLS and restraining refinement to amino acid residues 36–373 representing the CtGH5 module, 374–516 for the CtCBM6, 517–652 for CtCBM13, and 653–742 for CtFn3. Stereochemistry was assessed with COOT and PDBSUM (with 677 residues (96%) in preferred, 22 in allowed regions (3%), and 6 outliers (1%) in the Ramachandran plot). To obtain structures of CtGH5-CBM6 in complex with ligand the protein was crystallized using the sitting drop vapor phase diffusion method with an equal volume (100 nl) of protein and reservoir solution (unless otherwise stated), using the robotic nanodrop dispensing systems (mosquitoR LCP; TTPLabTech). Crystals of the protein (10 mg/ml) co-crystallized with arabinose (300 mm) were obtained in 1 m ammonium sulfate, 0.1 m Bis-Tris, pH 5.5, and 1% PEG 3350. Crystals with xylose (300 mm) grew in 100 mm sodium/potassium phosphate, 100 mm MES, pH 6.5, and 2 m sodium chloride. To obtain crystals of the arabinoxylanase in complex with an oligosaccharide, the nucleophile mutant E279S was used and mixed with a range of arabinoxylooligosaccharides that was generated by digestion of WAX with CtGH5-CBM6 (see above) and thereafter by 100 nm of the Cellvibrio japonicus GH43 exo-1,4-β-xylosidase. Only the inclusion of the largest purified oligosaccharide generated crystals of the arabinoxylanase. Crystals of CtGH5E279S-CBM6 were obtained by mixing an equal volume (100 nl) of the protein (11 mg/ml)/oligosaccharide (10 mm) solution and mother liquor solution consisting of 100 mm Tris-Bicine, pH 8.5, 12.5% (w/v) polyethylene glycol with an average molecular mass of 1,000 Da, 12.5% (w/v) polyethylene glycol with an average molecular mass of 3,350 Da and 12.5% (R,S)-2-methyl-2,4-pentanediol (racemic). Crystallographic data were collected on Beamlines IO2, IO4-1, and I24 at the DIAMOND Light Source (Harwell, UK). The data were processed using XDS The crystal structures were solved by molecular replacement using MolRep with CtGH5-CtCBM6 (PDB code 5AK1) as the search model. The refinement was done in RefMac5, and COOT was used for model (re)building. The final model were validated using Molprobity. The data collection and refinement statistics are listed in Table 2. Data collection and refinement statistics The values in parentheses are for highest resolution shell. \tCtXyl5A-D\tGH5-CBM6-Arap\tGH5-CBM6-Xylp\tGH5-CBM6- (Araf-Xylp4)\t \tData collection\t\t\t\t\t \t    Source\tESRF-ID14-1\tDiamond I04–1\tDiamond I24\tDiamond I02\t \t    Wavelength (Å)\t0.9334\t0.9173\t0.9772\t0.9791\t \t    Space group\tP21212\tP212121\tP212121\tP212121\t \t    Cell dimensions\t\t\t\t\t \t        a, b, c (Å)\t147.4, 191.7, 50.7\t67.1, 72.4, 109.1\t67.9, 72.5, 109.5\t76.3, 123.2, 125.4\t \t        α, β, γ (°)\t90, 90, 90\t90, 90, 90\t90, 90, 90\t90, 90, 90\t \t    No. of measured reflections\t244,475 (29,324)\t224,842 (11,281)\t152,004 (4,996)\t463,237 (23,068)\t \t    No. of independent reflections\t42246 (5,920)\t63,523 (3,175)\t42,716 (2,334)\t140,288 (6,879)\t \t    Resolution (Å)\t50.70–2.64 (2.78–2.64)\t44.85–1.65 (1.68–1.65)\t45.16–1.90 (1.94–1.90)\t48.43–1.65 (1.68–1.65)\t \t    Rmerge (%)\t16.5 (69.5)\t6.7 (65.1)\t2.8 (8.4)\t5.7 (74.9)\t \t    CC1/2\t0.985 (0.478)\t0.998 (0.594)\t0.999 (0.982)\t0.998 (0.484)\t \t    I/σI\t8.0 (2.0)\t13 (1.6)\t26.6 (8.0)\t11.2 (1.6)\t \t    Completeness (%)\t98.5 (96.4)\t98.5 (99.4)\t98.6 (85.0)\t98.8 (99.4)\t \t    Redundancy\t5.8 (5.0)\t3.5 (3.6)\t3.6 (2.1)\t3.3 (3.4)\t \t\t \tRefinement\t\t\t\t\t \t    Rwork/Rfree\t23.7/27.8\t12.2/17.0\t12.9/16.1\t14.5/19.9\t \t    No. atoms\t\t\t\t\t \t        Protein\t5446\t3790\t3729\t7333\t \t        Ligand\t19\t20\t20\t92\t \t        Water\t227\t579\t601\t923\t \t    B-factors\t\t\t\t\t \t        Protein\t41.6\t17.8\t15.8\t21.0\t \t        Ligand\t65.0\t19.4\t24.2\t39.5\t \t        Water\t35.4\t38.5\t32.2\t37.6\t \t    R.m.s deviations\t\t\t\t\t \t        Bond lengths (Å)\t0.008\t0.015\t0.012\t0.012\t \t        Bond angles (°)\t1.233\t1.502\t1.624\t1.554\t \t    Protein Data Bank code\t5G56\t5LA0\t5LA1\t2LA2\t \t Author Contributions A. L. obtained crystals of the GH5-CBM6 complex. L. I. C. analyzed the biochemistry of GH5 subfamilies. J. L. A. B. obtained crystals of CtXyl5A-D. A. J. analyzed the biochemistry of GH5-CBM6 and obtained crystals of GH5-CBM6. A. R. analyzed the biochemistry of GH5-CBM6 products. J. G. performed mass spectrometry. M. P. Y. provided the substrate. B. H. performed analysis of GH5 sequences. C. M. G. A. F. designed the experiments. H. J. G. designed the experiments, analyzed data, and contributed to writing the paper. S. N. solved the structure of CtXyl5A-D and contributed to writing the paper. A. B. used crystallography to solve GH5-CBM6 structures. F. C. analyzed the biochemistry of GH5-CBM6 mutants, obtained crystals of GH5-CBM6, and contributed to writing the paper. Supplementary Material This work was supported in part by European Research Council Grant 322820 (to H. J. G. and B. H.), Biotechnology and Biological Research Council Grants BB/K020358/1 and BB/K001949/1 (to H. J. G.), Wellcome Trust Grant RES/0120/7613 (to H. J. G.), Agence Nationale de la Recherche Grant ANR 12-BIME-0006-01 (to B. H.), and Fundação para a Ciência e Tecnologia Grants PTDC/BIAPRO/103980/2008 and PTDC/BIAMIC/5947/2014 (to C. M. G. A. F.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains supplemental Table S1 and Fig. S1. GH glycoside hydrolase CtXyl5A C. thermocellum arabinoxylanase CBM non-catalytic carbohydrate binding module Fn fibronectin WAX wheat arabinoxylan RAX rye arabinoxylan CX corn bran xylan HPAEC high performance anion exchange chromatography PDB Protein Data Bank BX birchwood xylan ESI electrospray ionization. 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Tracing protein chains Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard Coot: model-building tools for molecular graphics REFMAC5 for the refinement of macromolecular crystal structures PDBsum new things The structure and function of an arabinan-specific α-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases XDS MolRep: an automated program for molecular 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diff --git a/annotated_BioC_JSON/PMC5173035_ann.json b/annotated_BioC_JSON/PMC5173035_ann.json
new file mode 100644
index 0000000000000000000000000000000000000000..0f8a204003c2884907e9a79ae2bcfa9ebc24af53
--- /dev/null
+++ b/annotated_BioC_JSON/PMC5173035_ann.json
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+[{"sourceid":"5173035","sourcedb":"","project":"","target":"","text":"Biochemical and structural characterization of a DNA N6-adenine methyltransferase from Helicobacter pylori  DNA N6-methyladenine modification plays an important role in regulating a variety of biological functions in bacteria. However, the mechanism of sequence-specific recognition in N6-methyladenine modification remains elusive. M1.HpyAVI, a DNA N6-adenine methyltransferase from Helicobacter pylori, shows more promiscuous substrate specificity than other enzymes. Here, we present the crystal structures of cofactor-free and AdoMet-bound structures of this enzyme, which were determined at resolutions of 3.0 Å and 3.1 Å, respectively. The core structure of M1.HpyAVI resembles the canonical AdoMet-dependent MTase fold, while the putative DNA binding regions considerably differ from those of the other MTases, which may account for the substrate promiscuity of this enzyme. Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity. Taken together, our data revealed the structural basis underlying DNA N6-adenine methyltransferase substrate promiscuity. INTRODUCTION DNA methylation is a common form of modification on nucleic acids occurring in both prokaryotes and eukaryotes. Such a modification creates a signature motif recognized by DNA-interacting proteins and functions as a mechanism to regulate gene expression. DNA methylation is mediated by DNA methyltransferases (MTases), which catalyze the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to a given position of a particular DNA base within a specific DNA sequence. Three classes of DNA MTases have been identified to transfer a methyl group to different positions of DNA bases. C5-cytosine MTases, for example, methylate C5 of cytosine (m5C). In eukaryotes, m5C plays an important role in gene expression, chromatin organization, genome maintenance and parental imprinting, and is involved in a variety of human diseases including cancer. By contrast, the functions of the prokaryotic DNA cytosine MTase remain unknown. N4-cytosine MTases, which are frequently present in thermophilic or mesophilic bacteria, transfer a methyl group to the exocyclic amino group of cytosine (4mC). N4 methylation seems to be primarily a component of bacterial immune system against invasion by foreign DNA, such as conjugative plasmids and bacteriophages. The third group, N6-adenine MTases methylate the exocyclic amino groups of adenine (6mA), which exists in prokaryotes as a signal for genome defense, DNA replication and repair, regulation of gene expression, control of transposition and host-pathogen interactions. Recent studies utilizing new sequencing approaches have showed the existence of 6mA in several eukaryotic species. DNA 6mA modification is associated with important biological processes including nucleosome distribution close to the transcription start sites in Chlamydomonas, carrying heritable epigenetic information in C.elegans or controlling development of Drosophila. All the three types of methylation exist in prokaryotes, and most DNA MTases are components of the restriction-modification (R-M) systems. The R-M systems are composed of two enzymes displaying opposing activities. “R” stands for a restriction endonuclease cleaving specific DNA sequences, while “M” symbolizes a modification methyltransferase rendering these sequences resistant to cleavage. The cooperation of these two enzymes provides a defensive mechanism to protect bacteria from infection by bacteriophages. The R-M systems are classified into three types based on specific structural features, position of DNA cleavage and cofactor requirements. In types I and III, the DNA adenine or cytosine methyltransferase is part of a multi-subunit enzyme that catalyzes both restriction and modification. By contrast, two separate enzymes exist in type II systems, where a restriction endonuclease and a DNA adenine or cytosine methyltransferase recognize the same targets. To date, a number of bacterial DNA MTases have been structurally characterized, covering enzymes from all the three classes. All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base. Conserved amino acid motifs have been identified from reported structures, including ten motifs (I-X) in cytosine MTases and nine motifs (I-VIII and X) in adenine MTases, all of which are arranged in an almost constant order. According to the linear arrangement of three conserved domains, exocyclic amino MTases are subdivided into six groups (namely α, β, γ, ζ, δ and ε). N6-adenine and N4-cytosine MTases, in particular, are closely related by sharing common structural features. Despite the considerable similarity among bacterial MTases, some differences were observed among the enzymes from various species. For example, the structural regions of MTases beyond the catalytic domain are rather variable, such as the C-terminal domain of M.TaqI, the extended arm of M.MboIIA and M.RsrI, the helix bundle of EcoDam, and so on. DNA methylation is thought to influence bacterial virulence. DNA adenine methyltransferase has been shown to play a crucial role in colonization of deep tissue sites in Salmonella typhimurium and Aeromonas hydrophila. Importantly, DNA adenine methylation is a global regulator of genes expressed during infection and inhibitors of DNA adenine methylation are likely to have a broad antimicrobial action. Dam was considered a promising target for antimicrobial drug development. Helicobacter pylori is a Gram-negative bacterium that persistently colonizes in human stomach worldwide. It is a major pathogen of gastritis and peptic ulcer diseases as well as a cancer-causing factor for gastric cancer. H. pylori is involved in 90% of all gastric malignancies, infecting nearly 50% of the world's population and is the most crucial etiologic agent for gastric adenocarcinoma. H. pylori strains possess a few R-M systems like other bacteria to function as defensive systems. H. pylori 26695, for example, has 23 R-M systems. Methyltransferases were suggested to be involved in H. pylori pathogenicity. M1.HpyAVI is a DNA adenine MTase that belongs to the type II R-M system. This system contains two DNA MTases named M1.HpyAVI and M2.HpyAVI, and a putative restriction enzyme. M1.HpyAVI encoded by ORF hp0050 is an N6-adenine methyltransferase belonging to the β-class MTase. It has been reported recently that this enzyme recognizes the sequence of 5′-GAGG-3′, 5′-GGAG-3′ or 5′-GAAG-3′ and methylates adenines in these sequences. Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5′-GAAG-3′ seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5′-GAGG-3′. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.Here, we report the crystal structure of M1.HpyAVI from H. pylori 26695, which is the first determined N6-adenine MTase structure in H. pylori. The structure reveals a similar architecture as the canonical fold of homologous proteins, but displays several differences in the loop regions and TRD. Based on structural and biochemical analyses, we then identified two conserved amino acids, D29 at the catalytic site and E216 close to the C-terminus, as crucial residues for cofactor binding and methyltransferase activity of M1.HpyAVI. In addition, a non-conserved amino acid, P41, seems to play a key role in substrate recognition. RESULTS Overall structure Recombinant full-length M1.HpyAVI was produced as a soluble protein in Escherichia coli, but was quite unstable and tended to aggregate in low salt environment. The protein, however, remained fully soluble in a buffer containing higher concentration of sodium chloride (\u003e300 mM), which prompted that M1.HpyAVI is likely a halophilic protein. The cofactor-free and AdoMet-bound proteins were crystallized at different conditions. Both structures were determined by means of molecular replacement, and refined to 3.0 Å and 3.1 Å, respectively. Statistics of X-ray data collection and structure refinement were summarized in Table 1. Data collection and structure refinement statistics of M1.HpyAVI \tM1.HpyAVI\tM1.HpyAVI-AdoMet complex\t \tData collection\t\t\t \tWavelength (Å)\t1.0000\t0.97772\t \tSpace group\tP43212\tP65\t \tUnit-cell parameters (Å, ˚)\ta = b = 69.73, c = 532.75α = β = γ = 90\ta = b = 135.60, c = 265.15α = β = 90, γ = 120\t \tResolution range (Å) a\t49.09-3.00 (3.09-3.00)\t48.91-3.10 (3.18-3.10)\t \tUnique reflections a\t27243\t49833\t \tMultiplicity a\t3.7 (3.8)\t5.6 (4.0)\t \tCompleteness (%)a\t98.7 (98.9)\t99.7 (97.8)\t \tMean I/δ (I) a\t12.1 (3.4)\t14.0 (1.9)\t \tSolvent content (%)\t58.67\t61.96\t \tRmergea\t0.073 (0.378)\t0.106 (0.769)\t \tStructure refinement\t\t\t \tRwork\t0.251\t0.221\t \tRfree\t0.308\t0.276\t \tR.m.s.d., bond lengths (Å)\t0.007\t0.007\t \tR.m.s.d., bond angles (˚)\t1.408\t1.651\t \tRamachandran plot\t\t\t \tFavoured region (%)\t89.44\t91.44\t \tAllowed region (%)\t9.58\t7.11\t \tOutliers (%)\t0.99\t1.45\t \t Values in parentheses are statistics of the highest resolution shell. Four and eight protein monomers resided in the asymmetric units of the two crystal structures. Some amino acids, particularly those within two loops (residues 32-61 and 152-172) in both structures, were poorly defined in electron density and had to be omitted from the refined models. Details of invisible amino acids are given in Table S1. The two structures are very similar to each other (Figure 1) and could be well overlaid with an RMSD of 0.76 Å on 191 Cα atoms. The overall architecture of M1.HpyAVI revealed in these structures resembles the AdoMet-dependent MTase fold in which a twisted seven-stranded β-sheet flanked by six α-helices forms the structural core. Like the reported structures of the larger domain of MTases, three helices (αA, αB and αZ) are located at one face of the central β-sheet, while the other three αD, αE and αC sit at the other side. All these conserved structural motifs form a typical α/β Rossmann fold. The catalytic motif DPPY lies in a loop connecting αD and β4, and the cofactor AdoMet binds in a neighboring cavity. The loop (residues 136-166) located between β7 and αZ corresponds to a highly diverse region in other MTases that is involved in target DNA recognition. The hairpin loop (residues 101-133) bridging β6 and β7, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII. The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA. This loop, however, was well ordered in an M.TaqI-DNA complex structure and was shown to play a crucial role in DNA methylation by contacting the flipping adenine and recognizing specific DNA sequence. Overall structure of M1.HpyAVI A. Free form B. AdoMet-bound form. Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY. Rainbow coloring from blue through green to red indicates the N- to C-terminal position of the residues in the model. The α-helices and β-strands are labelled and numbered according to the commonly numbering rule for the known MTases. The AdoMet molecule is shown in green. Dimeric state of M1.HpyAVI in crystal and solution Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state. Increasing number of dimeric DNA MTases, however, has been identified from later studies. For instance, M.DpnII, M.RsrI, M.KpnI, and M.MboIIA have been found as dimers in solution. In addition, several MTases including M.MboIIA, M.RsrI and TTH0409 form tightly associated dimers in crystal structures. Nonetheless, some DNA MTases such as M.CcrMI and the Bacillus amyloliquefaciens MTase dissociate from dimer into monomer upon DNA-binding. According to the arrangement of the three conserved domains, M1.HpyAVI belongs to the β-subgroup, in which a conserved motif NXXTX9−11AXRXFSXXHX4WX6−9 YXFXLX3RX9−26NPX1−6NVWX29−34A has been identified at the dimerization interface in crystal structures. Most of conserved amino acids within that motif are present in the sequence of M1.HpyAVI (Figure 2A), implying dimerization of this protein. In agreement, a dimer of M1.HpyAVI was observed in our crystal structures with the two monomers related by a two-fold axis (Figure 2B and 2C). An area of ~1900 Å2 was buried at the dimeric interface, taking up ca 17% of the total area. The dimeric architecture was greatly stabilized by hydrogen bonds and salt bridges formed among residues R86, D93 and E96. In addition, comparison of the dimer structure of M1.HpyAVI with some other β-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3). M1.HpyAVI exists as dimer in crystal and solution A. A conserved interface area of β-class MTases is defined in M1.HpyAVI. Residues that involved are signed in red color; Dimerization of free-form M1.HpyAVI B. and cofactor-bound M1.HpyAVI C. The two monomers are marked in green and blue, AdoMet molecules are marked in magenta. D. Gel-filtration analysis revealed that M1.HpyAVI exist as a dimer in solution. FPLC system coupled to a Superdex 75 10/300 column. Elution profiles at 280 nm (blue) and 260 nm (red) are: different concentration (0.05, 0.1, 0.2, 0.5 mg/ml) of M1.HpyAVI protein. To probe the oligomeric form of M1.HpyAVI in solution, different concentrations of purified enzyme was loaded onto a Superdex 75 10/300 column. The protein was eluted at ~10 ml regardless of the protein concentrations, corresponding to a dimeric molecular mass of 54 kDa (Figure 2D). Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other β-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution. This variance might be caused by an addition of 100 mM arginine before cell lysis to keep protein solubility and also by later replacement of arginine with 30% glycerol by dialysis. These treatments probably changed protein conformation somehow and also the oligomeric state. Structure comparisons As a β-class N6 adenine MTase, the M1.HpyAVI structure displayed a good similarity with M.MboIIA (PDB ID 1G60) and M.RsrI (PDB ID 1NW7), which are falling into the same subgroup. Superimposition of M1.HpyAVI onto them gave RMSDs of 1.63 Å and 1.9 Å on 168 and 190 Cα atoms, respectively. The most striking structural difference was found to locate on the TRD region (residues 133-163 in M1.HpyAVI) (Figure 3A–3C), where the secondary structures vary among these structures. By comparison with the other two enzymes that possess protruding arms containing several α-helices and/or β-strands, the TRD of M1.HpyAVI is much shorter in length (Figure S1), wrapping more closely around the structural core and lacking apparent secondary structures. Given the proposed role of the TRD for DNA interaction at the major groove, some differences of DNA recognition mode can be expected. Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible. Structural comparisons between M1.HpyAVI and other DNA MTases A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange. N6-adenine and N4-cytosine MTases, in particular, are closely related by sharing common structural features. Structural comparison between M1.HpyAVI and a putative β-class N4 cytosine MTase named TTHA0409 (PDB ID 2ZIF) showed a good similarity as well, giving an RMSD of 1.73 Å on 164 Cα atoms (Figure 3D). Exactly like the above comparison, the most significant difference exists in the TRD, where the structures vary in terms of length and presence of α-helices (Figure S1). M1.HpyAVI displayed a considerable structural dissimilarity in comparison with N6-adenine MTases from other subgroups including the α-class DpnM (PDB ID 2DPM) and the γ-class M.TaqI (PDB ID 2ADM). Both comparisons gave RMSDs above 3.0 Å (Figure 3E and 3F). These two enzymes lack a counterpart loop present in the TRD of M1.HpyAVI, but instead rely on an extra domain for DNA binding and sequence recognition. Collectively, M1.HpyAVI possesses a long disordered TRD, which is in sharp contrast to the secondary structure-rich TRD in other β-class N6 adenine or N4 cytosine MTases or the extra DNA binding domain present in DNA MTases from other subgroups. This striking difference may be a significant determinant of the wider substrate spectrum of this H. pylori enzyme. AdoMet-binding pocket The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases. A hydrogen bond between D29 in the catalytic motif DPPY and the amino group of bound AdoMet is preserved as other MTase structures. Residues D8 and A9 from hydrogen-bonds with N6 and N1 of the purine ring, respectively, and E216 also locates at hydrogen bonding distance with O2′ and O3′ of the ribose. In addition, H168, T200 and S198 contact the terminal carboxyl of AdoMet. Superposition of M1.HpyAVI with the five structures shown in Figure 3 reveals that the orientation of cofactor is rather conserved except for M.TaqI (Figure 4B). The different conformation of the bound cofactor observed in M.TaqI might be attributable to the absence of corresponding residues of the conserved AdoMet-binding motif FXGXG in that structure. Structural and biochemical analyses define two conserved residues D29 and E216 to be the key sites for AdoMet binding A. The cofactor-binding cavity of M1.HpyAVI. Residues (yellow) that form direct hydrogen bonds with AdoMet (green) are indicated, distance of the hydrogen bond is marked. B. Superposition of AdoMet in the structures of M1.HpyAVI (green), DpnM (yellow) and M.TaqI (orange). The AdoMet terminal carboxyl of M.TaqI reveals different orientations. C. Cofactor binding affinity of wt-/mutants M1.HpyAVI proteins analyzed by microscale thermophoresis (MST). The binding affinity was determined between fluorescently labelled M1.HpyAVI protein and unlabeled AdoMet. The bound fraction is shown on the y-axis against the protein concentration. AdoMet (15 nM to 1 mM) was titrated into a fixed concentration of M1.HpyAVI wt/mutant proteins (800 nM). The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated. Measurements were made with 40% LED and 40% laser power at 25°C. D. DNA methyltransferase activity of wide type protein and the mutants is quantified using radioactive assay. [3H]-methyl transferred to duplex DNA containing 5′-GAGG-3′ was quantified by Beckman LS6500 for 10 min, experiments were repeated for three times and data were corrected by subtraction of the background. E. Superposition of M1.HpyAVI (green) with M.MboIIA (cyan) and M.RsrI (magenta). Residues D29 and E216 are conserved through all the DNA MTases mentioned in Figure 3 (not shown in Figure 4). To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay. As shown in Figure 4C, by contrast to the wild type enzyme, most mutants displayed variable reduction of KD value, among them the D29A and E216A mutants displayed no protein-AdoMet affinity at all. The results suggested that the hydrogen bonds formed by D29 and E216 with AdoMet were most crucial interactions for cofactor binding. Mutation of the two residues may directly prevent the methyl transfer reaction of M1.HpyAVI. The importance of D29 is preserved because it belongs to the catalytic active site DPPY, but the residue E216 has not been fully investigated even being a conserved amino acid throughout MTases (Figure 4E). E216 is the last residue of β2, which contacts the two hydroxyls of the ribose of AdoMet. Replacement of this residue by alanine completely abolishes the key hydrogen bonds for AdoMet-binding, and very likely blocks the methyl transfer reaction. To confirm this notion, [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the mutants. As shown in Figure 4D, the result of radiological assay agreed well with the MST measurement. The D29A and E216A mutants showed little or no methyl transfer activity, while other mutants exhibited reduced methyltransferase activity. As mentioned previously, FXGXG is a conserved AdoMet-binding motif of DNA MTases. We also made mutants of “FMGSG” to alanine for every amino acid, and found that the F195A mutant was insoluble probably due to decreasing the local hydrophobicity upon this mutation. We subsequently investigated the ligand binding affinity and methyl transfer reaction of the other mutants using MST and a radiological assay. We found that G197 played a crucial role in AdoMet-binding, while mutagenesis of M196 and G199 did not influence cofactor binding and catalytic activity (Figure S2A and B). G197 is a conserved residue throughout the DNA MTases, and replacing by alanine at this site likely change the local conformation of cofactor-binding pocket. Mutagenesis on this glycine residue in M.EcoKI or M.EcoP15I also abolished the AdoMet-binding activity. Although mutational study could not tell the role of F195 in ligand binding due to the insolubility of the F195A mutant, structural analysis suggested the importance of this residue in AdoMet-binding. The phenyl ring of F195 forms a perpendicular π-stacking interaction with the purine ring of AdoMet, which stabilizes the orientation of AdoMet bound in the pocket of M1.HpyAVI (Figure S2C). In a separate scenario, mutagenesis of this residue in M.EcoRV has been proven to play an important role in AdoMet binding. Potential DNA-binding sites The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58). The hairpin loop between β6 and β7 strands that carries a conserved HRRY sequence signature in the middle is proposed to insert into the minor groove of the bound DNA. As aforementioned, the TRD of M1.HpyAVI shows striking difference from the other DNA MTases, and the relaxed specificity of substrate recognition may be at least partially attributable to the disordered TRD. In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either. This loop, however, was largely stabilized upon DNA binding, as observed in the protein-DNA complex structures of M.TaqI (PDB ID 2IBS), M.HhaI (PDB ID 1MHT) and M.HaeIII (PDB ID 1DCT). The well-ordered loop in those structures directly contacts the flipping adenine and forms hydrogen bond with neighboring bases. These observations implied that the corresponding loop in other MTases, e.g. M1.HpyAVI, is likely responsible for reducing sequence recognition specificity and thus plays crucial roles in catalysis. Key residue for wider spectrum of substrate recognition Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment. Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A). Then, a [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the wide type protein and the mutants. As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI. Sequence alignment, structural analysis and radioactive methyl transfer activity define the key residue for wider substrate specificity of M1.HpyAVI A. Sequence alignment of M1.HpyAVI from 50 H. pylori strains including 26695 revealed several variant residues. Residues P41, N111, K165 and T166 of M1.HpyAVI from strain 26695 were chosen based on structural analysis and sequence alignment (shown in red arrow). Amino-acid conservation is depicted using WebLogo (Crooks et al, 2004). B., C., D. Methyl transfer reactions were performed using wt-M1.HpyAVI, M1.HpyAVI-P41S, M1.HpyAVI-N111T, and M1.HpyAVI-K165R T166V, respectively. Radioactivity incorporated into the duplex DNA containing 5′-GAGG-3′, 5′-GAAG-3′ or 5′-GGAG-3′ was quantified by Beckman LS6500 for 10 min. The experiments were repeated for three times and data were corrected by subtraction of the background. Our experimental data identified P41 as a key residue determining the recognition of GGAG of M1.HpyAVI. This amino acid locates in the highly flexible loop between residues 33 and 58, which is involved in DNA binding and substrate recognition as shown above. Replacement by serine at this position definitely changes the local conformation and hydrophobicity, and probably some structural properties of the whole loop, which may in turn result in reduced specificity for sequence recognition of the enzyme from strain 26695. DISCUSSION Although the DNA-bound structure of previous investigation on a γ-class N6-adenine MTase revealed that the target adenine was rotated out of DNA helix, details of the methyl transfer process were still unclear. Additionally, recent studies reported the importance of N6-methyladenine in some eukaryotic species, but until now there has not been any N6-adenine MTases being identified in eukaryotes. Biochemical and structural characterization of M1.HpyAVI provides a new model for uncovering the methyl transfer mechanism and for investigating the N6-methyladenine in eukaryotes. Oligomeric state of DNA MTases was long accepted as monomer, but our study indicated here that M1.HpyAVI exists as a dimer both in crystal and solution. Interestingly, some other β-class DNA exocyclic MTases showed similar oligomeric state in crystal and in solution, indicating that dimer may be the functional state shared by a subgroup of DNA MTases. The highly flexible region (residues 33-58) and TRD (residues 133-163) of M1.HpyAVI are supposed to interact with DNA at minor and major grooves, respectively. These two structural characteristics may account for the substrate promiscuity of this enzyme. And residue P41 might be a key residue partially determining the substrate spectrum of M1.HpyAVI. The missing loop between residues 33 and 58 may need DNA binding so as to form a stable conformation, which is similar to the condition of M.TaqI. Crystallization of M1.HpyAVI-DNA complex warrants future investigations, with the purpose of revealing the mechanism behind the wider substrate specificity of this enzyme. DNA methylation plays an important role in bacterial pathogenicity. DNA adenine methylation was known to regulate the expression of some virulence genes in bacteria including H.pylori. Inhibitors of DNA adenine methylation may have a broad antimicrobial action by targeting DNA adenine methyltransferase. As an important biological modification, DNA methylation directly influences bacterial survival. Knockout of M1.HpyAVI largely prevents the growth of H. pylori. Importantly, H. pylori is involved in 90% of all gastric malignancies. Appropriate antibiotic regimens could successfully cure gastric diseases caused by H.pylori infection. However, eradication of H. pylori infection remains a big challenge for the significantly increasing prevalence of its resistance to antibiotics. The development of new drugs targeting adenine MTases such as M1.HpyAVI offers a new opportunity for inhibition of H. pylori infection. Residues that play crucial roles for catalytic activity like D29 or E216 may influence the H.pylori survival. Small molecules targeting these highly conserved residues are likely to emerge less drug resistance. In summary, the structure of M1.HpyAVI is featured with a disordered TRD and a key residue P41that located in the putative DNA binding region that may associate with the wider substrate specificity. Residues D29 and E216 were identified to play a crucial role in cofactor binding. As the first crystal structure of N6-adenine MTase in H.pylori, this model may shed light on design of new antibiotics to interfere the growth and pathogenesis of H.pylori in human. MATERIALS AND METHODS Protein expression and purification The ORF encoding M1.HpyAVI was inserted into the expression plasmid pET22b (Novagen, Massachusetts, USA) to produce a recombinant protein containing a C-terminal His-tag. In order to produce soluble protein, a chaperone plasmid PG-KJE8 (TaKaRa, Dalian, China) was co-expressed with M1.HpyAVI. The recombinant protein was purified with a three-step chromatography protocol using a Ni-NTA affinity column, a HiLoad 16/60 Superdex 200 column and a mono-S HR 5/5 column (1ml) (GE Healthcare, Uppsala, Sweden). Mutants of M1.HpyAVI were generated using the Muta-direct Site-directed Mutagenesis kit (SBS Genetech, Beijing, China) and produced using the same protocol with wide type protein. Crystallization and data collection Crystallization trials were carried out for both the AdoMet-free and AdoMet-bound proteins using the hanging drop vapor diffusion technique. Crystals used for diffraction data collection of the apoprotein were grown under the condition of 1.0 M Bis-Tris, pH 9.0, 1.4 M ammonium tartrate, and the optimal crystallization condition for AdoMet-bound protein was 1.0 M Bis-Tris, pH 6.0, 14% PEG2000, 0.2 M lithium sulfate. X-ray diffraction data were collected at 100 K on beamline BL17U1 at the Shanghai Synchrotron Radiation Facility (SSRF) using an ADSC Quantum 315r CCD detector. All data were indexed, integrated and scaled using the XDS program. Structure determination and refinement The structure of ligand-free M1.HpyAVI was determined by means of molecular replacement using the M.MboIIA (PDB ID 1G60) as a search model. Automated structure determination using Phaser gave a solution showing four subunits sitting in the asymmetric unit. The model was refined using the COOT graphics package manually and phenix.refine. The AdoMet-bound structure was determined by means of molecular replacement using the refined model of the apoprotein, and refined in the same way. Statistics from the data collection and structure refinement are summarized in Table 1. All figures representing the M1.HpyAVI structures were generated using the molecular visualization program PyMol. Detection of protein dimerization The interface information of M1.HpyAVI free form and AdoMet-bound form structures were analyzed using the PDBePISA (Proteins, Interface, Structures and Assemblies) web server. The protein molecular weight was determined by gel filtration using a FPLC system coupled to a Superdex 75 HR 10 / 30 column. The sizing standard was calibrated using the gel filtration calibration kit LMW (GE Healthcare, Uppsala, Sweden). Binding affinity quantification via microscale thermophoresis (MST) Microscale thermophoresis was performed using the NT115 nanotemper technologies. M1.HpyAVI-wt and M1.HpyAVI-mutant proteins were fluorescently labeled using the protein label kit according to manufacturer's protocol. Affinity measurements were performed by using MST buffer (0.05% Tween-20 added as final concentration). A solution of unlabeled AdoMet was serially diluted from 1 mM to 15 nM. Equal volume of 0.8 μM labeled protein was mixed with the AdoMet and loaded into the silica capillaries. This binding curve can directly be fitted with the nonlinear solution of the law of mass action, with the dissociation constant (KD) as a result. Measurement was performed at 25°C using 40% LED power and 40%IR-laser power. The dissociation constant was calculated using the Nano-temper Analysis software. Radioactive methyltransferase analysis Several different DNA duplexes containing single site of 5′- GAGG-3′, 5′- GAAG-3′ or 5′-GGAG-3′ were used as substrate for methyl transfer reaction (Table S2). 0.1 μM of enzyme and 2 μM of S-[methyl-3H] adenosly methionine (China Isotope and Radiation Corporation, Beijing, China) were incubated at 37°C for 5 min, and then 5 μM of DNA substrate was added to initiate the reaction. Aliquots (20 μl) were taken out at 4-min time intervals and quenched with 2 N HCl. Subsequently, DNA of the mixture was purified using a DNA purification column (TIANGEN, Beijing, China) and the scintillation counting of tritiated DNA was quantified by Beckman LS6500 for 10 min. The background radioactivity was determined by omitting the enzyme from the reaction solution. All the reactions were performed in triplicate. SUPPLEMENTARY FIGURES AND TABLES CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest. ACCESSION CODES Atomic coordinates and structure factors for apo-M1.HpyAVI and cofactor-bound M1.HpyAVI have been deposited in the PDB, with accession codes 5HEK and 5HFJ respectively. REFERENCES Beyond Watson and Crick DNA methylation and molecular enzymology of DNA methyltransferases Crystal structure of MboIIA methyltransferase N6-methyl-adenine an epigenetic signal for DNA-protein interactions N4-methylcytosine as a minor base in bacterial DNA Molecular evolution of DNA-(cytosine-N4) methyltransferases evidence for their polyphyletic origin N6-methyladenine a potential epigenetic mark in eukaryotes DNA methylation and demethylation dynamics N6-methyldeoxyadenosine marks active transcription start sites in chlamydomonas DNA Methylation on N6-Adenine in C. elegans N6-methyladenine DNA modification in Drosophila Understanding the evolution of restriction-modification systems clues from sequence and structure comparisons Structure, function and mechanism of exocyclic DNA methyltransferases Diversity of DNA methyltransferases that recognize asymmetric target sequences Structure of the N6-adenine DNA methyltransferase M. 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diff --git a/annotated_BioC_JSON/PMC5603727_ann.json b/annotated_BioC_JSON/PMC5603727_ann.json
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+[{"sourceid":"5603727","sourcedb":"","project":"","target":"","text":"Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40 The RNA-binding protein Roquin is required to prevent autoimmunity. Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40). A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin. Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE). Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin. Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin.  Roquin is an RNA-binding protein that prevents autoimmunity by limiting expression of receptors such as Ox40. Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques. Posttranscriptional gene regulation is involved in a wide range of cellular functions and its critical importance has been described for many developmental and differentiation processes. Consistently, mutations of factors involved in posttranscriptional gene regulation pathways were found associated with a number of genetically inherited diseases. The Roquin protein is essential in T cells for the prevention of autoimmune disease. This is evident from the so-called sanroque mutation in Roquin-1, a single amino acid exchange from Met199 to Arg that causes the development of systemic lupus erythematosus-like symptoms in homozygous mice. The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells. Consistently, CD4+ and CD8+ T cells with the combined deletion of Roquin-encoding genes are spontaneously activated and CD4+ T-helper cells preferentially differentiate into the Th1, Tfh or Th17 subsets. Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs). We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA. The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin. At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain. The structural basis for CDE recognition by the Roquin-2 ROQ domain has also been recently reported. We found that the posttranscriptional activity of Roquin-1 and Roquin-2 is regulated through cleavage by the paracaspase MALT1 (refs). Enhanced MALT1-dependent cleavage and inactivation of Roquin, and thus less effective repression of target genes, result from increased strength of antigen recognition in T cells. These findings suggest that dependent on the strength of cognate antigen recognition differential gene expression and cell fate decisions can be established in naive T cells by a graded cleavage and inactivation of Roquin. In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology. Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain. We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments. Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells. Results SELEX identifies novel RNA ligands of Roquin-1 We set out to identify Roquin-bound RNA motifs in an unbiased manner by performing SELEX experiments. A biotinylated amino-terminal protein fragment of Roquin-1 (residues 2–440) was used to enrich RNAs from a library containing 47 random nucleotides over three sequential selection rounds. Next-generation sequencing (NGS) of the RNA before and after each selection round revealed that the starting pool represented about 99.6% unique reads in ∼4.2 × 106 sequences. Bioinformatic analysis of NGS data sets derived from the starting pool and enriched selection rounds revealed that the complexity was reduced to 78.6% unique reads in 3.7 × 106 sequences that were analysed after 3 rounds of selection and enrichment. For NGS data analysis, the COMPAS software (AptaIT, Munich, Germany) was applied. Enriched sequences were clustered into so-called patterns with highly homologous sequences. Hereby, the algorithm at first identified frequent motifs of five to eight nucleotides length and subsequently used iterative cycles of proto-pattern formation to cluster sequences bearing two of such frequent motifs. A final aptamer pattern was built up by sequences bearing two frequent motifs and, at the same time, having high similarities also in other sequence parts. Based on this so-called co-occurrence approach, patterns on the basis of frequent motifs were generated and were searched for prominent hexamer sequences (Supplementary Fig. 1a). We identified 5′-CGTTTT-3′, 5′-GCGTTT-3′, 5′-TGCGTT-3′ and 5′-GTTTTA-3′ motifs that were also reconfirmed in an independent experiment (Supplementary Fig. 1a) and are located within highly similar sequences (Fig. 1a and Supplementary Fig. 1b). Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b). Notably, our SELEX approach did not reveal the previously identified CDE sequence. We assume that the region of sequence identity in the CDE is too short for our sequence clustering algorithm. Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5–8 nt stem, with one base in the stem not being paired (Supplementary Fig. 1c). Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b). Importantly, this motif is present across species in the 3′-UTRs of respective mRNAs and showed highest conservation in the loop and the upper stem sequences with a drop of conservation towards the boundaries of the motif (Fig. 1c,d). The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d). NMR analysis of Roquin-bound SL RNAs We used NMR to analyse the secondary structure of Roquin-1-binding motifs derived from SELEX. Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs. Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson–Crick G–G base pair at this position (Fig. 2a). Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red). No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. In contrast, all expected base pairs are observed for the Ox40 CDE-like SL RNA (Fig. 2c; see also Supplementary Notes). Structures of ROQ bound to ADE SL RNAs To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements. The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively. In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b). The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e). As may be expected, the recognition of the hexaloop is significantly different from the triloop in the CDE RNA (Fig. 3b,c and Supplementary Fig. 2b). Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e). The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution. The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop. Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts. For example, the main chain atoms of Phe255 form two hydrogen bonds with the Watson–Crick face of the U11 base (Fig. 3d). Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). In addition, the Tyr250 main-chain carbonyl interacts with U13 imino proton (Fig. 3d,e). Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop. The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f). The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA). Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b). The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex. This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e). The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f). This is possible by exposing the base C12 of the Ox-40 ADE-like SL towards the solvent, which accordingly does not show any contacts to the protein. In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts. However, these involve an extended binding surface on the ROQ domain with a number of additional residues compared with the triloop RNA. NMR analysis of ROQ interactions with ADE SLs We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution. CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). This is fully consistent with the interactions observed in the crystal structure (Supplementary Fig. 2c–e) and indicates a similar binding surface. However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d). On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs. This is consistent with the very similar structural features and mode of RNA recognition of the ROQ domain with these RNAs (Supplementary Fig. 2a,d,e). Mutational analysis of the ROQ-ADE interaction To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4). Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2). We next tested a set of mutants (Supplementary Fig. 4), which were designed based on contacts observed in the crystal structure (Fig. 3) and the NMR CSPs (Fig. 4a,b). In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine. We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a). This underlines the central role of Tyr250 for stabilization of the hexaloop structure and recognition by stacking interactions (Fig. 3b,e). Mutation of Ser253, which shows large CSPs in the NMR titrations (Fig. 4a,b), does not significantly impair complex formation (Supplementary Fig. 4). The large chemical shift change is probably caused by ring current effects induced by the close proximity of the U11 and U13 bases. Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4). This indicates that replacement by Tyr does not strongly affect the RNA interaction, and that some conformational variations are tolerated. Thus, the mutational analysis is fully consistent with the recognition of the hexaloop observed in our crystal structures. To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells. Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion. The cells were then transduced with doxycycline-inducible retroviral vectors to reconstitute Roquin-1 expression (Fig. 5b). Depletion of Roquin proteins on tamoxifen treatment (Supplementary Fig. 5a) strongly increased surface expression of Ox40 and Icos (Fig. 5c). This increase in surface expression of both costimulatory receptors was partially corrected by the doxycycline-induced reconstitution with Roquin-1 WT protein (Fig. 5c left panels). Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels). The observed partial rescue may relate to the low, close to endogenous expression of these constructs (Supplementary Fig. 5b). However, it is also possible that continuous overexpression of targets following Roquin deletion induces a hyperactivated state in the T cells. This hyperactivation, compared with the actual posttranscriptional derepression, may contribute even stronger to the increased Icos and Ox40 expression levels. Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos. We also investigated the role of individual nucleotides in the Ox40 ADE-like SL for complex formation with the ROQ domain. We designed four mutants (Mut1–4, see Supplementary Fig. 6) that were expected to disrupt key interactions with the protein according to our co-crystal structure (Fig. 3d–f and Supplementary Fig. 2). NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6). We next used surface plasmon resonance experiments to determine dissociation constants for the ROQ-RNA interaction (Table 2 and Supplementary Fig. 7). Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. As intended, the mutation Mut 1 allows the formation of an additional base pair and thus leads to the formation of a tetraloop with a new G-C closing base pair (Supplementary Fig. 6a). Consistent with the structural analysis, we assume that this variant alters the hexaloop conformation and thus reduces the interaction with ROQ. Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7). No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d). Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ. Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7). Roquin binding to different SLs in the Ox40 3′-UTR We have recently shown that Roquin-1 binds to a CDE-like motif in the 3′-UTR of Ox40 mRNA (Figs 1d and 4c). We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR. The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2). The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2). This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR. We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a). These RNAs were then tested in EMSAs with the Roquin-1 N terminus (residues 2–440) (Fig. 6b). Gel shift assays show that binding to the wild-type 3′-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively. Consistent with this, both bands are strongly reduced when mutations are introduced that interfere with the formation of both SLs. Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL. We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR. Regulation of Ox40 expression via two motifs in its 3′-UTR To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells. Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts. Doxycycline treatment of cells from this cell line enabled ectopic Roquin-1 and co-translational mCherry expression due to the stable integration of an inducible lentiviral vector (Supplementary Fig. 8c). The expression of Ox40 in cells with and without doxycycline treatment was then quantified by flow cytometry (Supplementary Fig. 8c). Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c). In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40. In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c). To further analyse the functional consequences of Roquin binding to the 3′-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d). Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min). A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min). Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively. These findings underscore the importance of both structural motifs and reveal that they have an additive effect on the regulation of Ox40 mRNA expression in cells. Discussion Recent structural and functional studies have provided first insight into the RNA binding of Roquin. Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. Considering that the CDE RNA recognition is mostly structure specific and not sequence dependent, a wide spectrum of target mRNA might be recognized by Roquin. Some evidence for this is provided by a recent study by Landthaler and colleagues. Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes. Our experiments show that this SELEX-derived ADE shows functional activity comparable to the previously established CDE motif. The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop. Notably, the recognition of the respective RNA-helical stem regions by the ROQ domain is identical for the triloop and hexaloop motifs. However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). Of particular importance for the hexaloop recognition is Tyr250, which acts as a stabilizing element for the integrity of a defined loop conformation. It stacks with nucleotides in the hexaloop but not the CDE triloop (Fig. 3b,c). The functional role of Tyr250 for ADE-mediated mRNA regulation by Roquin-1 is thus explained by our experiments (Fig. 5b,c). The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL. Consistent with this, loss of ROQ binding is observed on replacement of U11 and U13 by other bases (Table 2). In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively. A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c). Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops. Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work. Most probably, the experimental setup of Murakawa et al. revealed both high- and low-affinity target motifs for Roquin, whereas our structural study reports on a high-affinity binding motif. Notably, Murakawa et al. neither found the Roquin-regulated Ox40 nor the Tnf 3′-UTRs, as both genes are not expressed in HEK 293 cells. However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement. It remains to be seen which exact features govern the recognition of the A20 SL by Roquin. The regulatory cis RNA elements in 3′-UTRs may also be targeted by additional trans-acting factors. We have recently identified the endonuclease Regnase-1 as a cofactor of Roquin function that shares an overlapping set of target mRNAs. In another study, the overlap in targets was confirmed, but a mutually exclusive regulation was proposed based on studies in lipopolysaccharide (LPS)-stimulated myeloid cells. In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target. In CD4+ T cells, Ox40 does not show derepression in individual knockouts of Roquin-1 or Roquin-2 encoding genes, but is strongly induced upon combined deficiency of both genes. In addition, conditional deletion of the Regnase-1-encoding gene induced Ox40 expression in these cells. Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes. However, in cultures of CD4+ T cells, Ox40 is translated on day 4–5 and is expressed much higher in T cells with combined deficiency of Roquin-1 and Roquin-2. At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c). Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays. Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3′-UTR by both proteins. In contrast, structural details on how Regnase-1 can interact with these SL RNAs are currently missing. Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene. Both were required for Regnase-1-mediated repression. These findings therefore raise the possibility that Regnase-1 interacts with ADE-like hexaloop structures either in a direct or indirect manner. Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation. The novel 3′-UTR loop motif that we have identified as a bona fide target of Roquin now expands this multilayer mode of co-regulation. We suggest that differential regulation of mRNA expression is not only achieved through multiple regulators with individual preferences for a given motif or variants thereof, but that regulators may also identify and use distinct motifs, as long as they exhibit some basic features regarding shape, size and sequence. The presence of distinct motifs in 3′-UTRs offers a broader variability for gene regulation by RNA cis elements. Their accessibility can be modulated by trans-acting factors that may bind regulatory motifs, unfold higher-order structures in the RNA or maintain a preference for duplex structures as was shown recently for mRNAs that are recognized by Staufen-1 (ref.). In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain. The exact stoichiometry of Roquin bound to the Ox40 3′-UTR is unknown. The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs. In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site. This opens the possibility that one Roquin molecule may cluster two motifs in a given 3′-UTR and/or cluster motifs from distinct 3′-UTRs to enhance downstream processing. Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA. We therefore hypothesize that the combination of multiple binding sites may be more commonly used to enhance the functional activity of Roquin. At the same time, the combination of cis elements may be important for differential gene regulation, as composite cis elements with lower affinity may be less sensitive to Roquin. This will lead to less effective repression in T cells when antigen recognition is of moderate signal strength and only incomplete cleavage of Roquin by MALT1 occurs. For understanding the intricate complexity of 3′-UTR regulation, future work will be necessary by combining large-scale approaches, such as cross-linking and immunoprecipitation experiments to identify RNA-binding sites, and structural biology to dissect the underlying molecular mechanisms. Methods SELEX experiments Selection of Roquin-1-bound RNAs from a random RNA library was performed in three rounds of selection with increased stringency of washing (3 × 100 μl, 4 × 100 μl and 5 × 100 μl washing steps) and with decreased protein concentrations (250, 150 and 50 nM). Before selection, 100 μg recombinant Roquin-1 and Roquin-1 M199R N-terminal protein (residues 2–440) were biotinylated: proteins were incubated for 30 min on ice with 10 × molar excess of EZ-link PEG4-NHS-Biotin (Pierce) in PBS (0.1 mg ml−1). Subsequently, the biotinylated protein was purified via gel filtration (MicroSpin column P6, BioRad) and the loss of protein during the biotinylation procedure was estimated by SDS–PAGE and Coomassie staining. The efficiency of the biotinylation reaction was evaluated after spotting of unlabelled and labelled proteins onto a nitrocellulose membrane. After blocking the membrane with 1% BSA in PBS, it was incubated in streptavidin–PE (R-Phycoerythrin) diluted 1:1,000 in PBS for 30 min at room temperature (RT). Subsequently, the membrane was washed three times with PBS and fluorescence intensity of PE bound to biotinylated protein was determined by fluoroimaging (Raytest, FLA5000, 473 nm, Y510 filter). The RNA startpool containing the 47-nt random sequence as well as the RNA pools for the second and third selection rounds were transcribed in vitro from double-stranded PCR DNA, and protein-bound RNA was isolated and reverse transcribed before PCR amplification, as previously described. Following transcription, the samples were separated on an 8% PAGE, the bands excised and RNA purified. Every round of selection started by combining the RNA pool (400 pmol) with biotinylated protein and incubating the mix for 30 min at 37 °C. Subsequently, binding buffer-equilibrated streptavidin-magnetic beads were added and incubated (10 min, 37 °C) to bind the protein–RNA complexes, followed by washes. By boiling the beads in 0.2 mM EDTA in water for 3 min, protein and RNA molecules were released. After removal of beads, the solution served as template for reverse transcription (One-Step RT-PCR Kit, Qiagen) and from the obtained complementary DNA the RNA pool of the next round of selection was transcribed. The cDNAs from every selection round (startpool, round 1, round 2 and round 3) were used for Index-PCRs to analyse the pool composition at every stage during selection. Comparable amounts of the PCR products were combined to one cDNA library and analysed by Solexa Illumina sequencing. Sequence motif and structural analysis To identify sequence motifs to which Roquin specifically binds, we counted the number of occurrences of each hexamer (46=4,096 motifs) in the sequences obtained by SELEX. We then generated a data set of randomized sequences of the same nucleotide composition as the SELEX-derived sequences, by permuting the SELEX-derived sequences with a custom script. Finally, we counted the number of occurrences of each hexamer in the set of randomized sequences and computed the log2 ratio of the number of occurrences of each motif in the real and randomized sequence sets. To identify a shared sequence motif in the SELEX patterns that showed the strongest enrichment in our selection experiments, the top 100 patterns were analysed with the Motif-based sequence analysis tool MEME (http://meme-suite.org) using the default settings. This analysis revealed three sequence motifs of which the first is shown in Fig. 1b. For the construction of sequence logos, we screened the obtained nucleotide sequences from SELEX replicate 1 and extracted the nucleotide sequences including the 7-nt flanking regions. Sequence logos were constructed with WebLogo 2.8.2 (http://weblogo.berkeley.edu/). For the Ox40 3′-UTR sequence alignment, we extracted Multiz alignments of 60 Vertebrates from the UCSC mouse GRCm38/mm10 assembly for the genomic region chr4:156,016,498–156,016,520. For each species contained in the alignment, we extracted genomic coordinates of the aligned sequence, extended the coordinates by 10 nt upstream and downstream, and retrieved the extended sequences from the corresponding genome assemblies. Finally, sequences were aligned with ClustalW 2.1 with standard settings and the alignment was visualized using Jalview. To evaluate the structural context the inferred motif is located in, we first appended to the nucleotide sequences obtained from the SELEX experiment the SELEX primers 5′-GGAGAGATGTGAACTT-3′ and 5′-AGTTTCGTGGATGCCAC-3′ to the 5′- and 3′-end, respectively. Next, we screened for sequences that contained the inferred motif and performed secondary structure prediction on those sequences with RNAfold from the ViennaRNA package version 1.8 with parameters '-p -d2'. Next, we used a custom Perl script to parse the base-pairing probability file generated by RNAfold and to calculate an average base-pair probability over all sequences that contained the inferred motif. Production of proteins Cloning of expression vectors for Roquin-1 ROQ (residues 147–326), ROQ (residues 171–326) and Roquin-1 N-term (residues 2–440) was carried out by standard procedures as described previously. Briefly, PCR-amplified fragments were put into pETM11 and pETTrx1a vectors based on pET24d as provided by the Protein Expression and Purification Facility at Helmholtz Zentrum München. All vectors contained tobacco etch virus (TEV) protease recognition sites for subsequent proteolytic removal of the tags. All length-variable Roquin-1 expression constructs were designed and cloned via restriction sites NcoI (5′) and XhoI (3′). ROQ domain RNA-binding mutants were cloned by Quick change PCR with high-fidelity Phusion DNA polymerase and subsequent treatment with DnpI. Alternatively, we used conventional cloning with a two-step PCR protocol and enzymatic restriction. The Roquin-1 fragments (147–326) and (171–326) were expressed as N-terminal His6-thioredoxin fusion proteins as recently described. Isotope-labelled protein for NMR studies was expressed in M9 minimal medium supplemented with 0.5 g l−1 15N ammonium chloride and 2 g l−1unlabelled or [U-13C] glucose. For the preparation of deuterated proteins, cells were adapted and grown as described previously. Briefly, we used a protocol with stepwise adaptation of cells to deuterium changing buffer from no D2O, low glucose to 50% D2O, low glucose and finally 99.5% D2O with deuterated glucose. The Roquin-1 N-terminal domain (residues 2–440) was expressed and purified essentially as described above for the ROQ domain, but no thioredoxin tag was used. For Roquin-1 N-terminal domain, all expression media and the final buffer contained 100 or 25 μM of zinc chloride, respectively. RNA preparation RNAs were synthesized and purchased from IBA GmbH (Göttingen, Germany), purified via PAGE followed by two steps of desalting. No major impurities were seen in NMR spectra. Complex formation for crystallography and NMR experiments was achieved by dissolving the lyophilized RNA in water or NMR buffer. This stock solution was snap-cooled by boiling at 95 °C for 5 min and transferred to an ice-cold bath for 10 min before aliquoting. All RNAs were stored at −80 °C, to avoid degradation and thermodynamically favoured duplex formation. Full length and fragments of Ox40 3′-UTR mRNA were produced by in vitro transcription (IVT) from DNA templates harbouring a T7 promoter site either with direct incorporation of α-32P-labelled UTP or subsequent 3′-labelling of purified RNA with γ-32P-labelled ATP. DNA templates were cloned by primer extension PCR. For IVT, 50–150 nM of DNA were incubated with 11 mM magnesium chloride, 8% (w/v) PEG8000, 1.25 mM of each NTP and 0.05 mg ml−1 of T7 polymerase in 1 × standard reaction buffer for 3–5 h at 37 °C. Labelled RNAs were produced in 50 μl reactions and purified via spin columns and directly subjected to EMSA assays. Unlabelled RNAs were produced in reactions of 500–5,000 μl. After IVT, the reactions were separated on 8% denaturing SDS–PAGEs, RNA of interest excised and eluted from the gel using the Elutrap kit (GE Healthcare). After elution, RNAs were dialysed against water and lyophilized. Subsequently, RNAs were dissolved in water and stocks generated by boiling them at 95 °C for 5 min and transferred to an ice-cold bath for 10 min before aliquoting. Labelling for EMSA assays was carried out as for short motifs and described recently. As a modification, dephosphorylation was performed for 30 min and 3′-phosphorylation with γ-32P-labelled ATP and T4 polynucleotide kinase for 90 min for higher efficiency, respectively. NMR spectroscopy NMR measurements of Roquin-1 ROQ (147–326) and ROQ (171–326) were performed in buffers as described, mixed with 10% D2O. Backbone chemical shift assignments of ROQ (171–326) with 1.1- to 1.2-fold excess of the Ox40 ADE-like SL motif or consensus ADE SL RNAs were recorded with protein concentrations of 350–400 μM. HNCA, HNCACB, HNCO, HNcaCO and 3D 15N-edited NOESY spectra were acquired at 298K on Bruker Avance III spectrometers at field strengths corresponding to 600 and 800 MHz proton Larmor frequency, equipped with TCI cryogenic probe heads. Spectra of ROQ in complex with Ox40 CDE-like SL RNA and the RNA alone have been reported before. Spectra were processed with Topspin3.2 and analysed with CCPNMR Analysis and Sparky. For RNA motifs, one- and two-dimensional imino NOESY spectra with water-flip-back WATERGATE were recorded at 600–900 MHz, at 278 and 298 K at 150–350 μM RNA concentrations. Sequential assignments were guided by secondary structure predictions with mfold and supported by 15N chemical shifts from natural abundance SOFAST-HMQC experiments. Electrophoretic mobility shift assays The EMSAs with ROQ domain and individual motifs were performed as described previously. In short, for the binding reaction a mastermix containing transfer RNA, 32P-labelled SL RNA and reaction buffer was prepared and then mixed with dilutions of the recombinant proteins to achieve the indicated protein concentrations. The binding was performed for 10 min at RT or 20 min on ice in 20 μl reaction volume in the presence of 2.5 μg μl−1 tRNA from baker’s yeast (Sigma), 500 pM 32P-labelled RNA, 20 mM HEPES (pH 7.4), 50 mM NaCl, 1 mM MgCl2, 1 mM dithiothreitol and 1 μg μl−1 BSA. For the binding reaction of Roquin-1 N-terminal with full-length Ox40 3′-UTRs or fragments thereof, ∼1 pmol of RNA was incubated with protein concentrations between 0 and 1,000 μM in a volume of 20 μl. RNP complexes were resolved by PAGE (6% polyacrylamide, 5% glycerol, 0.5 × TBE) at 120 V for 40 min at RT. Gels were then fixed, dried and exposed to a phosphor imager screen. X-ray crystallography The crystallization experiments for ROQ–RNA complexes were performed at the X-ray Crystallography Platform at Helmholtz Zentrum München. The crystals of both, Roquin-1 ROQ (171–326) with Ox40 ADE-like SL motif (22mer, 5′-UCCACACCGUUCUAGGUGCUGG-3′) and with the consensus SELEX-derived ADE SL motif (20mer, 5′-UGACUGCGUUUUAGGAGUUA-3′) were obtained from the same condition: 100 mM Bis-Tris buffer pH 5.5, 200 mM sodium chloride and 25% (v/w) PEG 3350. Crystallization was performed using the sitting-drop vapour-diffusion method at 292 K in 24-well plates and a protein concentration of 12 mg ml−1. The crystals appeared after 1 day. For the X-ray diffraction experiments, the crystals of both co-complexes were mounted in a nylon fibre loop and flash cooled to 100 K in liquid nitrogen. The cryoprotection was performed for 2 s in reservoir solution complemented with 20% (v/v) ethylene glycol. Diffraction data for ROQ Ox40 ADE-like motif was collected on the ID29 beamline (ESRF, Grenoble, France) using a Pilatus 6M at a wavelength of 1.25363 Å. Diffraction data for the ROQ-ADE complex were collected using Pilatus 2M detector at 1.00003 Å wavelength at PXIII beamline at SLS (Villigen, Switzerland). All data sets were indexed and integrated using XDS and scaled using SCALA. Intensities were converted to structure–factor amplitudes using the programme TRUNCATE. Table 1 summarizes data collection and processing statistics for both data sets. Structure determination and refinement The structure of both ROQ-Ox40 ADE-like SL and ROQ-ADE SL were solved by molecular replacement using the native Roquin-1 ROQ (147–326) structure as a search model (PDB: 4QI0 (ref.)). Model building was performed in COOT. RNA molecules were modelled manually. The refinement of both structures was done in REFMAC5 (ref.) using the maximum-likelihood target function including translation, libration and screw-rotation displacements of a pseudo-rigid body (TLS). For the ROQ-ADE SL structure, non-crystallographic symmetry (NCS) averaging was implemented. The final models are characterized by R and Rfree factors of 21.8 and 25.7% for ROQ-Ox40 ADE-like SL, and 18.6 and 23.4% for ROQ-ADE SL (Table 1), respecively. The stereochemical analysis of both final models was done in PROCHECK and MolProbity. It indicates that there are no residues with generously allowed or unfavourable backbone dihedral angles, and that 99.4% (for ROQ-Ox40 ADE-like SL structure) and 92.3% (for ROQ-ADE SL structure) of all residues are in the core region of the Ramachandran plot. Functional assays Functional assays determining the Roquin-mediated regulation of Ox40 with different 3′-UTR variants were performed as described previously. In brief, Rc3h1/2−/− mouse embryonic fibroblast (MEF) cells, stably transduced with a doxycycline-inducible Roquin-1-p2A-mCherry construct, were retrovirally infected with Ox40 constructs of different 3′-UTR length or mutation, which led to the expression of Ox40 on the cell surface (CDE-like mutation changing nt 14–16 GCA to CGT, ADE-like mutation changing nt 15–17 from GGT to CCA). Forty-eight hours after infection, the cells were split and one half of the cells was treated with doxycycline (1 μg ml−1), to induce expression of Roquin-1 and mCherry, connected via the self-cleaving peptide p2A. Thus, Roquin-expressing cells were marked by mCherry expression. Sixteen to 20 h after induction, the cells were harvested, stained with allophycocyanin (APC)-conjugated anti-Ox40 and analysed by flow cytometry. To compare the Ox40 expression levels achieved by different constructs, the relative Ox40 mean fluorescence intensity (MFI) was determined by dividing the MFI of treated (mCherry+) cells by the MFI of untreated (mCherry−) cells. Mouse experiments Compound mutant mice with the Rc3h1fl/fl (ref.) and Rc3h2fl/fl (ref.) (denoted Rc3h1/2fl/fl), as well as Cd4-Cre-ERT2 (ref.) and Gt(ROSA)26Sortm1(rtTA*M2)Jae alleles were maintained on a C57BL/6 genetic background. All animals were housed in a pathogen-free barrier facility in accordance with the Ludwig-Maximilians-University München institutional, state and federal guidelines. Generation of overexpression vectors Expression constructs of Roquin-1 and Ox40 were cloned into a modified pRetroX-Tight vector (Clontech). The puromycine-resistance cassette was removed and a cassette containing attR1-ccdB-attR2 was inserted, to generate a Gateway destination vector. Roquin-1 and Ox40 constructs were inserted by LR reaction (Invitrogen). Any mutants thereof were generated by site-directed mutagenesis. Virus production Replication-deficient retrovirus production and T-cell transduction was performed as previously described. Briefly, retroviral and packaging plasmids were introduced into HEK293T cells by calcium-phosphate transfection. Forty-eight hours after transfection, cell culture supernatants containing the retrovirus particles were harvested, passed through 0.45-μm filters and stored at −80 °C. Cell isolation and culture Splenocytes were isolated from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice. CD4+ T cells were isolated by negative selection with magnetic beads according to the manufacturer’s instructions (Stem Cell Technologies). CD4+ T cells were cultured in DMEM medium supplemented with 10% (vol/vol) fetal bovine serum, 1 × nonessential amino acids (Lonza), 10 mM HEPES pH 7.4 (Invitrogen), 50 μM β-mercaptoethanol (Invitrogen) and 100 U ml−1 penicillin–streptomycin (Invitrogen). Rc3h1/2fl/fl deletion was induced by addition of 4′OH-Tamoxifen (0.3 μM) for 24 h. For TH1 differentiation, CD4+ T cells were cultured in six-well plates pre-coated with goat anti-hamster IgG (MP Biochemicals) and DMEM medium further supplemented with anti-CD3 (0,25 μg ml−1), anti-CD28 (2,5 μg ml−1), IL-12 (10 ng ml−1) and anti-IL-4 (10 μg ml−1) for 40 h. Cells were then infected with retroviral constructs, allowing reconstitution with either Roquin-1, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A, and cultured in IL-2 containing media (20 U ml−1). Forty-eight hours after transduction, the cells were split and one half of cells was treated with doxycycline (1 μg ml−1), to induce expression of Roquin-1 WT and Roquin-1 mutants. Twenty-four hours after induction, the cells were harvested for analysis by immunoblot and flow cytometry with the indicated antibodies (1:200 anti-mouse Icos-biotin clone 7E–17G9 (eBioscience); 1:200 Streptavidin-PerCP (Becton Dickinson); 1:200 anti-mouse Ox40-PE clone OX-86 (eBioscience)). Immunoblot analysis CD4+ T cells were incubated for 15 min on ice with lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.25% (vol/vol) Nonidet-P40, 1.5 mM MgCl2 and protease inhibitor mix without EDTA (Roche) and 1 mM dithiothreitol). Lysate was cleared by centrifugation (10 min, 10 000 g, 4 °C). Immunoblotting was performed by standard protocols with hybridoma supernatants containing monoclonal antibody recognizing Roquin-1 and Roquin-2 (anti-roquin, clone 3F12). mRNA decay experiments Hela Tet-Off Advanced Cells (Clontech 631156) were stably transduced with retroviruses expressing different Ox40 constructs. FACS analysis 41 h post transduction revealed similar Ox40 surface expression levels on all five cell samples. After transduction, the cell lines were initially cultured for at least 48 h without doxycycline, to ensure high Ox40-expression levels. For each time point, 400 000 cells were spread on one well in a six-well plate. To switch off Ox40-transcription, doxycycline was supplied with the medium at time point 0. After one washing step with PBS, cells were directly harvested from each well with Trizol before Dox application (0 h), as well as 2, 3 and 4 h after Dox application. RNA was isolated using standard Trizol protocols. Reverse transcription was performed with the Qiagen Quantitect Reverse Transcription Kit following the manufacturer’s protocols. Quantitative PCR was carried out on a Roche Light Cycler 480 using the Light Cycler 480 Probes Master Mix and primer-/probe-combinations from Roches Universal Probe Library. Relative mRNA expression levels were calculated by normalization to the housekeeper gene ywhaz. Surface plasmon resonance ROQ–RNA binding experiments were performed on a BIACORE 3000 instrument (Biacore Inc.). ROQ domain was diluted to a final concentration of 35 μg ml−1 in 10 mM HEPES pH 7.0 and chemically immobilized (amine coupling) onto CM5 sensor chips (Biacore Inc.). The RNA samples were diluted in the running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 2 mM MgCl2 and 0.005% Tween 20) to the final concentration of 31.25, 62.5, 125, 250 and 500 nM, and 1 and 2 μM, and injected over the sensor chip surface at 30 μl min−1 at 10 °C. The RNA samples were injected onto the sensor chip from the lowest to the highest concentration. Each RNA-type sample was tested three times with the exception of Mut1–3 two times. Injection of 250 nM RNA was always performed in duplicate within each experiment. To subtract any background noise from each data set, all samples were also run over an unmodified sensor chip surface. Data were analysed using BIAevaluation programme (Biacore Inc.) (Supplementary Fig. 7). For each measurement, the equilibrium dissociation constant was calculated (KD) from steady state binding. The KD from three independent experiments were used to calculate the mean values of these variables and the s.e.m. The results for all tested RNA samples are compared in Table 2. Additional information Accession codes: Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5F5H and 5F5F for the ROQ-Ox40 ADE-like SL and ROQ-ADE SL, respectively. Chemical shifts of the ROQ-Ox40 ADE-like SL and ROQ-ADE SL have been deposited in the Biological Magnetic Resonance Data Bank under accession codes 26587 and 26588, respectively. How to cite this article: Janowski, R. et al. Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40. Nat. Commun. 7:11032 doi: 10.1038/ncomms11032 (2016). Supplementary Material Regulation of lymphocyte development and function by RNA-binding proteins Post-transcriptional coordination of immunological responses by RNA-binding proteins A RING-type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity Loss of Roquin induces early death and immune deregulation but not autoimmunity Molecular control of Tfh-cell differentiation by Roquin family proteins Roquin-2 shares functions with its paralog Roquin-1 in the repression of mRNAs controlling T follicular helper cells and systemic inflammation Roquin paralogs 1 and 2 redundantly repress the Icos and Ox40 costimulator mRNAs and control follicular helper T cell differentiation Cleavage of roquin and regnase-1 by the paracaspase MALT1 releases their cooperatively repressed targets to promote T(H)17 differentiation Roquin promotes constitutive mRNA decay via a conserved class of stem-loop recognition motifs Roquin represses autoimmunity by limiting inducible T-cell co-stimulator messenger RNA Structural basis for RNA recognition in roquin-mediated post-transcriptional gene regulation The ROQ domain of Roquin recognizes mRNA constitutive-decay element and double-stranded RNA Structure of human Roquin-2 and its complex with constitutive-decay element RNA Uncoupling Malt1 threshold function from paracaspase activity results in destructive autoimmune inflammation The ROQUIN family of proteins localizes to stress granules via the ROQ domain and binds target mRNAs A xenon-129 biosensor for monitoring MHC-peptide interactions RC3H1 post-transcriptionally regulates A20 mRNA and modulates the activity of the IKK/NF-kappaB pathway Regnase-1 and Roquin regulate a common element in inflammatory mRNAs by spatiotemporally distinct mechanisms Malt1-induced cleavage of regnase-1 in CD4(+) helper T cells regulates immune activation Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1 RNA recognition by Roquin in post-transcriptional gene regulation Deciphering the protein-RNA recognition code: combining large-scale quantitative methods with structural biology In vitro selection of RNA aptamers that inhibit the activity of type A botulinum neurotoxin Fitting a mixture model by expectation maximization to discover motifs in biopolymers Aligning multiple genomic sequences with the threaded blockset aligner Bidirectional binding of invariant chain peptides to an MHC class II molecule Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients The CCPN data model for NMR spectroscopy: development of a software pipeline  Mfold web server for nucleic acid folding and hybridization prediction Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds Xds Scaling and assessment of data quality Overview of the CCP4 suite and current developments On the treatment of negative intensity observations Features and development of Coot Refinement of macromolecular structures by the maximum-likelihood method Use of TLS parameters to model anisotropic displacements in macromolecular refinement PROCHECK: a program to check the stereochemical quality of protein structures MolProbity: all-atom structure validation for macromolecular crystallography TGF-beta signalling is required for CD4(+) T cell homeostasis but dispensable for regulatory T cell function Roquin binds inducible costimulator mRNA and effectors of mRNA decay to induce microRNA-independent post-transcriptional repression The authors declare no competing financial interests. Author contributions A.S. carried out cloning, protein expression and purification, and NMR experiments. R.J. performed crystallization and structure determination. G.A.H. carried out EMSA assays and SELEX experiments, and functional studies were performed by G.A.H, N.W. and S.B. M.B. and R.B. helped setting up the SELEX experiments and identified patterns from NGS data, on the basis of which A.G. and M.Z. analysed the motifs and secondary structures. T.B. contributed unpublished reagents and advice. A.S., G.A.H., R.J., V.H., D.N. and M.S. designed the project and wrote the paper. All authors discussed the results and commented on the manuscript. SELEX identifies a novel SL RNA ligand of Roquin-1. (a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences. (c) Conservation of the motif found in Ox40 3′-UTRs for various species as indicated. The labels correspond to the versions of the genome assemblies in the UCSC server (see Method section). rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL. The broken line between the G–G base pair in the ADE SL indicates a putative non-Watson–Crick pairing. The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison. See also Supplementary Fig. 1. NMR analysis of the SL RNAs used in this study. Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). The respective SL RNAs and their base pairs are indicated. Red asterisks indicate NMR signals of the protein. Black asterisks in a indicate a second conformation (see Supplementary Notes). Green lines in the secondary structure schemes on the left refer to visible imino NMR signals and thus experimental confirmation of the base pairs indicated. Red nucleotides indicate significant chemical shift changes observed. The dotted green line between G6 and G15 in a highlights a G–G base pair. Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA. (a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta). Selected RNA bases and protein secondary structure elements are labelled. (b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green). Only RNA-interacting residues that are different in both structures are shown. Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA. The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257). The protein chain is shown in turquoise and the RNA is shown in grey. Atoms are colour coded according to charge. (e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop. U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247. The side chain of Tyr250 makes hydrophobic interactions with the pyrimidine side chain of U10 on one side and U11 on the other side. Lys259 interacts with the phosphate groups of U10 and U11. (f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11. In d – f, amino acids are shown in turquoise and blue, nucleotides in grey colour. See also Supplementary Notes and Supplementary Fig. 2. NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA. (a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red). Selected shifts of amide resonances are indicated. (b) Plot of chemical shift change versus residue number in the ROQ domain (residues 171–326) from a. Grey negative bars indicate missing assignments in one of the spectra. Gaps indicate prolines. (c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green). See also Supplementary Notes and Supplementary Fig. 3. Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR. (a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5). Functional importance of Roquin-1 target motifs in cells. (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). Arrows indicate the individual binding events to either motif. It is noteworthy that the higher bands observed at large protein concentrations are probably additional nonspecific, lower-affinity interactions of Roquin-1 with the 3′-UTR or protein aggregates. (c) Relative Ox40 MFI normalized to expression levels from the Ox40 CDS construct. Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments. Statistical significance was calculated by one-way analysis of variance (ANOVA) Kruskal–Wallis test followed by Dunn’s multiple comparison test (**P\u003c0.01). (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). Error bars represent the mean of technical duplicates in one experiment. mRNA half-life times were calculated with Graph Pad Prism. Data are representative of two experiments with similar results. Data collection and refinement statistics.  \tROQ-Ox40ADE-like SL\tROQ-ADE SL\t \tData collection\t \t space group\tP21212\tP212121\t \t \t \t \t \t Cell dimensions\t \t a, b, c (Å)\t89.66, 115.79, 42.61\t72.90, 89.30, 144.70\t \t α, β, γ (°)\t90, 90, 90\t90, 90, 90\t \t Resolution (Å)\t50–2.23 (2.29–2.23)\t50–3.0 (3.08–3.00)\t \t Rmerge\t5.9 (68.3)\t14.8 (93.8)\t \t I/σI\t14.9 (2.1)\t16.7 (3.1)\t \t Completeness (%)\t98.7 (97.7)\t99.9 (99.9)\t \t Redundancy\t3.9 (3.7)\t13.2 (12.7)\t \t \t \t \t \tRefinement\t \t Resolution (Å)\t2.23\t3.00\t \t No. reflections\t21,018\t18,598\t \t Rwork/Rfree\t21.8/25.7\t18.6/23.4\t \t \t \t \t \t No. atoms\t \t Protein\t2,404\t4,820\t \t Ligand/ion\t894\t1,708\t \t Water\t99\t49\t \t B-factor overall\t47.2\t60.4\t \t \t \t \t \tRoot mean squared deviations\t \t Bond lengths (Å)\t0.006\t0.014\t \t Bond angles (°)\t1.07\t1.77\t \t \t \t \t \tRamachandran plot\t \t Most favoured (%)\t98.6\t99.8\t \t Additional allowed (%)\t1.4\t0.2\t \t ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop. For each data set, only one crystal has been used. *Values in parentheses are for highest-resolution shell. KD for selected RNAs obtained from SPR measurements with immobilized ROQ domain of 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diff --git a/annotated_BioC_XML/PMC4806292_ann.xml b/annotated_BioC_XML/PMC4806292_ann.xml
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@@ -0,0 +1,7759 @@
+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection>
+  <source>PMC</source>
+  <date>20201216</date>
+  <key>pmc.key</key>
+  <document>
+    <id>4806292</id>
+    <infon key="license">CC BY</infon>
+    <infon key="tt_curatable">no</infon>
+    <infon key="tt_version">2</infon>
+    <infon key="tt_round">2</infon>
+    <passage>
+      <infon key="article-id_doi">10.1038/srep23641</infon>
+      <infon key="article-id_pii">srep23641</infon>
+      <infon key="article-id_pmc">4806292</infon>
+      <infon key="article-id_pmid">27009356</infon>
+      <infon key="elocation-id">23641</infon>
+      <infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon>
+      <infon key="name_0">surname:Klima;given-names:Martin</infon>
+      <infon key="name_1">surname:Tóth;given-names:Dániel J.</infon>
+      <infon key="name_10">surname:Humpolickova;given-names:Jana</infon>
+      <infon key="name_11">surname:Nencka;given-names:Radim</infon>
+      <infon key="name_12">surname:Veverka;given-names:Vaclav</infon>
+      <infon key="name_13">surname:Balla;given-names:Tamas</infon>
+      <infon key="name_14">surname:Boura;given-names:Evzen</infon>
+      <infon key="name_2">surname:Hexnerova;given-names:Rozalie</infon>
+      <infon key="name_3">surname:Baumlova;given-names:Adriana</infon>
+      <infon key="name_4">surname:Chalupska;given-names:Dominika</infon>
+      <infon key="name_5">surname:Tykvart;given-names:Jan</infon>
+      <infon key="name_6">surname:Rezabkova;given-names:Lenka</infon>
+      <infon key="name_7">surname:Sengupta;given-names:Nivedita</infon>
+      <infon key="name_8">surname:Man;given-names:Petr</infon>
+      <infon key="name_9">surname:Dubankova;given-names:Anna</infon>
+      <infon key="section_type">TITLE</infon>
+      <infon key="type">front</infon>
+      <infon key="volume">6</infon>
+      <infon key="year">2016</infon>
+      <offset>0</offset>
+      <text>Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein</text>
+      <annotation id="1">
+        <infon key="score">0.99856466</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:08Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="24" length="23"/>
+        <text>in vitro reconstitution</text>
+      </annotation>
+      <annotation id="2">
+        <infon key="score">0.9986492</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="88" length="5"/>
+        <text>human</text>
+      </annotation>
+      <annotation id="3">
+        <infon key="score">0.990704</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="94" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="4">
+        <infon key="score">0.9947903</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:19Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="107" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">ABSTRACT</infon>
+      <infon key="type">abstract</infon>
+      <offset>121</offset>
+      <text>Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.</text>
+      <annotation id="5">
+        <infon key="score">0.9951973</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="121" length="34"/>
+        <text>Phosphatidylinositol 4-kinase beta</text>
+      </annotation>
+      <annotation id="6">
+        <infon key="score">0.99757165</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="157" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="7">
+        <infon key="score">0.99863964</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="179" length="5"/>
+        <text>human</text>
+      </annotation>
+      <annotation id="8">
+        <infon key="score">0.99822444</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="185" length="4"/>
+        <text>PI4K</text>
+      </annotation>
+      <annotation id="9">
+        <infon key="score">0.99892217</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:47Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="212" length="32"/>
+        <text>phosphatidylinositol 4-phosphate</text>
+      </annotation>
+      <annotation id="10">
+        <infon key="score">0.99903166</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:52Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="246" length="4"/>
+        <text>PI4P</text>
+      </annotation>
+      <annotation id="11">
+        <infon key="score">0.9924697</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T17:19:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="305" length="10"/>
+        <text>eukaryotic</text>
+      </annotation>
+      <annotation id="12">
+        <infon key="score">0.9989231</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="358" length="5"/>
+        <text>PI4Ks</text>
+      </annotation>
+      <annotation id="13">
+        <infon key="score">0.9871787</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="419" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="14">
+        <infon key="score">0.99880505</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:52Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="500" length="4"/>
+        <text>PI4P</text>
+      </annotation>
+      <annotation id="15">
+        <infon key="score">0.9945003</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="555" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="16">
+        <infon key="score">0.99744546</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="624" length="3"/>
+        <text>NMR</text>
+      </annotation>
+      <annotation id="17">
+        <infon key="score">0.9976865</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="628" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="18">
+        <infon key="score">0.99748504</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="656" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="19">
+        <infon key="score">0.89974385</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T17:18:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="691" length="21"/>
+        <text>Golgi adaptor protein</text>
+      </annotation>
+      <annotation id="20">
+        <infon key="score">0.9647868</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:03Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="713" length="51"/>
+        <text>acyl-coenzyme A binding domain containing protein 3</text>
+      </annotation>
+      <annotation id="21">
+        <infon key="score">0.998719</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="766" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="22">
+        <infon key="score">0.99585414</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="787" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="23">
+        <infon key="score">0.9966311</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="818" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="24">
+        <infon key="score">0.9964915</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="897" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="25">
+        <infon key="score">0.99765295</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="906" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="721">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:44:10Z</infon>
+        <location offset="926" length="18"/>
+        <text>enzymatic activity</text>
+      </annotation>
+      <annotation id="26">
+        <infon key="score">0.99838036</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:16Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="958" length="11"/>
+        <text>ACBD3:PI4KB</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>1035</offset>
+      <text>Phosphatidylinositol 4-kinase beta (PI4KB, also known as PI4K IIIβ) is a soluble cytosolic protein yet its function is to phosphorylate membrane lipids. It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P). PI4P is an essential lipid found in various membrane compartments including the Golgi and trans-Golgi network (TGN), the plasma membrane and the endocytic compartments. In these locations, PI4P plays an important role in cell signaling and lipid transport, and serves as a precursor for higher phosphoinositides or as a docking site for clathrin adaptor or lipid transfer proteins. A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories. These structures are essential for effective viral replication. Recently, highly specific PI4KB inhibitors were developed as potential antivirals.</text>
+      <annotation id="27">
+        <infon key="score">0.9937843</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="1035" length="34"/>
+        <text>Phosphatidylinositol 4-kinase beta</text>
+      </annotation>
+      <annotation id="28">
+        <infon key="score">0.9973278</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="1071" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="29">
+        <infon key="score">0.9963335</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:41Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="1092" length="9"/>
+        <text>PI4K IIIβ</text>
+      </annotation>
+      <annotation id="30">
+        <infon key="score">0.99854964</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1206" length="5"/>
+        <text>human</text>
+      </annotation>
+      <annotation id="31">
+        <infon key="score">0.9986753</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1212" length="4"/>
+        <text>PI4K</text>
+      </annotation>
+      <annotation id="32">
+        <infon key="score">0.9987841</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:47Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1244" length="20"/>
+        <text>phosphatidylinositol</text>
+      </annotation>
+      <annotation id="33">
+        <infon key="score">0.9988022</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1266" length="2"/>
+        <text>PI</text>
+      </annotation>
+      <annotation id="34">
+        <infon key="score">0.9988311</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1282" length="32"/>
+        <text>phosphatidylinositol 4-phosphate</text>
+      </annotation>
+      <annotation id="35">
+        <infon key="score">0.9987827</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:52Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1316" length="4"/>
+        <text>PI4P</text>
+      </annotation>
+      <annotation id="36">
+        <infon key="score">0.9987464</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:52Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1323" length="4"/>
+        <text>PI4P</text>
+      </annotation>
+      <annotation id="37">
+        <infon key="score">0.99876595</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:52Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1512" length="4"/>
+        <text>PI4P</text>
+      </annotation>
+      <annotation id="38">
+        <infon key="score">0.99704933</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T17:18:20Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1617" length="17"/>
+        <text>phosphoinositides</text>
+      </annotation>
+      <annotation id="616">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:58:00Z</infon>
+        <location offset="1660" length="8"/>
+        <text>clathrin</text>
+      </annotation>
+      <annotation id="39">
+        <infon key="score">0.75636566</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T17:17:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1721" length="42"/>
+        <text>positive-sense single-stranded RNA viruses</text>
+      </annotation>
+      <annotation id="40">
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+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T17:17:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1765" length="12"/>
+        <text>+RNA viruses</text>
+      </annotation>
+      <annotation id="41">
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1814" length="5"/>
+        <text>human</text>
+      </annotation>
+      <annotation id="42">
+        <infon key="score">0.9986339</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1838" length="5"/>
+        <text>human</text>
+      </annotation>
+      <annotation id="43">
+        <infon key="score">0.991351</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:58:30Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="1844" length="5"/>
+        <text>PI4KA</text>
+      </annotation>
+      <annotation id="44">
+        <infon key="score">0.9915263</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="1853" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="45">
+        <infon key="score">0.99670714</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:52Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1888" length="4"/>
+        <text>PI4P</text>
+      </annotation>
+      <annotation id="692">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:16:05Z</infon>
+        <location offset="1968" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="46">
+        <infon key="score">0.9973713</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:57:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2007" length="5"/>
+        <text>viral</text>
+      </annotation>
+      <annotation id="47">
+        <infon key="score">0.94735247</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2052" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>2112</offset>
+      <text>PI4K kinases must be recruited to the correct membrane type to fulfill their enzymatic functions. Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins. In contrast, type III PI4Ks (PI4KA and PI4KB) are soluble cytosolic proteins that are recruited to appropriate membranes indirectly via protein-protein interactions. The recruitment of PI4KA to the plasma membrane by EFR3 and TTC7 is relatively well understood even at the structural level, but, the actual molecular mechanism of PI4KB recruitment to the Golgi is still poorly understood.</text>
+      <annotation id="613">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:37Z</infon>
+        <location offset="2112" length="4"/>
+        <text>PI4K</text>
+      </annotation>
+      <annotation id="619">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:59:37Z</infon>
+        <location offset="2117" length="7"/>
+        <text>kinases</text>
+      </annotation>
+      <annotation id="617">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:58:25Z</infon>
+        <location offset="2210" length="13"/>
+        <text>Type II PI4Ks</text>
+      </annotation>
+      <annotation id="48">
+        <infon key="score">0.9979887</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:58:06Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2225" length="6"/>
+        <text>PI4K2A</text>
+      </annotation>
+      <annotation id="49">
+        <infon key="score">0.99793905</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:58:10Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2236" length="6"/>
+        <text>PI4K2B</text>
+      </annotation>
+      <annotation id="50">
+        <infon key="score">0.99160933</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T17:20:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2248" length="21"/>
+        <text>heavily palmitoylated</text>
+      </annotation>
+      <annotation id="621">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T18:50:50Z</infon>
+        <location offset="2289" length="17"/>
+        <text>membrane proteins</text>
+      </annotation>
+      <annotation id="618">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:59:08Z</infon>
+        <location offset="2321" length="14"/>
+        <text>type III PI4Ks</text>
+      </annotation>
+      <annotation id="51">
+        <infon key="score">0.9966485</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:58:30Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2337" length="5"/>
+        <text>PI4KA</text>
+      </annotation>
+      <annotation id="52">
+        <infon key="score">0.99594134</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2347" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="53">
+        <infon key="score">0.9957268</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:58:30Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2493" length="5"/>
+        <text>PI4KA</text>
+      </annotation>
+      <annotation id="54">
+        <infon key="score">0.99851316</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:58:39Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2525" length="4"/>
+        <text>EFR3</text>
+      </annotation>
+      <annotation id="55">
+        <infon key="score">0.9985258</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:58:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2534" length="4"/>
+        <text>TTC7</text>
+      </annotation>
+      <annotation id="56">
+        <infon key="score">0.9931477</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2638" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>2697</offset>
+      <text>Acyl-coenzyme A binding domain containing protein 3 (ACBD3, also known as GCP60 and PAP7) is a Golgi resident protein. Its membrane localization is mediated by the interaction with the Golgi integral protein golgin B1/giantin. ACBD3 functions as an adaptor protein and signaling hub across cellular signaling pathways. ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2. ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus.</text>
+      <annotation id="57">
+        <infon key="score">0.997024</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:04Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2697" length="51"/>
+        <text>Acyl-coenzyme A binding domain containing protein 3</text>
+      </annotation>
+      <annotation id="58">
+        <infon key="score">0.998771</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2750" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="59">
+        <infon key="score">0.99883205</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:00:26Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2771" length="5"/>
+        <text>GCP60</text>
+      </annotation>
+      <annotation id="60">
+        <infon key="score">0.998755</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:00:30Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2781" length="4"/>
+        <text>PAP7</text>
+      </annotation>
+      <annotation id="61">
+        <infon key="score">0.9971978</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:00:36Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2905" length="9"/>
+        <text>golgin B1</text>
+      </annotation>
+      <annotation id="62">
+        <infon key="score">0.99844754</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:00:41Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2915" length="7"/>
+        <text>giantin</text>
+      </annotation>
+      <annotation id="63">
+        <infon key="score">0.99773693</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2924" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="64">
+        <infon key="score">0.9977296</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3016" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="65">
+        <infon key="score">0.99771476</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:01:06Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3071" length="9"/>
+        <text>golgin A3</text>
+      </annotation>
+      <annotation id="66">
+        <infon key="score">0.99874544</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:01:10Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3081" length="10"/>
+        <text>golgin-160</text>
+      </annotation>
+      <annotation id="67">
+        <infon key="score">0.8595108</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:01:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3115" length="13"/>
+        <text>Numb proteins</text>
+      </annotation>
+      <annotation id="68">
+        <infon key="score">0.9956681</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:01:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3195" length="17"/>
+        <text>metal transporter</text>
+      </annotation>
+      <annotation id="69">
+        <infon key="score">0.99869007</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:01:19Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3213" length="4"/>
+        <text>DMT1</text>
+      </annotation>
+      <annotation id="622">
+        <infon key="type">oligomeric_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:01:50Z</infon>
+        <location offset="3222" length="9"/>
+        <text>monomeric</text>
+      </annotation>
+      <annotation id="70">
+        <infon key="score">0.9980881</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:01:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3232" length="9"/>
+        <text>G protein</text>
+      </annotation>
+      <annotation id="71">
+        <infon key="score">0.9987012</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:01:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3242" length="7"/>
+        <text>Dexras1</text>
+      </annotation>
+      <annotation id="72">
+        <infon key="score">0.82419854</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:01:43Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3262" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="73">
+        <infon key="score">0.99800915</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:01:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3288" length="12"/>
+        <text>lipid kinase</text>
+      </annotation>
+      <annotation id="74">
+        <infon key="score">0.99757916</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3301" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="75">
+        <infon key="score">0.99777836</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3338" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="76">
+        <infon key="score">0.7060604</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:02:16Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="3476" length="20"/>
+        <text>polyglutamine repeat</text>
+      </annotation>
+      <annotation id="77">
+        <infon key="score">0.9988851</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:38:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3508" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="78">
+        <infon key="score">0.99929535</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:01:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3515" length="10"/>
+        <text>huntingtin</text>
+      </annotation>
+      <annotation id="79">
+        <infon key="score">0.662374</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:01:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3553" length="9"/>
+        <text>monomeric</text>
+      </annotation>
+      <annotation id="80">
+        <infon key="score">0.9979733</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:01:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3563" length="9"/>
+        <text>G protein</text>
+      </annotation>
+      <annotation id="81">
+        <infon key="score">0.9988242</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:02:24Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3573" length="4"/>
+        <text>Rhes</text>
+      </annotation>
+      <annotation id="82">
+        <infon key="score">0.99851507</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:02:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3578" length="7"/>
+        <text>Dexras2</text>
+      </annotation>
+      <annotation id="83">
+        <infon key="score">0.9976471</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3587" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="84">
+        <infon key="score">0.9963929</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:57:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3617" length="5"/>
+        <text>viral</text>
+      </annotation>
+      <annotation id="623">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:02:53Z</infon>
+        <location offset="3623" length="26"/>
+        <text>non-structural 3A proteins</text>
+      </annotation>
+      <annotation id="85">
+        <infon key="score">0.991055</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:03:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3679" length="14"/>
+        <text>picornaviruses</text>
+      </annotation>
+      <annotation id="86">
+        <infon key="score">0.99691606</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:03:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3704" length="10"/>
+        <text>poliovirus</text>
+      </annotation>
+      <annotation id="87">
+        <infon key="score">0.9906332</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:03:30Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3716" length="17"/>
+        <text>coxsackievirus B3</text>
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+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:03:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3739" length="11"/>
+        <text>Aichi virus</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>3754</offset>
+      <text>We present a biochemical and structural characterization of the molecular complex composed of the ACBD3 protein and the PI4KB enzyme. We show that ACBD3 can recruit PI4KB to model membranes as well as redirect PI4KB to cellular membranes where it is not naturally found. Our data also show that ACBD3 regulates the enzymatic activity of PI4KB kinase through membrane recruitment rather than allostery.</text>
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+        <infon key="score">0.9988179</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:03:42Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>biochemical and structural characterization</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>ACBD3</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>PI4KB</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <text>PI4KB</text>
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+        <infon key="identifier">PR:</infon>
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+        <text>ACBD3</text>
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+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:44:10Z</infon>
+        <location offset="4069" length="18"/>
+        <text>enzymatic activity</text>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
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+        <text>PI4KB</text>
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+      <annotation id="624">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:04:05Z</infon>
+        <location offset="4097" length="6"/>
+        <text>kinase</text>
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+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_1</infon>
+      <offset>4156</offset>
+      <text>Results</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
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+      <text>ACBD3 and PI4KB interact with 1:1 stoichiometry with submicromolar affinity</text>
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+        <text>ACBD3</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="4174" length="5"/>
+        <text>PI4KB</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>4240</offset>
+      <text>In order to verify the interactions between ACBD3 and PI4KB we expressed and purified both proteins. To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423–522) was removed (Fig. 1A). This internal deletion does not significantly affect the kinase activity(SI Fig. 1A) or interaction with ACBD3 (SI Fig. 1B,C). In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction. Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain. We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel).</text>
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+        <text>ACBD3</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>expressed and purified</text>
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:04:34Z</infon>
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+        <text>bacterial expression</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:04:55Z</infon>
+        <location offset="4388" length="31"/>
+        <text>intrinsically disordered region</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4423" length="5"/>
+        <text>PI4KB</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>423–522</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:05:37Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>removed</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:05:34Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4485" length="8"/>
+        <text>deletion</text>
+      </annotation>
+      <annotation id="625">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:04:05Z</infon>
+        <location offset="4528" length="6"/>
+        <text>kinase</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4576" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:05:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4604" length="22"/>
+        <text>in vitro binding assay</text>
+      </annotation>
+      <annotation id="107">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4628" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
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+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:05:30Z</infon>
+        <location offset="4634" length="38"/>
+        <text>co-purified with the NiNTA-immobilized</text>
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+        <infon key="score">0.7604731</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:05:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4684" length="14"/>
+        <text>His6GB1-tagged</text>
+      </annotation>
+      <annotation id="109">
+        <infon key="score">0.997761</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4699" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="110">
+        <infon key="score">0.9989556</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:05:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4769" length="26"/>
+        <text>mammalian two-hybrid assay</text>
+      </annotation>
+      <annotation id="738">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T16:06:50Z</infon>
+        <location offset="4821" length="9"/>
+        <text>localized</text>
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+      <annotation id="111">
+        <infon key="score">0.96972895</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4855" length="8"/>
+        <text>Q domain</text>
+      </annotation>
+      <annotation id="112">
+        <infon key="score">0.99847716</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4867" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="113">
+        <infon key="score">0.99634355</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:08:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4913" length="9"/>
+        <text>glutamine</text>
+      </annotation>
+      <annotation id="674">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:08:36Z</infon>
+        <location offset="4941" length="17"/>
+        <text>N-terminal region</text>
+      </annotation>
+      <annotation id="114">
+        <infon key="score">0.99838805</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4962" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="115">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:05:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4982" length="14"/>
+        <text>helical domain</text>
+      </annotation>
+      <annotation id="116">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:05:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5001" length="9"/>
+        <text>expressed</text>
+      </annotation>
+      <annotation id="117">
+        <infon key="score">0.8818047</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:06Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5015" length="8"/>
+        <text>Q domain</text>
+      </annotation>
+      <annotation id="118">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="5027" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="119">
+        <infon key="score">0.99775714</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:05:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5043" length="7"/>
+        <text>241–308</text>
+      </annotation>
+      <annotation id="675">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:08:36Z</infon>
+        <location offset="5060" length="17"/>
+        <text>N-terminal region</text>
+      </annotation>
+      <annotation id="120">
+        <infon key="score">0.99809617</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="5081" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="121">
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+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:05:51Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5097" length="4"/>
+        <text>1–68</text>
+      </annotation>
+      <annotation id="122">
+        <infon key="score">0.9982603</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:05:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5106" length="7"/>
+        <text>E. coli</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>5275</offset>
+      <text>Because it has been reported that ACBD3 can dimerize in a mammalian two-hybrid assay, we were interested in determining the stoichiometry of the ACBD3:PI4KB protein complex. The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively. This result suggests that both proteins are monomeric and the stoichiometry of the ACBD3: PI4KB protein complex is 1:1 (Fig. 1C, left panel). Similar results were obtained for the complex of the Q domain of ACBD3 and the N-terminal region of PI4KB (Fig. 1C, right panel). We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR). SPR measurements revealed a strong interaction with a Kd value of 320 +/−130 nM (Fig. 1D, SI Fig. 1D). We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range.</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="5309" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="124">
+        <infon key="score">0.9589814</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:56Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5319" length="8"/>
+        <text>dimerize</text>
+      </annotation>
+      <annotation id="125">
+        <infon key="score">0.99886334</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:05:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5333" length="26"/>
+        <text>mammalian two-hybrid assay</text>
+      </annotation>
+      <annotation id="126">
+        <infon key="score">0.9991631</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:16Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="5420" length="11"/>
+        <text>ACBD3:PI4KB</text>
+      </annotation>
+      <annotation id="127">
+        <infon key="score">0.9983687</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5453" length="26"/>
+        <text>sedimentation coefficients</text>
+      </annotation>
+      <annotation id="128">
+        <infon key="score">0.9965487</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
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+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:41Z</infon>
+        <location offset="5499" length="5"/>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:16Z</infon>
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+      </annotation>
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+        <infon key="type">experimental_method</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>analytical ultracentrifugation</text>
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:08:09Z</infon>
+        <location offset="5647" length="17"/>
+        <text>molecular weights</text>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>monomeric</text>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:07:34Z</infon>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:08:36Z</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6068" length="11"/>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6090" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
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+        <infon key="score">0.9989314</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:07:05Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>surface plasmon resonance</text>
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+      <annotation id="140">
+        <infon key="score">0.9986051</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:07:09Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:07:09Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:07:52Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>Kd</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <text>PI4KB</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6314" length="5"/>
+        <text>ACBD3</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:08:36Z</infon>
+        <location offset="6328" length="17"/>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6349" length="5"/>
+        <text>PI4KB</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:08:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6384" length="21"/>
+        <text>dissociation constant</text>
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+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>6446</offset>
+      <text>Structural analysis of the ACBD3:PI4KB complex</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:09:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6446" length="19"/>
+        <text>Structural analysis</text>
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+        <infon key="score">0.9992986</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:16Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="6473" length="11"/>
+        <text>ACBD3:PI4KB</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>6493</offset>
+      <text>Full length ACBD3 and PI4KB both contain large intrinsically disordered regions that impede crystallization. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of the complex to determine which parts of the complex are well folded (SI Fig. 2). However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB.</text>
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+        <infon key="score">0.9989967</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:07:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6493" length="11"/>
+        <text>Full length</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6505" length="5"/>
+        <text>ACBD3</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <text>PI4KB</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:09:37Z</infon>
+        <location offset="6540" length="32"/>
+        <text>intrinsically disordered regions</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:09:43Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6610" length="45"/>
+        <text>hydrogen-deuterium exchange mass spectrometry</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:09:48Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6657" length="6"/>
+        <text>HDX-MS</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:10:09Z</infon>
+        <location offset="6733" length="11"/>
+        <text>well folded</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:10:37Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6792" length="8"/>
+        <text>crystals</text>
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:10:31Z</infon>
+        <location offset="6831" length="9"/>
+        <text>truncated</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6875" length="5"/>
+        <text>ACBD3</text>
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+      <annotation id="647">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <location offset="6881" length="8"/>
+        <text>Q domain</text>
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+      <annotation id="677">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:08:36Z</infon>
+        <location offset="6898" length="17"/>
+        <text>N-terminal region</text>
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+        <infon key="score">0.9982198</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6919" length="5"/>
+        <text>PI4KB</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>6926</offset>
+      <text>For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization. As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules. Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously. Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44. Over 93% of non-exchangeable side-chain signals were assigned for the free ACBD3 Q domain. Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances. The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB). Assignments obtained for non-exchangeable side-chain signals were over 99% complete. The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation. Structural statistics for the final water-refined sets of structures are shown in SI Table 1.</text>
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+        <infon key="updated_at">2023-09-21T14:10:45Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6958" length="20"/>
+        <text>isotopically labeled</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6979" length="5"/>
+        <text>ACBD3</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
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+        <infon key="updated_at">2023-09-21T14:10:45Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7019" length="5"/>
+        <text>ACBD3</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <location offset="7025" length="8"/>
+        <text>Q domain</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7034" length="5"/>
+        <text>PI4KB</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:08:36Z</infon>
+        <location offset="7040" length="17"/>
+        <text>N-terminal region</text>
+      </annotation>
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+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:11:28Z</infon>
+        <location offset="7083" length="16"/>
+        <text>NMR spectroscopy</text>
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+        <infon key="updated_at">2023-09-21T14:08:36Z</infon>
+        <location offset="7140" length="17"/>
+        <text>N-terminal region</text>
+      </annotation>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:11:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7190" length="13"/>
+        <text>co-expression</text>
+      </annotation>
+      <annotation id="164">
+        <infon key="score">0.99493366</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:12:01Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7285" length="3"/>
+        <text>15N</text>
+      </annotation>
+      <annotation id="165">
+        <infon key="score">0.9964359</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:12:05Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7289" length="3"/>
+        <text>13C</text>
+      </annotation>
+      <annotation id="688">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:12:45Z</infon>
+        <location offset="7293" length="8"/>
+        <text>labelled</text>
+      </annotation>
+      <annotation id="166">
+        <infon key="score">0.9989409</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:12:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7381" length="4"/>
+        <text>free</text>
+      </annotation>
+      <annotation id="167">
+        <infon key="score">0.9986345</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7386" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="650">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <location offset="7392" length="8"/>
+        <text>Q domain</text>
+      </annotation>
+      <annotation id="690">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:15:09Z</infon>
+        <location offset="7440" length="14"/>
+        <text>2D 15N/1H HSQC</text>
+      </annotation>
+      <annotation id="168">
+        <infon key="score">0.99490064</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:11:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7455" length="7"/>
+        <text>spectra</text>
+      </annotation>
+      <annotation id="686">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:11:53Z</infon>
+        <location offset="7528" length="28"/>
+        <text>triple-resonance experiments</text>
+      </annotation>
+      <annotation id="169">
+        <infon key="score">0.99697316</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:12:01Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7607" length="3"/>
+        <text>15N</text>
+      </annotation>
+      <annotation id="170">
+        <infon key="score">0.97899354</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:12:10Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7615" length="2"/>
+        <text>1H</text>
+      </annotation>
+      <annotation id="171">
+        <infon key="score">0.9988808</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:12:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7627" length="4"/>
+        <text>free</text>
+      </annotation>
+      <annotation id="172">
+        <infon key="score">0.9985446</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7632" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="651">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <location offset="7638" length="8"/>
+        <text>Q domain</text>
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+      <annotation id="689">
+        <infon key="type">residue_range</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:13:11Z</infon>
+        <location offset="7726" length="9"/>
+        <text>Met1-Lys4</text>
+      </annotation>
+      <annotation id="173">
+        <infon key="score">0.9995875</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:13:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7741" length="5"/>
+        <text>Gln44</text>
+      </annotation>
+      <annotation id="174">
+        <infon key="score">0.99882644</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:12:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7818" length="4"/>
+        <text>free</text>
+      </annotation>
+      <annotation id="175">
+        <infon key="score">0.9986002</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7823" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="652">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <location offset="7829" length="8"/>
+        <text>Q domain</text>
+      </annotation>
+      <annotation id="176">
+        <infon key="score">0.9974669</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:15:33Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7924" length="3"/>
+        <text>Gln</text>
+      </annotation>
+      <annotation id="177">
+        <infon key="score">0.99632865</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:15:41Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7935" length="3"/>
+        <text>Gln</text>
+      </annotation>
+      <annotation id="178">
+        <infon key="score">0.9996074</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:13:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7951" length="5"/>
+        <text>Gln44</text>
+      </annotation>
+      <annotation id="179">
+        <infon key="score">0.9996132</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:14:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7972" length="5"/>
+        <text>Gln48</text>
+      </annotation>
+      <annotation id="180">
+        <infon key="score">0.9880802</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:13:29Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8073" length="9"/>
+        <text>glutamine</text>
+      </annotation>
+      <annotation id="181">
+        <infon key="score">0.907675</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:11:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8159" length="7"/>
+        <text>spectra</text>
+      </annotation>
+      <annotation id="182">
+        <infon key="score">0.99920267</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:15:49Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8261" length="3"/>
+        <text>Gln</text>
+      </annotation>
+      <annotation id="183">
+        <infon key="score">0.9972893</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8266" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="184">
+        <infon key="score">0.99957603</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:14:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8277" length="4"/>
+        <text>Ala2</text>
+      </annotation>
+      <annotation id="185">
+        <infon key="score">0.9949039</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8283" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="186">
+        <infon key="score">0.907263</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:12:01Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8401" length="3"/>
+        <text>15N</text>
+      </annotation>
+      <annotation id="187">
+        <infon key="score">0.95269054</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:12:06Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8406" length="3"/>
+        <text>13C</text>
+      </annotation>
+      <annotation id="687">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:12:10Z</infon>
+        <location offset="8414" length="2"/>
+        <text>1H</text>
+      </annotation>
+      <annotation id="188">
+        <infon key="score">0.9967265</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:16:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8475" length="4"/>
+        <text>NOEs</text>
+      </annotation>
+      <annotation id="691">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:15:21Z</infon>
+        <location offset="8498" length="20"/>
+        <text>3D 15N/1H NOESY-HSQC</text>
+      </annotation>
+      <annotation id="189">
+        <infon key="score">0.9968702</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:15:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8523" length="17"/>
+        <text>13C/1H HMQC-NOESY</text>
+      </annotation>
+      <annotation id="190">
+        <infon key="score">0.98942995</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:11:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8541" length="7"/>
+        <text>spectra</text>
+      </annotation>
+      <annotation id="693">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:16:25Z</infon>
+        <location offset="8580" length="22"/>
+        <text>structural calculation</text>
+      </annotation>
+      <annotation id="191">
+        <infon key="score">0.7945851</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:16:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8604" length="21"/>
+        <text>Structural statistics</text>
+      </annotation>
+      <annotation id="192">
+        <infon key="score">0.94309074</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:16:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8662" length="10"/>
+        <text>structures</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>8698</offset>
+      <text>This structure revealed that the Q domain forms a two helix hairpin. The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A). Preceding the kinase helix are three ordered residues (Val42, Ile43, and Asp44) that also contribute to the interaction (Fig. 2B). The remaining part of the PI4KB N-termini, however, is disordered (SI Fig. 5). Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C).</text>
+      <annotation id="193">
+        <infon key="score">0.998018</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8703" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="194">
+        <infon key="score">0.99866307</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8731" length="8"/>
+        <text>Q domain</text>
+      </annotation>
+      <annotation id="195">
+        <infon key="score">0.9987442</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:25:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8748" length="17"/>
+        <text>two helix hairpin</text>
+      </annotation>
+      <annotation id="694">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:25:28Z</infon>
+        <location offset="8777" length="5"/>
+        <text>helix</text>
+      </annotation>
+      <annotation id="695">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:25:28Z</infon>
+        <location offset="8813" length="5"/>
+        <text>helix</text>
+      </annotation>
+      <annotation id="196">
+        <infon key="score">0.98671126</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:26:21Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8851" length="18"/>
+        <text>three helix bundle</text>
+      </annotation>
+      <annotation id="197">
+        <infon key="score">0.972546</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:25:28Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8900" length="5"/>
+        <text>helix</text>
+      </annotation>
+      <annotation id="198">
+        <infon key="score">0.99828124</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8913" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="199">
+        <infon key="score">0.9977347</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:26:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8940" length="5"/>
+        <text>44–64</text>
+      </annotation>
+      <annotation id="200">
+        <infon key="score">0.9990201</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:25:41Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8985" length="12"/>
+        <text>kinase helix</text>
+      </annotation>
+      <annotation id="201">
+        <infon key="score">0.9991654</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:25:42Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9024" length="12"/>
+        <text>kinase helix</text>
+      </annotation>
+      <annotation id="202">
+        <infon key="score">0.99960405</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:26:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9065" length="5"/>
+        <text>Val42</text>
+      </annotation>
+      <annotation id="203">
+        <infon key="score">0.9996008</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:26:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9072" length="5"/>
+        <text>Ile43</text>
+      </annotation>
+      <annotation id="204">
+        <infon key="score">0.99959177</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:26:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9083" length="5"/>
+        <text>Asp44</text>
+      </annotation>
+      <annotation id="205">
+        <infon key="score">0.9983835</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9167" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:27:13Z</infon>
+        <infon key="identifier">GO:</infon>
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+        <text>PI4KB:ACBD3</text>
+      </annotation>
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:27:37Z</infon>
+        <location offset="9250" length="28"/>
+        <text>interactions are hydrophobic</text>
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+        <infon key="score">0.9969462</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:27:40Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9301" length="14"/>
+        <text>hydrogen bonds</text>
+      </annotation>
+      <annotation id="208">
+        <infon key="score">0.9955851</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9343" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="209">
+        <infon key="score">0.99958163</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:27:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9349" length="6"/>
+        <text>Tyr261</text>
+      </annotation>
+      <annotation id="210">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9360" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:27:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9366" length="5"/>
+        <text>His63</text>
+      </annotation>
+      <annotation id="212">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9402" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="213">
+        <infon key="score">0.99957126</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:27:56Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9408" length="6"/>
+        <text>Tyr288</text>
+      </annotation>
+      <annotation id="214">
+        <infon key="score">0.9939761</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9423" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="215">
+        <infon key="score">0.99950206</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:26:44Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9439" length="5"/>
+        <text>Asp44</text>
+      </annotation>
+      <annotation id="216">
+        <infon key="score">0.99854785</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9490" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="217">
+        <infon key="score">0.9942094</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:25:28Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9496" length="5"/>
+        <text>helix</text>
+      </annotation>
+      <annotation id="218">
+        <infon key="score">0.99885774</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:28:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9505" length="11"/>
+        <text>amphipathic</text>
+      </annotation>
+      <annotation id="219">
+        <infon key="score">0.991676</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:28:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9525" length="19"/>
+        <text>hydrophobic surface</text>
+      </annotation>
+      <annotation id="220">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9558" length="8"/>
+        <text>Q domain</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>9580</offset>
+      <text>To corroborate the structural data, we introduced a number of point mutations and validated their effect on complex formation using an in vitro pull-down assay (Fig. 2D). Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:28:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9599" length="15"/>
+        <text>structural data</text>
+      </annotation>
+      <annotation id="222">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:28:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9619" length="10"/>
+        <text>introduced</text>
+      </annotation>
+      <annotation id="223">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:28:25Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9642" length="15"/>
+        <text>point mutations</text>
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+      <annotation id="224">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:28:38Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9715" length="24"/>
+        <text>in vitro pull-down assay</text>
+      </annotation>
+      <annotation id="225">
+        <infon key="score">0.99917686</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:28:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9751" length="9"/>
+        <text>Wild type</text>
+      </annotation>
+      <annotation id="226">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9761" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="227">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T16:15:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9775" length="11"/>
+        <text>co-purified</text>
+      </annotation>
+      <annotation id="228">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:28:28Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9823" length="11"/>
+        <text>His6-tagged</text>
+      </annotation>
+      <annotation id="229">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:28:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9835" length="9"/>
+        <text>wild type</text>
+      </annotation>
+      <annotation id="230">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9845" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="231">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9871" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="232">
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+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:29:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9877" length="4"/>
+        <text>V42A</text>
+      </annotation>
+      <annotation id="233">
+        <infon key="score">0.9988329</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:29:09Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9886" length="4"/>
+        <text>V47A</text>
+      </annotation>
+      <annotation id="234">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:28:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9891" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="235">
+        <infon key="score">0.8356905</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:28:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9913" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="236">
+        <infon key="score">0.9989662</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:28:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9941" length="17"/>
+        <text>binding interface</text>
+      </annotation>
+      <annotation id="237">
+        <infon key="score">0.99909496</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:28:53Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9960" length="4"/>
+        <text>I43A</text>
+      </annotation>
+      <annotation id="238">
+        <infon key="score">0.9991035</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:28:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9966" length="4"/>
+        <text>V55A</text>
+      </annotation>
+      <annotation id="239">
+        <infon key="score">0.9991026</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:29:00Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9972" length="4"/>
+        <text>L56A</text>
+      </annotation>
+      <annotation id="240">
+        <infon key="score">0.9991299</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:28:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9993" length="9"/>
+        <text>wild type</text>
+      </annotation>
+      <annotation id="241">
+        <infon key="score">0.99872416</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10003" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="242">
+        <infon key="score">0.99819034</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10029" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="243">
+        <infon key="score">0.9990381</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:29:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10035" length="5"/>
+        <text>Y266A</text>
+      </annotation>
+      <annotation id="244">
+        <infon key="score">0.9991756</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:38:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10041" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="245">
+        <infon key="score">0.99897647</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:29:18Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10070" length="5"/>
+        <text>Y285A</text>
+      </annotation>
+      <annotation id="246">
+        <infon key="score">0.99898046</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:38:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10076" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="247">
+        <infon key="score">0.9989837</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:29:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10101" length="5"/>
+        <text>F258A</text>
+      </annotation>
+      <annotation id="248">
+        <infon key="score">0.9990701</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:29:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10108" length="5"/>
+        <text>H284A</text>
+      </annotation>
+      <annotation id="249">
+        <infon key="score">0.9990722</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:29:30Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10119" length="5"/>
+        <text>Y288A</text>
+      </annotation>
+      <annotation id="250">
+        <infon key="score">0.99653506</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:28:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10125" length="7"/>
+        <text>mutants</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>10144</offset>
+      <text>ACBD3 efficiently recruits the PI4KB enzyme to membranes</text>
+      <annotation id="251">
+        <infon key="score">0.9836066</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10144" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="252">
+        <infon key="score">0.9976108</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10175" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>10201</offset>
+      <text>We next sought to determine if the ACBD3:PI4KB interaction drives membrane localization of the PI4KB enzyme. To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate – the PI lipid. We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A).</text>
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+        <infon key="score">0.9657908</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:16Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="10236" length="11"/>
+        <text>ACBD3:PI4KB</text>
+      </annotation>
+      <annotation id="254">
+        <infon key="score">0.99302506</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10296" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="255">
+        <infon key="score">0.92459184</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T16:15:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10346" length="36"/>
+        <text>in vitro membrane recruitment system</text>
+      </annotation>
+      <annotation id="256">
+        <infon key="score">0.68418074</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:30:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10389" length="26"/>
+        <text>Giant Unilamellar Vesicles</text>
+      </annotation>
+      <annotation id="257">
+        <infon key="score">0.94216317</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:30:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10417" length="4"/>
+        <text>GUVs</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10438" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
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+        <infon key="score">0.99724686</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <text>PI</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10487" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="261">
+        <infon key="score">0.92744774</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:04:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10493" length="6"/>
+        <text>kinase</text>
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+      <annotation id="739">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T16:06:50Z</infon>
+        <location offset="10517" length="9"/>
+        <text>localized</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:30:28Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>GUVs</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <text>ACBD3</text>
+      </annotation>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:30:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10637" length="4"/>
+        <text>GUVs</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10665" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="700">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:35:53Z</infon>
+        <location offset="10679" length="18"/>
+        <text>DGS-NTA (Ni) lipid</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10716" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>10759</offset>
+      <text>We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel). We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B). Given this result, overexpression of the Q domain should also interfere with the PI4KB dependent Golgi functions. Ceramide transport and accumulation in Golgi is a well-known PI4KB dependent process. We have used fluorescently labeled ceramide and analyzed its trafficking in non-transfected cells and cell overexpressing the Q domain. As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi.</text>
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+        <infon key="identifier">PR:</infon>
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+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="701">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:36:14Z</infon>
+        <location offset="10814" length="20"/>
+        <text>localization signals</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:35:07Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>overexpression</text>
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+      <annotation id="653">
+        <infon key="type">structure_element</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>kinase</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="10983" length="5"/>
+        <text>PI4KB</text>
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+      <annotation id="703">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:36:55Z</infon>
+        <location offset="11018" length="14"/>
+        <text>overexpressing</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:34:35Z</infon>
+        <location offset="11037" length="3"/>
+        <text>GFP</text>
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+      <annotation id="654">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <location offset="11043" length="8"/>
+        <text>Q domain</text>
+      </annotation>
+      <annotation id="706">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:37:30Z</infon>
+        <location offset="11110" length="6"/>
+        <text>signal</text>
+      </annotation>
+      <annotation id="626">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:04:05Z</infon>
+        <location offset="11152" length="6"/>
+        <text>kinase</text>
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+      <annotation id="707">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:37:39Z</infon>
+        <location offset="11264" length="6"/>
+        <text>signal</text>
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+      <annotation id="272">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:35:06Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11279" length="14"/>
+        <text>overexpression</text>
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+      <annotation id="273">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:42:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11297" length="3"/>
+        <text>GFP</text>
+      </annotation>
+      <annotation id="274">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:34:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11312" length="11"/>
+        <text>non-binding</text>
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+      <annotation id="275">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11324" length="8"/>
+        <text>Q domain</text>
+      </annotation>
+      <annotation id="276">
+        <infon key="score">0.737417</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:38:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11333" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="702">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:36:44Z</infon>
+        <location offset="11361" length="12"/>
+        <text>localization</text>
+      </annotation>
+      <annotation id="277">
+        <infon key="score">0.9970674</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11392" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="278">
+        <infon key="score">0.9735685</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:35:07Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11428" length="14"/>
+        <text>overexpression</text>
+      </annotation>
+      <annotation id="655">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <location offset="11450" length="8"/>
+        <text>Q domain</text>
+      </annotation>
+      <annotation id="279">
+        <infon key="score">0.99336</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11490" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="280">
+        <infon key="score">0.9975262</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:37:51Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11523" length="8"/>
+        <text>Ceramide</text>
+      </annotation>
+      <annotation id="281">
+        <infon key="score">0.52904665</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11584" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="282">
+        <infon key="score">0.81602323</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:37:56Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11622" length="21"/>
+        <text>fluorescently labeled</text>
+      </annotation>
+      <annotation id="283">
+        <infon key="score">0.998357</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:37:51Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11644" length="8"/>
+        <text>ceramide</text>
+      </annotation>
+      <annotation id="705">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T18:50:57Z</infon>
+        <location offset="11716" length="14"/>
+        <text>overexpressing</text>
+      </annotation>
+      <annotation id="656">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <location offset="11735" length="8"/>
+        <text>Q domain</text>
+      </annotation>
+      <annotation id="284">
+        <infon key="score">0.998293</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:37:50Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11784" length="8"/>
+        <text>ceramide</text>
+      </annotation>
+      <annotation id="704">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:37:01Z</infon>
+        <location offset="11819" length="10"/>
+        <text>expressing</text>
+      </annotation>
+      <annotation id="285">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:35:24Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11834" length="2"/>
+        <text>wt</text>
+      </annotation>
+      <annotation id="657">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <location offset="11837" length="8"/>
+        <text>Q domain</text>
+      </annotation>
+      <annotation id="286">
+        <infon key="score">0.9462286</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T17:18:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11869" length="3"/>
+        <text>RFP</text>
+      </annotation>
+      <annotation id="287">
+        <infon key="score">0.99905616</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:38:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11880" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="658">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <location offset="11887" length="8"/>
+        <text>Q domain</text>
+      </annotation>
+      <annotation id="288">
+        <infon key="score">0.9983005</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:37:51Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11908" length="8"/>
+        <text>ceramide</text>
+      </annotation>
+      <annotation id="289">
+        <infon key="score">0.9988522</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:16Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="11952" length="11"/>
+        <text>ACBD3:PI4KB</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>12033</offset>
+      <text>We further analyzed the function of ACBD3:PI4KB interaction in membrane recruitment of PI4KB in living cells using fluorescently tagged proteins. We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system. We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP. The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D). We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin. As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase. In every case the ACDB3 Q domain was rapidly (within 5 minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2). Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel). However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel).</text>
+      <annotation id="615">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:16Z</infon>
+        <location offset="12069" length="11"/>
+        <text>ACBD3:PI4KB</text>
+      </annotation>
+      <annotation id="290">
+        <infon key="score">0.9982387</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12120" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
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+      <text>To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate. We observed no effect on the kinase activity of PI4KB (Fig. 4A) suggesting that ACBD3 does not directly affect the enzyme (e.g. induction of a conformation change). However, in vivo ACBD3 is located at the Golgi membranes, whereas in this experiment, ACBD3 was located in the solution and PI is provided as micelles. We therefore designed a more physiologically relevant experiment. For this, we again turned to the GUV system with ACBD3 localized to the GUV membrane. The GUVs contained 10% PI to serve as a substrate for PI4KB kinase. The buffer also contained CFP-SidC, which binds to PI4P with nanomolar affinity. This enabled visualization of the kinase reaction using a confocal microscope. We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3. Reaction was also performed in the absence of ATP as a negative control (Fig. 4B). These experiments showed that PI4KB enzymatic activity increases when ACBD3 is membrane localized (Fig. 4C, SI Fig. 6). We conclude that enzyme activation proceeds through a membrane recruitment mechanism.</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <text>PI4P</text>
+      </annotation>
+      <annotation id="633">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:04:05Z</infon>
+        <location offset="14184" length="6"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:43:41Z</infon>
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+        <text>confocal microscope</text>
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+        <text>phosphorylation</text>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="updated_at">2023-09-21T14:04:05Z</infon>
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+        <text>alone</text>
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+        <infon key="type">protein_type</infon>
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+        <infon key="updated_at">2023-09-21T14:04:05Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <text>absence of</text>
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+        <infon key="identifier">CHEBI:</infon>
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+        <text>ATP</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:44:11Z</infon>
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+        <infon key="identifier">PR:</infon>
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+        <text>ACBD3</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">title_1</infon>
+      <offset>14664</offset>
+      <text>Discussion</text>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>14675</offset>
+      <text>Membrane recruitment of PI4KB enzyme is crucial to ensure its proper function at the Golgi and TGN. However, the molecular mechanism and structural basis for PI4KB interaction with the membrane is poorly understood. In principle, any of the binding partners of PI4KB could play a role in membrane recruitment. To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.</text>
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+        <text>PI4KB</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="14833" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="14936" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="357">
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="358">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="15063" length="13"/>
+        <text>small GTPases</text>
+      </annotation>
+      <annotation id="359">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="15077" length="5"/>
+        <text>Rab11</text>
+      </annotation>
+      <annotation id="360">
+        <infon key="score">0.99702436</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="15087" length="4"/>
+        <text>Arf1</text>
+      </annotation>
+      <annotation id="361">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="15112" length="36"/>
+        <text>acyl-CoA binding domain containing 3</text>
+      </annotation>
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+        <infon key="type">protein</infon>
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+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="15150" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="363">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:44:49Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="15166" length="25"/>
+        <text>neuronal calcium sensor-1</text>
+      </annotation>
+      <annotation id="364">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:44:53Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="15193" length="5"/>
+        <text>NCS-1</text>
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+      <annotation id="365">
+        <infon key="score">0.9992748</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:45:19Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="15213" length="9"/>
+        <text>frequenin</text>
+      </annotation>
+      <annotation id="366">
+        <infon key="score">0.9982541</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:45:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15226" length="5"/>
+        <text>yeast</text>
+      </annotation>
+      <annotation id="367">
+        <infon key="score">0.99290615</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:44:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15241" length="15"/>
+        <text>14-3-3 proteins</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>15258</offset>
+      <text>The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase. Although Rab11 does not appear to be required for recruitment of PI4KB to the Golgi, PI4KB is required for Golgi recruitment of Rab11. Arf1, the other small GTP binding protein, is known to influence the activity and localization of PI4KB, but it does not appear to interact directly with PI4KB (our unpublished data). The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear. NCS-1 is an N-terminally myristoylated protein that participates in exocytosis. It is expressed only in certain cell types, suggesting that if it contributes to PI4KB membrane recruitment, it does so in a tissues specific manner. The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation. However, 14-3-3 proteins do not appear to interfere with membrane recruitment of this kinase. ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase. For these reasons we focused on the interaction of the PI4KB enzyme with the Golgi resident ACBD3 protein in this study.</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+      <text>Here we present the mechanism for membrane recruitment of PI4KB by the Golgi resident ACBD3 protein. We show that these proteins interact directly with a Kd value in the submicromolar range. The interaction is sufficient to recruit PI4KB to model membranes in vitro as well as to the mitochondria where PI4KB is never naturally found. To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain. The Q domain adopts a helical hairpin fold that is further stabilized upon binding the kinase helix (Fig. 2A). Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase. Based on our and previously published structures we built a pseudoatomic model of PI4KB multi-protein assembly on the membrane (Fig. 5) that illustrates how the enzyme is recruited and positioned towards its lipidic substrate and how it in turn recruits Rab11.</text>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>solution structure</text>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:16Z</infon>
+        <infon key="identifier">GO:</infon>
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+        <text>ACBD3:PI4KB</text>
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+        <infon key="updated_at">2023-09-21T14:08:36Z</infon>
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+        <infon key="identifier">SO:</infon>
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+        <text>short amphipatic helix</text>
+      </annotation>
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+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T17:22:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17321" length="5"/>
+        <text>44–64</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17343" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
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+        <infon key="score">0.99783504</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17349" length="8"/>
+        <text>Q domain</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17363" length="8"/>
+        <text>Q domain</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:47:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17381" length="20"/>
+        <text>helical hairpin fold</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="17446" length="12"/>
+        <text>kinase helix</text>
+      </annotation>
+      <annotation id="425">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>kinase</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17643" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="427">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:04:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17684" length="6"/>
+        <text>kinase</text>
+      </annotation>
+      <annotation id="638">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:04:05Z</infon>
+        <location offset="17741" length="6"/>
+        <text>kinase</text>
+      </annotation>
+      <annotation id="428">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="17847" length="6"/>
+        <text>kinase</text>
+      </annotation>
+      <annotation id="429">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:16:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17893" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="430">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:47:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17915" length="18"/>
+        <text>pseudoatomic model</text>
+      </annotation>
+      <annotation id="431">
+        <infon key="score">0.99829084</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17937" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="432">
+        <infon key="score">0.9989454</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:45:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18109" length="5"/>
+        <text>Rab11</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>18118</offset>
+      <text>+RNA viruses replicate at specific PI4P-enriched membranous compartments. These are called replication factories (because they enhance viral replication) or membranous webs (because of their appearance under the electron microscope). To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid. Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3. Our data suggest that they could do this via 3A:ACBD3:PI4KB complex formation. The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication. This could eventually have implications for therapeutic intervention to combat picornaviruses-mediated diseases ranging from polio to the common cold.</text>
+      <annotation id="433">
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+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:47:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18118" length="12"/>
+        <text>+RNA viruses</text>
+      </annotation>
+      <annotation id="434">
+        <infon key="score">0.99769825</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:52Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18153" length="4"/>
+        <text>PI4P</text>
+      </annotation>
+      <annotation id="435">
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+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:57:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18253" length="5"/>
+        <text>viral</text>
+      </annotation>
+      <annotation id="436">
+        <infon key="score">0.99788505</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:48:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18387" length="7"/>
+        <text>viruses</text>
+      </annotation>
+      <annotation id="614">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:38Z</infon>
+        <location offset="18437" length="4"/>
+        <text>PI4K</text>
+      </annotation>
+      <annotation id="620">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:59:38Z</infon>
+        <location offset="18442" length="7"/>
+        <text>kinases</text>
+      </annotation>
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+        <infon key="score">0.9990773</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:52Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18480" length="4"/>
+        <text>PI4P</text>
+      </annotation>
+      <annotation id="438">
+        <infon key="score">0.699083</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T17:18:55Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18485" length="5"/>
+        <text>lipid</text>
+      </annotation>
+      <annotation id="759">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T17:21:20Z</infon>
+        <location offset="18492" length="26"/>
+        <text>Non-structural 3A proteins</text>
+      </annotation>
+      <annotation id="439">
+        <infon key="score">0.99718</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:03:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18529" length="14"/>
+        <text>picornaviruses</text>
+      </annotation>
+      <annotation id="440">
+        <infon key="score">0.9856564</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:48:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18553" length="11"/>
+        <text>Enterovirus</text>
+      </annotation>
+      <annotation id="441">
+        <infon key="score">0.99433774</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:48:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18571" length="10"/>
+        <text>poliovirus</text>
+      </annotation>
+      <annotation id="725">
+        <infon key="type">species</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:49:06Z</infon>
+        <location offset="18583" length="17"/>
+        <text>coxsackievirus-B3</text>
+      </annotation>
+      <annotation id="726">
+        <infon key="type">species</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:49:20Z</infon>
+        <location offset="18602" length="13"/>
+        <text>rhinovirus-14</text>
+      </annotation>
+      <annotation id="442">
+        <infon key="score">0.7567454</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:49:24Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18621" length="9"/>
+        <text>Kobuvirus</text>
+      </annotation>
+      <annotation id="443">
+        <infon key="score">0.9271165</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:49:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18637" length="13"/>
+        <text>Aichi virus-1</text>
+      </annotation>
+      <annotation id="444">
+        <infon key="score">0.99703985</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18682" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="445">
+        <infon key="score">0.99915993</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:49:48Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="18734" length="14"/>
+        <text>3A:ACBD3:PI4KB</text>
+      </annotation>
+      <annotation id="446">
+        <infon key="score">0.99662316</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18772" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="447">
+        <infon key="score">0.99881554</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18789" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="448">
+        <infon key="score">0.9772059</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:06:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18795" length="8"/>
+        <text>Q domain</text>
+      </annotation>
+      <annotation id="449">
+        <infon key="score">0.99930453</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:25:42Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18812" length="12"/>
+        <text>kinase helix</text>
+      </annotation>
+      <annotation id="450">
+        <infon key="score">0.9953868</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18905" length="5"/>
+        <text>ACBD3</text>
+      </annotation>
+      <annotation id="451">
+        <infon key="score">0.9890894</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:55:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18912" length="5"/>
+        <text>PI4KB</text>
+      </annotation>
+      <annotation id="452">
+        <infon key="score">0.99866444</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:17Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="18927" length="11"/>
+        <text>ACBD3:PI4KB</text>
+      </annotation>
+      <annotation id="453">
+        <infon key="score">0.9974644</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:50:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18954" length="12"/>
+        <text>picornaviral</text>
+      </annotation>
+      <annotation id="454">
+        <infon key="score">0.9958832</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:03:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19059" length="14"/>
+        <text>picornaviruses</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_1</infon>
+      <offset>19131</offset>
+      <text>Materials and Methods</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>19153</offset>
+      <text>Plasmid construction, protein expression, and purification</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>19212</offset>
+      <text>All proteins used in this study were recombinant and were expressed in E. coli using previously developed protocols. Briefly, full-length human ACBD3 (UniProtKB entry Q9H3P7) and PI4KB (UniProtKB entry Q9UBF8, isoform 1) lipid kinase and their deletion mutants were cloned into a previously modified pRSFD vector (Novagen) that already contained an N-terminal 6xHis tag followed by a GB1 solubility tag and TEV protease cleavage site. Mutations were generated using the Phusion Site-Directed Mutagenesis Kit (Thermo Scientific). The plasmids used are listed in the SI (SI Table 2). The proteins were expressed in E. coli BL21 Star cells as previously described. Upon overnight expression in autoinduction media bacterial cells were harvested and lysed in lysis buffer (50 mM Tris pH 8, 300 mM NaCl, 3 mM β-mercaptoethanol, 20 mM imidazole, 10% glycerol). The lysate was incubated with the Ni-NTA resin (Macherey-Nagel) and then extensively washed with the lysis buffer. The protein was eluted with the lysis buffer supplemented with 300 mM imidazole. When appropriate, tags were removed with TEV protease, and the protein was further purified using the size exclusion chromatography on Superdex 75 or Superdex 200 columns (GE Healthcare) in SEC buffer (10 mM Tris pH 8, 200 mM NaCl, 3 mM β-ME). Proteins were concentrated to 1–5 mg/ml (measured spectroscopically) and stored at −80 °C until needed.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>20638</offset>
+      <text>In vitro pull-downs</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>20658</offset>
+      <text>Ni-NTA sepharose beads (Macherey-Nagel) were mixed with both binding partners (one of which was tagged with an N-terminal 6xHis tag) at a final concentration of 1 μM in a final volume of 200 μL binding buffer (30 mM Tris pH 8, 200 mM NaCl, 10 mM imidazole, and 1 mM TCEP). After 30 min incubation at 4 °C the beads were washed twice with 200 μL of the binding buffer, and total protein was directly eluted with the Laemmli sample buffer and analyzed by SDS-PAGE.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>21143</offset>
+      <text>SPR (Surface plasmon resonance) and AUC (Analytical ultracentrifugation)</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>21216</offset>
+      <text>PI4KB was chip-immobilized as detailed in the SI. Afterwards, the ACBD3 protein was injected in a series of concentrations for 3 min and then dissociation was monitored for another 5 min. The data were fit to a single-exponential model. Rate constants of association and dissociation were obtained by fitting the observed change in resonance signal using the following equations:</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>21600</offset>
+      <text>where c is the protein concentration, t is time, kon is the association rate constant, koff is the dissociation rate constant, D1 and D2 are the linear drift terms, and Ras, Rdis, R0, R1, and Rmax are corresponding changes in the relative response signal.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>21856</offset>
+      <text>AUC was used to perform sedimentation velocity experiments using a ProteomeLab XL-I Beckman Coulter analytical ultracentrifuge equipped with an AN50Ti rotor. All measurements were performed in 10 mM Tris pH 8, 200 mM NaCl, and 3 mM β-mercaptoethanol at 20 °C and 48000 rpm. All data were collected using an absorbance (230 nm and 280 nm) optical system. Data analysis was performed with the SEDFIT package and data were analyzed using a sedimentation coefficient distribution model c(s).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>22360</offset>
+      <text>In vitro kinase assay</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>22382</offset>
+      <text>In vitro kinase activity was measured using a bioluminescent ADP-Glo assay (Promega) as described previously. Briefly, reactions were carried out in a total volume of 5 μL in 384-well plates by diluting the indicated amounts of the PI4KB enzyme and/or ACBD3 protein into the kinase buffer (20 mM Tris pH 7.5, 5 mM MgCl2, 0.2% Triton-X100, 0.1 mg/mL BSA, 2 mM DTT, 50 μM phosphatidylinositol). Reaction was initiated by adding ATP to a final concentration of 100 μM. Samples were incubated for 60 min at 25 °C and the amount of hydrolyzed ATP was measured according to the manufacturer’s protocol using a TECAN infinite M 1000 plate reader.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>23046</offset>
+      <text>NMR spectroscopy</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>23063</offset>
+      <text>NMR spectra were acquired at 25 °C on a 600 MHz and 850 MHz Bruker Avance spectrometers, both of which were equipped with a triple-resonance (15N/13C/1H) cryoprobe. The sample volume was 0.35 mL, with a 280 μM concentration for the free Q domain and a 470 μM concentration for the ACBD3:PI4KB complex in the NMR buffer (25 mM sodium phosphate pH 6.5, 100 mM NaCl, 1 mM TCEP, 0.01% NaN3), 5% D2O/95% H2O. A series of double- and triple-resonance spectra were recorded to determine essentially complete sequence-specific resonance backbone and side-chain assignments. Constraints for 1H-1H distance required to calculate the structure of free Q domain and ACBD3:PI4KB complex were derived from 3D 15N/1H NOESY-HSQC and 13C/1H NOESY-HMQC, which were acquired using a NOE mixing time of 100 ms.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>23877</offset>
+      <text>The families of converged structures for the ACBD3:PI4KB complex and free Q domain were calculated using standard software as detailed in the SI. The structures with the lowest total energy were selected and validated. The statistics for the resulting structures are summarized in SI Table 1.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>24170</offset>
+      <text>Protein labeling with fluorescent dyes</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>24209</offset>
+      <text>PI4KB was labeled on native cysteine residues. Briefly, pure recombinant protein was incubated overnight at 4 °C with a 3x molar excess of Alexa 488 C5 maleimide (Life Technologies). The reaction was quenched by adding 10 mM β-mercaptoethanol (βME) and the protein was repurified by size exclusion chromatography.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>24530</offset>
+      <text>Giant Unilamellar Vesicle Preparation and Imaging</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>24580</offset>
+      <text>Giant Unilamellar Vesicles (GUVs) composed of POPC (54.9 mol %), POPS (10 mol %), cholesterol (20 mol %), PI (10 mol %), DGS-NTA(Ni) [1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) ] (5 mol %) (Avanti Polar lipids), and ATTO647N-DOPE (0.1 mol %) (ATTO-TEC GmbH) were prepared by electroformation as described previously, please see SI.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>24979</offset>
+      <text>Live Cell Imaging</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>24997</offset>
+      <text>COS-7 cells were plated onto 29-mm-diameter poly-L-Lysine coated glass bottom dishes (In Vitro Scientific) at 100,000 cells/well density and transfected using the Lipofectamine2000 reagent (Invitrogen) with plasmid DNAs (0.5–1 mg/well) according to manufacturer’s instructions. The plasmids are described in SI Table 2. 24 hr post transfection, COS-7 cells were washed with a modified Krebs-Ringer solution (10 mM Na-HEPES pH 7.4, 120 mM NaCl, 4.7 mM KCl, 2 mM CaCl2, 0.7 mM MgSO4, 10 mM glucose) in the same dish and were imaged using an LSM 710 confocal microscope (Carl Zeiss MicroImaging) with a 63 × 1.4-numerical-aperture planapochromatic objective. For ceramide uptake experiments, COS-7 cells were loaded with 0.05 μM BODIPY® FL C5-Ceramide (Molecular Probes, ThermoFisher Scientific) complexed with BSA in modified Krebs-Ringer solution at room temperature for 20 min. Cells were then washed three times and imaged using the above mentioned settings.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>25988</offset>
+      <text>Immunofluorescent imaging</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>26014</offset>
+      <text>COS-7 cells were plated onto 25-mm-diameter poly-L-Lysine coated circular glass coverslips in six-well plates (100,000 cells/well), and transfected using the Lipofectamine2000 reagent (Invitrogen) with plasmid DNAs (0.5–1 mg/well) according to manufacturer’s instructions. Twenty four hours post transfection, cells were washed with PBS, fixed with 4% paraformaldehyde, stained with mouse anti-PI4KB primary antibody (BD Transduction Laboratories, 1:500 dilution) and then after washing with PBS stained with Alexa Fluor 647 conjugated donkey anti-mouse secondary antibody (Molecular Probes, ThermoFisher Scientific, 1:500 dilution). Cover slips were mounted and observed with the above mentioned microscopy settings.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>26738</offset>
+      <text>HD exchange</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>26750</offset>
+      <text>Hydrogen/deuterium exchange was performed as previously described with the following modifications. The exchange was done in 10 mM Tris-HCl pD 8.0, 200 mM NaCl at 20 °C. Protein concentration during the exchange was 1 μM. Aliquots (50 μL) were removed after 10, 20, 60, 120, 600, 1800, and 3600 s and the exchange was quenched by the addition of 50 μL of 0.25 M glycine-HCl pH 2.3 and rapid freezing in liquid nitrogen.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>27188</offset>
+      <text>Prior to the analysis each sample was quickly thawed and injected onto an immobilized rhizopuspepsin column (bed volume 66 μL). Digestion was driven by a flow of 0.4% formic acid in water at a flow rate of 100 μL/min (LC-20AD pump, Shimadzu). The resulting peptides were trapped and desalted online on a peptide microtrap (Optimize Technologies). After a desalting step (3 min), peptides were separated using a linear gradient of 10–25% buffer B for 2 min, followed by a quick jump to 99% buffer B (buffer A = 0.4% formic acid/2% acetonitrile in water; buffer B = 95% acetonitrile/0.4% formic acid in water). The outlet of the LC system was interfaced to an electrospray ionization source of a Fourier transform ion cyclotron resonance mass spectrometer (12 T SolariX XR, Bruker Daltonics). Exchange was followed on 32 peptides from PI4KB (N) and 26 peptides from ACBD3(Q), covering in both cases 100% of the protein sequence. Peptides were identified by LC-MS/MS and MASCOT search against a database containing the sequences of the studied proteins. Data from H/D exchange were analyzed by program DeutEx written in the laboratory (unpublished).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_1</infon>
+      <offset>28355</offset>
+      <text>Additional Information</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>28378</offset>
+      <text>Accession codes: The structures and assigned chemical shifts for the free Q domain and the ACBD3:PI4KB complex were deposited in PDB database under accession codes 2N72 and 2N73, and BMRB database under accession codes 25790 and 25791.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>28614</offset>
+      <text>How to cite this article: Klima, M. et al. Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein. Sci. Rep. 6, 23641; doi: 10.1038/srep23641 (2016).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">title_1</infon>
+      <offset>28830</offset>
+      <text>Supplementary Material</text>
+    </passage>
+    <passage>
+      <infon key="fpage">136</infon>
+      <infon key="lpage">145</infon>
+      <infon key="name_0">surname:Boura;given-names:E.</infon>
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+      <infon key="pub-id_doi">10.1016/j.yexcr.2015.03.028</infon>
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+      <text>The authors declare no competing financial interests.</text>
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+      <text>Author Contributions M.K. and A.D. carried out DNA cloning, M.K., A.B., D.C. and E.B. carried out protein expression and purification, M.K. performed pull-down assays, L.R. carried out analytical ultracentrifugation, M.K. and J.T. performed S.P.R. experiments, R.H. and V.V. carried out NMR experiments, structure refinement, and deposition, A.B. and P.M. performed HDX/MS experiments, D.C. carried out in vitro kinase assay, E.B. performed protein labeling, E.B. and J.H. carried out GUV preparation and imaging, D.T. and N.S. performed some of the cloning and the cell-based experiments, E.B. supervised the project, E.B., M.K., M.N., V.V. and T.B. wrote the manuscript, all authors contributed to data analysis and commented on the manuscript.</text>
+    </passage>
+    <passage>
+      <infon key="file">srep23641-f1.jpg</infon>
+      <infon key="id">f1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>34270</offset>
+      <text>Biochemical characterization of the ACBD3:PI4KB complex.</text>
+      <annotation id="727">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T15:58:09Z</infon>
+        <location offset="34270" length="28"/>
+        <text>Biochemical characterization</text>
+      </annotation>
+      <annotation id="455">
+        <infon key="score">0.99930733</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:56:17Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="34306" length="11"/>
+        <text>ACBD3:PI4KB</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">srep23641-f1.jpg</infon>
+      <infon key="id">f1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>34327</offset>
+      <text>(A) Schematic representation of the ACBD3 and PI4KB constructs used for the experiments. ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD). PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes. (B) In vitro pull-down assay. Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3. The inputs and bound proteins were analyzed on SDS gels stained with Coomassie Blue. The asterisks mark the bands corresponding to specific interactions. Cropped gels ran the same experimental conditions are shown. Please, see SI Fig. 9 for original full-length gels. (C) Analytical Ultracentrifugation. AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance. SPR analysis of the PI4KB binding to immobilized ACBD3. Sensorgrams for four concentrations of PI4KB are shown.</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="39357" length="23"/>
+        <text>mean membrane intensity</text>
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+      <annotation id="593">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
+        <location offset="39518" length="3"/>
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+      <annotation id="744">
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T16:08:20Z</infon>
+        <location offset="39548" length="3"/>
+        <text>SEM</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">srep23641-f5.jpg</infon>
+      <infon key="id">f5</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>39617</offset>
+      <text>Pseudoatomic model of the PI4KB multiprotein complex assembly.</text>
+      <annotation id="594">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T16:09:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="39617" length="18"/>
+        <text>Pseudoatomic model</text>
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+        <location offset="39643" length="5"/>
+        <text>PI4KB</text>
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+    </passage>
+    <passage>
+      <infon key="file">srep23641-f5.jpg</infon>
+      <infon key="id">f5</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>39680</offset>
+      <text>PI4KB in orange, Rab11 in purple, ACBD3 in blue. The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server. The GOLD domain is tethered to the membrane by GolginB1 (also known as Giantin) which is not shown for clarity. Intrinsically disordered linkers are modeled in an arbitrary but physically plausible conformation.</text>
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+        <infon key="identifier">PR:</infon>
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+        <text>Rab11</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="39755" length="3"/>
+        <text>NMR</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>crystal structure</text>
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+      <annotation id="602">
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T16:09:37Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="39817" length="11"/>
+        <text>PI4KB:Rab11</text>
+      </annotation>
+      <annotation id="603">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T17:22:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="39854" length="4"/>
+        <text>ACBD</text>
+      </annotation>
+      <annotation id="604">
+        <infon key="score">0.9989673</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T17:22:13Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="39863" length="4"/>
+        <text>GOLD</text>
+      </annotation>
+      <annotation id="605">
+        <infon key="score">0.9982494</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T16:09:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="39880" length="16"/>
+        <text>homology modeled</text>
+      </annotation>
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+        <infon key="score">0.9897456</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:16:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="39929" length="10"/>
+        <text>structures</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T16:09:39Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="39956" length="6"/>
+        <text>Phyre2</text>
+      </annotation>
+      <annotation id="608">
+        <infon key="score">0.9989318</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T17:22:16Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="39979" length="4"/>
+        <text>GOLD</text>
+      </annotation>
+      <annotation id="609">
+        <infon key="score">0.9989569</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T17:17:19Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="40022" length="8"/>
+        <text>GolginB1</text>
+      </annotation>
+      <annotation id="610">
+        <infon key="score">0.9990276</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T14:00:41Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="40046" length="7"/>
+        <text>Giantin</text>
+      </annotation>
+      <annotation id="745">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T16:09:58Z</infon>
+        <location offset="40087" length="32"/>
+        <text>Intrinsically disordered linkers</text>
+      </annotation>
+    </passage>
+  </document>
+</collection>
diff --git a/annotated_BioC_XML/PMC4817029_ann.xml b/annotated_BioC_XML/PMC4817029_ann.xml
new file mode 100644
index 0000000000000000000000000000000000000000..76995bdbdf826909792db9b7f5f8e53caa6ce53b
--- /dev/null
+++ b/annotated_BioC_XML/PMC4817029_ann.xml
@@ -0,0 +1,9515 @@
+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection>
+  <source>PMC</source>
+  <date>20201215</date>
+  <key>pmc.key</key>
+  <document>
+    <id>4817029</id>
+    <infon key="license">CC BY</infon>
+    <infon key="tt_curatable">no</infon>
+    <infon key="tt_version">2</infon>
+    <infon key="tt_round">2</infon>
+    <passage>
+      <infon key="article-id_doi">10.1038/srep23473</infon>
+      <infon key="article-id_pii">srep23473</infon>
+      <infon key="article-id_pmc">4817029</infon>
+      <infon key="article-id_pmid">27032335</infon>
+      <infon key="elocation-id">23473</infon>
+      <infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon>
+      <infon key="name_0">surname:Liberato;given-names:Marcelo V.</infon>
+      <infon key="name_1">surname:Silveira;given-names:Rodrigo L.</infon>
+      <infon key="name_10">surname:Polikarpov;given-names:Igor</infon>
+      <infon key="name_2">surname:Prates;given-names:Érica T.</infon>
+      <infon key="name_3">surname:de Araujo;given-names:Evandro A.</infon>
+      <infon key="name_4">surname:Pellegrini;given-names:Vanessa O. A.</infon>
+      <infon key="name_5">surname:Camilo;given-names:Cesar M.</infon>
+      <infon key="name_6">surname:Kadowaki;given-names:Marco A.</infon>
+      <infon key="name_7">surname:Neto;given-names:Mario de O.</infon>
+      <infon key="name_8">surname:Popov;given-names:Alexander</infon>
+      <infon key="name_9">surname:Skaf;given-names:Munir S.</infon>
+      <infon key="section_type">TITLE</infon>
+      <infon key="type">front</infon>
+      <infon key="volume">6</infon>
+      <infon key="year">2016</infon>
+      <offset>0</offset>
+      <text>Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism</text>
+      <annotation id="793">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:39:48Z</infon>
+        <location offset="32" length="28"/>
+        <text>family 5 glycoside hydrolase</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">ABSTRACT</infon>
+      <infon key="type">abstract</infon>
+      <offset>105</offset>
+      <text>Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.</text>
+      <annotation id="1">
+        <infon key="score">0.9987004</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:39:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="105" length="20"/>
+        <text>Glycoside hydrolases</text>
+      </annotation>
+      <annotation id="2">
+        <infon key="score">0.99921453</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="127" length="3"/>
+        <text>GHs</text>
+      </annotation>
+      <annotation id="3">
+        <infon key="score">0.99916106</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="229" length="11"/>
+        <text>full-length</text>
+      </annotation>
+      <annotation id="4">
+        <infon key="score">0.9978618</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="241" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="5">
+        <infon key="score">0.9992067</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:18Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="256" length="9"/>
+        <text>cellulase</text>
+      </annotation>
+      <annotation id="6">
+        <infon key="score">0.99826276</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="271" length="22"/>
+        <text>Bacillus licheniformis</text>
+      </annotation>
+      <annotation id="7">
+        <infon key="score">0.99894685</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="295" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="8">
+        <infon key="score">0.99763244</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:38Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="321" length="15"/>
+        <text>GH5 subfamily 4</text>
+      </annotation>
+      <annotation id="9">
+        <infon key="score">0.9979503</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:46Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="375" length="17"/>
+        <text>ancillary modules</text>
+      </annotation>
+      <annotation id="10">
+        <infon key="score">0.99924576</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:51Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="394" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="11">
+        <infon key="score">0.9989945</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="413" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="12">
+        <infon key="score">0.99890447</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="450" length="21"/>
+        <text>X-ray crystallography</text>
+      </annotation>
+      <annotation id="13">
+        <infon key="score">0.99891025</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="473" length="28"/>
+        <text>small-angle X-ray scattering</text>
+      </annotation>
+      <annotation id="14">
+        <infon key="score">0.9987839</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="506" length="30"/>
+        <text>molecular dynamics simulations</text>
+      </annotation>
+      <annotation id="15">
+        <infon key="score">0.9945221</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="569" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="16">
+        <infon key="score">0.99930584</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="602" length="16"/>
+        <text>catalytic domain</text>
+      </annotation>
+      <annotation id="17">
+        <infon key="score">0.99936837</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:25Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="620" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="18">
+        <infon key="score">0.9930973</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="639" length="16"/>
+        <text>fully functional</text>
+      </annotation>
+      <annotation id="19">
+        <infon key="score">0.999096</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:34Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="656" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="20">
+        <infon key="score">0.9993052</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="770" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="21">
+        <infon key="score">0.98631513</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="834" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="22">
+        <infon key="score">0.99929404</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="852" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="23">
+        <infon key="score">0.9990971</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:42:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="878" length="15"/>
+        <text>binding subsite</text>
+      </annotation>
+      <annotation id="24">
+        <infon key="score">0.9990859</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="938" length="2"/>
+        <text>GH</text>
+      </annotation>
+      <annotation id="25">
+        <infon key="score">0.99607617</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="983" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="26">
+        <infon key="score">0.9482441</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1009" length="23"/>
+        <text>catalytically competent</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>1053</offset>
+      <text>The production of biofuels from renewable sources is an important element of the global strategy for generating sustainable energy with reduced environmental impact. Current technologies for obtaining liquid biofuels and green chemicals rely on the enzymatic digestion of lignocellulosic biomass from a variety of feedstocks. Plant biomass-the most abundant source of carbohydrates on Earth-is primarily composed of cellulose microfibrils surrounded by a hydrated heteropolymeric matrix of hemicellulose and lignin. Plant biomass may be subjected to thermo-chemical pretreatments and enzymatic reactions to produce soluble fermentable sugars.</text>
+      <annotation id="27">
+        <infon key="score">0.9980286</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:42:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1379" length="5"/>
+        <text>Plant</text>
+      </annotation>
+      <annotation id="28">
+        <infon key="score">0.9926569</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:42:25Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1421" length="13"/>
+        <text>carbohydrates</text>
+      </annotation>
+      <annotation id="798">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:00Z</infon>
+        <location offset="1469" length="9"/>
+        <text>cellulose</text>
+      </annotation>
+      <annotation id="29">
+        <infon key="score">0.75858444</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:42:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1543" length="13"/>
+        <text>hemicellulose</text>
+      </annotation>
+      <annotation id="30">
+        <infon key="score">0.8075788</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:42:38Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1561" length="6"/>
+        <text>lignin</text>
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+      <annotation id="31">
+        <infon key="score">0.9981589</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:42:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1569" length="5"/>
+        <text>Plant</text>
+      </annotation>
+      <annotation id="32">
+        <infon key="score">0.98998487</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:42:43Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1688" length="6"/>
+        <text>sugars</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>1696</offset>
+      <text>The canonical model of hydrolytic degradation of cellulose requires at least three classes of enzymes. Cellobiohydrolases (CBHs) processively cleave the glycosidic bonds at the reducing and non-reducing ends of cellulose chains in crystalline regions to produce cellobiose. Endoglucanases (EGs) introduce random cuts in the amorphous regions of cellulose and create new chain extremities for CBH attack; thus, these enzymes act synergistically. The released cellobiose molecules are then enzymatically converted into glucose by β-glucosidases.</text>
+      <annotation id="33">
+        <infon key="score">0.6507011</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:01Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1745" length="9"/>
+        <text>cellulose</text>
+      </annotation>
+      <annotation id="34">
+        <infon key="score">0.9988967</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1799" length="18"/>
+        <text>Cellobiohydrolases</text>
+      </annotation>
+      <annotation id="35">
+        <infon key="score">0.9992205</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1819" length="4"/>
+        <text>CBHs</text>
+      </annotation>
+      <annotation id="36">
+        <infon key="score">0.877582</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:01Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1907" length="9"/>
+        <text>cellulose</text>
+      </annotation>
+      <annotation id="37">
+        <infon key="score">0.99929</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:53:00Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1958" length="10"/>
+        <text>cellobiose</text>
+      </annotation>
+      <annotation id="38">
+        <infon key="score">0.99881196</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:45:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1970" length="14"/>
+        <text>Endoglucanases</text>
+      </annotation>
+      <annotation id="39">
+        <infon key="score">0.9992318</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1986" length="3"/>
+        <text>EGs</text>
+      </annotation>
+      <annotation id="40">
+        <infon key="score">0.7187887</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:01Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2041" length="9"/>
+        <text>cellulose</text>
+      </annotation>
+      <annotation id="41">
+        <infon key="score">0.81927955</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:27Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2088" length="3"/>
+        <text>CBH</text>
+      </annotation>
+      <annotation id="42">
+        <infon key="score">0.9992581</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:53:06Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2154" length="10"/>
+        <text>cellobiose</text>
+      </annotation>
+      <annotation id="43">
+        <infon key="score">0.9990037</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:11Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2213" length="7"/>
+        <text>glucose</text>
+      </annotation>
+      <annotation id="44">
+        <infon key="score">0.99811155</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:43Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2224" length="14"/>
+        <text>β-glucosidases</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>2243</offset>
+      <text>The molecular architecture of glycoside hydrolases (GHs) frequently consists of a catalytic domain (CD), where hydrolysis occurs, and one or more ancillary modules (AMs), which are usually connected by less structured linkers. The most common type of AMs are carbohydrate-binding modules (CBMs), which are able to recognize and bind specific carbohydrate chains. Generally distinct and independent structural domains, the CBMs facilitate carbohydrate hydrolysis by increasing the local concentration of enzymes at the surface of insoluble substrates, thereby targeting the CD component to its cognate ligands. CBMs might also disrupt the crystalline structure of cellulose microfibrils, although the underlying mechanism remains poorly understood. Thus, CBMs enhance the accessibility of CDs to carbohydrate chains to improve enzymatic activity, making them important candidates for the development of effective biomass-degrading enzymes in industrial settings.</text>
+      <annotation id="45">
+        <infon key="score">0.99880946</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:39:56Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2273" length="20"/>
+        <text>glycoside hydrolases</text>
+      </annotation>
+      <annotation id="46">
+        <infon key="score">0.9992569</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2295" length="3"/>
+        <text>GHs</text>
+      </annotation>
+      <annotation id="47">
+        <infon key="score">0.9993674</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:21Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2325" length="16"/>
+        <text>catalytic domain</text>
+      </annotation>
+      <annotation id="48">
+        <infon key="score">0.99939394</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2343" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="49">
+        <infon key="score">0.99907243</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2389" length="17"/>
+        <text>ancillary modules</text>
+      </annotation>
+      <annotation id="50">
+        <infon key="score">0.99794024</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2408" length="3"/>
+        <text>AMs</text>
+      </annotation>
+      <annotation id="51">
+        <infon key="score">0.81550145</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2445" length="15"/>
+        <text>less structured</text>
+      </annotation>
+      <annotation id="52">
+        <infon key="score">0.99828774</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2461" length="7"/>
+        <text>linkers</text>
+      </annotation>
+      <annotation id="53">
+        <infon key="score">0.97873074</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2494" length="3"/>
+        <text>AMs</text>
+      </annotation>
+      <annotation id="54">
+        <infon key="score">0.99920225</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:37Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2502" length="28"/>
+        <text>carbohydrate-binding modules</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <text>CBMs</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:26Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2585" length="12"/>
+        <text>carbohydrate</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <text>CBMs</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:26Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <text>carbohydrate</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2816" length="2"/>
+        <text>CD</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3038" length="12"/>
+        <text>carbohydrate</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>3205</offset>
+      <text>Although there are examples of active GHs that lack AMs, the majority of the enzymes depend on AMs for activity. In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B). A pioneer work of Sakon et al. revealed that rigid structural extension of the GH9 CD by a type C CBM3 imprints a processive mode of action to this endoglucanase. Further publications showed that CBM-based structural extensions of the active site are important for substrate engagement and recognition.</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="3384" length="22"/>
+        <text>substrate-binding site</text>
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+        <infon key="type">protein_type</infon>
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+        <infon key="updated_at">2023-09-21T09:45:36Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3423" length="27"/>
+        <text>carbohydrate-active enzymes</text>
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+      <annotation id="74">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:45:48Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3460" length="17"/>
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+        <infon key="type">protein</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <location offset="3486" length="18"/>
+        <text>Thermobifida fusca</text>
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+        <infon key="type">protein</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="3523" length="19"/>
+        <text>Serratia marcescens</text>
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+        <infon key="type">protein_type</infon>
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+        <infon key="updated_at">2023-09-21T09:46:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3546" length="18"/>
+        <text>starch phosphatase</text>
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:46:36Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3570" length="20"/>
+        <text>Arabidopsis thaliana</text>
+      </annotation>
+      <annotation id="81">
+        <infon key="score">0.8692663</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:39Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3597" length="15"/>
+        <text>GH5 subfamily 4</text>
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+      <annotation id="808">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:47:14Z</infon>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="3621" length="13"/>
+        <text>endoglucanase</text>
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+      <annotation id="83">
+        <infon key="score">0.99876606</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:47:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3640" length="19"/>
+        <text>Bacillus halodurans</text>
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+      <annotation id="84">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="3661" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+      <annotation id="85">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:48:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3750" length="3"/>
+        <text>GH9</text>
+      </annotation>
+      <annotation id="86">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>CD</text>
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+      <annotation id="820">
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:48:46Z</infon>
+        <location offset="3762" length="11"/>
+        <text>type C CBM3</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="3819" length="13"/>
+        <text>endoglucanase</text>
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+      <annotation id="88">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:49:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="3867" length="3"/>
+        <text>CBM</text>
+      </annotation>
+      <annotation id="89">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="3906" length="11"/>
+        <text>active site</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>3974</offset>
+      <text>Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module. Removal of the CBM46 caused a ~60-fold reduction of the activity of the enzyme against β-glucans, but showed little or no effect against xyloglucan hydrolysis. Moreover, the CBM46 mediated a significant increase in the BhCel5B activity in plant cell wall settings. Modeling of cellotriose in the negative subsites of the active site of BhCel5B demonstrated the structural conservation of the -1 position, but provided little information about direct interactions between CBM46 and the substrate. It was speculated that β-1,3 kink of the β-glucan might allow the ligand to reach for the CBM46, whereas pure β-1,4 linkages in the backbone of xyloglucan chains would restrict binding to the CD, thus explaining the lack of influence of the CBM46 on the enzymatic activity of BhCel5B against xyloglucans in solution. It was also argued that the CBM46 could potentialize the activity by driving BhCel5B towards xyloglucan-rich regions in the context of the plant cell walls, but no large-scale conformational adjustments of the AMs have been shown to occur or suggested to take part in the enzymatic activity.</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:49:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4013" length="15"/>
+        <text>X-ray structure</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:39:32Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4036" length="11"/>
+        <text>tri-modular</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:47:14Z</infon>
+        <location offset="4048" length="5"/>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:48:53Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4054" length="13"/>
+        <text>endoglucanase</text>
+      </annotation>
+      <annotation id="93">
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:47:36Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4073" length="19"/>
+        <text>Bacillus halodurans</text>
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+      <annotation id="94">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4119" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <location offset="4138" length="5"/>
+        <text>CBM46</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4161" length="11"/>
+        <text>active site</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4189" length="2"/>
+        <text>CD</text>
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+      <annotation id="97">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:49:49Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4199" length="31"/>
+        <text>immunoglobulin (Ig)-like module</text>
+      </annotation>
+      <annotation id="98">
+        <infon key="score">0.9758551</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:49:52Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4232" length="10"/>
+        <text>Removal of</text>
+      </annotation>
+      <annotation id="99">
+        <infon key="score">0.9956642</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4247" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="100">
+        <infon key="score">0.99816173</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:49:44Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4319" length="9"/>
+        <text>β-glucans</text>
+      </annotation>
+      <annotation id="101">
+        <infon key="score">0.9977476</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:49:58Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4369" length="10"/>
+        <text>xyloglucan</text>
+      </annotation>
+      <annotation id="102">
+        <infon key="score">0.9944535</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4406" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="103">
+        <infon key="score">0.9991316</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4451" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+      <annotation id="104">
+        <infon key="score">0.99774253</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:42:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4471" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="105">
+        <infon key="score">0.9915229</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:50:15Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4497" length="8"/>
+        <text>Modeling</text>
+      </annotation>
+      <annotation id="106">
+        <infon key="score">0.9992712</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:50:05Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4509" length="11"/>
+        <text>cellotriose</text>
+      </annotation>
+      <annotation id="107">
+        <infon key="score">0.9989642</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:50:25Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4528" length="17"/>
+        <text>negative subsites</text>
+      </annotation>
+      <annotation id="108">
+        <infon key="score">0.9991369</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4553" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="109">
+        <infon key="score">0.9991585</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4568" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+      <annotation id="110">
+        <infon key="score">0.9542444</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:50:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4593" length="23"/>
+        <text>structural conservation</text>
+      </annotation>
+      <annotation id="111">
+        <infon key="score">0.8204445</infon>
+        <infon key="type">residue_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:50:44Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4624" length="2"/>
+        <text>-1</text>
+      </annotation>
+      <annotation id="112">
+        <infon key="score">0.98499066</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4703" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="113">
+        <infon key="score">0.99886227</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:51:19Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4769" length="8"/>
+        <text>β-glucan</text>
+      </annotation>
+      <annotation id="114">
+        <infon key="score">0.99476916</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4818" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="115">
+        <infon key="score">0.99862576</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:49:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4872" length="10"/>
+        <text>xyloglucan</text>
+      </annotation>
+      <annotation id="116">
+        <infon key="score">0.9993088</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4920" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="117">
+        <infon key="score">0.9971175</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4969" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="118">
+        <infon key="score">0.9991604</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="5004" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+      <annotation id="119">
+        <infon key="score">0.9982805</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:51:06Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5020" length="11"/>
+        <text>xyloglucans</text>
+      </annotation>
+      <annotation id="120">
+        <infon key="score">0.9978167</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5073" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="121">
+        <infon key="score">0.9991603</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="5122" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+      <annotation id="122">
+        <infon key="score">0.99331254</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:51:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5138" length="23"/>
+        <text>xyloglucan-rich regions</text>
+      </annotation>
+      <annotation id="123">
+        <infon key="score">0.99859613</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:42:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5184" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="124">
+        <infon key="score">0.9228424</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5255" length="3"/>
+        <text>AMs</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>5349</offset>
+      <text>The mechanisms of ligand binding mediated by large-scale conformational changes in proteins following the induced-fit or conformational selection models have recently attracted considerable attention. Although initially introduced as contradictory theories, these two limiting cases can be unified considering the flux description concept or the extended conformational selection model. While local ligand-induced conformational adjustments have been reported for carbohydrate-active enzymes, cognate ligands recognition and hydrolysis mediated by a large-scale conformational mobility of distinct domains in multidomain settings is uncommon for endoglucanases.</text>
+      <annotation id="858">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:17:58Z</infon>
+        <location offset="5695" length="8"/>
+        <text>extended</text>
+      </annotation>
+      <annotation id="125">
+        <infon key="score">0.99613124</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:51:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5813" length="27"/>
+        <text>carbohydrate-active enzymes</text>
+      </annotation>
+      <annotation id="126">
+        <infon key="score">0.999311</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:45:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5995" length="14"/>
+        <text>endoglucanases</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>6011</offset>
+      <text>Here, we report the crystal structure of a full-length GH5_4 enzyme from Bacillus licheniformis (BlCel5B) that exhibits two AMs (Ig-like module and CBM46) appended to the CD. We structurally and functionally characterize the enzyme using a combination of protein crystallography, small-angle X-ray scattering (SAXS), molecular dynamics computer simulations and site-directed mutagenesis, and show that the AMs and their conformational mobility are essential for the enzymatic activity of BlCel5B. We find that the large-scale conformational adjustments of the distal CBM46 mediated by the Ig-like hinge domain are crucial in active-site assembly for optimal substrate binding and hydrolysis. We propose that the BlCel5B conformational selection/induced-fit mechanism of hydrolysis represents a novel paradigm that applies to several GH5_4 members and, possibly, to a number of other multidomain GHs.</text>
+      <annotation id="127">
+        <infon key="score">0.9939641</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6031" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="128">
+        <infon key="score">0.99908876</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6054" length="11"/>
+        <text>full-length</text>
+      </annotation>
+      <annotation id="810">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:47:14Z</infon>
+        <location offset="6066" length="5"/>
+        <text>GH5_4</text>
+      </annotation>
+      <annotation id="129">
+        <infon key="score">0.9983735</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6084" length="22"/>
+        <text>Bacillus licheniformis</text>
+      </annotation>
+      <annotation id="130">
+        <infon key="score">0.99898714</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6108" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="131">
+        <infon key="score">0.9986204</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6135" length="3"/>
+        <text>AMs</text>
+      </annotation>
+      <annotation id="132">
+        <infon key="score">0.9992919</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6140" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="133">
+        <infon key="score">0.998767</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6159" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="134">
+        <infon key="score">0.9994461</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6182" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="135">
+        <infon key="score">0.9962609</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:12Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6189" length="42"/>
+        <text>structurally and functionally characterize</text>
+      </annotation>
+      <annotation id="136">
+        <infon key="score">0.99858046</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6266" length="23"/>
+        <text>protein crystallography</text>
+      </annotation>
+      <annotation id="137">
+        <infon key="score">0.9989625</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6291" length="28"/>
+        <text>small-angle X-ray scattering</text>
+      </annotation>
+      <annotation id="138">
+        <infon key="score">0.99831426</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:19Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6321" length="4"/>
+        <text>SAXS</text>
+      </annotation>
+      <annotation id="139">
+        <infon key="score">0.99881864</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6328" length="39"/>
+        <text>molecular dynamics computer simulations</text>
+      </annotation>
+      <annotation id="140">
+        <infon key="score">0.9988634</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:25Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6372" length="25"/>
+        <text>site-directed mutagenesis</text>
+      </annotation>
+      <annotation id="141">
+        <infon key="score">0.99568814</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6417" length="3"/>
+        <text>AMs</text>
+      </annotation>
+      <annotation id="142">
+        <infon key="score">0.9990846</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6499" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="143">
+        <infon key="score">0.99801886</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6578" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="144">
+        <infon key="score">0.9992043</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:31Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6600" length="20"/>
+        <text>Ig-like hinge domain</text>
+      </annotation>
+      <annotation id="145">
+        <infon key="score">0.99893785</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6636" length="11"/>
+        <text>active-site</text>
+      </annotation>
+      <annotation id="146">
+        <infon key="score">0.99904066</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6723" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="811">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:47:14Z</infon>
+        <location offset="6844" length="5"/>
+        <text>GH5_4</text>
+      </annotation>
+      <annotation id="147">
+        <infon key="score">0.9993019</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6906" length="3"/>
+        <text>GHs</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_1</infon>
+      <offset>6911</offset>
+      <text>Results</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>6919</offset>
+      <text>BlCel5B Crystal Structure</text>
+      <annotation id="148">
+        <infon key="score">0.9960616</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6919" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="149">
+        <infon key="score">0.99812376</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6927" length="17"/>
+        <text>Crystal Structure</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>6945</offset>
+      <text>BlCel5B crystals in the substrate-free form and complexed with cellopentaose (C5) were obtained and diffracted to 1.7 Å and 1.75 Å resolutions, respectively (Supplementary Table 1). The substrate-free and complexed structures exhibited no substantial conformational differences (with the exception of the substrate). Because of minor variations in the loops located distal to the substrate-binding site, a root mean squared deviation (rmsd) of 0.33 Å between the complexed and substrate-free structures was observed. A single protein chain occupies the asymmetric unit, and most of the residues were built, with the exception of the first 17 residues and those in the loop between L398 and P405 due to weak electron density.</text>
+      <annotation id="150">
+        <infon key="score">0.99899524</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6945" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="151">
+        <infon key="score">0.99850273</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6953" length="8"/>
+        <text>crystals</text>
+      </annotation>
+      <annotation id="152">
+        <infon key="score">0.9985334</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6969" length="14"/>
+        <text>substrate-free</text>
+      </annotation>
+      <annotation id="153">
+        <infon key="score">0.99859357</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:24Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6993" length="14"/>
+        <text>complexed with</text>
+      </annotation>
+      <annotation id="154">
+        <infon key="score">0.9993591</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7008" length="13"/>
+        <text>cellopentaose</text>
+      </annotation>
+      <annotation id="155">
+        <infon key="score">0.9694119</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7023" length="2"/>
+        <text>C5</text>
+      </annotation>
+      <annotation id="156">
+        <infon key="score">0.9989707</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7131" length="14"/>
+        <text>substrate-free</text>
+      </annotation>
+      <annotation id="157">
+        <infon key="score">0.99924445</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7150" length="9"/>
+        <text>complexed</text>
+      </annotation>
+      <annotation id="158">
+        <infon key="score">0.9981719</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:46:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7160" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="159">
+        <infon key="score">0.9984168</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:46Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7297" length="5"/>
+        <text>loops</text>
+      </annotation>
+      <annotation id="160">
+        <infon key="score">0.9989159</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:45:11Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7325" length="22"/>
+        <text>substrate-binding site</text>
+      </annotation>
+      <annotation id="161">
+        <infon key="score">0.9982372</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7351" length="27"/>
+        <text>root mean squared deviation</text>
+      </annotation>
+      <annotation id="162">
+        <infon key="score">0.9980987</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:54:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7380" length="4"/>
+        <text>rmsd</text>
+      </annotation>
+      <annotation id="163">
+        <infon key="score">0.9992434</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:30Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7408" length="9"/>
+        <text>complexed</text>
+      </annotation>
+      <annotation id="164">
+        <infon key="score">0.9989364</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7422" length="14"/>
+        <text>substrate-free</text>
+      </annotation>
+      <annotation id="165">
+        <infon key="score">0.9980007</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:54:36Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7437" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="166">
+        <infon key="score">0.94741315</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:54:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7578" length="17"/>
+        <text>first 17 residues</text>
+      </annotation>
+      <annotation id="167">
+        <infon key="score">0.99886227</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7613" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="168">
+        <infon key="score">0.99858093</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:54:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7626" length="4"/>
+        <text>L398</text>
+      </annotation>
+      <annotation id="169">
+        <infon key="score">0.9983217</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:54:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7635" length="4"/>
+        <text>P405</text>
+      </annotation>
+      <annotation id="170">
+        <infon key="score">0.9979699</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:54:25Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7652" length="16"/>
+        <text>electron density</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>7679</offset>
+      <text>The BlCel5B structure comprises three distinct domains: an N-terminal CD (residues 18 to 330), an Ig-like module (residues 335 to 428) and a family 46 CBM (residues 432 to 533) (Fig. 1A,B). Similarly to other members of the GH5 family, the CD of BlCel5B has a typical TIM barrel fold with eight inner β-strands and eight outer α helices that are interconnected by loops and three short α helices. Very short linkers, D429-D430-P431 and V331-P332-N333-A334, connect the CBM46 to the Ig-like module and the Ig-like module to the CD, respectively. Both Ig-like module and CBM46 have a β-sandwich fold composed of two β-sheets of four and three antiparallel β-strands interconnected by loops and a short α helix between strands β3 and β4 (Fig. 1C). A structural comparison between the Ig-like module and the CBM46 using the Dali server yielded an rmsd of 2.3 Å and a Z-score of 10.2. However, despite their structural resemblance, these modules share only 17% sequence identity. A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd = 1.3 Å and Z-score = 15.3. The CBM46 from BhCel5B is the most structurally similar to BlCel5B CBM46, with rmsd = 1.6 Å and Z-score = 12.4. The sequence identity relative to BhCel5B, however, is low (28% for Ig-like and 25% for CBM46).</text>
+      <annotation id="171">
+        <infon key="score">0.99880683</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7683" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="172">
+        <infon key="score">0.9972167</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:55:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7691" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="173">
+        <infon key="score">0.9994228</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7749" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="174">
+        <infon key="score">0.9975144</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:54:45Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7762" length="9"/>
+        <text>18 to 330</text>
+      </annotation>
+      <annotation id="175">
+        <infon key="score">0.9992367</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7777" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="176">
+        <infon key="score">0.9974611</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:54:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7802" length="10"/>
+        <text>335 to 428</text>
+      </annotation>
+      <annotation id="177">
+        <infon key="score">0.9041701</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:55:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7820" length="13"/>
+        <text>family 46 CBM</text>
+      </annotation>
+      <annotation id="178">
+        <infon key="score">0.9974942</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:54:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7844" length="10"/>
+        <text>432 to 533</text>
+      </annotation>
+      <annotation id="804">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:46:54Z</infon>
+        <location offset="7903" length="3"/>
+        <text>GH5</text>
+      </annotation>
+      <annotation id="179">
+        <infon key="score">0.9994931</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7919" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="180">
+        <infon key="score">0.9988254</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7925" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="181">
+        <infon key="score">0.9976907</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:55:23Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7947" length="15"/>
+        <text>TIM barrel fold</text>
+      </annotation>
+      <annotation id="182">
+        <infon key="score">0.9976674</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:55:27Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7980" length="9"/>
+        <text>β-strands</text>
+      </annotation>
+      <annotation id="183">
+        <infon key="score">0.99879986</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:55:32Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8006" length="9"/>
+        <text>α helices</text>
+      </annotation>
+      <annotation id="184">
+        <infon key="score">0.9226088</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8043" length="5"/>
+        <text>loops</text>
+      </annotation>
+      <annotation id="821">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:55:33Z</infon>
+        <location offset="8065" length="9"/>
+        <text>α helices</text>
+      </annotation>
+      <annotation id="799">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:59Z</infon>
+        <location offset="8087" length="7"/>
+        <text>linkers</text>
+      </annotation>
+      <annotation id="185">
+        <infon key="score">0.9010072</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:56:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8096" length="14"/>
+        <text>D429-D430-P431</text>
+      </annotation>
+      <annotation id="822">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:57:03Z</infon>
+        <location offset="8115" length="19"/>
+        <text>V331-P332-N333-A334</text>
+      </annotation>
+      <annotation id="186">
+        <infon key="score">0.99080867</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8148" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="187">
+        <infon key="score">0.9991174</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8161" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="188">
+        <infon key="score">0.99914384</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8184" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="189">
+        <infon key="score">0.99945945</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8206" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="190">
+        <infon key="score">0.99923486</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8229" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="191">
+        <infon key="score">0.9880321</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8248" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="192">
+        <infon key="score">0.99734473</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:49:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8261" length="15"/>
+        <text>β-sandwich fold</text>
+      </annotation>
+      <annotation id="193">
+        <infon key="score">0.89907354</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:57:49Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8293" length="8"/>
+        <text>β-sheets</text>
+      </annotation>
+      <annotation id="194">
+        <infon key="score">0.99596715</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:57:16Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8320" length="22"/>
+        <text>antiparallel β-strands</text>
+      </annotation>
+      <annotation id="195">
+        <infon key="score">0.9775023</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8361" length="5"/>
+        <text>loops</text>
+      </annotation>
+      <annotation id="823">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:58:14Z</infon>
+        <location offset="8379" length="7"/>
+        <text>α helix</text>
+      </annotation>
+      <annotation id="196">
+        <infon key="score">0.96771604</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:57:21Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8395" length="7"/>
+        <text>strands</text>
+      </annotation>
+      <annotation id="197">
+        <infon key="score">0.99835396</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:57:39Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8403" length="2"/>
+        <text>β3</text>
+      </annotation>
+      <annotation id="198">
+        <infon key="score">0.9979388</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:57:43Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8410" length="2"/>
+        <text>β4</text>
+      </annotation>
+      <annotation id="199">
+        <infon key="score">0.9986167</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:49:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8426" length="21"/>
+        <text>structural comparison</text>
+      </annotation>
+      <annotation id="200">
+        <infon key="score">0.99914145</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8460" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="201">
+        <infon key="score">0.9686031</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8483" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="202">
+        <infon key="score">0.9988082</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:49:41Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8499" length="11"/>
+        <text>Dali server</text>
+      </annotation>
+      <annotation id="203">
+        <infon key="score">0.9985506</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:54:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8522" length="4"/>
+        <text>rmsd</text>
+      </annotation>
+      <annotation id="204">
+        <infon key="score">0.99829483</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:58:35Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8542" length="7"/>
+        <text>Z-score</text>
+      </annotation>
+      <annotation id="205">
+        <infon key="score">0.9988423</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:49:47Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8656" length="22"/>
+        <text>structure-based search</text>
+      </annotation>
+      <annotation id="206">
+        <infon key="score">0.99914235</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8727" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="207">
+        <infon key="score">0.9991908</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8760" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="894">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:49:55Z</infon>
+        <location offset="8791" length="6"/>
+        <text>solved</text>
+      </annotation>
+      <annotation id="208">
+        <infon key="score">0.99865067</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8798" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="209">
+        <infon key="score">0.85789466</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:39:32Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8821" length="11"/>
+        <text>tri-modular</text>
+      </annotation>
+      <annotation id="812">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:47:14Z</infon>
+        <location offset="8833" length="5"/>
+        <text>GH5_4</text>
+      </annotation>
+      <annotation id="210">
+        <infon key="score">0.99749374</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:47:41Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8851" length="19"/>
+        <text>Bacillus halodurans</text>
+      </annotation>
+      <annotation id="211">
+        <infon key="score">0.990575</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8872" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+      <annotation id="212">
+        <infon key="score">0.9986528</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:54:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8886" length="4"/>
+        <text>rmsd</text>
+      </annotation>
+      <annotation id="213">
+        <infon key="score">0.9982287</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:58:35Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8903" length="7"/>
+        <text>Z-score</text>
+      </annotation>
+      <annotation id="214">
+        <infon key="score">0.99091774</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8923" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="215">
+        <infon key="score">0.9590935</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8934" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+      <annotation id="216">
+        <infon key="score">0.99646795</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8978" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="217">
+        <infon key="score">0.9937691</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8986" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="218">
+        <infon key="score">0.99858934</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:54:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8998" length="4"/>
+        <text>rmsd</text>
+      </annotation>
+      <annotation id="219">
+        <infon key="score">0.99820775</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:58:35Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9015" length="7"/>
+        <text>Z-score</text>
+      </annotation>
+      <annotation id="220">
+        <infon key="score">0.9766565</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9065" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+      <annotation id="221">
+        <infon key="score">0.9958629</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:49:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9099" length="7"/>
+        <text>Ig-like</text>
+      </annotation>
+      <annotation id="222">
+        <infon key="score">0.9557978</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9119" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>9161</offset>
+      <text>The Ig-like module, adjacent to the CD, contains only one tyrosine (Y367) exposed to solvent and no tryptophan residues. Because aromatic residues play a major role in glucose recognition, this observation suggests that substrate binding may not be the primary function of Ig-like module. In contrast, the CBM46 has three tryptophan residues, two of which face the CD substrate binding site (Fig. 1A), indicating that it may be actively engaged in the carbohydrate binding.</text>
+      <annotation id="223">
+        <infon key="score">0.9992533</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9165" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="224">
+        <infon key="score">0.99951124</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9197" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="225">
+        <infon key="score">0.99710685</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:58:50Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9219" length="8"/>
+        <text>tyrosine</text>
+      </annotation>
+      <annotation id="226">
+        <infon key="score">0.9993705</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:59:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9229" length="4"/>
+        <text>Y367</text>
+      </annotation>
+      <annotation id="227">
+        <infon key="score">0.9975345</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:58:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9261" length="10"/>
+        <text>tryptophan</text>
+      </annotation>
+      <annotation id="228">
+        <infon key="score">0.9978206</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:11Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9329" length="7"/>
+        <text>glucose</text>
+      </annotation>
+      <annotation id="229">
+        <infon key="score">0.9991925</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9434" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="230">
+        <infon key="score">0.991456</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9467" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="231">
+        <infon key="score">0.9973424</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:58:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9483" length="10"/>
+        <text>tryptophan</text>
+      </annotation>
+      <annotation id="232">
+        <infon key="score">0.9994098</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9526" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="233">
+        <infon key="score">0.9986832</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:59:13Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9529" length="22"/>
+        <text>substrate binding site</text>
+      </annotation>
+      <annotation id="234">
+        <infon key="score">0.9937337</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:27Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9613" length="12"/>
+        <text>carbohydrate</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>9635</offset>
+      <text>Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule. The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end. The ability of BlCel5B to cleave C5 into glucose and C4 molecules in solution was demonstrated by enzymatic product profile mass spectrometry analysis (Fig. 2A). The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D). These residues belong to the CD and are conserved in the GH5 family.</text>
+      <annotation id="235">
+        <infon key="score">0.9983968</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:59:27Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9635" length="21"/>
+        <text>Electron density maps</text>
+      </annotation>
+      <annotation id="827">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:01:00Z</infon>
+        <location offset="9676" length="11"/>
+        <text>presence of</text>
+      </annotation>
+      <annotation id="236">
+        <infon key="score">0.9993093</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:01:13Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9690" length="13"/>
+        <text>cellotetraose</text>
+      </annotation>
+      <annotation id="237">
+        <infon key="score">0.803788</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:01:09Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9705" length="2"/>
+        <text>C4</text>
+      </annotation>
+      <annotation id="238">
+        <infon key="score">0.99934393</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:36Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9726" length="13"/>
+        <text>cellopentaose</text>
+      </annotation>
+      <annotation id="239">
+        <infon key="score">0.8238797</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:40Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9741" length="2"/>
+        <text>C5</text>
+      </annotation>
+      <annotation id="240">
+        <infon key="score">0.99800676</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9752" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="241">
+        <infon key="score">0.99895775</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:51:14Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9755" length="35"/>
+        <text>negative substrate-binding subsites</text>
+      </annotation>
+      <annotation id="242">
+        <infon key="score">0.99930835</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9818" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="243">
+        <infon key="score">0.99286747</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:59:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+      <annotation id="825">
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+        <infon key="updated_at">2023-09-21T10:00:23Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:11Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9968" length="7"/>
+        <text>glucose</text>
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+      <annotation id="248">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:40Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9999" length="2"/>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:59:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10034" length="9"/>
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+      <annotation id="826">
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="updated_at">2023-09-21T10:00:59Z</infon>
+        <location offset="10063" length="11"/>
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+        <location offset="10077" length="5"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:11Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10083" length="7"/>
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+        <infon key="score">0.9905514</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <location offset="10132" length="18"/>
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+        <infon key="score">0.999263</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10186" length="4"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:31Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10287" length="2"/>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:43:11Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10295" length="7"/>
+        <text>glucose</text>
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+        <infon key="score">0.9217632</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:01:09Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="score">0.9981227</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:50:09Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10352" length="43"/>
+        <text>enzymatic product profile mass spectrometry</text>
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+      <annotation id="259">
+        <infon key="score">0.4754696</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:01:09Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10420" length="2"/>
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+      <annotation id="794">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:31Z</infon>
+        <location offset="10439" length="7"/>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:01:49Z</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="10463" length="11"/>
+        <text>coordinated</text>
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+        <infon key="score">0.9969641</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:02:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10478" length="14"/>
+        <text>hydrogen bonds</text>
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+      <annotation id="262">
+        <infon key="score">0.9992698</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:02:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10505" length="3"/>
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+        <infon key="score">0.99924517</infon>
+        <infon key="type">residue_name_number</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10510" length="4"/>
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+        <infon key="type">residue_name_number</infon>
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+        <location offset="10516" length="4"/>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:02:27Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10522" length="4"/>
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+      <annotation id="266">
+        <infon key="score">0.9993518</infon>
+        <infon key="type">residue_name_number</infon>
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+        <infon key="updated_at">2023-09-21T10:02:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10528" length="4"/>
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+      <annotation id="267">
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+        <infon key="type">residue_name_number</infon>
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+        <location offset="10538" length="4"/>
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+      <annotation id="833">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:02:54Z</infon>
+        <location offset="10552" length="16"/>
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+        <infon key="type">residue_name_number</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10582" length="3"/>
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+      <annotation id="269">
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+        <infon key="type">structure_element</infon>
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+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10626" length="2"/>
+        <text>CD</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:03:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10637" length="9"/>
+        <text>conserved</text>
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+      <annotation id="805">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:46:54Z</infon>
+        <location offset="10654" length="3"/>
+        <text>GH5</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>10667</offset>
+      <text>BlCel5B enzymatic activity</text>
+      <annotation id="271">
+        <infon key="score">0.9967526</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10667" length="7"/>
+        <text>BlCel5B</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>10694</offset>
+      <text>BlCel5B exhibits optimum activity toward carboxymethylcellulose (CMC; 8.7 U/mg) at a pH of 4.0 and 55 °C and retains approximately half of its maximum activity at 80 °C, demonstrating considerable thermal stability (Fig. 2B,C). BlCel5B is also active on β-glucan (34 U/mg), lichenan (17.8 U/mg) and xyloglucan (15.7 U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-β-mannan or the insoluble substrate Azo-Avicel. Kinetic parameters were calculated assuming Michaelis-Menten behavior with CMC as substrate: KM = 1.78 g L−1 and Vmax = 1.41 × 10−4 g s−1 mg protein−1 (Fig. 2D). Although BlCel5B is not a highly active enzyme against one specific substrate as compared to others GH5_4, it has the advantage of being active against different substrates with β-1,3 and/or β-1,4 glycosidic linkages.</text>
+      <annotation id="272">
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+        <infon key="type">protein</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="10694" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="273">
+        <infon key="score">0.9986595</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:03:26Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10735" length="22"/>
+        <text>carboxymethylcellulose</text>
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+      <annotation id="274">
+        <infon key="score">0.99820554</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:03:31Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10759" length="3"/>
+        <text>CMC</text>
+      </annotation>
+      <annotation id="275">
+        <infon key="score">0.99701774</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10922" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="800">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:58Z</infon>
+        <location offset="10938" length="6"/>
+        <text>active</text>
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+        <infon key="score">0.9989734</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:51:20Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10948" length="8"/>
+        <text>β-glucan</text>
+      </annotation>
+      <annotation id="277">
+        <infon key="score">0.998659</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:03:38Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10968" length="8"/>
+        <text>lichenan</text>
+      </annotation>
+      <annotation id="278">
+        <infon key="score">0.99895394</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:49:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10993" length="10"/>
+        <text>xyloglucan</text>
+      </annotation>
+      <annotation id="279">
+        <infon key="score">0.9986243</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:03:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11074" length="13"/>
+        <text>galactomannan</text>
+      </annotation>
+      <annotation id="280">
+        <infon key="score">0.9518401</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:04:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11089" length="3"/>
+        <text>rye</text>
+      </annotation>
+      <annotation id="281">
+        <infon key="score">0.99827766</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:04:11Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11093" length="12"/>
+        <text>arabinoxylan</text>
+      </annotation>
+      <annotation id="282">
+        <infon key="score">0.99909824</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:04:07Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11107" length="12"/>
+        <text>1,4-β-mannan</text>
+      </annotation>
+      <annotation id="283">
+        <infon key="score">0.9968621</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:04:17Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11147" length="10"/>
+        <text>Azo-Avicel</text>
+      </annotation>
+      <annotation id="836">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:04:39Z</infon>
+        <location offset="11203" length="25"/>
+        <text>Michaelis-Menten behavior</text>
+      </annotation>
+      <annotation id="284">
+        <infon key="score">0.99784744</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:03:31Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11234" length="3"/>
+        <text>CMC</text>
+      </annotation>
+      <annotation id="285">
+        <infon key="score">0.9709084</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:04:30Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11252" length="2"/>
+        <text>KM</text>
+      </annotation>
+      <annotation id="286">
+        <infon key="score">0.98771733</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:04:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11272" length="4"/>
+        <text>Vmax</text>
+      </annotation>
+      <annotation id="287">
+        <infon key="score">0.99737585</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11330" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="801">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:58Z</infon>
+        <location offset="11354" length="6"/>
+        <text>active</text>
+      </annotation>
+      <annotation id="813">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:47:14Z</infon>
+        <location offset="11421" length="5"/>
+        <text>GH5_4</text>
+      </annotation>
+      <annotation id="802">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:58Z</infon>
+        <location offset="11458" length="6"/>
+        <text>active</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>11580</offset>
+      <text>To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A. These mutants were expressed and purified as described for the wild-type enzyme. Strikingly, neither of the deletion variants exhibited detectable activity toward any of the substrates tested using full-length BlCel5B (Table 1), demonstrating that the Ig-like module and the CBM46 are essential for BlCel5B activity. Thermal shift assays were conducted to confirm structural stability of the mutants (Supplementary Fig. 1). All of the constructs showed similar melting temperatures: 62 °C for BlCel5B, 58 °C for BlCel5BΔCBM46, 56 °C for BlCel5BΔIg-CBM46, 65 °C for BlCel5BW479A and 59 °C for BlCel5BW479A, thus confirming their proper overall fold.</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:47Z</infon>
+        <location offset="11616" length="17"/>
+        <text>ancillary modules</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11638" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="289">
+        <infon key="score">0.99880445</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:05:43Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11656" length="16"/>
+        <text>enzymatic assays</text>
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+      <annotation id="839">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:07:23Z</infon>
+        <location offset="11708" length="7"/>
+        <text>mutants</text>
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+      <annotation id="290">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11719" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="291">
+        <infon key="score">0.5755105</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:05:45Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11725" length="8"/>
+        <text>deletion</text>
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+        <infon key="score">0.9981463</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:06:07Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11735" length="6"/>
+        <text>ΔCBM46</text>
+      </annotation>
+      <annotation id="837">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:06:35Z</infon>
+        <location offset="11750" length="7"/>
+        <text>Ig-like</text>
+      </annotation>
+      <annotation id="293">
+        <infon key="score">0.97647095</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11760" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="294">
+        <infon key="score">0.7081323</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:05:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11766" length="8"/>
+        <text>deletion</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:06:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11776" length="9"/>
+        <text>ΔIg-CBM46</text>
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+      <annotation id="296">
+        <infon key="score">0.99609196</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:05:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11798" length="15"/>
+        <text>point mutations</text>
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+      <annotation id="297">
+        <infon key="score">0.3921337</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11821" length="5"/>
+        <text>CBM46</text>
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+      <annotation id="298">
+        <infon key="score">0.9528272</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:06:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11850" length="5"/>
+        <text>W479A</text>
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+        <infon key="score">0.9781924</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:07:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11860" length="5"/>
+        <text>W481A</text>
+      </annotation>
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+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:07:23Z</infon>
+        <location offset="11873" length="7"/>
+        <text>mutants</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:05:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11886" length="22"/>
+        <text>expressed and purified</text>
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+        <infon key="score">0.99896145</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:07:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11930" length="9"/>
+        <text>wild-type</text>
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+      <annotation id="841">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:07:46Z</infon>
+        <location offset="11975" length="17"/>
+        <text>deletion variants</text>
+      </annotation>
+      <annotation id="302">
+        <infon key="score">0.9990125</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12065" length="11"/>
+        <text>full-length</text>
+      </annotation>
+      <annotation id="303">
+        <infon key="score">0.9991658</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12077" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="304">
+        <infon key="score">0.99874085</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12119" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="305">
+        <infon key="score">0.94216156</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12142" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="306">
+        <infon key="score">0.9989291</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12166" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="307">
+        <infon key="score">0.9990215</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:05:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12184" length="20"/>
+        <text>Thermal shift assays</text>
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+      <annotation id="308">
+        <infon key="score">0.70613146</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:07:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12259" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="309">
+        <infon key="score">0.99822545</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:05:51Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12328" length="20"/>
+        <text>melting temperatures</text>
+      </annotation>
+      <annotation id="310">
+        <infon key="score">0.9989581</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12360" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="311">
+        <infon key="score">0.9986719</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:07:55Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12379" length="13"/>
+        <text>BlCel5BΔCBM46</text>
+      </annotation>
+      <annotation id="312">
+        <infon key="score">0.9987348</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:08:06Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12404" length="16"/>
+        <text>BlCel5BΔIg-CBM46</text>
+      </annotation>
+      <annotation id="313">
+        <infon key="score">0.99815816</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:08:12Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12432" length="12"/>
+        <text>BlCel5BW479A</text>
+      </annotation>
+      <annotation id="314">
+        <infon key="score">0.9983828</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:08:11Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12459" length="12"/>
+        <text>BlCel5BW479A</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>12539</offset>
+      <text>We also examined the function of the CBM46 inner surface residues W479 and W481 (Fig. 1A) in BlCel5B activity by performing enzymatic assays with W479A and W481A mutants. Both mutations reduced enzymatic activity toward all tested substrates (Table 1), with W481A having a stronger effect than W479A (~64% vs. 79% activity relative to wt BlCel5B using β-glucan and ~10% vs. 50% using CMC). This indicates that CBM46 must interact with the substrate via residues W479 and W481. However, since the BlCel5B crystal structure exhibits no close contact between these residues and the substrate, these results suggest the existence of large-amplitude interdomain motions that may enable direct interactions between CBM46 and the carbohydrate.</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12576" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="316">
+        <infon key="score">0.5860964</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:08:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12588" length="7"/>
+        <text>surface</text>
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+      <annotation id="317">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:08:24Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12605" length="4"/>
+        <text>W479</text>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:08:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12614" length="4"/>
+        <text>W481</text>
+      </annotation>
+      <annotation id="319">
+        <infon key="score">0.9991447</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12632" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="320">
+        <infon key="score">0.99821043</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:08:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12663" length="16"/>
+        <text>enzymatic assays</text>
+      </annotation>
+      <annotation id="321">
+        <infon key="score">0.9988212</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:06:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12685" length="5"/>
+        <text>W479A</text>
+      </annotation>
+      <annotation id="322">
+        <infon key="score">0.998841</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:07:04Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12695" length="5"/>
+        <text>W481A</text>
+      </annotation>
+      <annotation id="323">
+        <infon key="score">0.9983955</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:07:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12701" length="7"/>
+        <text>mutants</text>
+      </annotation>
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+        <infon key="score">0.88579583</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:08:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12715" length="9"/>
+        <text>mutations</text>
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+      <annotation id="325">
+        <infon key="score">0.9985268</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:07:04Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12797" length="5"/>
+        <text>W481A</text>
+      </annotation>
+      <annotation id="326">
+        <infon key="score">0.99865025</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:06:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12833" length="5"/>
+        <text>W479A</text>
+      </annotation>
+      <annotation id="327">
+        <infon key="score">0.99929404</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:08:44Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12874" length="2"/>
+        <text>wt</text>
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+        <infon key="score">0.99914324</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12877" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="329">
+        <infon key="score">0.99908346</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:51:20Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12891" length="8"/>
+        <text>β-glucan</text>
+      </annotation>
+      <annotation id="330">
+        <infon key="score">0.9982147</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:03:31Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12923" length="3"/>
+        <text>CMC</text>
+      </annotation>
+      <annotation id="331">
+        <infon key="score">0.9895173</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12949" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="332">
+        <infon key="score">0.99948907</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:08:25Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13001" length="4"/>
+        <text>W479</text>
+      </annotation>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:08:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="13035" length="7"/>
+        <text>BlCel5B</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13043" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="868">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:21:54Z</infon>
+        <location offset="13073" length="5"/>
+        <text>close</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13248" length="5"/>
+        <text>CBM46</text>
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+      <annotation id="337">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:27Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13262" length="12"/>
+        <text>carbohydrate</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>13279</offset>
+      <text>BlCelB5 dynamics and binding-site architecture</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <text>BlCelB5</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:51:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13300" length="12"/>
+        <text>binding-site</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>13326</offset>
+      <text>Molecular dynamics (MD) simulations were performed to investigate the conformational mobility of BlCel5B. In the simulations of the crystal structure for BlCel5B bound to C4, the substrate dissociates from the protein within the first 100 ns of the simulation time (Supplementary Fig. 2A). This observation suggests that cellotetraose does not exhibit detectable affinity for this specific BlCel5B conformation in solution, as one might otherwise expect for a reaction product. No changes beyond local fluctuations were observed in any of the three BlCel5B domains within the time scale of these runs (400 ns; Supplementary Fig. 2B). However, the CBM46 and Ig-like domains did exhibit rigid body-like motions relative to the CD, with rmsd values around 2.3 Å and 1.8 Å, respectively, suggesting that BlCel5B may execute large-amplitude interdomain motions over longer time scales (Supplementary Fig. 2B,C).</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:09:00Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>Molecular dynamics</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:09:04Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>MD</text>
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+        <infon key="score">0.99540097</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:09:09Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>simulations</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="13423" length="7"/>
+        <text>BlCel5B</text>
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+      <annotation id="344">
+        <infon key="score">0.9986778</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:09:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="13439" length="11"/>
+        <text>simulations</text>
+      </annotation>
+      <annotation id="345">
+        <infon key="score">0.9987263</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13458" length="17"/>
+        <text>crystal structure</text>
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+      <annotation id="346">
+        <infon key="score">0.99925226</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="13480" length="7"/>
+        <text>BlCel5B</text>
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+      <annotation id="842">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:09:34Z</infon>
+        <location offset="13488" length="8"/>
+        <text>bound to</text>
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+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:01:09Z</infon>
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+        <text>C4</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:09:15Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="13575" length="10"/>
+        <text>simulation</text>
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+      <annotation id="348">
+        <infon key="score">0.9992524</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:01:14Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13647" length="13"/>
+        <text>cellotetraose</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="13716" length="7"/>
+        <text>BlCel5B</text>
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+      <annotation id="350">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="13875" length="7"/>
+        <text>BlCel5B</text>
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+      <annotation id="351">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13973" length="5"/>
+        <text>CBM46</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:49:14Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13983" length="15"/>
+        <text>Ig-like domains</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="14051" length="2"/>
+        <text>CD</text>
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+      <annotation id="354">
+        <infon key="score">0.99847704</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:54:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>rmsd</text>
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+      <annotation id="355">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="14126" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>14243</offset>
+      <text>Accordingly, simulations were then performed using accelerated molecular dynamics (aMD) techniques to probe BlCel5B interdomain motions. aMD enhances conformational sampling by raising the basins of the dihedral potential energy surface without affecting the general form of the atomistic potential, thereby increasing transition rates between different local minima. aMD trajectories corresponding to more than 1.0 μs of conventional MD runs were generated. During these simulations, we observed occlusive conformations between CBM46 and CD that resulted in a rearrangement of the enzyme’s architecture around the active site (Video S1). Figure 3A shows BlCel5B in the crystallographic conformation (red) and in a selected configuration obtained with aMD (blue) in the absence of the substrate. Interdomain motions were gauged by the time evolution of the distance between the α carbons of residues I120 and E477 (represented as spheres in Fig. 3A), belonging to the CD and CBM46, respectively. Figure 3C shows that the I120-E477 distance (red curve) gradually decreases from ~35 Å to ~7 Å within the first half of the 1.0 μs aMD trajectory, indicating a transition between the semi-open (crystallographic) and occluded (aMD sampled) configurations. During the second half of the aMD simulation, the full-length enzyme remained in the closed conformation, with the CBM46 covering the carbohydrate-binding site. These results suggest that BlCel5B undergoes large-scale interdomain movements that enable interactions between CBM46 and the substrate bound to the CD.</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:09:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14256" length="11"/>
+        <text>simulations</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:09:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14294" length="30"/>
+        <text>accelerated molecular dynamics</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14326" length="3"/>
+        <text>aMD</text>
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+      <annotation id="359">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="14351" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14380" length="3"/>
+        <text>aMD</text>
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+      <annotation id="361">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:47:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14446" length="33"/>
+        <text>dihedral potential energy surface</text>
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+      <annotation id="362">
+        <infon key="score">0.9982533</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14611" length="3"/>
+        <text>aMD</text>
+      </annotation>
+      <annotation id="363">
+        <infon key="score">0.98539543</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:47:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14615" length="12"/>
+        <text>trajectories</text>
+      </annotation>
+      <annotation id="364">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:09:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14678" length="2"/>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:09:51Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14715" length="11"/>
+        <text>simulations</text>
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+      <annotation id="366">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="14772" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="367">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="14782" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="368">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="14858" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="369">
+        <infon key="score">0.99922407</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="14898" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="370">
+        <infon key="score">0.982692</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:46:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14913" length="16"/>
+        <text>crystallographic</text>
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+      <annotation id="371">
+        <infon key="score">0.9982843</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14995" length="3"/>
+        <text>aMD</text>
+      </annotation>
+      <annotation id="372">
+        <infon key="score">0.9991255</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:59:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15013" length="10"/>
+        <text>absence of</text>
+      </annotation>
+      <annotation id="844">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:10Z</infon>
+        <location offset="15100" length="8"/>
+        <text>distance</text>
+      </annotation>
+      <annotation id="373">
+        <infon key="score">0.99943405</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:14:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15143" length="4"/>
+        <text>I120</text>
+      </annotation>
+      <annotation id="374">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:14:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15152" length="4"/>
+        <text>E477</text>
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+      <annotation id="375">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="15211" length="2"/>
+        <text>CD</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="15218" length="5"/>
+        <text>CBM46</text>
+      </annotation>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:14:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15264" length="4"/>
+        <text>I120</text>
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+      <annotation id="378">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:14:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15269" length="4"/>
+        <text>E477</text>
+      </annotation>
+      <annotation id="379">
+        <infon key="score">0.59275645</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15274" length="8"/>
+        <text>distance</text>
+      </annotation>
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+        <infon key="score">0.99833626</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15370" length="3"/>
+        <text>aMD</text>
+      </annotation>
+      <annotation id="381">
+        <infon key="score">0.9909917</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15374" length="10"/>
+        <text>trajectory</text>
+      </annotation>
+      <annotation id="382">
+        <infon key="score">0.9992021</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15422" length="9"/>
+        <text>semi-open</text>
+      </annotation>
+      <annotation id="383">
+        <infon key="score">0.6727852</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:44Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15433" length="16"/>
+        <text>crystallographic</text>
+      </annotation>
+      <annotation id="384">
+        <infon key="score">0.9992982</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15455" length="8"/>
+        <text>occluded</text>
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+      <annotation id="843">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:02Z</infon>
+        <location offset="15465" length="3"/>
+        <text>aMD</text>
+      </annotation>
+      <annotation id="850">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:12:52Z</infon>
+        <location offset="15524" length="14"/>
+        <text>aMD simulation</text>
+      </annotation>
+      <annotation id="385">
+        <infon key="score">0.99914116</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15544" length="11"/>
+        <text>full-length</text>
+      </annotation>
+      <annotation id="386">
+        <infon key="score">0.9993155</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15579" length="6"/>
+        <text>closed</text>
+      </annotation>
+      <annotation id="387">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="15609" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="388">
+        <infon key="score">0.99907833</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:11:12Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="15628" length="25"/>
+        <text>carbohydrate-binding site</text>
+      </annotation>
+      <annotation id="389">
+        <infon key="score">0.99925894</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="15682" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="390">
+        <infon key="score">0.38964275</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="15767" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="391">
+        <infon key="score">0.9977906</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:09:35Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15791" length="8"/>
+        <text>bound to</text>
+      </annotation>
+      <annotation id="392">
+        <infon key="score">0.9989761</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="15804" length="2"/>
+        <text>CD</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>15823</offset>
+      <text>To study the interactions of BlCel5B with a non-hydrolyzed glucan chain, we built a model structure with a cellooctaose (C8) chain spanning the entire positive (+1 to +4) and negative (−4 to −1) subsites of the enzyme. Starting from the crystallographic BlCel5B conformation, the C8 molecule deviated significantly from the active site and assumed a non-productive binding mode (Supplementary Fig. 2D). This observation suggests that the open conformation of BlCel5B is not able to hold the substrate in a position suitable for hydrolysis (Supplementary Fig. 2E). However, after subjecting the BlCel5B-C8 complex to a 0.5 μs aMD simulation with harmonic restraints on the C8 chain to prevent it from deviating from the productive binding mode, the CBM46 readily closed over the CD and trapped the C8 chain in position for hydrolysis (Fig. 3B). In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C). This BlCel5B-C8 configuration remains stable over an additional 500 ns of conventional MD simulation with no restraints (Fig. 3C cyan line, Supplementary Fig. 2E,F).</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="15852" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="394">
+        <infon key="score">0.9978961</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:53:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15882" length="6"/>
+        <text>glucan</text>
+      </annotation>
+      <annotation id="395">
+        <infon key="score">0.61043835</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:11:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15913" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="396">
+        <infon key="score">0.9992067</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:11:50Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15930" length="12"/>
+        <text>cellooctaose</text>
+      </annotation>
+      <annotation id="397">
+        <infon key="score">0.9925861</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:11:54Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15944" length="2"/>
+        <text>C8</text>
+      </annotation>
+      <annotation id="895">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:52:17Z</infon>
+        <location offset="15974" length="19"/>
+        <text>positive (+1 to +4)</text>
+      </annotation>
+      <annotation id="896">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:52:36Z</infon>
+        <location offset="15998" length="19"/>
+        <text>negative (−4 to −1)</text>
+      </annotation>
+      <annotation id="398">
+        <infon key="score">0.9882339</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:52:42Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="16018" length="8"/>
+        <text>subsites</text>
+      </annotation>
+      <annotation id="399">
+        <infon key="score">0.99916506</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16077" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="400">
+        <infon key="score">0.89470834</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:11:55Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16103" length="2"/>
+        <text>C8</text>
+      </annotation>
+      <annotation id="401">
+        <infon key="score">0.9989809</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="16147" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="402">
+        <infon key="score">0.99927276</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:13:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16261" length="4"/>
+        <text>open</text>
+      </annotation>
+      <annotation id="403">
+        <infon key="score">0.99922884</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16282" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="404">
+        <infon key="score">0.9962482</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:12:06Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="16417" length="10"/>
+        <text>BlCel5B-C8</text>
+      </annotation>
+      <annotation id="405">
+        <infon key="score">0.8329121</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:51:35Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16448" length="14"/>
+        <text>aMD simulation</text>
+      </annotation>
+      <annotation id="406">
+        <infon key="score">0.923577</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:11:55Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16495" length="2"/>
+        <text>C8</text>
+      </annotation>
+      <annotation id="407">
+        <infon key="score">0.901596</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="16571" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="408">
+        <infon key="score">0.98974496</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16585" length="6"/>
+        <text>closed</text>
+      </annotation>
+      <annotation id="409">
+        <infon key="score">0.99818546</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="16601" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="410">
+        <infon key="score">0.9084791</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:11:55Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16620" length="2"/>
+        <text>C8</text>
+      </annotation>
+      <annotation id="828">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:01:00Z</infon>
+        <location offset="16674" length="11"/>
+        <text>presence of</text>
+      </annotation>
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+        <infon key="score">0.6347472</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="16701" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="851">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:13:43Z</infon>
+        <location offset="16760" length="26"/>
+        <text>crystallographic structure</text>
+      </annotation>
+      <annotation id="412">
+        <infon key="score">0.9988453</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16812" length="14"/>
+        <text>substrate-free</text>
+      </annotation>
+      <annotation id="413">
+        <infon key="score">0.9980185</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16827" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="414">
+        <infon key="score">0.99516326</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:12:39Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16835" length="15"/>
+        <text>aMD simulations</text>
+      </annotation>
+      <annotation id="415">
+        <infon key="score">0.99944776</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:14:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16879" length="4"/>
+        <text>I120</text>
+      </annotation>
+      <annotation id="416">
+        <infon key="score">0.99935466</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:14:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16884" length="4"/>
+        <text>E477</text>
+      </annotation>
+      <annotation id="845">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:11Z</infon>
+        <location offset="16889" length="8"/>
+        <text>distance</text>
+      </annotation>
+      <annotation id="417">
+        <infon key="score">0.99933016</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16933" length="6"/>
+        <text>closed</text>
+      </annotation>
+      <annotation id="418">
+        <infon key="score">0.8814976</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:11:55Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16969" length="2"/>
+        <text>C8</text>
+      </annotation>
+      <annotation id="419">
+        <infon key="score">0.9988757</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17006" length="14"/>
+        <text>substrate-free</text>
+      </annotation>
+      <annotation id="420">
+        <infon key="score">0.9991295</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17021" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="421">
+        <infon key="score">0.9085049</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:12:07Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="17046" length="10"/>
+        <text>BlCel5B-C8</text>
+      </annotation>
+      <annotation id="854">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:14:05Z</infon>
+        <location offset="17128" length="13"/>
+        <text>MD simulation</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>17220</offset>
+      <text>A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D. Together, these results indicate that CBM46 is a key component of the catalytic active complex, providing an explanation as to why CBM46 is essential for the enzymatic activity of BlCel5B.</text>
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+        <infon key="score">0.99593264</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17330" length="5"/>
+        <text>CBM46</text>
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+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:58:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17336" length="10"/>
+        <text>tryptophan</text>
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+        <infon key="type">residue_name_number</infon>
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+        <text>CD</text>
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+        <infon key="type">residue_name</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
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+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
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+      <text>To enable substantially longer time scales compared to atomistic simulations, we further explored the dynamics of BlCel5B using coarse-grained MD (CG-MD) simulations. We performed three independent ~120 μs CG-MD simulations, for a total of approximately 360 μs of sampling. The distance between the α carbons of two residues centrally positioned in the CD and CBM46 (Fig. 4A) was monitored, and the results shown in Fig. 4B indicate that the wide-amplitude events described above frequently appear in this time scale. The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18105" length="2"/>
+        <text>CD</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:15:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>computed distance distribution</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>closed</text>
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+      <annotation id="444">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18442" length="14"/>
+        <text>substrate-free</text>
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:12:40Z</infon>
+        <location offset="18457" length="15"/>
+        <text>aMD simulations</text>
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+      <annotation id="445">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18483" length="5"/>
+        <text>CBM46</text>
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+      <annotation id="446">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18508" length="2"/>
+        <text>CD</text>
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+      <annotation id="447">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:59:14Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18524" length="22"/>
+        <text>substrate binding site</text>
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+      <annotation id="448">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18556" length="9"/>
+        <text>semi-open</text>
+      </annotation>
+      <annotation id="852">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:13:43Z</infon>
+        <location offset="18595" length="26"/>
+        <text>crystallographic structure</text>
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+      <annotation id="449">
+        <infon key="score">0.9993492</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:17:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18633" length="8"/>
+        <text>extended</text>
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+      <annotation id="450">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18642" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="451">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>CD</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>CBM46</text>
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+      <annotation id="453">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18725" length="17"/>
+        <text>crystal structure</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>18751</offset>
+      <text>BlCel5B conformers fit the SAXS envelope</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18751" length="7"/>
+        <text>BlCel5B</text>
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+      <annotation id="455">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:19Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18778" length="4"/>
+        <text>SAXS</text>
+      </annotation>
+      <annotation id="456">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:16:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18783" length="8"/>
+        <text>envelope</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>18792</offset>
+      <text>SAXS experiments were conducted to assess BlCel5B conformational states in solution, and the results revealed the enzyme in its monomeric form, with average values of Rg = 27.17 Å and Dmax = 87.59 Å (Supplementary Table 2). The ab initio dummy atom model (DAM) demonstrated that the SAXS-derived BlCel5B molecular envelope could not be single-handedly filled by any of the main conformational states encountered in the simulations (Fig. 4D).</text>
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+        <infon key="identifier">MESH:</infon>
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+        <text>SAXS</text>
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+        <infon key="identifier">PR:</infon>
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+        <text>BlCel5B</text>
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+      <annotation id="459">
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+        <infon key="type">oligomeric_state</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18920" length="9"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:16:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="19020" length="26"/>
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+        <infon key="type">experimental_method</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>DAM</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19075" length="4"/>
+        <text>SAXS</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="19088" length="7"/>
+        <text>BlCel5B</text>
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+      <annotation id="856">
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:16:19Z</infon>
+        <location offset="19106" length="8"/>
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+        <infon key="updated_at">2023-09-21T10:09:51Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19211" length="11"/>
+        <text>simulations</text>
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+    </passage>
+    <passage>
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+      <infon key="type">paragraph</infon>
+      <offset>19248</offset>
+      <text>It is known that a Kratky plot exhibits a peak with an elevated baseline at high q for a monodisperse system composed of multi-domain particles with flexible extensions. Indeed, an elevation of the baseline toward a hyperbolic-like curve was observed for BlCel5B, indicating a considerable degree of molecular mobility in solution (Supplementary Fig. 3). Thus, the conformational heterogeneity of the enzyme can be decomposed in structural terms as a combination of conformational states identified in our crystallographic and MD studies. We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope. The resulting average scattering curve from this model fits the experimental protein scattering intensity, with χ = 1.89 (Supplementary Fig. 3).</text>
+      <annotation id="467">
+        <infon key="score">0.99568725</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:16:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19267" length="11"/>
+        <text>Kratky plot</text>
+      </annotation>
+      <annotation id="468">
+        <infon key="score">0.99910104</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="19503" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="857">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:17:13Z</infon>
+        <location offset="19754" length="31"/>
+        <text>crystallographic and MD studies</text>
+      </annotation>
+      <annotation id="469">
+        <infon key="score">0.9935033</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19805" length="4"/>
+        <text>SAXS</text>
+      </annotation>
+      <annotation id="470">
+        <infon key="score">0.9407316</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:16:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19810" length="8"/>
+        <text>envelope</text>
+      </annotation>
+      <annotation id="471">
+        <infon key="score">0.998749</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:17:41Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19862" length="15"/>
+        <text>superimposition</text>
+      </annotation>
+      <annotation id="472">
+        <infon key="score">0.999127</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="19939" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="473">
+        <infon key="score">0.99936813</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19960" length="6"/>
+        <text>closed</text>
+      </annotation>
+      <annotation id="872">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:41:06Z</infon>
+        <location offset="19970" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="873">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:41:16Z</infon>
+        <location offset="19976" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="874">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:41:26Z</infon>
+        <location offset="19979" length="8"/>
+        <text>occluded</text>
+      </annotation>
+      <annotation id="474">
+        <infon key="score">0.9985977</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:09:51Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20020" length="11"/>
+        <text>simulations</text>
+      </annotation>
+      <annotation id="475">
+        <infon key="score">0.99917656</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20065" length="9"/>
+        <text>semi-open</text>
+      </annotation>
+      <annotation id="476">
+        <infon key="score">0.9984336</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20107" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="477">
+        <infon key="score">0.9993529</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:17:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20154" length="8"/>
+        <text>extended</text>
+      </annotation>
+      <annotation id="478">
+        <infon key="score">0.9984976</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:09:51Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20185" length="11"/>
+        <text>simulations</text>
+      </annotation>
+      <annotation id="479">
+        <infon key="score">0.98378396</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20232" length="4"/>
+        <text>SAXS</text>
+      </annotation>
+      <annotation id="480">
+        <infon key="score">0.9104051</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:16:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20237" length="8"/>
+        <text>envelope</text>
+      </annotation>
+      <annotation id="481">
+        <infon key="score">0.9977047</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:17:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20261" length="24"/>
+        <text>average scattering curve</text>
+      </annotation>
+      <annotation id="482">
+        <infon key="score">0.99628973</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:18:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20332" length="20"/>
+        <text>scattering intensity</text>
+      </annotation>
+      <annotation id="483">
+        <infon key="score">0.994159</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:18:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20359" length="1"/>
+        <text>χ</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>20397</offset>
+      <text>GH5_4 phylogenetic analysis</text>
+      <annotation id="814">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:47:14Z</infon>
+        <location offset="20397" length="5"/>
+        <text>GH5_4</text>
+      </annotation>
+      <annotation id="484">
+        <infon key="score">0.99817145</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:18:29Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20403" length="21"/>
+        <text>phylogenetic analysis</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>20425</offset>
+      <text>To date, there are 427 sequences classified as subfamily 4 members in the CAZy database. After the exclusion of partial sequences and the suppression of highly identical members (higher than 90% identity), 144 sequences containing between 277 and 400 residues were aligned and used to construct a phylogenetic tree (Supplementary Fig. 4A). According to PFAM database conserved domain classification, 128 GH5 enzymes have an architecture consisting of an N-terminal catalytic module, a CBM_X2 module and an unknown module of approximately 100 residues at the C-terminus (Supplementary Fig. 4B). Of these, 12 enzymes have an additional CBM1, and 5 have a CBM2 at the N-terminal region. Based on this PFAM architecture and CAZy subfamily classification, all the 144 enzymes (including BlCel5B) belong to the GH5_4 subfamily and group together in the same branch of the phylogenetic tree, evidencing a common ancestor. These results support the hypothesis that the enzymes may employ the same mechanism by which ligand binding is mediated by an extensive conformational breathing of the enzyme that involves the large-scale movement of CBM46 around the Ig-like module (CBM_X2) as a structural hinge.</text>
+      <annotation id="485">
+        <infon key="score">0.8797403</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:18:44Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20664" length="11"/>
+        <text>277 and 400</text>
+      </annotation>
+      <annotation id="486">
+        <infon key="score">0.99440295</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:18:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20690" length="7"/>
+        <text>aligned</text>
+      </annotation>
+      <annotation id="862">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:20:09Z</infon>
+        <location offset="20722" length="17"/>
+        <text>phylogenetic tree</text>
+      </annotation>
+      <annotation id="806">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:46:54Z</infon>
+        <location offset="20829" length="3"/>
+        <text>GH5</text>
+      </annotation>
+      <annotation id="487">
+        <infon key="score">0.99921066</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:19:02Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="20890" length="16"/>
+        <text>catalytic module</text>
+      </annotation>
+      <annotation id="860">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:19:29Z</infon>
+        <location offset="20910" length="6"/>
+        <text>CBM_X2</text>
+      </annotation>
+      <annotation id="488">
+        <infon key="score">0.99906105</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:19:34Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="21059" length="4"/>
+        <text>CBM1</text>
+      </annotation>
+      <annotation id="489">
+        <infon key="score">0.9988931</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:19:39Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="21078" length="4"/>
+        <text>CBM2</text>
+      </annotation>
+      <annotation id="490">
+        <infon key="score">0.99761593</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="21207" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="815">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:47:14Z</infon>
+        <location offset="21230" length="5"/>
+        <text>GH5_4</text>
+      </annotation>
+      <annotation id="861">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:20:08Z</infon>
+        <location offset="21291" length="17"/>
+        <text>phylogenetic tree</text>
+      </annotation>
+      <annotation id="491">
+        <infon key="score">0.99684453</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="21557" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="492">
+        <infon key="score">0.9991983</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="21574" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="863">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:20:35Z</infon>
+        <location offset="21590" length="6"/>
+        <text>CBM_X2</text>
+      </annotation>
+      <annotation id="493">
+        <infon key="score">0.9989972</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:20:38Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="21603" length="16"/>
+        <text>structural hinge</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">title_1</infon>
+      <offset>21621</offset>
+      <text>Discussion</text>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>21632</offset>
+      <text>Growing interest in biotechnological applications of enzymes exhibiting activity toward lignocellulosic biomass has sparked efforts in the discovery and development of novel enzymes, as well as the search for a deeper understanding of their mechanisms of action. Here, we elucidate the trimodular molecular architecture of the full-length BlCel5B, a member of the GH5_4 subfamily, for which large-scale conformational dynamics appears to play a central role in its enzymatic activity. Full-length BlCel5B is active on both cellulosic and hemicellulosic substrates and auxiliary modules are crucial for its activity.</text>
+      <annotation id="869">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:37:12Z</infon>
+        <location offset="21918" length="10"/>
+        <text>trimodular</text>
+      </annotation>
+      <annotation id="494">
+        <infon key="score">0.999111</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21959" length="11"/>
+        <text>full-length</text>
+      </annotation>
+      <annotation id="495">
+        <infon key="score">0.99920964</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="21971" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="816">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:47:14Z</infon>
+        <location offset="21996" length="5"/>
+        <text>GH5_4</text>
+      </annotation>
+      <annotation id="496">
+        <infon key="score">0.9991419</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22117" length="11"/>
+        <text>Full-length</text>
+      </annotation>
+      <annotation id="497">
+        <infon key="score">0.9992415</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="22129" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="498">
+        <infon key="score">0.98844063</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22140" length="6"/>
+        <text>active</text>
+      </annotation>
+      <annotation id="865">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:21:10Z</infon>
+        <location offset="22155" length="10"/>
+        <text>cellulosic</text>
+      </annotation>
+      <annotation id="866">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:21:21Z</infon>
+        <location offset="22170" length="14"/>
+        <text>hemicellulosic</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>22248</offset>
+      <text>Most carbohydrate-active enzymes are modular and consist of a catalytic domain appended to one or more separate AMs. AMs, such as CBMs, typically recognize carbohydrates and target their cognate catalytic domains toward the substrate. Because the structural analysis of the protein is challenging if the linkers connecting the structural subunits of the enzyme are long and flexible, the standard approach is to study the domains separately. In this work, a combination of protein crystallography, computational molecular dynamics, and SAXS analyses enabled the identification of a new conformational selection-based molecular mechanism that involves GH5 catalytic domain and two AMs in full-length BlCel5B. We observed that the BlCel5B distal CBM46 is directly involved in shaping the local architecture of the substrate-binding site. Although the CD alone appears unable to bind the substrate for catalysis, the AMs exhibit open-close motions that allow the substrate to be captured in a suitable position for hydrolysis. Here, we advocate that large-amplitude motions of AMs are crucial for assembling the enzyme into its active conformation, highlighting a new function of CBMs. This mechanism of substrate binding closely resembles the extended conformational selection model, with the induced-fit mechanism of reaction as its limiting case. To the best of our knowledge, this enzymatic mechanism has not been proposed previously for any GH.</text>
+      <annotation id="499">
+        <infon key="score">0.99675375</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:21:34Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22253" length="27"/>
+        <text>carbohydrate-active enzymes</text>
+      </annotation>
+      <annotation id="500">
+        <infon key="score">0.99907744</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:21Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22310" length="16"/>
+        <text>catalytic domain</text>
+      </annotation>
+      <annotation id="501">
+        <infon key="score">0.5816129</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22360" length="3"/>
+        <text>AMs</text>
+      </annotation>
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+        <infon key="score">0.7359264</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:05Z</infon>
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+        <text>AMs</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:42Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>CBMs</text>
+      </annotation>
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+        <infon key="score">0.9982529</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:42:25Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="22404" length="13"/>
+        <text>carbohydrates</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:49:19Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>catalytic domains</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="22552" length="7"/>
+        <text>linkers</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="22721" length="23"/>
+        <text>protein crystallography</text>
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+      <annotation id="509">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:51:04Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>computational molecular dynamics</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22784" length="4"/>
+        <text>SAXS</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:46:54Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>GH5</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:21Z</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>full-length</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="22947" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="516">
+        <infon key="score">0.99905556</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="22977" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="517">
+        <infon key="score">0.9524748</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22992" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="518">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:45:11Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>substrate-binding site</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23097" length="2"/>
+        <text>CD</text>
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+      <annotation id="520">
+        <infon key="score">0.9879859</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:46:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23100" length="5"/>
+        <text>alone</text>
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+      <annotation id="521">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23162" length="3"/>
+        <text>AMs</text>
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+      <annotation id="522">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:13:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23174" length="4"/>
+        <text>open</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:21:53Z</infon>
+        <location offset="23179" length="5"/>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:05Z</infon>
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+        <text>AMs</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>CBMs</text>
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+      <annotation id="859">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:17:58Z</infon>
+        <location offset="23489" length="8"/>
+        <text>extended</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="23691" length="2"/>
+        <text>GH</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>23695</offset>
+      <text>The CD binding site of BlCel5B is open and relatively flat and is thus barely able to properly hold the substrate in position for catalysis without assistance from the CBM46. In contrast, other GH5s belonging to subfamily 4 listed in the Protein Data Bank exhibit a deep binding cleft or tunnel that can effectively entrap the substrate for catalysis (Fig. 5). Due to the marked interdomain conformational rearrangement observed in our simulations, the CBM46 generates a confined binding site in BlCel5B that resembles the binding site architecture of the other GH5 enzymes that lack AMs. Thus, BlCel5B appears to have adopted a strategy of CBM46-mediated interactions for proper functioning. Although the homologous BhCel5B has the same domain architecture of BlCel5B and belongs to the same subfamily (a comparison of the sequence and structure of BlCel5B and BhCel5B is presented in Supplementary Fig. 5), its binding site exhibits important differences that may impact the catalytic mechanism. The BhCel5B binding site is V-shaped and deeper than the BlCel5B binding site (Figs 5 and 6). This is due to the loop between residues F177 and R185 from BhCel5B (absent in the BlCel5B), which contains residue W181 that forms part of the binding cleft (Fig. 6). Consistently, although BhCel5B CBM46 is important for β-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates. A closer inspection of results of the phylogenetic analysis, more specifically of the clade composed by GH5_4 enzymes with trimodular architecture (Supplementary Fig. 4C), reveals subclades whose main characteristic is the varying length of the loop located between residues 161 and 163 (BlCel5B residue numbering). Therefore, our results show that BlCel5B represents a smaller group of enzymes that are completely dependent on its AMs for hydrolysis of plant cell wall polysaccharides, and that the underlying mechanism may rely on large-scale interdomain motions.</text>
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+        <infon key="identifier">SO:</infon>
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+        <text>CD binding site</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23863" length="5"/>
+        <text>CBM46</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:45:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23889" length="4"/>
+        <text>GH5s</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:12Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23966" length="13"/>
+        <text>binding cleft</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:52:49Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23983" length="6"/>
+        <text>tunnel</text>
+      </annotation>
+      <annotation id="533">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:09:51Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="24131" length="11"/>
+        <text>simulations</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:35:47Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>binding site</text>
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+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24191" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:35:46Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24218" length="12"/>
+        <text>binding site</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:46:54Z</infon>
+        <location offset="24257" length="3"/>
+        <text>GH5</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:45:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24274" length="4"/>
+        <text>lack</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:05Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>AMs</text>
+      </annotation>
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+        <infon key="score">0.9986505</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24290" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
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+        <infon key="score">0.7155743</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24336" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="542">
+        <infon key="score">0.9988024</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24412" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+      <annotation id="543">
+        <infon key="score">0.9987122</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24456" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="544">
+        <infon key="score">0.9529958</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:35:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>structure</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="24557" length="7"/>
+        <text>BhCel5B</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:35:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24608" length="12"/>
+        <text>binding site</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24697" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+      <annotation id="549">
+        <infon key="score">0.9989014</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:35:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24705" length="12"/>
+        <text>binding site</text>
+      </annotation>
+      <annotation id="550">
+        <infon key="score">0.97640747</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:37:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24721" length="8"/>
+        <text>V-shaped</text>
+      </annotation>
+      <annotation id="551">
+        <infon key="score">0.99807286</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24750" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="552">
+        <infon key="score">0.9989003</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:35:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24758" length="12"/>
+        <text>binding site</text>
+      </annotation>
+      <annotation id="553">
+        <infon key="score">0.99921954</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24806" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="554">
+        <infon key="score">0.99956995</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24828" length="4"/>
+        <text>F177</text>
+      </annotation>
+      <annotation id="555">
+        <infon key="score">0.9995683</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24837" length="4"/>
+        <text>R185</text>
+      </annotation>
+      <annotation id="556">
+        <infon key="score">0.9985153</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24847" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+      <annotation id="557">
+        <infon key="score">0.9065534</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:37:31Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24856" length="6"/>
+        <text>absent</text>
+      </annotation>
+      <annotation id="558">
+        <infon key="score">0.99850297</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24870" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="559">
+        <infon key="score">0.9995596</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24903" length="4"/>
+        <text>W181</text>
+      </annotation>
+      <annotation id="560">
+        <infon key="score">0.9990684</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:12Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24931" length="13"/>
+        <text>binding cleft</text>
+      </annotation>
+      <annotation id="561">
+        <infon key="score">0.9984578</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24978" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+      <annotation id="562">
+        <infon key="score">0.99243915</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24986" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="563">
+        <infon key="score">0.9990066</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:37:25Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="25009" length="16"/>
+        <text>β-1,3-1,4-glucan</text>
+      </annotation>
+      <annotation id="564">
+        <infon key="score">0.99781764</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25038" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+      <annotation id="803">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:58Z</infon>
+        <location offset="25068" length="6"/>
+        <text>active</text>
+      </annotation>
+      <annotation id="565">
+        <infon key="score">0.99545187</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:46:44Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25075" length="7"/>
+        <text>without</text>
+      </annotation>
+      <annotation id="566">
+        <infon key="score">0.89027363</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25083" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="567">
+        <infon key="score">0.99849916</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:37:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25095" length="9"/>
+        <text>truncated</text>
+      </annotation>
+      <annotation id="568">
+        <infon key="score">0.6794937</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25126" length="6"/>
+        <text>active</text>
+      </annotation>
+      <annotation id="569">
+        <infon key="score">0.99904317</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:49:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="25141" length="10"/>
+        <text>xyloglucan</text>
+      </annotation>
+      <annotation id="570">
+        <infon key="score">0.9831435</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25173" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="571">
+        <infon key="score">0.9978708</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:18:30Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="25285" length="21"/>
+        <text>phylogenetic analysis</text>
+      </annotation>
+      <annotation id="817">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:47:14Z</infon>
+        <location offset="25351" length="5"/>
+        <text>GH5_4</text>
+      </annotation>
+      <annotation id="572">
+        <infon key="score">0.97137105</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:37:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25370" length="10"/>
+        <text>trimodular</text>
+      </annotation>
+      <annotation id="573">
+        <infon key="score">0.9991372</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25492" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="574">
+        <infon key="score">0.9713052</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:37:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25522" length="11"/>
+        <text>161 and 163</text>
+      </annotation>
+      <annotation id="575">
+        <infon key="score">0.9986671</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25535" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="576">
+        <infon key="score">0.9987871</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25596" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="577">
+        <infon key="score">0.98740965</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25679" length="3"/>
+        <text>AMs</text>
+      </annotation>
+      <annotation id="578">
+        <infon key="score">0.9983164</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:42:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25701" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="579">
+        <infon key="score">0.9876605</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:37:18Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="25717" length="15"/>
+        <text>polysaccharides</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>25816</offset>
+      <text>The amino acid sequence of the BlCel5B Ig-like module is recognized by BLASTP as belonging to CBM_X2, a poorly described group that has been compared with CBM-like accessory modules without a defined function. Despite the similarity of BlCel5B Ig-like module to CBMs, it lacks an identifiable aromatic residue-rich carbohydrate-binding site. Nonetheless, according to our results, the Ig-like module seems to play an important function as a structural hinge, dynamically holding the CBM46 and CD in positions that are appropriate for enzymatic activity.</text>
+      <annotation id="580">
+        <infon key="score">0.9989795</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25847" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="581">
+        <infon key="score">0.99937105</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25855" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="582">
+        <infon key="score">0.99841034</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:51:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="25887" length="6"/>
+        <text>BLASTP</text>
+      </annotation>
+      <annotation id="864">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:20:35Z</infon>
+        <location offset="25910" length="6"/>
+        <text>CBM_X2</text>
+      </annotation>
+      <annotation id="870">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:38:57Z</infon>
+        <location offset="25971" length="26"/>
+        <text>CBM-like accessory modules</text>
+      </annotation>
+      <annotation id="583">
+        <infon key="score">0.999084</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="26052" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="584">
+        <infon key="score">0.99936295</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26060" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="585">
+        <infon key="score">0.9975055</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:42Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26078" length="4"/>
+        <text>CBMs</text>
+      </annotation>
+      <annotation id="586">
+        <infon key="score">0.99744666</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:11:13Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26131" length="25"/>
+        <text>carbohydrate-binding site</text>
+      </annotation>
+      <annotation id="587">
+        <infon key="score">0.99939424</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26201" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="588">
+        <infon key="score">0.9986139</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:38:24Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26257" length="16"/>
+        <text>structural hinge</text>
+      </annotation>
+      <annotation id="589">
+        <infon key="score">0.89083683</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26299" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="590">
+        <infon key="score">0.9985557</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26309" length="2"/>
+        <text>CD</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>26370</offset>
+      <text>Based on the results of our crystallographic, computer simulation, and SAXS structural analyses, as well as site-directed mutagenesis and activity assays, we propose a molecular mechanism for BlCel5B substrate binding, which might apply to other GH5_4 subfamily enzymes that share this tri-modular architecture. BlCel5B can be found in several different conformational states ranging from CBM46/CD closed (or occluded) to extended conformations (Fig. 7). In extended configurations, the substrate may dock at the shallow substrate binding site of CD in one of the semi-closed conformations of the enzyme; however, its binding is properly stabilized for hydrolysis only with the aid of induced-fit repositioning mediated by CBM46. After cleavage, the intrinsic dynamics of BlCel5B would eventually allow the opening of the active site for product release. The proposed mechanism is consistent with our mutagenesis and enzymatic activity assays, which show that the Ig-like module and CBM46 are indispensable for BlCel5B catalytic activity and, together with the CD, form the unique catalytic domain of the enzyme. These experiments reveal a novel function for CBMs in which they are intimately involved in the assembly of the active site and catalytic process. Computer simulations suggest that large-scale motions of the CBM46 and Ig-like domains mediate conformational selection and final induced-fit adjustments to trap the substrate at the active site and promote hydrolysis. SAXS data support the modeling results, providing compelling evidence for highly mobile domains in solution.</text>
+      <annotation id="591">
+        <infon key="score">0.99876994</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:39:08Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26398" length="37"/>
+        <text>crystallographic, computer simulation</text>
+      </annotation>
+      <annotation id="592">
+        <infon key="score">0.91871595</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:39:11Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26441" length="24"/>
+        <text>SAXS structural analyses</text>
+      </annotation>
+      <annotation id="593">
+        <infon key="score">0.99891764</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:39:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26478" length="25"/>
+        <text>site-directed mutagenesis</text>
+      </annotation>
+      <annotation id="594">
+        <infon key="score">0.9507582</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:39:15Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26508" length="15"/>
+        <text>activity assays</text>
+      </annotation>
+      <annotation id="595">
+        <infon key="score">0.9991353</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="26562" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="818">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:47:14Z</infon>
+        <location offset="26616" length="5"/>
+        <text>GH5_4</text>
+      </annotation>
+      <annotation id="871">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:39:32Z</infon>
+        <location offset="26656" length="11"/>
+        <text>tri-modular</text>
+      </annotation>
+      <annotation id="596">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="26682" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="597">
+        <infon key="score">0.9973775</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26759" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="598">
+        <infon key="score">0.9993305</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26765" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="599">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26768" length="6"/>
+        <text>closed</text>
+      </annotation>
+      <annotation id="600">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26779" length="8"/>
+        <text>occluded</text>
+      </annotation>
+      <annotation id="601">
+        <infon key="score">0.9993074</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:17:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26792" length="8"/>
+        <text>extended</text>
+      </annotation>
+      <annotation id="602">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:17:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+      </annotation>
+      <annotation id="603">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:59:14Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26891" length="22"/>
+        <text>substrate binding site</text>
+      </annotation>
+      <annotation id="604">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>CD</text>
+      </annotation>
+      <annotation id="605">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:39:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26934" length="11"/>
+        <text>semi-closed</text>
+      </annotation>
+      <annotation id="606">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27093" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="607">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="27142" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="608">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:35Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>active site</text>
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+      <annotation id="609">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:40:45Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27271" length="41"/>
+        <text>mutagenesis and enzymatic activity assays</text>
+      </annotation>
+      <annotation id="610">
+        <infon key="score">0.99924946</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27334" length="14"/>
+        <text>Ig-like module</text>
+      </annotation>
+      <annotation id="611">
+        <infon key="score">0.9819795</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27353" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="612">
+        <infon key="score">0.9991903</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="27381" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="613">
+        <infon key="score">0.9994134</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27431" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="614">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:46:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27444" length="6"/>
+        <text>unique</text>
+      </annotation>
+      <annotation id="615">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:21Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27451" length="16"/>
+        <text>catalytic domain</text>
+      </annotation>
+      <annotation id="616">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:44:42Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27529" length="4"/>
+        <text>CBMs</text>
+      </annotation>
+      <annotation id="617">
+        <infon key="score">0.9988718</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27595" length="11"/>
+        <text>active site</text>
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+      <annotation id="618">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:40:27Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27630" length="20"/>
+        <text>Computer simulations</text>
+      </annotation>
+      <annotation id="619">
+        <infon key="score">0.95659685</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27691" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="620">
+        <infon key="score">0.999217</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:49:23Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27701" length="15"/>
+        <text>Ig-like domains</text>
+      </annotation>
+      <annotation id="621">
+        <infon key="score">0.99876976</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27813" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="622">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27849" length="4"/>
+        <text>SAXS</text>
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+      <annotation id="623">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:40:29Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27871" length="8"/>
+        <text>modeling</text>
+      </annotation>
+      <annotation id="624">
+        <infon key="score">0.9989699</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:46:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27923" length="13"/>
+        <text>highly mobile</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_1</infon>
+      <offset>27958</offset>
+      <text>Methods</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>27966</offset>
+      <text>Cloning, Expression and Purification</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>28003</offset>
+      <text>The gene encoding BlCel5B (GenBank: AAU23417.1) was amplified from Bacillus licheniformis genomic DNA (ATCC 14580) without the predicted signal peptide sequence (nucleotides 1 to 81) using the primers Blcel5B_Fw and Blcel5B_Rv (Supplementary Table 3). The fragment was cloned into the expression vector pETTRXA-1a/LIC by ligation-independent cloning (LIC), as described elsewhere.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>28384</offset>
+      <text>The same method was used for construction of domain deletions. For Ig-like + CBM46 deletion, Δ(Ig-CBM46), the fragment encoding the CD (nucleotides 82 to 1086) was amplified using the primers Blcel5B_Fw and Blcel5BΔ1087-1683_Rv. For CBM46 deletion, ΔCBM46, the fragment encoding the CD + Ig-like (nucleotides 82 to 1377) was amplified using the primers Blcel5B_Fw and Blcel5BΔ1378-1683_Rv (Supplementary Table 3). Both fragments were cloned into pETTRXA-1a/LIC.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>28862</offset>
+      <text>The wt protein BlCel5B, mutated proteins and AM deletions were expressed in E. coli Rosetta2 (DE3) strain. The cells were grown at 37 °C and 150 RPM in Luria Bertani Broth medium supplemented with 50 μg/mL kanamycin to an A600 of 1.5–2.0, after which the temperature was reduced to 20 °C and protein expression was induced with 1 mM IPTG for 6 h.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>29224</offset>
+      <text>The extract was then loaded onto a NiNTA resin (Qiagen) equilibrated with a washing buffer (5 mM imidazole, 100 mM NaCl, 50 mM Tris-HCl, pH 8.0). Non-absorbed material was washed with ten times column volume with washing buffer and the purified protein was eluted with 200 mM imidazole, 100 mM NaCl, 50 mM Tris-HCl at pH = 7.0. His6 tag was removed by overnight digestion with TEV (Tobacco Etch Virus) at 4 °C, and untagged protein was purified by gel filtration through a HiLoad 16/60 Superdex 200 column in buffer containing 50 mM NaCl, 25 mM Tris-HCl at pH 7.0.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>29812</offset>
+      <text>Site-directed Mutagenesis</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>29838</offset>
+      <text>The BlCel5B point mutations W479A and W481A were obtained by the inverse PCR method of site-directed mutagenesis. Phusion® “High-Fidelity” DNA polymerase (NEB, USA) was used for amplifications with the plasmid pETTRXA-1a/LIC-Blcel5B as a template. Mutagenic primers Blcel5BW479A_Fw/Rv and Blcel5BW481A_Fw/Rv (Supplementary Table 3) were generated by HTP-OligoDesigner tool (http://www.ifsc.usp.br/htpoligo/).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>30252</offset>
+      <text>Activity Assays</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>30268</offset>
+      <text>Enzymatic activity assays were performed by a colorimetric method using the 3,5-dinitrosalicylic acid (DNS), with glucose being a standard for the calibration curves. Assays of optimal temperature and pH were performed in triplicate with 1% medium-viscosity CMC as the substrate. For optimal temperature, the reaction mixture containing 10 μL of enzyme at 0.1 mg/mL, 50 μL of 1% (w/v) CMC and 40 μL of 50 mM sodium citrate buffer (pH 5.0) was incubated at 30 to 80 °C for 15 min and stopped by adding 100 μL of DNS solution. After this, the mixture was incubated again for 5 min at 100 °C and the absorbance was measured at 540 nm with a spectrophotometer. For optimal pH determination, the same amount of enzyme and substrate were diluted in 40 mM acetate/borate/phosphate buffer (ABF) with different pH values ranging from 2.0 to 10.0. The reactions were carried out under the predetermined optimal temperature.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>31212</offset>
+      <text>The substrate specificity of the enzyme was determind using rye arabinan, xyloglucan, β-glucan, galactomannan, lichenan, β-mannan, Azo-Avicel and CMC as substrates. The substrates were diluted in water to 1% (w/v), and the reaction mixture was composed of 10 mL of purified enzyme at a concentration of 0.1 mg/mL, 0.4 mL of 50 mM sodium citrate buffer at pH 5.0, and 0.5 mL of 1% (w/v) substrate aqueous solution. The reaction was incubated at 50 °C for 15 min, followed by treatment with DNS as mentioned above. Enzyme unit was defined as the amount of enzyme that produces 1.0 μM of glucose in one minute for each substrate.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>31859</offset>
+      <text>The kinetic parameters were determined by increasing concentrations of CMC. Reactions were performed in 50 mM sodium citrate buffer (pH = 4.0) at 50 °C, and measured by DNS method as well. Kinetic constants were determined by non-linear regression using OriginPro 8.0.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>32137</offset>
+      <text>Thermal Shift Assays</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>32158</offset>
+      <text>The thermal denaturation assays were performed using a Real Time PCR Machine (Stratagene Mx3005P) as described by Dupeux and co-workers. Briefly, the enzymes were diluted to 10 μM in 50 mM sodium citrate buffer (pH = 4.0) containing 1x SYPRO orange dye (Thermo Fisher Scientific). The fluorescence emission of the probe was monitored (excitation and emission at 492 and 516 nm, respectively) varying the temperature between 25 and 75 °C with the rate of 1 °C/min.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>32639</offset>
+      <text>Cellopentaose Cleavage Experiment</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>32673</offset>
+      <text>The full-length BlCel5B and AM deletion constructs were tested for product formation from cellopentaose. Cellopentaose (1.0 mM) was incubated with 25 μg of purified enzyme in 10 mM ammonium bicarbonate buffer (pH 7.0) in a total volume of 50 μL. The reaction was incubated for 90 min at 50 °C and then stopped by treatment at 100 °C for 5 min. After centrifugation for 10 min at 16,000 g the samples were subjected to MALDI/TOF-MS. Samples were supplemented with NaCl to a final concentration of 20 mM and 1 μL of the supernatant was co-crystallized with 1 μL 2,5-dihydroxybenzoic acid (10 mg/mL) in acetonitrile 30% and spotted on the target plate. The products were analyzed on Microflex LT MALDI-TOF (Bruker Daltonics) operating in positive ion mode. A single spectrum was obtained by averaging four independent spectra generated by 300 laser shots at 60% potency.</text>
+      <annotation id="879">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:37:00Z</infon>
+        <location offset="33436" length="8"/>
+        <text>spectrum</text>
+      </annotation>
+      <annotation id="878">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:36:55Z</infon>
+        <location offset="33488" length="7"/>
+        <text>spectra</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>33575</offset>
+      <text>Crystallization, Data Collection, and Structure Determination</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>33637</offset>
+      <text>After purification, BlCel5B was concentrated to 10 mg/mL for crystallization trials. Crystallization screens were set up using the sitting-drop vapor-diffusion method on a Cartesian PixSys 4200 (Genomic Solutions, United Kingdom) in a 96-well plate with drops formed by 100 nL protein solution plus 100 nL reservoir solution. The commercial kits Crystal Screen and Index (Hampton) were used as initial conditions. Crystals were grown at 18 °C between 3 and 7 days, and screened for diffraction.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>34141</offset>
+      <text>Crystals were supplemented with cryoprotection solution, flash cooled in liquid nitrogen and diffraction data were collected at 100 K, at beamline ID23-1 (wavelength of 0.97 Å) from the European Synchrotron Radiation Facility (Grenoble, France). A crystal grown in condition containing 22.5% PEG 4000, 14% isopropanol and 0.1 M sodium citrate, pH 6.0, was selected to collect diffraction data to 1.7 Å resolution. The complex of the enzyme with substrate was obtained by crystal soaking with five times molar excess of cellopentaose for 24 hours. Diffraction data for the complexed enzyme were collected at 1.75 Å resolution.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>34782</offset>
+      <text>Data were integrated with iMosflm and scaled with Aimless. The structure was solved by molecular replacement with Phaser using an endoglucanase from Clostridium cellulovoran (PDB code: 3NDY) as the search model. Coot was used for density fitting, and refinement was performed with PHENIX.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>35071</offset>
+      <text>Atomistic simulations</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>35093</offset>
+      <text>We took the BlCel5B structure complexed with cellotetraose as the starting configuration for the MD simulations. The missing residues were taken from the apo BlCel5B structure after structural alignment using the LovoAlign server. Hydrogen atoms were then added according to the protonation states determined at the optimum pH of 4.0 using the H + + server. The following residues were considered protonated: H55, H77, D89, E96, E103, H114, E129, E159, E197, D198, E202, H205, E208, D211, H220, E245, E248, E260, H278, H292, D306, E312, E371, E375, E476, H416, E477, E489, D497, and E524. The remaining protonatable residues were considered in the standard protonation state. The BlCel5B-cellotetraose complex was then immersed in a rectangular simulation box of dimensions such that a solvent layer at least 16 Å thick surrounded the protein. The simulation box, built with Packmol, also contained 0.10 M NaCl aqueous solution with excess counter ions to keep the system electrically neutral. The final system comprised approximately 85500 atoms.</text>
+      <annotation id="891">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:43:11Z</infon>
+        <location offset="35247" length="3"/>
+        <text>apo</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>36152</offset>
+      <text>The simulations were performed using NAMD with the CHARMM force field and the TIP3P water model. Periodic boundary conditions were employed, using particle mesh Ewald to handle electrostatics and a 12-Å cutoff radius for truncating short-range potentials. Bonds involving hydrogen atoms were constrained at their equilibrium lengths and a time step of 2 fs was used to integrate the equations of motion. The simulations were carried out under constant pressure and temperature of 1 atm and 310 K, respectively, employing the Langevin barostat and thermostat.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>36714</offset>
+      <text>Accelerated Molecular Dynamics</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>36745</offset>
+      <text>In accelerated molecular dynamics, the trajectory is propagated on a modified potential aimed to enhance conformational sampling. Whenever the potential energy drops below a given threshold E, a boost ΔV(r) is applied, so that the escaping rates of local minima increase. When the potential energy gets over the threshold E, the system evolves on the original energy surface. This method has the advantage of conserving the general shape of the potential energy surface and of requiring no prior definition of reaction coordinates, so the system is allowed to explore freely its conformational space. Here, we restricted the energy boost only to the dihedral potential energy, as changes in torsion angles are the main source of conformational changes in proteins. The energy boost assumes the form of equation (1) that depends on the energy threshold E and on the parameter α – which modulates the shape of the potential energy surface where the boost is applied. We set the parameters E and α according to previous studies, which recommend that E equals the average dihedral energy obtained from a conventional MD simulation plus 4 kcal/mol times the number of residues, and α equals 0.8 kcal/mol times the number of residues. The average dihedral energy was 2275.5 kcal/mol and the BlCel5B has 516 residues, so we set E = 2275.5 + 4 × 516 = 4339.5 kcal/mol and α = 0.8 × 516 = 418.8 kcal/mol.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>38188</offset>
+      <text>Simulation procedures</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>38210</offset>
+      <text>BlCel5B-cellotetraose – Having built the system, we carried out the following steps for equilibration: (i) 1000 steps of energy minimization followed by 100 ps of MD simulation with all non-solvent heavy atoms fixed; (ii) same as (i), but with only the α carbons fixed; (iii) 5 ns of MD with all atoms free. After these preliminary steps, a trajectory lasting 400 ns was generated using conventional MD and then the aMD dihedral boost was applied for additional 1.0 μs. After 100 ns of conventional MD, the cellotetraose dissociated and the simulation began to represent the dynamics of unbound state of BlCel5B.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>38835</offset>
+      <text>BlCel5B-cellooctaose</text>
+      <annotation id="884">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:39:16Z</infon>
+        <location offset="38835" length="20"/>
+        <text>BlCel5B-cellooctaose</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>38856</offset>
+      <text>To build the cellooctaose chain, we extended the original cellotetraose chain in the crystal structure with 4 additional glucose residues spanning regions around the BlCel5B positive subsites. Then, we submitted the system to the following procedure: (i) 1000 steps of energy minimization followed by 1 ns of MD keeping all the non-solvent heavy atoms fixed, except the 4 modeled glucose residues of the cellooctaose chain; (ii) same as step (i), but with only the α carbons fixed; (iii) 1 ns of MD with only the non-modeled glucose residues fixed. We then performed a 200-ns-long MD with three harmonic potentials involving cellooctaose chain: first, between C3 atom (CHARMM atom names) of the second glucose residue from the cellooctaose non-reducing end and the CD2 atom of the W47 tryptophan residue; second, between the OH3 atom of the forth glucose residue from the cellooctaose non-reducing end and HE2 atom of the H113 histidine residue; and third, between the HE2 atom of the catalytic residue E159 and O4 glycosidic oxygen between the fourth and fifth glucose unit of the cellooctaose chain. After these preliminary relaxation steps, the harmonic potentials were removed and the trajectory was propagated by 400 ns using MD. To get a model of the BlCel5B-cellooctaose complex in the closed conformation, we took the configuration after 80 ns of the restrained 200-ns MD simulation as the starting point for a 500-ns-long restrained aMD simulation, in which the CBM46 moved towards the CD in the presence of the harmonically-restrained cellooctaose chain. After this procedure, we released the restraints and propagated the closed BlCel5B-cellooctaose complex for additional 500 ns of conventional, restraint-free MD simulation.</text>
+      <annotation id="885">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:39:16Z</infon>
+        <location offset="40113" length="20"/>
+        <text>BlCel5B-cellooctaose</text>
+      </annotation>
+      <annotation id="886">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:39:16Z</infon>
+        <location offset="40496" length="20"/>
+        <text>BlCel5B-cellooctaose</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>40605</offset>
+      <text>Coarse-grained MD simulations</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>40635</offset>
+      <text>The coarse-grained model was constructed from the minimized all-atom protein. We have used the domELNEDIN CG model for the protein. In this representation, an elastic network is used within each domain as a structural scaffold in order to maintain the overall shape of the protein, and a slightly modified version of MARTINI CG model describes the interactions involving beads not connected by harmonic springs.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>41047</offset>
+      <text>The delimitation of each domain was quite clear considering the short linkers connecting them and the recognition of their structural patterns in databases. We assumed CD, Ig-like module, and CBM46 as consisted of residues 18–331, 332–430, and 431–533, respectively. Therefore, there were elastic network bonds only within these domains (domELNEDIN CG model in Supplementary Fig. 6A).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>41438</offset>
+      <text>The protonation state of each residue bead in the protein was the same adopted in the atomistic simulations. The system was then solvated by 10000 standard MARTINI CG water beads, including 10% of antifreeze particles. Also, 58 chloride and 48 sodium ions were added for charge neutrality. The size of final system was 109 Å × 109 Å × 109 Å.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>41799</offset>
+      <text>Preliminary simulations were performed to test the elastic network (EN) parameters. We have tested six different ENs in 100 ns of simulations, using combinations of cut-off distance (Rc) of 8 Å and 9 Å with spring force constant (ks) of 500, 800 and 1000 kJ mol−1 nm−2. The time evolution of root mean square deviation relative to the crystal structure as well as the mobility profile of the protein in these simulations were compared to the correspondent data from a 100 ns atomistic simulation. From this procedure, the parameters Rc = 9 Å and ks = 500 kJ mol−1 nm−2 resulted in the best match between atomistic and coarse-grained simulations (Supplementary Fig. 6B).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>42500</offset>
+      <text>The coarse-grained simulations were carried out using GROMACS. Periodic boundary conditions were employed. Van der Waals interactions were shifted to zero in the range 0.9–1.2 nm, and the electrostatic interactions, in the range 0.0–1.2 nm. The simulations were performed in the isothermal-isobaric ensemble (NpT), employing the Berendsen thermostat and barostat for temperature and pressure control, respectively, with time constants τT = 0.5 ps and τp = 1.2 ps.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>42982</offset>
+      <text>The CG simulations were carried out using the following protocol: the system was first minimized for 1000 steps using the steepest descent method. Then, it was submitted to a relaxation procedure comprising gradual increasing in time step or temperature. In the first stage of relaxation, the protein beads were restrained with a 1000 kJ mol−1 nm−2 force constant and a 50 ps simulation was carried out at 50 K, using the short time step of 1 fs. In the second stage, the time step was increased up to 5 ps lasting 1000 ps of simulation time. In the last stage of relaxation, all the system is released to move and it underwent a gradual increase in temperature, consisting on five segments of 100 ps at 50, 100, 150, 200 and 310 K. After achieving the desired temperature of 310 K, we performed three production simulations using 20-fs timestep. We have used a random number generator for assigning velocities to generate three independent simulations.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>43950</offset>
+      <text>In general, smoothing of the energy surface in CG model makes the time scales faster. A speed up factor of 4 is typically employed to rescale the time scale of MARTINI CG systems. Therefore, all CG simulations times described here and in the main text are effective times, i.e., 4× simulation time.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>44250</offset>
+      <text>Small Angle X-ray Scattering</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>44279</offset>
+      <text>SAXS data were collected at the SAXS2 beamline of the Brazilian Synchrotron Light Laboratory-LNLS (Campinas, Brazil) on a bi-dimensional position sensitive CCD detector (MarResearch, USA) using the radiation wavelength 1.54 Å. The sample-detector distance of 1000 mm allowed covering the momentum transfer range 0.01 Å−1 &lt; q &lt; 0.35 Å−1 (q = 4πsin θ/λ, where 2θ is the scattering angle).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>44697</offset>
+      <text>The protein samples were prepared in McIlvaine’s buffer at 50 mM, pH 5 and 20 °C. In each measurement, two successive frames of 300s were recorded for each sample at 1 and 2 mg/mL to monitor radiation damage. The patterns were integrated using the FIT2D program. The comparative analysis for each scattering curve at 1 and 2 mg/mL of BlCel5B (data not shown), as well as the radius of gyration values (Rg), indicated that concentration and aggregation effects did not exist. The linearity of the Guinier plot indicated that the preparation was monodisperse.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>45266</offset>
+      <text>The radius of gyration of the molecules (Rg) was estimated by two methods, using the Guinier equation-I(q) = I(0).exp[(−q2.Rg2)/3], q.Rg &lt; 1.3- and also with the inverse Fourier transform in GNOM. The same program was used to obtain the distance distribution function P(r) and the maximum diameter Dmax. Ten independent dummy atom models (DAMs) were restored by the ab initio proceeding implemented in DAMMIN package. The best model, selected using normalized spatial discrepancy parameter computed by DAMAVER program, was superimposed on the crystallographic model with the SUPCOMB.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>45860</offset>
+      <text>Then, based on the enzyme conformations reported by MD and protein crystallography, the computed X-ray scattering profile was fitted to a given experimental SAXS data by minimizing the χ function in the FOXS program.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>46079</offset>
+      <text>To assess the inter-domain information, the contribution of individual conformer and the flexibility of BlCel5B, we proceed in two approaches. First, the theoretical profiles and experimental data comparison was performed to infer the best-fit conformation of the ensemble-based analysis by the ensemble optimization method – EOM, which assumes coexistence of a range of conformations in solution for which an average scattering intensity fits the experimental SAXS data; all models were generated with the three individual domains (Ig-like, CBM46, and CD) free to randomly move in order to cover the entire conformational space. The second approach was based on a fractional volume calculation from three conformation members extracted from the MD simulations, each with a distinct scatter curve. OLIGOMER provided solution of a system of linear equations between the experimental and generated conformations by MD.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>46998</offset>
+      <text>The simulated scattering curves from the MD and crystallographic models were obtained using the CRYSOL.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>47102</offset>
+      <text>Phylogenetic assignment</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>47126</offset>
+      <text>Sequences for all GH5 members, in which only the catalytic domain were considered, were downloaded from PFAM database and their classification into subfamilies were obtained within the CAZy database. The sequences belonging to subfamily 4 were selected and those that had over 90% identity or represented partial coverage were rejected. Based on their multiple sequence alignment, the phylogenetic tree was constructed using the maximum likelihood method implemented in the MEGA program version 6.06. One hundred Bootstrap replications were performed to examine the reliability of the phylogenetic tree.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_1</infon>
+      <offset>47730</offset>
+      <text>Additional Information</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>47753</offset>
+      <text>How to cite this article: Liberato, M. V. et al. Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism. Sci. Rep. 6, 23473; doi: 10.1038/srep23473 (2016).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">title_1</infon>
+      <offset>47959</offset>
+      <text>Supplementary Material</text>
+    </passage>
+    <passage>
+      <infon key="fpage">121</infon>
+      <infon key="lpage">45</infon>
+      <infon key="name_0">surname:Chundawat;given-names:S. P. S.</infon>
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+      <infon key="name_2">surname:Himmel;given-names:M. E.</infon>
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+      <text>Family 46 Carbohydrate-binding Modules Contribute to the Enzymatic Hydrolysis of Xyloglucan and β-1,3-1,4-Glucans through Distinct Mechanisms</text>
+    </passage>
+    <passage>
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+      <offset>48853</offset>
+      <text>The conformation and function of a multimodular glycogen-degrading pneumococcal virulence factor</text>
+    </passage>
+    <passage>
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+      <text>Author Contributions M.V.L. and I.P. designed the experiments; C.M.C. performed the gene mutations and cloning; M.V.L. and V.O.A.P. expressed and purified the enzymes and characterized the enzymatic activities; M.V.L. crystallized and determined the crystal structures, with data collection supervised by A.P.; M.A.K. performed mass spectrometry experiment; E.A.A. and M.O.N. collected and treated SAXS data. R.L.S., E.T.P. and M.S.S. designed the computer simulations; R.L.S. performed Accelerated Molecular Dynamics; E.T.P. performed Coarse-grained MD Simulations; M.V.L., R.L.S., M.S.S. and I.P. wrote the manuscript with the input from all the other authors; M.S.S. and I.P. supervised the project.</text>
+    </passage>
+    <passage>
+      <infon key="file">srep23473-f1.jpg</infon>
+      <infon key="id">f1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>53941</offset>
+      <text>Crystal models of BlCel5B.</text>
+      <annotation id="625">
+        <infon key="score">0.99844503</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:35:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="53941" length="14"/>
+        <text>Crystal models</text>
+      </annotation>
+      <annotation id="626">
+        <infon key="score">0.99846846</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="53959" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">srep23473-f1.jpg</infon>
+      <infon key="id">f1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>53968</offset>
+      <text>Complete structure is shown as a cartoon illustration in (a) and a van der Waals surface in (b). The CD module (red) has a typical TIM-barrel fold, and its substrate-binding site is adjacent to CBM46 (blue). Despite the proximity of the binding site in the crystallographic model, the CBM46 residues W479 and W481 are distant from the substrate cellotetraose (yellow). The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray.</text>
+      <annotation id="627">
+        <infon key="score">0.99364626</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:47:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="53977" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="797">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:27Z</infon>
+        <location offset="54069" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="628">
+        <infon key="score">0.99158597</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:35:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="54099" length="15"/>
+        <text>TIM-barrel fold</text>
+      </annotation>
+      <annotation id="629">
+        <infon key="score">0.9988049</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:45:11Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="54124" length="22"/>
+        <text>substrate-binding site</text>
+      </annotation>
+      <annotation id="630">
+        <infon key="score">0.76680785</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="54162" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="631">
+        <infon key="score">0.99875355</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:35:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="54205" length="12"/>
+        <text>binding site</text>
+      </annotation>
+      <annotation id="632">
+        <infon key="score">0.88893646</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="54253" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="633">
+        <infon key="score">0.9994466</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:08:25Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="54268" length="4"/>
+        <text>W479</text>
+      </annotation>
+      <annotation id="634">
+        <infon key="score">0.99945384</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:08:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="54277" length="4"/>
+        <text>W481</text>
+      </annotation>
+      <annotation id="635">
+        <infon key="score">0.9993051</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:01:14Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="54313" length="13"/>
+        <text>cellotetraose</text>
+      </annotation>
+      <annotation id="636">
+        <infon key="score">0.99909556</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:49:28Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="54341" length="14"/>
+        <text>Ig-like domain</text>
+      </annotation>
+      <annotation id="637">
+        <infon key="score">0.9991934</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:27Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="54423" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="638">
+        <infon key="score">0.8081565</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="54430" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="639">
+        <infon key="score">0.99850583</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:36:19Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="54443" length="13"/>
+        <text>superposition</text>
+      </annotation>
+      <annotation id="640">
+        <infon key="score">0.99915457</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:49:32Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="54464" length="14"/>
+        <text>Ig-like domain</text>
+      </annotation>
+      <annotation id="641">
+        <infon key="score">0.9668259</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="54483" length="5"/>
+        <text>CBM46</text>
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+      <annotation id="642">
+        <infon key="score">0.999071</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="54585" length="4"/>
+        <text>loop</text>
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+      <annotation id="643">
+        <infon key="score">0.999288</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:01:14Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="54623" length="13"/>
+        <text>Cellotetraose</text>
+      </annotation>
+      <annotation id="644">
+        <infon key="score">0.93913716</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:36:28Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="54646" length="17"/>
+        <text>subsites -1 to -3</text>
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+      <annotation id="832">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:01:57Z</infon>
+        <location offset="54681" length="11"/>
+        <text>coordinated</text>
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+    <passage>
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+      <infon key="id">f2</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>54730</offset>
+      <text>BlCel5B enzymatic activity characterization.</text>
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+        <infon key="score">0.99579805</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="54730" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="877">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:36:47Z</infon>
+        <location offset="54738" length="35"/>
+        <text>enzymatic activity characterization</text>
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+    </passage>
+    <passage>
+      <infon key="file">srep23473-f2.jpg</infon>
+      <infon key="id">f2</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>54775</offset>
+      <text>(a) MALDI/TOF-MS spectra of the products released after incubation of BlCel5B and its two deletion constructs (ΔCBM46 and ΔIg-CBM46) with the substrate cellopentaose (C5). The first three spectra show the substrate, enzyme and buffer controls. The forth spectrum reveals that full length BlCel5B is capable of enzymatic hydrolysis of C5 into smaller oligosaccharides such as C4, C3 and C2. The last two spectra show that the C-terminal deletions eliminate the enzyme activity. BlCel5B activities on CMC as functions of pH and temperature are shown in (b) and (c), respectively. The enzyme exhibits optimal pH of 4.0 and optimal temperature of 55 °C, retaining about 50% of its activity at 80 °C. (d) Michaelis-Menten curve using CMC as a substrate.</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:36:51Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="54779" length="12"/>
+        <text>MALDI/TOF-MS</text>
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+      <annotation id="647">
+        <infon key="score">0.9872946</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:36:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="54792" length="7"/>
+        <text>spectra</text>
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+      <annotation id="648">
+        <infon key="score">0.99913824</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:33Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="54845" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="883">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:38:40Z</infon>
+        <location offset="54865" length="19"/>
+        <text>deletion constructs</text>
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+      <annotation id="649">
+        <infon key="score">0.9989943</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:06:08Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="54886" length="6"/>
+        <text>ΔCBM46</text>
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+      <annotation id="650">
+        <infon key="score">0.99894</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:06:51Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="54897" length="9"/>
+        <text>ΔIg-CBM46</text>
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+      <annotation id="651">
+        <infon key="score">0.99934024</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:36Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="54927" length="13"/>
+        <text>cellopentaose</text>
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+      <annotation id="652">
+        <infon key="score">0.97373194</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:40Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="54942" length="2"/>
+        <text>C5</text>
+      </annotation>
+      <annotation id="653">
+        <infon key="score">0.86749804</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:36:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="54963" length="7"/>
+        <text>spectra</text>
+      </annotation>
+      <annotation id="654">
+        <infon key="score">0.7187502</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:36:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55029" length="8"/>
+        <text>spectrum</text>
+      </annotation>
+      <annotation id="655">
+        <infon key="score">0.9990318</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:37:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55051" length="11"/>
+        <text>full length</text>
+      </annotation>
+      <annotation id="656">
+        <infon key="score">0.9991542</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:33Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="55063" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="657">
+        <infon key="score">0.86857474</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:40Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="55109" length="2"/>
+        <text>C5</text>
+      </annotation>
+      <annotation id="658">
+        <infon key="score">0.9976169</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:53:37Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="55125" length="16"/>
+        <text>oligosaccharides</text>
+      </annotation>
+      <annotation id="659">
+        <infon key="score">0.50656426</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:01:09Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="55150" length="2"/>
+        <text>C4</text>
+      </annotation>
+      <annotation id="880">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:37:19Z</infon>
+        <location offset="55154" length="2"/>
+        <text>C3</text>
+      </annotation>
+      <annotation id="881">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:37:32Z</infon>
+        <location offset="55161" length="2"/>
+        <text>C2</text>
+      </annotation>
+      <annotation id="660">
+        <infon key="score">0.83065146</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:36:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55178" length="7"/>
+        <text>spectra</text>
+      </annotation>
+      <annotation id="882">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:38:25Z</infon>
+        <location offset="55221" length="29"/>
+        <text>eliminate the enzyme activity</text>
+      </annotation>
+      <annotation id="661">
+        <infon key="score">0.9991924</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:33Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="55252" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="662">
+        <infon key="score">0.99763143</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:03:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="55274" length="3"/>
+        <text>CMC</text>
+      </annotation>
+      <annotation id="663">
+        <infon key="score">0.99861073</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:37:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55475" length="22"/>
+        <text>Michaelis-Menten curve</text>
+      </annotation>
+      <annotation id="664">
+        <infon key="score">0.9977623</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:03:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="55504" length="3"/>
+        <text>CMC</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">srep23473-f3.jpg</infon>
+      <infon key="id">f3</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>55532</offset>
+      <text>Open-close transitions of BlCel5B.</text>
+      <annotation id="665">
+        <infon key="score">0.752593</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:13:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55532" length="4"/>
+        <text>Open</text>
+      </annotation>
+      <annotation id="666">
+        <infon key="score">0.92618257</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:21:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55537" length="5"/>
+        <text>close</text>
+      </annotation>
+      <annotation id="667">
+        <infon key="score">0.9987601</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:33Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="55558" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">srep23473-f3.jpg</infon>
+      <infon key="id">f3</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>55567</offset>
+      <text>(a) BlCel5B in the absence of substrate and (b) in the presence of cellooctaose, as observed in our aMD simulations. The distance between the α carbon of residues I120 (CD) and E477 (CBM46), illustrated as spheres in (a), is plotted in (c), revealing a transition by the decrease in the distance from 40 Å to 7 Å (substrate-free) or 20 Å (in presence of cellooctaose). For the substrate-free enzyme, the red line refers to a 1 μs-long aMD; for the BlCel5B-cellooctaose complex, the first 500 ns refers to aMD (in blue) and the second 500 ns to conventional MD (in turquoise). (d) A snapshot of the BlCel5B-cellooctaose complex, highlighting the tryptophan residues that interact with the glucan chain in subsites −4 to +4. Residues W479 and W481 belong to CBM46 and only become available for substrate interactions in the closed configuration of BlCel5B.</text>
+      <annotation id="668">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:33Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="55571" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="824">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:59:48Z</infon>
+        <location offset="55586" length="10"/>
+        <text>absence of</text>
+      </annotation>
+      <annotation id="669">
+        <infon key="score">0.9991783</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:01:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55622" length="11"/>
+        <text>presence of</text>
+      </annotation>
+      <annotation id="670">
+        <infon key="score">0.99471444</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:11:51Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="55634" length="12"/>
+        <text>cellooctaose</text>
+      </annotation>
+      <annotation id="849">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:12:40Z</infon>
+        <location offset="55667" length="15"/>
+        <text>aMD simulations</text>
+      </annotation>
+      <annotation id="846">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:11Z</infon>
+        <location offset="55688" length="8"/>
+        <text>distance</text>
+      </annotation>
+      <annotation id="671">
+        <infon key="score">0.9995372</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:14:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55730" length="4"/>
+        <text>I120</text>
+      </annotation>
+      <annotation id="672">
+        <infon key="score">0.9992906</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:27Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="55736" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="673">
+        <infon key="score">0.99951124</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:14:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55744" length="4"/>
+        <text>E477</text>
+      </annotation>
+      <annotation id="674">
+        <infon key="score">0.99878496</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="55750" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="847">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:11Z</infon>
+        <location offset="55854" length="8"/>
+        <text>distance</text>
+      </annotation>
+      <annotation id="675">
+        <infon key="score">0.99880093</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55881" length="14"/>
+        <text>substrate-free</text>
+      </annotation>
+      <annotation id="676">
+        <infon key="score">0.9990532</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:01:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55909" length="11"/>
+        <text>presence of</text>
+      </annotation>
+      <annotation id="677">
+        <infon key="score">0.9680609</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:11:51Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="55921" length="12"/>
+        <text>cellooctaose</text>
+      </annotation>
+      <annotation id="678">
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:47:28Z</infon>
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+      <text>(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).</text>
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+        <infon key="score">0.9975522</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:33Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="57534" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="889">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:41:13Z</infon>
+        <location offset="57549" length="16"/>
+        <text>crystallographic</text>
+      </annotation>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:10:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="57570" length="6"/>
+        <text>closed</text>
+      </annotation>
+      <annotation id="726">
+        <infon key="score">0.9984163</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:47:47Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57596" length="19"/>
+        <text>Bacillus halodurans</text>
+      </annotation>
+      <annotation id="727">
+        <infon key="score">0.99806625</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:48:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="57616" length="5"/>
+        <text>Cel5B</text>
+      </annotation>
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+        <infon key="score">0.9970697</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="57623" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+      <annotation id="729">
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:41:19Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57651" length="21"/>
+        <text>Piromyces rhizinflata</text>
+      </annotation>
+      <annotation id="730">
+        <infon key="score">0.9514452</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:46:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57673" length="3"/>
+        <text>GH5</text>
+      </annotation>
+      <annotation id="731">
+        <infon key="score">0.9991021</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:48:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57677" length="13"/>
+        <text>endoglucanase</text>
+      </annotation>
+      <annotation id="732">
+        <infon key="score">0.9984065</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:41:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57711" length="26"/>
+        <text>Clostridium cellulolyticum</text>
+      </annotation>
+      <annotation id="733">
+        <infon key="score">0.9659778</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:46:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57738" length="3"/>
+        <text>GH5</text>
+      </annotation>
+      <annotation id="734">
+        <infon key="score">0.9990024</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:48:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57742" length="13"/>
+        <text>endoglucanase</text>
+      </annotation>
+      <annotation id="735">
+        <infon key="score">0.9983325</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:41:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57776" length="25"/>
+        <text>Clostridium cellulovorans</text>
+      </annotation>
+      <annotation id="736">
+        <infon key="score">0.8675645</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:46:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57802" length="3"/>
+        <text>GH5</text>
+      </annotation>
+      <annotation id="737">
+        <infon key="score">0.9990245</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:48:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57806" length="13"/>
+        <text>endoglucanase</text>
+      </annotation>
+      <annotation id="738">
+        <infon key="score">0.99848115</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:41:27Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57840" length="18"/>
+        <text>Bacteroides ovatus</text>
+      </annotation>
+      <annotation id="739">
+        <infon key="score">0.85893065</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:46:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57859" length="3"/>
+        <text>GH5</text>
+      </annotation>
+      <annotation id="740">
+        <infon key="score">0.9991406</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:42:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57863" length="13"/>
+        <text>xyloglucanase</text>
+      </annotation>
+      <annotation id="741">
+        <infon key="score">0.9982492</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:41:30Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57897" length="20"/>
+        <text>Paenibacillus pabuli</text>
+      </annotation>
+      <annotation id="742">
+        <infon key="score">0.9038993</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:46:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57918" length="3"/>
+        <text>GH5</text>
+      </annotation>
+      <annotation id="743">
+        <infon key="score">0.9991715</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:42:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57922" length="13"/>
+        <text>xyloglucanase</text>
+      </annotation>
+      <annotation id="744">
+        <infon key="score">0.9983506</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:41:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57956" length="19"/>
+        <text>Prevotella bryantii</text>
+      </annotation>
+      <annotation id="745">
+        <infon key="score">0.6447121</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:46:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57976" length="3"/>
+        <text>GH5</text>
+      </annotation>
+      <annotation id="746">
+        <infon key="score">0.9989398</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:48:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57980" length="13"/>
+        <text>endoglucanase</text>
+      </annotation>
+      <annotation id="747">
+        <infon key="score">0.99841285</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:41:34Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="58014" length="30"/>
+        <text>Ruminiclostridium thermocellum</text>
+      </annotation>
+      <annotation id="748">
+        <infon key="score">0.8743299</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:46:55Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="58061" length="3"/>
+        <text>GH5</text>
+      </annotation>
+      <annotation id="749">
+        <infon key="score">0.9991216</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:19Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="58065" length="9"/>
+        <text>cellulase</text>
+      </annotation>
+      <annotation id="750">
+        <infon key="score">0.99889106</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:41:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="58076" length="8"/>
+        <text>xylanase</text>
+      </annotation>
+      <annotation id="751">
+        <infon key="score">0.9972579</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:41:45Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="58089" length="7"/>
+        <text>mannase</text>
+      </annotation>
+      <annotation id="752">
+        <infon key="score">0.7350698</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:41:51Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="58117" length="23"/>
+        <text>Bacteroidetes bacterium</text>
+      </annotation>
+      <annotation id="753">
+        <infon key="score">0.7986666</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:42:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="58141" length="4"/>
+        <text>AC2a</text>
+      </annotation>
+      <annotation id="754">
+        <infon key="score">0.9983577</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:42:06Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="58146" length="13"/>
+        <text>endocellulase</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">srep23473-f6.jpg</infon>
+      <infon key="id">f6</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>58176</offset>
+      <text>Comparison of the binding cleft of the BlCel5B and BhCel5B.</text>
+      <annotation id="890">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:42:33Z</infon>
+        <location offset="58176" length="10"/>
+        <text>Comparison</text>
+      </annotation>
+      <annotation id="755">
+        <infon key="score">0.9991272</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:13Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="58194" length="13"/>
+        <text>binding cleft</text>
+      </annotation>
+      <annotation id="756">
+        <infon key="score">0.9568515</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:33Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="58215" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="757">
+        <infon key="score">0.9822322</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="58227" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">srep23473-f6.jpg</infon>
+      <infon key="id">f6</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>58236</offset>
+      <text>The main difference between BlCel5B and BhCel5B is that the latter exhibits a deeper cleft due to the presence of residue W181 in the loop between F177 and R185. We conjecture that this difference in the binding site architecture relates to the importance that the CBM46 plays in the BlCel5B enzymatic mechanism.</text>
+      <annotation id="758">
+        <infon key="score">0.9989668</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:33Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="58264" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="759">
+        <infon key="score">0.99901545</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="58276" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+      <annotation id="760">
+        <infon key="score">0.9974975</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:42:41Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="58321" length="5"/>
+        <text>cleft</text>
+      </annotation>
+      <annotation id="829">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:01:00Z</infon>
+        <location offset="58338" length="11"/>
+        <text>presence of</text>
+      </annotation>
+      <annotation id="761">
+        <infon key="score">0.9995627</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="58358" length="4"/>
+        <text>W181</text>
+      </annotation>
+      <annotation id="762">
+        <infon key="score">0.9989575</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:53:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="58370" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="763">
+        <infon key="score">0.99955577</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="58383" length="4"/>
+        <text>F177</text>
+      </annotation>
+      <annotation id="764">
+        <infon key="score">0.9995542</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:36:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="58392" length="4"/>
+        <text>R185</text>
+      </annotation>
+      <annotation id="765">
+        <infon key="score">0.99892116</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:35:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="58440" length="12"/>
+        <text>binding site</text>
+      </annotation>
+      <annotation id="766">
+        <infon key="score">0.9979352</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="58501" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="767">
+        <infon key="score">0.9990663</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:33Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="58520" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">srep23473-f7.jpg</infon>
+      <infon key="id">f7</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>58549</offset>
+      <text>Proposed molecular mechanism of BlCel5B conformational selection.</text>
+      <annotation id="768">
+        <infon key="score">0.9990453</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:33Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="58581" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">srep23473-f7.jpg</infon>
+      <infon key="id">f7</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>58615</offset>
+      <text>As suggested by the simulations and SAXS data, BlCel5B spans multiple conformations ranging from closed to extended CBM46/CD states. In a given open state, the substrate may reach the active site and become entrapped by the capping of CBM46 onto CD and induced-fit conformational adjustments. After hydrolysis, the reaction product is released to yield apo-BlCel5B, which becomes ready for a new cycle.</text>
+      <annotation id="769">
+        <infon key="score">0.9985917</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:09:51Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="58635" length="11"/>
+        <text>simulations</text>
+      </annotation>
+      <annotation id="770">
+        <infon key="score">0.99830294</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:52:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="58651" length="4"/>
+        <text>SAXS</text>
+      </annotation>
+      <annotation id="771">
+        <infon key="score">0.99919575</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:33Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="58662" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+      <annotation id="772">
+        <infon key="score">0.9993125</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <location offset="58712" length="6"/>
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+      <annotation id="773">
+        <infon key="score">0.9993204</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <location offset="58722" length="8"/>
+        <text>extended</text>
+      </annotation>
+      <annotation id="774">
+        <infon key="score">0.9992793</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="58731" length="5"/>
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+      <annotation id="775">
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:13:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="58759" length="4"/>
+        <text>open</text>
+      </annotation>
+      <annotation id="777">
+        <infon key="score">0.99899673</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="58799" length="11"/>
+        <text>active site</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="58850" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="779">
+        <infon key="score">0.99932384</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:41:27Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="58861" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="780">
+        <infon key="score">0.99932706</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:43:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="58968" length="3"/>
+        <text>apo</text>
+      </annotation>
+      <annotation id="781">
+        <infon key="score">0.99923134</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:33Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="58972" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">t1.xml</infon>
+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_title_caption</infon>
+      <offset>59018</offset>
+      <text>Activity of BlCel5B constructs against tested substrates.</text>
+      <annotation id="782">
+        <infon key="score">0.9987154</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:40:33Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="59030" length="7"/>
+        <text>BlCel5B</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">t1.xml</infon>
+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table</infon>
+      <infon key="xml">&lt;?xml version="1.0" encoding="UTF-8"?&gt;
+&lt;table frame="hsides" rules="groups" border="1"&gt;&lt;colgroup&gt;&lt;col align="left"/&gt;&lt;col align="center"/&gt;&lt;col align="center"/&gt;&lt;col align="center"/&gt;&lt;col align="center"/&gt;&lt;col align="center"/&gt;&lt;/colgroup&gt;&lt;thead valign="bottom"&gt;&lt;tr&gt;&lt;th rowspan="2" align="left" valign="bottom" charoff="50"&gt;Substrate (1%)&lt;/th&gt;&lt;th colspan="5" align="center" valign="top" charoff="50"&gt;Relative Activity (%)&lt;/th&gt;&lt;/tr&gt;&lt;tr&gt;&lt;th align="center" valign="top" charoff="50"&gt;WT&lt;xref ref-type="fn" rid="t1-fn1"&gt;*&lt;/xref&gt;&lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;W479A&lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;W481A&lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;ΔCBM46&lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;ΔIg-CBM46&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign="top"&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;β-glucan&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;100&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;79.1&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;63.6&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;CMC&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;25.5&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;12.2&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;2.4&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;Lichenan&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;52.4&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;41&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;28.6&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;Xyloglucan&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;45.2&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;41.2&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;30.8&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;Azo-Avicel&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;xref ref-type="fn" rid="t1-fn2"&gt;**&lt;/xref&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;Arabinoxylan&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;Galactomannan&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;1,4-β-mannan&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;nd&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon>
+      <offset>59076</offset>
+      <text>Substrate (1%)	Relative Activity (%)	 	WT*	W479A	W481A	ΔCBM46	ΔIg-CBM46	 	β-glucan	100	79.1	63.6	nd	nd	 	CMC	25.5	12.2	2.4	nd	nd	 	Lichenan	52.4	41	28.6	nd	nd	 	Xyloglucan	45.2	41.2	30.8	nd	nd	 	Azo-Avicel	nd**	nd	nd	nd	nd	 	Arabinoxylan	nd	nd	nd	nd	nd	 	Galactomannan	nd	nd	nd	nd	nd	 	1,4-β-mannan	nd	nd	nd	nd	nd	 	</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="59115" length="2"/>
+        <text>WT</text>
+      </annotation>
+      <annotation id="838">
+        <infon key="type">mutant</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:06:59Z</infon>
+        <location offset="59119" length="5"/>
+        <text>W479A</text>
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+      <annotation id="784">
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+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:07:04Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="59125" length="5"/>
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+      </annotation>
+      <annotation id="892">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:44:36Z</infon>
+        <location offset="59131" length="6"/>
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+      </annotation>
+      <annotation id="893">
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:44:51Z</infon>
+        <location offset="59138" length="9"/>
+        <text>ΔIg-CBM46</text>
+      </annotation>
+      <annotation id="785">
+        <infon key="score">0.9988532</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:51:20Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="59150" length="8"/>
+        <text>β-glucan</text>
+      </annotation>
+      <annotation id="834">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:03:32Z</infon>
+        <location offset="59181" length="3"/>
+        <text>CMC</text>
+      </annotation>
+      <annotation id="786">
+        <infon key="score">0.99829346</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:03:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="59207" length="8"/>
+        <text>Lichenan</text>
+      </annotation>
+      <annotation id="787">
+        <infon key="score">0.9985032</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T09:49:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="59237" length="10"/>
+        <text>Xyloglucan</text>
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+      <annotation id="835">
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:04:18Z</infon>
+        <location offset="59271" length="10"/>
+        <text>Azo-Avicel</text>
+      </annotation>
+      <annotation id="788">
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:04:12Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="59301" length="12"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:03:49Z</infon>
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+        <text>Galactomannan</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:04:08Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="59362" length="12"/>
+        <text>1,4-β-mannan</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">t1.xml</infon>
+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_footnote</infon>
+      <offset>59407</offset>
+      <text>*WT = wild type.</text>
+      <annotation id="791">
+        <infon key="score">0.90642285</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T10:08:45Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="59408" length="2"/>
+        <text>WT</text>
+      </annotation>
+      <annotation id="792">
+        <infon key="score">0.99874187</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T13:45:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="59413" length="9"/>
+        <text>wild type</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">t1.xml</infon>
+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_footnote</infon>
+      <offset>59428</offset>
+      <text>**nd = not detected.</text>
+    </passage>
+  </document>
+</collection>
diff --git a/annotated_BioC_XML/PMC4980666_ann.xml b/annotated_BioC_XML/PMC4980666_ann.xml
new file mode 100644
index 0000000000000000000000000000000000000000..dc7e82db959b6c4d86ce0ec64e3e9770dc7e81bf
--- /dev/null
+++ b/annotated_BioC_XML/PMC4980666_ann.xml
@@ -0,0 +1,7170 @@
+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection>
+  <source>PMC</source>
+  <date>20230107</date>
+  <key>pmc.key</key>
+  <document>
+    <id>4980666</id>
+    <infon key="license">CC BY</infon>
+    <infon key="tt_curatable">no</infon>
+    <infon key="tt_version">2</infon>
+    <infon key="tt_round">2</infon>
+    <passage>
+      <infon key="article-id_doi">10.1038/srep31500</infon>
+      <infon key="article-id_pii">srep31500</infon>
+      <infon key="article-id_pmc">4980666</infon>
+      <infon key="article-id_pmid">27510745</infon>
+      <infon key="elocation-id">31500</infon>
+      <infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon>
+      <infon key="name_0">surname:Carcelli;given-names:Mauro</infon>
+      <infon key="name_1">surname:Rogolino;given-names:Dominga</infon>
+      <infon key="name_2">surname:Gatti;given-names:Anna</infon>
+      <infon key="name_3">surname:De Luca;given-names:Laura</infon>
+      <infon key="name_4">surname:Sechi;given-names:Mario</infon>
+      <infon key="name_5">surname:Kumar;given-names:Gyanendra</infon>
+      <infon key="name_6">surname:White;given-names:Stephen W.</infon>
+      <infon key="name_7">surname:Stevaert;given-names:Annelies</infon>
+      <infon key="name_8">surname:Naesens;given-names:Lieve</infon>
+      <infon key="section_type">TITLE</infon>
+      <infon key="type">front</infon>
+      <infon key="volume">6</infon>
+      <infon key="year">2016</infon>
+      <offset>0</offset>
+      <text>N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes</text>
+      <annotation id="1">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:56Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="0" length="15"/>
+        <text>N-acylhydrazone</text>
+      </annotation>
+      <annotation id="605">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:45Z</infon>
+        <location offset="30" length="9"/>
+        <text>influenza</text>
+      </annotation>
+      <annotation id="585">
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <location offset="40" length="5"/>
+        <text>virus</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:00Z</infon>
+        <location offset="46" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="516">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:46Z</infon>
+        <location offset="49" length="12"/>
+        <text>endonuclease</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">ABSTRACT</infon>
+      <infon key="type">abstract</infon>
+      <offset>97</offset>
+      <text>Influenza virus PA endonuclease has recently emerged as an attractive target for the development of novel antiviral therapeutics. This is an enzyme with divalent metal ion(s) (Mg2+ or Mn2+) in its catalytic site: chelation of these metal cofactors is an attractive strategy to inhibit enzymatic activity. Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays. Several N-acylhydrazones were found to have promising anti-influenza activity in the low micromolar concentration range and good selectivity. Computational docking studies are carried on to investigate the key features that determine inhibition of the endonuclease enzyme by N-acylhydrazones. Moreover, we here describe the crystal structure of PA-Nter in complex with one of the most active inhibitors, revealing its interactions within the protein’s active site.</text>
+      <annotation id="606">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:45Z</infon>
+        <location offset="97" length="9"/>
+        <text>Influenza</text>
+      </annotation>
+      <annotation id="586">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <location offset="107" length="5"/>
+        <text>virus</text>
+      </annotation>
+      <annotation id="525">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:00Z</infon>
+        <location offset="113" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="517">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:46Z</infon>
+        <location offset="116" length="12"/>
+        <text>endonuclease</text>
+      </annotation>
+      <annotation id="2">
+        <infon key="score">0.9991626</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:02Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="273" length="4"/>
+        <text>Mg2+</text>
+      </annotation>
+      <annotation id="570">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:17Z</infon>
+        <location offset="281" length="4"/>
+        <text>Mn2+</text>
+      </annotation>
+      <annotation id="3">
+        <infon key="score">0.9989165</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="294" length="14"/>
+        <text>catalytic site</text>
+      </annotation>
+      <annotation id="644">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:48Z</infon>
+        <location offset="310" length="9"/>
+        <text>chelation</text>
+      </annotation>
+      <annotation id="4">
+        <infon key="score">0.99873513</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:46Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="445" length="16"/>
+        <text>N-acylhydrazones</text>
+      </annotation>
+      <annotation id="5">
+        <infon key="score">0.9988744</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:40Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="468" length="15"/>
+        <text>enzymatic assay</text>
+      </annotation>
+      <annotation id="526">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="489" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="548">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="492" length="4"/>
+        <text>Nter</text>
+      </annotation>
+      <annotation id="6">
+        <infon key="score">0.9952571</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="497" length="12"/>
+        <text>endonuclease</text>
+      </annotation>
+      <annotation id="581">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:35Z</infon>
+        <location offset="525" length="40"/>
+        <text>cell-based influenza vRNP reconstitution</text>
+      </annotation>
+      <annotation id="7">
+        <infon key="score">0.8346987</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="570" length="18"/>
+        <text>virus yield assays</text>
+      </annotation>
+      <annotation id="8">
+        <infon key="score">0.998764</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:46Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="598" length="16"/>
+        <text>N-acylhydrazones</text>
+      </annotation>
+      <annotation id="571">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:43Z</infon>
+        <location offset="649" length="9"/>
+        <text>influenza</text>
+      </annotation>
+      <annotation id="9">
+        <infon key="score">0.9989174</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:55:35Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="732" length="29"/>
+        <text>Computational docking studies</text>
+      </annotation>
+      <annotation id="10">
+        <infon key="score">0.9984523</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="842" length="12"/>
+        <text>endonuclease</text>
+      </annotation>
+      <annotation id="11">
+        <infon key="score">0.9986233</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:46Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="865" length="16"/>
+        <text>N-acylhydrazones</text>
+      </annotation>
+      <annotation id="12">
+        <infon key="score">0.9964547</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:52Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="914" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="527">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="935" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="549">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="938" length="4"/>
+        <text>Nter</text>
+      </annotation>
+      <annotation id="13">
+        <infon key="score">0.9980684</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="943" length="15"/>
+        <text>in complex with</text>
+      </annotation>
+      <annotation id="14">
+        <infon key="score">0.9990225</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="1042" length="11"/>
+        <text>active site</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>1057</offset>
+      <text>Influenza virus is an enveloped virus with a segmented negative-oriented single-stranded RNA genome, belonging to the Orthomyxoviridae. Seasonal influenza A and B viruses affect each year approximately 5–10% of the adult and 20–30% of the paediatric population, and there is a permanent risk of sudden influenza pandemics, such as the notorious ‘Spanish flu’ in 1918 and the swine-origin H1N1 pandemic in 2009. Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir). The M2 inhibitors have limited clinical utility due to their central nervous system side effects and widespread resistance, as in the case of the 2009 pandemic H1N1 virus; resistance is also a growing concern for oseltamivir. Therefore, there is an urgent need for new antiviral drugs with an entirely different mode of action.</text>
+      <annotation id="604">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:44Z</infon>
+        <location offset="1057" length="9"/>
+        <text>Influenza</text>
+      </annotation>
+      <annotation id="584">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:15Z</infon>
+        <location offset="1067" length="5"/>
+        <text>virus</text>
+      </annotation>
+      <annotation id="15">
+        <infon key="score">0.8894888</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:47:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1079" length="15"/>
+        <text>enveloped virus</text>
+      </annotation>
+      <annotation id="16">
+        <infon key="score">0.6531854</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:50:31Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1112" length="37"/>
+        <text>negative-oriented single-stranded RNA</text>
+      </annotation>
+      <annotation id="17">
+        <infon key="score">0.9807248</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:31Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1175" length="16"/>
+        <text>Orthomyxoviridae</text>
+      </annotation>
+      <annotation id="18">
+        <infon key="score">0.70304847</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1202" length="11"/>
+        <text>influenza A</text>
+      </annotation>
+      <annotation id="19">
+        <infon key="score">0.4334353</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1218" length="1"/>
+        <text>B</text>
+      </annotation>
+      <annotation id="603">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:31Z</infon>
+        <location offset="1220" length="7"/>
+        <text>viruses</text>
+      </annotation>
+      <annotation id="572">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:43Z</infon>
+        <location offset="1359" length="9"/>
+        <text>influenza</text>
+      </annotation>
+      <annotation id="608">
+        <infon key="type">species</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:48:53Z</infon>
+        <location offset="1445" length="4"/>
+        <text>H1N1</text>
+      </annotation>
+      <annotation id="20">
+        <infon key="score">0.652398</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1488" length="9"/>
+        <text>influenza</text>
+      </annotation>
+      <annotation id="587">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <location offset="1498" length="5"/>
+        <text>virus</text>
+      </annotation>
+      <annotation id="21">
+        <infon key="score">0.99333996</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:47:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1539" length="5"/>
+        <text>viral</text>
+      </annotation>
+      <annotation id="607">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:48:08Z</infon>
+        <location offset="1545" length="14"/>
+        <text>M2 ion-channel</text>
+      </annotation>
+      <annotation id="22">
+        <infon key="score">0.9990307</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:48:18Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1561" length="10"/>
+        <text>amantadine</text>
+      </annotation>
+      <annotation id="23">
+        <infon key="score">0.9990896</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:48:22Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1576" length="11"/>
+        <text>rimantadine</text>
+      </annotation>
+      <annotation id="24">
+        <infon key="score">0.9936174</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:47:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1599" length="5"/>
+        <text>viral</text>
+      </annotation>
+      <annotation id="25">
+        <infon key="score">0.87974066</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:48:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1605" length="13"/>
+        <text>neuraminidase</text>
+      </annotation>
+      <annotation id="26">
+        <infon key="score">0.99916184</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:48:27Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1620" length="9"/>
+        <text>zanamivir</text>
+      </annotation>
+      <annotation id="27">
+        <infon key="score">0.9991722</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:48:31Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1634" length="11"/>
+        <text>oseltamivir</text>
+      </annotation>
+      <annotation id="28">
+        <infon key="score">0.8850591</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:49:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1652" length="2"/>
+        <text>M2</text>
+      </annotation>
+      <annotation id="609">
+        <infon key="type">species</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:48:54Z</infon>
+        <location offset="1808" length="4"/>
+        <text>H1N1</text>
+      </annotation>
+      <annotation id="588">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <location offset="1813" length="5"/>
+        <text>virus</text>
+      </annotation>
+      <annotation id="29">
+        <infon key="score">0.99908054</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:48:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1861" length="11"/>
+        <text>oseltamivir</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>1984</offset>
+      <text>The influenza virus polymerase complex is composed of three subunits: PB1, PB2 and PA. The PA subunit performs the ‘cap-snatching’ endonuclease reaction, the PB2 subunit is responsible for initial binding of the capped RNAs, while the actual RNA synthesis is performed by the PB1 protein.</text>
+      <annotation id="573">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:43Z</infon>
+        <location offset="1988" length="9"/>
+        <text>influenza</text>
+      </annotation>
+      <annotation id="589">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <location offset="1998" length="5"/>
+        <text>virus</text>
+      </annotation>
+      <annotation id="30">
+        <infon key="score">0.7705644</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:49:34Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2004" length="10"/>
+        <text>polymerase</text>
+      </annotation>
+      <annotation id="31">
+        <infon key="score">0.9870968</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:49:39Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2054" length="3"/>
+        <text>PB1</text>
+      </annotation>
+      <annotation id="32">
+        <infon key="score">0.9846103</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:49:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2059" length="3"/>
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+      </annotation>
+      <annotation id="33">
+        <infon key="score">0.9803749</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2067" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="34">
+        <infon key="score">0.9600598</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2075" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="615">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:02Z</infon>
+        <location offset="2078" length="7"/>
+        <text>subunit</text>
+      </annotation>
+      <annotation id="518">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:46Z</infon>
+        <location offset="2115" length="12"/>
+        <text>endonuclease</text>
+      </annotation>
+      <annotation id="35">
+        <infon key="score">0.98064315</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:49:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2142" length="3"/>
+        <text>PB2</text>
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+      <annotation id="614">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:01Z</infon>
+        <location offset="2146" length="7"/>
+        <text>subunit</text>
+      </annotation>
+      <annotation id="610">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:50:11Z</infon>
+        <location offset="2196" length="11"/>
+        <text>capped RNAs</text>
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+      <annotation id="611">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:50:21Z</infon>
+        <location offset="2226" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="36">
+        <infon key="score">0.98972344</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:49:39Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2260" length="3"/>
+        <text>PB1</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>2277</offset>
+      <text>Given its crucial role in the viral life cycle, the influenza virus polymerase is widely recognized as a superior target for antiviral drug development and, in particular, inhibition of the PA endonuclease has deserved much attention in recent years.</text>
+      <annotation id="37">
+        <infon key="score">0.99807453</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:47:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2307" length="5"/>
+        <text>viral</text>
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+      <annotation id="574">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:43Z</infon>
+        <location offset="2329" length="9"/>
+        <text>influenza</text>
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+      <annotation id="590">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <location offset="2339" length="5"/>
+        <text>virus</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:49:34Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2345" length="10"/>
+        <text>polymerase</text>
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+      <annotation id="528">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="2467" length="2"/>
+        <text>PA</text>
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+      <annotation id="519">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:46Z</infon>
+        <location offset="2470" length="12"/>
+        <text>endonuclease</text>
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+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>2528</offset>
+      <text>The endonuclease catalytic site resides in the N-terminal domain of PA (PA-Nter; residues 1~195). It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions. The two-metal-ion model is consistent with numerous biochemical findings. Since the intracellular concentration of Mg2+ is at least 1000-fold higher than that of Mn2+, magnesium may be more biologically relevant. A controversy about number and type of metal ions exists also for the active site of HIV-1 integrase. HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2532" length="12"/>
+        <text>endonuclease</text>
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+      <annotation id="40">
+        <infon key="score">0.998888</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2545" length="14"/>
+        <text>catalytic site</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:15Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2575" length="17"/>
+        <text>N-terminal domain</text>
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+        <infon key="score">0.45754546</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2596" length="2"/>
+        <text>PA</text>
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+      <annotation id="529">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="2600" length="2"/>
+        <text>PA</text>
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+      <annotation id="550">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="2603" length="4"/>
+        <text>Nter</text>
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+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:28Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2618" length="5"/>
+        <text>1~195</text>
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+      <annotation id="44">
+        <infon key="score">0.9974348</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:33Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2641" length="9"/>
+        <text>histidine</text>
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+      <annotation id="45">
+        <infon key="score">0.99958926</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:36Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2652" length="5"/>
+        <text>His41</text>
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+      <annotation id="46">
+        <infon key="score">0.9985449</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2682" length="18"/>
+        <text>strictly conserved</text>
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+      <annotation id="47">
+        <infon key="score">0.8895876</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2701" length="6"/>
+        <text>acidic</text>
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+      <annotation id="48">
+        <infon key="score">0.999592</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2718" length="5"/>
+        <text>Glu80</text>
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+      <annotation id="49">
+        <infon key="score">0.9995907</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2725" length="6"/>
+        <text>Asp108</text>
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+      <annotation id="50">
+        <infon key="score">0.99958843</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2733" length="6"/>
+        <text>Glu119</text>
+      </annotation>
+      <annotation id="51">
+        <infon key="score">0.72042996</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2748" length="10"/>
+        <text>coordinate</text>
+      </annotation>
+      <annotation id="52">
+        <infon key="score">0.9995765</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2774" length="6"/>
+        <text>Ile120</text>
+      </annotation>
+      <annotation id="53">
+        <infon key="score">0.99908924</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:07Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2801" length="9"/>
+        <text>manganese</text>
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+      <annotation id="54">
+        <infon key="score">0.99902713</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:11Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2814" length="9"/>
+        <text>magnesium</text>
+      </annotation>
+      <annotation id="55">
+        <infon key="score">0.99921817</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:20Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2945" length="4"/>
+        <text>Mg2+</text>
+      </annotation>
+      <annotation id="56">
+        <infon key="score">0.9946342</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:24Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2992" length="5"/>
+        <text>Mn2+,</text>
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+      <annotation id="57">
+        <infon key="score">0.9991565</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:11Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2998" length="9"/>
+        <text>magnesium</text>
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+      <annotation id="58">
+        <infon key="score">0.999073</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="3113" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="59">
+        <infon key="score">0.9471887</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:34Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3128" length="5"/>
+        <text>HIV-1</text>
+      </annotation>
+      <annotation id="60">
+        <infon key="score">0.88152456</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3134" length="9"/>
+        <text>integrase</text>
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+      <annotation id="61">
+        <infon key="score">0.8539974</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:34Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3145" length="5"/>
+        <text>HIV-1</text>
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+      <annotation id="62">
+        <infon key="score">0.9976342</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:47Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3151" length="9"/>
+        <text>integrase</text>
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+        <infon key="score">0.96459323</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3258" length="5"/>
+        <text>metal</text>
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+        <infon key="score">0.9967907</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:47:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3279" length="5"/>
+        <text>viral</text>
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+        <infon key="score">0.5193359</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3313" length="2"/>
+        <text>PA</text>
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+        <infon key="score">0.99583817</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3387" length="9"/>
+        <text>influenza</text>
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+      <annotation id="67">
+        <infon key="score">0.9984561</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3397" length="12"/>
+        <text>endonuclease</text>
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+        <infon key="score">0.99910116</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:53:01Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3441" length="22"/>
+        <text>2,4-dioxobutanoic acid</text>
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+      <annotation id="69">
+        <infon key="score">0.99924695</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:53:07Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3477" length="9"/>
+        <text>flutimide</text>
+      </annotation>
+      <annotation id="70">
+        <infon key="score">0.9988958</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:53:12Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3508" length="21"/>
+        <text>2-hydroxyphenyl amide</text>
+      </annotation>
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+        <infon key="score">0.99888647</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:53:18Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3554" length="14"/>
+        <text>tetramic acids</text>
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+      <annotation id="72">
+        <infon key="score">0.9990219</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:53:24Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3570" length="24"/>
+        <text>5-hydroxypyrimidin-4-one</text>
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+      <annotation id="73">
+        <infon key="score">0.9982033</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:53:28Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <text>EGCG</text>
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+    <passage>
+      <infon key="section_type">INTRO</infon>
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+      <offset>3693</offset>
+      <text>In recent years, we focused our research on chemical scaffolds that are able to chelate metal ions of PA-Nter, resulting in inhibition of influenza virus replication. N-acylhydrazones represent an appealing class of chelating ligands with a broad spectrum of biological activities, such as activity against HIV, hepatitis A, vaccinia and influenza virus. In the present work, we report the biological activity of a series of N-acylhydrazones (Fig. 2), as determined in an enzymatic assay with PA-Nter endonuclease as well as in cell-based influenza viral ribonucleoprotein (vRNP) reconstitution and virus yield assays. Several N-acylhydrazones were found to have promising anti-influenza activity with 50% effective concentration values (EC50) in the range of 3–20 μM and good selectivity (Table 1 and Fig. 3). Computational docking studies of two candidate ligands in the PA-Nter active site gave information about the features that could determine inhibition of endonuclease activity. Moreover, we describe the X-ray crystal structure of PA-Nter in complex with one of the most active inhibitors.</text>
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+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:56:07Z</infon>
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+        <text>cell-based influenza viral ribonucleoprotein (vRNP) reconstitution</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:55:19Z</infon>
+        <location offset="4292" length="18"/>
+        <text>virus yield assays</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:46Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4320" length="16"/>
+        <text>N-acylhydrazones</text>
+      </annotation>
+      <annotation id="577">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:43Z</infon>
+        <location offset="4371" length="9"/>
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+      <annotation id="85">
+        <infon key="score">0.6208835</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:56:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>50% effective concentration</text>
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+      <annotation id="86">
+        <infon key="score">0.99088424</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:56:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>EC50</text>
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+        <infon key="score">0.997997</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:55:35Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4504" length="29"/>
+        <text>Computational docking studies</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="score">0.99886763</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4574" length="11"/>
+        <text>active site</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4657" length="12"/>
+        <text>endonuclease</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:56:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4706" length="23"/>
+        <text>X-ray crystal structure</text>
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+      <annotation id="533">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="4733" length="2"/>
+        <text>PA</text>
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+      <annotation id="554">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="4736" length="4"/>
+        <text>Nter</text>
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+        <infon key="score">0.99766636</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4741" length="15"/>
+        <text>in complex with</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_1</infon>
+      <offset>4797</offset>
+      <text>Results and Discussion</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>4820</offset>
+      <text>Chemistry</text>
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+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>4830</offset>
+      <text>N-acylhydrazones 1–27 (Fig. 2) were prepared in high yields by following literature methods (Fig. 2A); they were characterized by spectroscopic tools, mass spectrometry and elemental analysis. Even if isomerism around the C = N bond is possible, 1–27 are present in the E form in solution, as evidenced by the chemical shift values of the HC = N and NH protons in the 1H-NMR spectrum. Exceptions are represented by the alkyl-derivatives 3 and 4 (2:1 and 5:3 E:Z ratio, respectively).</text>
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+        <infon key="identifier">CHEBI:</infon>
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+        <text>N-acylhydrazones</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:57:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <text>1–27</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:57:29Z</infon>
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+        <text>mass spectrometry</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:57:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5003" length="18"/>
+        <text>elemental analysis</text>
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+        <infon key="score">0.90843266</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:57:51Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5076" length="4"/>
+        <text>1–27</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:57:33Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5198" length="6"/>
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+      <annotation id="621">
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:57:23Z</infon>
+        <location offset="5205" length="8"/>
+        <text>spectrum</text>
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+        <infon key="score">0.8405048</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:57:56Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5267" length="1"/>
+        <text>3</text>
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+        <infon key="score">0.9325075</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:57:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5273" length="1"/>
+        <text>4</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>5326</offset>
+      <text>If R’ (Fig. 2A) is a 2-hydroxy substituted phenyl ring, the corresponding acylhydrazones can coordinate one or, depending on denticity, two metal centers (modes A and B in Fig. 4). Starting from N’-(2,3-dihydroxybenzylidene)-semicarbazide (1) and its methoxy-analogue (2), we modified the acylhydrazonic substituent R” (3–8, 18, 19, Fig. 2A). In 18 and 19, also the gallic moiety can be involved in the chelation of the metal cofactors (mode C, Fig. 4). In order to investigate the role of hydroxyl substituents 9–11, 13–17, 20–23 and 27 were also synthesized. Compound 12 was synthesized in order to confirm the crucial influence of the gallic moiety. Finally, 26 was here considered, because it is an inhibitor of HIV RNase H, another enzyme with two magnesium ions in its active site.</text>
+      <annotation id="100">
+        <infon key="score">0.99670947</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:06Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5400" length="14"/>
+        <text>acylhydrazones</text>
+      </annotation>
+      <annotation id="101">
+        <infon key="score">0.87742805</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5419" length="10"/>
+        <text>coordinate</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:16Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <text>N’-(2,3-dihydroxybenzylidene)-semicarbazide</text>
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+        <infon key="score">0.9352348</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:19Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5566" length="1"/>
+        <text>1</text>
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+        <infon key="score">0.92682606</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:21Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <text>2</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:27Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5646" length="3"/>
+        <text>3–8</text>
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+        <infon key="score">0.9970642</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5651" length="2"/>
+        <text>18</text>
+      </annotation>
+      <annotation id="107">
+        <infon key="score">0.9953459</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5655" length="2"/>
+        <text>19</text>
+      </annotation>
+      <annotation id="108">
+        <infon key="score">0.9968497</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5672" length="2"/>
+        <text>18</text>
+      </annotation>
+      <annotation id="109">
+        <infon key="score">0.9944232</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:38Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5679" length="2"/>
+        <text>19</text>
+      </annotation>
+      <annotation id="110">
+        <infon key="score">0.9775343</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:40Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5692" length="6"/>
+        <text>gallic</text>
+      </annotation>
+      <annotation id="645">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:48Z</infon>
+        <location offset="5729" length="9"/>
+        <text>chelation</text>
+      </annotation>
+      <annotation id="111">
+        <infon key="score">0.84397453</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:54Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5838" length="4"/>
+        <text>9–11</text>
+      </annotation>
+      <annotation id="112">
+        <infon key="score">0.8735382</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:51Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5844" length="5"/>
+        <text>13–17</text>
+      </annotation>
+      <annotation id="113">
+        <infon key="score">0.96470314</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5851" length="5"/>
+        <text>20–23</text>
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+      <annotation id="114">
+        <infon key="score">0.99696344</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:47Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5861" length="2"/>
+        <text>27</text>
+      </annotation>
+      <annotation id="115">
+        <infon key="score">0.9877781</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:44Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5896" length="2"/>
+        <text>12</text>
+      </annotation>
+      <annotation id="116">
+        <infon key="score">0.9775613</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5964" length="6"/>
+        <text>gallic</text>
+      </annotation>
+      <annotation id="117">
+        <infon key="score">0.9980586</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:58:56Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5988" length="2"/>
+        <text>26</text>
+      </annotation>
+      <annotation id="118">
+        <infon key="score">0.8888434</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:45:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6042" length="3"/>
+        <text>HIV</text>
+      </annotation>
+      <annotation id="119">
+        <infon key="score">0.99612683</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:59:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6046" length="7"/>
+        <text>RNase H</text>
+      </annotation>
+      <annotation id="120">
+        <infon key="score">0.9988881</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:11Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="6079" length="9"/>
+        <text>magnesium</text>
+      </annotation>
+      <annotation id="121">
+        <infon key="score">0.9984977</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6101" length="11"/>
+        <text>active site</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>6128</offset>
+      <text>Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+. Different reaction conditions were used (1:1 and 1:2 metal to ligand ratio, up to 4 equivalents of triethylamine), but in any case the same chemical species Mg(HL)2∙4H2O was recovered and conveniently characterized. The use of a coordinating solvent as d6-DMSO causes partial decoordination of the ligand, but the 1H-NMR spectrum in MeOD, instead, shows only the signals attributable to the complex. In the 13C-NMR spectrum, the signal of the C = O quaternary carbon is practically unaffected by complexation, suggesting that the C = O group is weakly involved in the coordination to the metal ion. This is confirmed, in the IR spectrum, by the shift of about 20 cm−1 of the C = O absorption, while a shift of 30–50 cm−1 is expected when the carbonylic oxygen is tightly bound to the metal ion. ESI-mass spectra and elemental analysis confirmed the formula Mg(HL)2∙4H2O.</text>
+      <annotation id="122">
+        <infon key="score">0.9984599</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:46Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="6165" length="16"/>
+        <text>N-acylhydrazones</text>
+      </annotation>
+      <annotation id="646">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:48Z</infon>
+        <location offset="6202" length="9"/>
+        <text>chelation</text>
+      </annotation>
+      <annotation id="123">
+        <infon key="score">0.93400145</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:59:38Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="6228" length="5"/>
+        <text>metal</text>
+      </annotation>
+      <annotation id="124">
+        <infon key="score">0.9970854</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6253" length="9"/>
+        <text>influenza</text>
+      </annotation>
+      <annotation id="534">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="6263" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="555">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="6266" length="4"/>
+        <text>Nter</text>
+      </annotation>
+      <annotation id="125">
+        <infon key="score">0.99884176</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6271" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="126">
+        <infon key="score">0.9987612</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:59:45Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="6354" length="2"/>
+        <text>19</text>
+      </annotation>
+      <annotation id="127">
+        <infon key="score">0.99912053</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:59:47Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="6358" length="3"/>
+        <text>H2L</text>
+      </annotation>
+      <annotation id="128">
+        <infon key="score">0.9987998</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:59:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="6371" length="4"/>
+        <text>Mg2+</text>
+      </annotation>
+      <annotation id="129">
+        <infon key="score">0.9989767</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:59:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="6476" length="13"/>
+        <text>triethylamine</text>
+      </annotation>
+      <annotation id="130">
+        <infon key="score">0.9799634</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:59:21Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="6534" length="12"/>
+        <text>Mg(HL)2∙4H2O</text>
+      </annotation>
+      <annotation id="131">
+        <infon key="score">0.98885256</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:59:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="6630" length="7"/>
+        <text>d6-DMSO</text>
+      </annotation>
+      <annotation id="132">
+        <infon key="score">0.9973355</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:57:33Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6691" length="6"/>
+        <text>1H-NMR</text>
+      </annotation>
+      <annotation id="133">
+        <infon key="score">0.9893941</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:57:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6698" length="8"/>
+        <text>spectrum</text>
+      </annotation>
+      <annotation id="134">
+        <infon key="score">0.9976766</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:00:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6784" length="7"/>
+        <text>13C-NMR</text>
+      </annotation>
+      <annotation id="135">
+        <infon key="score">0.898486</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:57:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6792" length="8"/>
+        <text>spectrum</text>
+      </annotation>
+      <annotation id="136">
+        <infon key="score">0.6472191</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:00:07Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7002" length="2"/>
+        <text>IR</text>
+      </annotation>
+      <annotation id="137">
+        <infon key="score">0.91154146</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:57:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7005" length="8"/>
+        <text>spectrum</text>
+      </annotation>
+      <annotation id="138">
+        <infon key="score">0.9450806</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:00:12Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7172" length="8"/>
+        <text>ESI-mass</text>
+      </annotation>
+      <annotation id="139">
+        <infon key="score">0.7451172</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:44:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7181" length="7"/>
+        <text>spectra</text>
+      </annotation>
+      <annotation id="140">
+        <infon key="score">0.9956361</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:57:38Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7193" length="18"/>
+        <text>elemental analysis</text>
+      </annotation>
+      <annotation id="141">
+        <infon key="score">0.96558464</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:59:21Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7234" length="12"/>
+        <text>Mg(HL)2∙4H2O</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>7274</offset>
+      <text>The interaction between the N-acylhydrazone ligands and the magnesium cation was investigated also by means of UV-visible spectroscopy (UV-visible titrations of 23 and 19 with increasing amount of Mg(CH3COO)2 are shown in Figure S1). The spectrum of 19 includes a band at 313 nm assignable to n-π* transitions of the C = N and C = O groups. By adding increasing equivalents of Mg(CH3COO)2, the absorption around 400 nm increases, and a new band appears with a maximum at 397 nm. The opposite trend is observed in the range 300–350 nm, where an isosbestic point is present close to 335 nm. When the same experiment was performed with 23, a different behavior was observed. Increasing concentration of Mg2+, in fact, caused a diminution in the maximum absorption, an isosbestic point is visible at about 345 nm, but a new band at 400 nm does not appear. Ligands 19 and 23 coordinate the Mg2+ ions in different ways: 19 chelates the metal ion by using the deprotonated salicyl oxygen and the iminic nitrogen, while for 23, the gallic moiety is supposed to be involved (Fig. 4A,B versus C), leading to different, less extensive, modifications of the UV spectrum. These results will be revisited during the discussion of the biological activity.</text>
+      <annotation id="142">
+        <infon key="score">0.9965074</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7302" length="15"/>
+        <text>N-acylhydrazone</text>
+      </annotation>
+      <annotation id="143">
+        <infon key="score">0.9987508</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:11Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7334" length="9"/>
+        <text>magnesium</text>
+      </annotation>
+      <annotation id="144">
+        <infon key="score">0.9987264</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:00:27Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7385" length="23"/>
+        <text>UV-visible spectroscopy</text>
+      </annotation>
+      <annotation id="145">
+        <infon key="score">0.99818856</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:00:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7410" length="21"/>
+        <text>UV-visible titrations</text>
+      </annotation>
+      <annotation id="146">
+        <infon key="score">0.99549353</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:00:36Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7435" length="2"/>
+        <text>23</text>
+      </annotation>
+      <annotation id="147">
+        <infon key="score">0.9924136</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:00:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7442" length="2"/>
+        <text>19</text>
+      </annotation>
+      <annotation id="148">
+        <infon key="score">0.50728977</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:00:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7450" length="17"/>
+        <text>increasing amount</text>
+      </annotation>
+      <annotation id="149">
+        <infon key="score">0.99768364</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:00:22Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7471" length="11"/>
+        <text>Mg(CH3COO)2</text>
+      </annotation>
+      <annotation id="623">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:57:23Z</infon>
+        <location offset="7512" length="8"/>
+        <text>spectrum</text>
+      </annotation>
+      <annotation id="150">
+        <infon key="score">0.99344337</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:00:45Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7524" length="2"/>
+        <text>19</text>
+      </annotation>
+      <annotation id="151">
+        <infon key="score">0.9985274</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:00:22Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7651" length="11"/>
+        <text>Mg(CH3COO)2</text>
+      </annotation>
+      <annotation id="152">
+        <infon key="score">0.9965898</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:00:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7907" length="2"/>
+        <text>23</text>
+      </annotation>
+      <annotation id="153">
+        <infon key="score">0.9798636</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:00:51Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7974" length="5"/>
+        <text>Mg2+,</text>
+      </annotation>
+      <annotation id="154">
+        <infon key="score">0.99524873</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:00:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8134" length="2"/>
+        <text>19</text>
+      </annotation>
+      <annotation id="155">
+        <infon key="score">0.99277425</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:00:55Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8141" length="2"/>
+        <text>23</text>
+      </annotation>
+      <annotation id="616">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:58Z</infon>
+        <location offset="8144" length="10"/>
+        <text>coordinate</text>
+      </annotation>
+      <annotation id="156">
+        <infon key="score">0.99888766</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:00:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8159" length="4"/>
+        <text>Mg2+</text>
+      </annotation>
+      <annotation id="157">
+        <infon key="score">0.99557614</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:01:08Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8188" length="2"/>
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+        <infon key="score">0.9941742</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:01:03Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8290" length="2"/>
+        <text>23</text>
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+      <annotation id="625">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:01:14Z</infon>
+        <location offset="8420" length="2"/>
+        <text>UV</text>
+      </annotation>
+      <annotation id="624">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:57:23Z</infon>
+        <location offset="8423" length="8"/>
+        <text>spectrum</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>8540</offset>
+      <text>Inhibition of the PA-Nter enzyme</text>
+      <annotation id="535">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="8558" length="2"/>
+        <text>PA</text>
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+      <annotation id="556">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="8561" length="4"/>
+        <text>Nter</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>8573</offset>
+      <text>All the compounds were tested for their ability to inhibit the influenza endonuclease in an enzymatic plasmid-based assay with recombinant PA-Nter, as well as in cell-based influenza methods (i.e. virus yield and vRNP reconstitution assays). The results are shown in Table 1 and summarized in Fig. 3 to visualize the structure-activity relationships; Figure S2 shows the dose-response curves for three representative compounds (i.e. 10, 13 and 23) in either the PA-enzyme or vRNP reconstitution assay.</text>
+      <annotation id="159">
+        <infon key="score">0.98925996</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8636" length="9"/>
+        <text>influenza</text>
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+      <annotation id="160">
+        <infon key="score">0.9990226</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8646" length="12"/>
+        <text>endonuclease</text>
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+      <annotation id="161">
+        <infon key="score">0.9988707</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:01:41Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8665" length="29"/>
+        <text>enzymatic plasmid-based assay</text>
+      </annotation>
+      <annotation id="536">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="8712" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="557">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="8715" length="4"/>
+        <text>Nter</text>
+      </annotation>
+      <annotation id="626">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:01:59Z</infon>
+        <location offset="8735" length="28"/>
+        <text>cell-based influenza methods</text>
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+      <annotation id="627">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:10Z</infon>
+        <location offset="8770" length="42"/>
+        <text>virus yield and vRNP reconstitution assays</text>
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+      <annotation id="162">
+        <infon key="score">0.9907825</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8944" length="20"/>
+        <text>dose-response curves</text>
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+      <annotation id="163">
+        <infon key="score">0.99843997</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:21Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9006" length="2"/>
+        <text>10</text>
+      </annotation>
+      <annotation id="164">
+        <infon key="score">0.9982728</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:23Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9010" length="2"/>
+        <text>13</text>
+      </annotation>
+      <annotation id="165">
+        <infon key="score">0.99854183</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:26Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9017" length="2"/>
+        <text>23</text>
+      </annotation>
+      <annotation id="628">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:41Z</infon>
+        <location offset="9035" length="38"/>
+        <text>PA-enzyme or vRNP reconstitution assay</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>9075</offset>
+      <text>The moderate activity (IC50 = 24 μM) of N’-2,3-dihydroxybenzylidene semicarbazide (1) was completely lost when the NH2 moiety was replaced by a hydrophobic heptyl chain (3), but it is less affected when a phenyl or a 2-hydroxyphenyl is present (5 and 7, IC50 = 84 and 54 μM, respectively). When the hydroxyl in position 3 on R1 (2,3-dihydroxybenzylidene) was replaced by a methoxy group (2-hydroxy-3-methoxybenzylidene), the activity disappeared (compounds 2, 4, 6 and 8). The activity is unaffected (IC50 values ranging from 45 to 75 μM) when going from two hydroxyls in R1 (7) to compounds with three hydroxyls (i.e. 9, 10 and 11). Similarly, 11 (R1 = 3,4,5-trihydroxyphenyl, R2 = 2-hydroxyphenyl) had comparable activity as 27 (R1 = 3,4,5-trihydroxyphenyl, R2 = NH2). Within the series carrying a 2-hydroxyphenyl R2 group, the activity of 11 is particularly intriguing. 11 does not have the possibility to chelate in a tridentate ONO fashion (mode A in Fig. 4), but it can coordinate two cations by means of its three OH groups in R1 (mode C, Fig. 4). Note that a similar chelating mode was observed in a crystal structure, solved by Cusack and coworkers, of PA-Nter endonuclease in complex with the inhibitor EGCG.</text>
+      <annotation id="166">
+        <infon key="score">0.9975967</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9098" length="4"/>
+        <text>IC50</text>
+      </annotation>
+      <annotation id="167">
+        <infon key="score">0.99913883</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:52Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9115" length="41"/>
+        <text>N’-2,3-dihydroxybenzylidene semicarbazide</text>
+      </annotation>
+      <annotation id="168">
+        <infon key="score">0.8099994</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:40:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9158" length="1"/>
+        <text>1</text>
+      </annotation>
+      <annotation id="635">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:04:51Z</infon>
+        <location offset="9245" length="1"/>
+        <text>3</text>
+      </annotation>
+      <annotation id="169">
+        <infon key="score">0.78925484</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:40:24Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9320" length="1"/>
+        <text>5</text>
+      </annotation>
+      <annotation id="170">
+        <infon key="score">0.7282984</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:40:28Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9326" length="1"/>
+        <text>7</text>
+      </annotation>
+      <annotation id="171">
+        <infon key="score">0.99741757</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9329" length="4"/>
+        <text>IC50</text>
+      </annotation>
+      <annotation id="172">
+        <infon key="score">0.998898</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:15:13Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9404" length="24"/>
+        <text>2,3-dihydroxybenzylidene</text>
+      </annotation>
+      <annotation id="173">
+        <infon key="score">0.99892056</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:15:18Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9463" length="30"/>
+        <text>2-hydroxy-3-methoxybenzylidene</text>
+      </annotation>
+      <annotation id="631">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:03:39Z</infon>
+        <location offset="9532" length="1"/>
+        <text>2</text>
+      </annotation>
+      <annotation id="632">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:03:55Z</infon>
+        <location offset="9535" length="1"/>
+        <text>4</text>
+      </annotation>
+      <annotation id="633">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:04:06Z</infon>
+        <location offset="9538" length="1"/>
+        <text>6</text>
+      </annotation>
+      <annotation id="634">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:04:15Z</infon>
+        <location offset="9544" length="1"/>
+        <text>8</text>
+      </annotation>
+      <annotation id="174">
+        <infon key="score">0.9979487</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9576" length="4"/>
+        <text>IC50</text>
+      </annotation>
+      <annotation id="175">
+        <infon key="score">0.6382894</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:04:24Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9651" length="1"/>
+        <text>7</text>
+      </annotation>
+      <annotation id="176">
+        <infon key="score">0.98685247</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:04:28Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9694" length="1"/>
+        <text>9</text>
+      </annotation>
+      <annotation id="177">
+        <infon key="score">0.9808538</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:04:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9697" length="2"/>
+        <text>10</text>
+      </annotation>
+      <annotation id="178">
+        <infon key="score">0.9842451</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:40:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9704" length="2"/>
+        <text>11</text>
+      </annotation>
+      <annotation id="179">
+        <infon key="score">0.9821738</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:40:37Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9720" length="2"/>
+        <text>11</text>
+      </annotation>
+      <annotation id="180">
+        <infon key="score">0.9929395</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:40:41Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9802" length="2"/>
+        <text>27</text>
+      </annotation>
+      <annotation id="181">
+        <infon key="score">0.93515146</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:40:43Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9917" length="2"/>
+        <text>11</text>
+      </annotation>
+      <annotation id="182">
+        <infon key="score">0.9458555</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:40:46Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9948" length="2"/>
+        <text>11</text>
+      </annotation>
+      <annotation id="183">
+        <infon key="score">0.78353983</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10051" length="10"/>
+        <text>coordinate</text>
+      </annotation>
+      <annotation id="184">
+        <infon key="score">0.9987627</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:52Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10183" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="537">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="10237" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="558">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="10240" length="4"/>
+        <text>Nter</text>
+      </annotation>
+      <annotation id="185">
+        <infon key="score">0.5075802</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10245" length="12"/>
+        <text>endonuclease</text>
+      </annotation>
+      <annotation id="186">
+        <infon key="score">0.99833864</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10258" length="15"/>
+        <text>in complex with</text>
+      </annotation>
+      <annotation id="187">
+        <infon key="score">0.9987085</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:53:50Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10288" length="4"/>
+        <text>EGCG</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>10329</offset>
+      <text>The PA-Nter inhibitory activity strongly depends on the number and position of hydroxyl substituents in R1 and R2: this is clearly highlighted by the data obtained with compounds 13–23, in which R2 is a 3,4,5-trihydroxyphenyl (gallic) group, the most active scaffold in our series. The analogue carrying an unsubstituted aromatic ring as R1 (compound 13) had moderate activity (IC50 = 69 μM). When one OH was added at position 2 of the R1 ring (14), the activity was lost. Adding a second OH substituent at position 5 resulted in strong activity (compound 15, IC50 = 9 μM); medium activity for a 3-OH (18; IC50 = 83 μM), and marginal activity when the second OH is at position 4 (17, IC50 ≥ 370 μM). The addition of a 3-methoxy group (19) abolished all inhibitory activity. This cannot be related to variations in the chelating features displayed by the R1 moiety, since compounds 14–19 all have, in theory, the capacity to chelate one metal ion through the ortho-OH and iminic nitrogen (mode A in Fig. 4). Moreover, compound 18 can, in principle, chelate the two M2+ ions in the active site according to mode B (Fig. 4), yet it (IC50 = 83 μM) has nine-fold lower activity than 15, that does not possess this two-metal chelating feature. Therefore, we hypothesized that the inhibitory activity of the series containing the gallic moiety is determined by: (i) the capacity of the moiety R2 to chelate two metal ions in the active site of the enzyme, according to mode C (Fig. 4); and (ii) the presence and position of one or more hydroxyl substituents in R1, which may possibly result in ligand-protein interactions (e.g. through hydrogen bonds). This assumption was supported by molecular docking calculations and X-ray analysis of inhibitor 23 in complex with PA-Nter (vide infra). At this point, change of the substituents in R1 represents the next logical step. Substitution of the 5-hydroxyl in 15 by a methoxy group (16) causes a dramatic drop in activity (IC50 = 9 and 454 μM for 15 and 16, respectively). When two or three OH groups are present in R1, their spatial disposition greatly affects the activity. In particular, all the compounds with a trihydroxylated phenyl group as R1 (i.e. 20, 21, 22 and 23) were able to inhibit PA-Nter quite potently. The lowest IC50 values were obtained for 21 and 23 (IC50 = 13 and 7 μM, respectively), which both have one of their three hydroxyl groups at position 5. The most active compound in this series was 23, which lacks the hydroxyl group at position 2 of R1, further confirming that this function is undesirable or even detrimental for inhibitory activity against PA-Nter, as already noticed above for 14.</text>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="10333" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="559">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="10336" length="4"/>
+        <text>Nter</text>
+      </annotation>
+      <annotation id="636">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:06:47Z</infon>
+        <location offset="10508" length="5"/>
+        <text>13–23</text>
+      </annotation>
+      <annotation id="188">
+        <infon key="score">0.68469006</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:40:50Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10680" length="2"/>
+        <text>13</text>
+      </annotation>
+      <annotation id="189">
+        <infon key="score">0.99552864</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10707" length="4"/>
+        <text>IC50</text>
+      </annotation>
+      <annotation id="190">
+        <infon key="score">0.9441393</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:40:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10774" length="2"/>
+        <text>14</text>
+      </annotation>
+      <annotation id="191">
+        <infon key="score">0.98732704</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:40:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10885" length="2"/>
+        <text>15</text>
+      </annotation>
+      <annotation id="192">
+        <infon key="score">0.98189247</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10889" length="4"/>
+        <text>IC50</text>
+      </annotation>
+      <annotation id="193">
+        <infon key="score">0.9942561</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:41:00Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10931" length="2"/>
+        <text>18</text>
+      </annotation>
+      <annotation id="194">
+        <infon key="score">0.99097943</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10935" length="4"/>
+        <text>IC50</text>
+      </annotation>
+      <annotation id="195">
+        <infon key="score">0.9926069</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:41:03Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11009" length="2"/>
+        <text>17</text>
+      </annotation>
+      <annotation id="196">
+        <infon key="score">0.98846984</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11013" length="4"/>
+        <text>IC50</text>
+      </annotation>
+      <annotation id="197">
+        <infon key="score">0.99281037</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:41:06Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11064" length="2"/>
+        <text>19</text>
+      </annotation>
+      <annotation id="663">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:41:20Z</infon>
+        <location offset="11210" length="5"/>
+        <text>14–19</text>
+      </annotation>
+      <annotation id="198">
+        <infon key="score">0.9937058</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:41:25Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11355" length="2"/>
+        <text>18</text>
+      </annotation>
+      <annotation id="199">
+        <infon key="score">0.9915317</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:07:46Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11393" length="3"/>
+        <text>M2+</text>
+      </annotation>
+      <annotation id="200">
+        <infon key="score">0.9978527</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11409" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="201">
+        <infon key="score">0.996293</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11459" length="4"/>
+        <text>IC50</text>
+      </annotation>
+      <annotation id="202">
+        <infon key="score">0.987648</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:41:31Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11507" length="2"/>
+        <text>15</text>
+      </annotation>
+      <annotation id="203">
+        <infon key="score">0.99806106</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11751" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="204">
+        <infon key="score">0.99656165</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:08:11Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11958" length="14"/>
+        <text>hydrogen bonds</text>
+      </annotation>
+      <annotation id="205">
+        <infon key="score">0.99873257</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:08:27Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12008" length="30"/>
+        <text>molecular docking calculations</text>
+      </annotation>
+      <annotation id="206">
+        <infon key="score">0.99835294</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:08:25Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12043" length="14"/>
+        <text>X-ray analysis</text>
+      </annotation>
+      <annotation id="207">
+        <infon key="score">0.9963385</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:41:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12071" length="2"/>
+        <text>23</text>
+      </annotation>
+      <annotation id="208">
+        <infon key="score">0.9927866</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12074" length="15"/>
+        <text>in complex with</text>
+      </annotation>
+      <annotation id="539">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="12090" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="560">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="12093" length="4"/>
+        <text>Nter</text>
+      </annotation>
+      <annotation id="209">
+        <infon key="score">0.98792946</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:41:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12228" length="2"/>
+        <text>15</text>
+      </annotation>
+      <annotation id="210">
+        <infon key="score">0.99426925</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:41:45Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12251" length="2"/>
+        <text>16</text>
+      </annotation>
+      <annotation id="211">
+        <infon key="score">0.99376845</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12291" length="4"/>
+        <text>IC50</text>
+      </annotation>
+      <annotation id="212">
+        <infon key="score">0.9953263</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:41:38Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12315" length="2"/>
+        <text>15</text>
+      </annotation>
+      <annotation id="213">
+        <infon key="score">0.9959229</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:41:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12322" length="2"/>
+        <text>16</text>
+      </annotation>
+      <annotation id="214">
+        <infon key="score">0.9899739</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:41:52Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12525" length="2"/>
+        <text>20</text>
+      </annotation>
+      <annotation id="215">
+        <infon key="score">0.98735577</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:42:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12529" length="2"/>
+        <text>21</text>
+      </annotation>
+      <annotation id="666">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:42:18Z</infon>
+        <location offset="12533" length="2"/>
+        <text>22</text>
+      </annotation>
+      <annotation id="667">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:42:25Z</infon>
+        <location offset="12540" length="2"/>
+        <text>23</text>
+      </annotation>
+      <annotation id="540">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="12565" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="561">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="12568" length="4"/>
+        <text>Nter</text>
+      </annotation>
+      <annotation id="629">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:48Z</infon>
+        <location offset="12600" length="4"/>
+        <text>IC50</text>
+      </annotation>
+      <annotation id="664">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:42:01Z</infon>
+        <location offset="12630" length="2"/>
+        <text>21</text>
+      </annotation>
+      <annotation id="665">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:42:09Z</infon>
+        <location offset="12637" length="2"/>
+        <text>23</text>
+      </annotation>
+      <annotation id="630">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:48Z</infon>
+        <location offset="12641" length="4"/>
+        <text>IC50</text>
+      </annotation>
+      <annotation id="668">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:42:33Z</infon>
+        <location offset="12786" length="2"/>
+        <text>23</text>
+      </annotation>
+      <annotation id="541">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="12947" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="562">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="12950" length="4"/>
+        <text>Nter</text>
+      </annotation>
+      <annotation id="669">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:42:41Z</infon>
+        <location offset="12985" length="2"/>
+        <text>14</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>13044</offset>
+      <text>Consistent with a crucial role of the R2 gallic moiety in metal chelation, the strong activity of 15 was completely lost in its 3,4,5-trimethoxy analogue 12. On the other hand, the R2 gallic containing compounds displayed moderate activity (IC50 values around 40 μM) when R1 was absent (i.e. the 3,4,5-trihydroxybenzohydrazide 28, Fig. 2), or composed of an extended ring system (26) or a pyrrole ring (25). Still lower activity was seen with the pyridine analogue 24. Evidently, the 3,4,5-trihydroxybenzyl moiety at R2 is fundamental but not sufficient to ensure potent PA-Nter endonuclease inhibition, since the interactions of R1 with the amino acid side chains of the protein appear crucial in modulating activity.</text>
+      <annotation id="647">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:48Z</infon>
+        <location offset="13108" length="9"/>
+        <text>chelation</text>
+      </annotation>
+      <annotation id="216">
+        <infon key="score">0.99587184</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:42:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13142" length="2"/>
+        <text>15</text>
+      </annotation>
+      <annotation id="217">
+        <infon key="score">0.99225724</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:42:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13198" length="2"/>
+        <text>12</text>
+      </annotation>
+      <annotation id="218">
+        <infon key="score">0.99825305</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13285" length="4"/>
+        <text>IC50</text>
+      </annotation>
+      <annotation id="219">
+        <infon key="score">0.9990565</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:43:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13340" length="30"/>
+        <text>3,4,5-trihydroxybenzohydrazide</text>
+      </annotation>
+      <annotation id="220">
+        <infon key="score">0.9956102</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:43:07Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13371" length="2"/>
+        <text>28</text>
+      </annotation>
+      <annotation id="221">
+        <infon key="score">0.99730444</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:43:10Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13424" length="2"/>
+        <text>26</text>
+      </annotation>
+      <annotation id="222">
+        <infon key="score">0.9970535</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:43:12Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13447" length="2"/>
+        <text>25</text>
+      </annotation>
+      <annotation id="223">
+        <infon key="score">0.9971583</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:43:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13509" length="2"/>
+        <text>24</text>
+      </annotation>
+      <annotation id="542">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="13615" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="563">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="13618" length="4"/>
+        <text>Nter</text>
+      </annotation>
+      <annotation id="520">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:46Z</infon>
+        <location offset="13623" length="12"/>
+        <text>endonuclease</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>13766</offset>
+      <text>Inhibition of vRNP activity or virus replication in cells</text>
+      <annotation id="224">
+        <infon key="score">0.9976355</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:10:07Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="13780" length="4"/>
+        <text>vRNP</text>
+      </annotation>
+      <annotation id="225">
+        <infon key="score">0.9874249</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13797" length="5"/>
+        <text>virus</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>13824</offset>
+      <text>To determine the anti-influenza virus activity of compounds 1–28 in cell culture, we performed an influenza vRNP reconstitution assay in human embryonic kidney 293 T (HEK293T) cells, then subjected the active compounds (i.e. EC50 &lt; 100 μM) to a virus yield assay in influenza virus-infected Madin-Darby canine kidney (MDCK) cells (Table 1 and Fig. 3). For some N-acylhydrazone compounds, we observed quite potent and selective activity in the vRNP reconstitution assay. This indicates that they are able to inhibit viral RNA synthesis and suggests that they could be classified as original PA inhibitors. Values for EC50 (vRNP) or EC90 (virus yield) in the range of 0.4–18 μM were obtained for compounds 15 and 20–23, which all carry a 3,4,5-trihydroxyphenyl as R2, and possess either two (15) or three (20–23) hydroxyl substituents in the R1 moiety. As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.</text>
+      <annotation id="226">
+        <infon key="score">0.6290952</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13846" length="9"/>
+        <text>influenza</text>
+      </annotation>
+      <annotation id="593">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <location offset="13856" length="5"/>
+        <text>virus</text>
+      </annotation>
+      <annotation id="227">
+        <infon key="score">0.9537156</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:11:38Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13884" length="4"/>
+        <text>1–28</text>
+      </annotation>
+      <annotation id="228">
+        <infon key="score">0.943576</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:10:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="13922" length="35"/>
+        <text>influenza vRNP reconstitution assay</text>
+      </annotation>
+      <annotation id="229">
+        <infon key="score">0.99535996</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:10:29Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="13961" length="5"/>
+        <text>human</text>
+      </annotation>
+      <annotation id="230">
+        <infon key="score">0.9930876</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:56:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14049" length="4"/>
+        <text>EC50</text>
+      </annotation>
+      <annotation id="231">
+        <infon key="score">0.9428053</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:10:38Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14069" length="17"/>
+        <text>virus yield assay</text>
+      </annotation>
+      <annotation id="232">
+        <infon key="score">0.8899718</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14090" length="9"/>
+        <text>influenza</text>
+      </annotation>
+      <annotation id="594">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <location offset="14100" length="5"/>
+        <text>virus</text>
+      </annotation>
+      <annotation id="233">
+        <infon key="score">0.9984858</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14185" length="15"/>
+        <text>N-acylhydrazone</text>
+      </annotation>
+      <annotation id="234">
+        <infon key="score">0.9985456</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:10:41Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14267" length="25"/>
+        <text>vRNP reconstitution assay</text>
+      </annotation>
+      <annotation id="235">
+        <infon key="score">0.9965383</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:47:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14339" length="5"/>
+        <text>viral</text>
+      </annotation>
+      <annotation id="612">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:50:22Z</infon>
+        <location offset="14345" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="236">
+        <infon key="score">0.5546336</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="14414" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="237">
+        <infon key="score">0.9939942</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:56:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14440" length="4"/>
+        <text>EC50</text>
+      </annotation>
+      <annotation id="238">
+        <infon key="score">0.7582195</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:44:46Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="14446" length="4"/>
+        <text>vRNP</text>
+      </annotation>
+      <annotation id="239">
+        <infon key="score">0.9961493</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:38:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14455" length="4"/>
+        <text>EC90</text>
+      </annotation>
+      <annotation id="595">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <location offset="14461" length="5"/>
+        <text>virus</text>
+      </annotation>
+      <annotation id="240">
+        <infon key="score">0.9972523</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:11:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14528" length="2"/>
+        <text>15</text>
+      </annotation>
+      <annotation id="241">
+        <infon key="score">0.9963448</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:11:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14535" length="5"/>
+        <text>20–23</text>
+      </annotation>
+      <annotation id="242">
+        <infon key="score">0.9966973</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:11:45Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14614" length="2"/>
+        <text>15</text>
+      </annotation>
+      <annotation id="243">
+        <infon key="score">0.9963566</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:11:47Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14628" length="2"/>
+        <text>20</text>
+      </annotation>
+      <annotation id="244">
+        <infon key="score">0.9705081</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:11:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14631" length="2"/>
+        <text>23</text>
+      </annotation>
+      <annotation id="637">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:11:30Z</infon>
+        <location offset="14685" length="24"/>
+        <text>enzymatic PA-Nter assays</text>
+      </annotation>
+      <annotation id="245">
+        <infon key="score">0.9964953</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:11:54Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14763" length="2"/>
+        <text>21</text>
+      </annotation>
+      <annotation id="246">
+        <infon key="score">0.9962042</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:11:56Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14767" length="2"/>
+        <text>22</text>
+      </annotation>
+      <annotation id="247">
+        <infon key="score">0.9944267</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:11:51Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14774" length="2"/>
+        <text>23</text>
+      </annotation>
+      <annotation id="248">
+        <infon key="score">0.9941497</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:11:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14861" length="1"/>
+        <text>9</text>
+      </annotation>
+      <annotation id="249">
+        <infon key="score">0.9900468</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:12:01Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14864" length="2"/>
+        <text>10</text>
+      </annotation>
+      <annotation id="250">
+        <infon key="score">0.99025047</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:12:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14871" length="2"/>
+        <text>11</text>
+      </annotation>
+      <annotation id="251">
+        <infon key="score">0.9884703</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:12:06Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14876" length="2"/>
+        <text>10</text>
+      </annotation>
+      <annotation id="252">
+        <infon key="score">0.9921761</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:12:08Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14883" length="2"/>
+        <text>22</text>
+      </annotation>
+      <annotation id="253">
+        <infon key="score">0.996451</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:56:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14914" length="4"/>
+        <text>EC50</text>
+      </annotation>
+      <annotation id="254">
+        <infon key="score">0.99858147</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:43:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14926" length="25"/>
+        <text>vRNP reconstitution assay</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>14990</offset>
+      <text>The hydrazide 28 displayed weak (virus yield) to moderate (vRNP reconstitution) activity, albeit less than the most active molecules in the 3,4,5-trihydroxyphenyl series (i.e. 18 and 21–23). Even if there are no data indicating that the compounds reported in the paper are subject to hydrolysis, the activity of 28 could raise the concern that for some N-acylhydrazones the antiviral activity in cell culture may be related to their intracellular hydrolysis. However, this is unlikely, since the antiviral potency showed large differences (i.e. EC50 values between 0.42 and 29 μM) for compounds with the same R2 but different R1 groups, meaning that R1 does play a role in modulating the antiviral effect.</text>
+      <annotation id="255">
+        <infon key="score">0.9966097</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:12:16Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14994" length="9"/>
+        <text>hydrazide</text>
+      </annotation>
+      <annotation id="256">
+        <infon key="score">0.9948773</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:12:20Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15004" length="2"/>
+        <text>28</text>
+      </annotation>
+      <annotation id="596">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <location offset="15023" length="5"/>
+        <text>virus</text>
+      </annotation>
+      <annotation id="638">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:13:04Z</infon>
+        <location offset="15049" length="19"/>
+        <text>vRNP reconstitution</text>
+      </annotation>
+      <annotation id="257">
+        <infon key="score">0.9985487</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:13:22Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15166" length="2"/>
+        <text>18</text>
+      </annotation>
+      <annotation id="258">
+        <infon key="score">0.9972357</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:13:24Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15173" length="5"/>
+        <text>21–23</text>
+      </annotation>
+      <annotation id="259">
+        <infon key="score">0.9956599</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:13:26Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15302" length="2"/>
+        <text>28</text>
+      </annotation>
+      <annotation id="260">
+        <infon key="score">0.99789184</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:46Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15343" length="16"/>
+        <text>N-acylhydrazones</text>
+      </annotation>
+      <annotation id="261">
+        <infon key="score">0.9966673</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:56:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15535" length="4"/>
+        <text>EC50</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>15701</offset>
+      <text>Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8. Two notable exceptions are 18 and 19 (containing a 2,3-dihydroxybenzylidene or 2-hydroxy-3-methoxybenzylidene R1, respectively) which were not cytotoxic at 200 μM and displayed favorable antiviral selectivity.</text>
+      <annotation id="262">
+        <infon key="score">0.99871427</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:13:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15733" length="24"/>
+        <text>2,3-dihydroxybenzylidene</text>
+      </annotation>
+      <annotation id="263">
+        <infon key="score">0.9609433</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:13:38Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15764" length="1"/>
+        <text>3</text>
+      </annotation>
+      <annotation id="264">
+        <infon key="score">0.9444579</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:13:40Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15767" length="1"/>
+        <text>5</text>
+      </annotation>
+      <annotation id="265">
+        <infon key="score">0.9343455</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:13:43Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15773" length="1"/>
+        <text>7</text>
+      </annotation>
+      <annotation id="266">
+        <infon key="score">0.9988452</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:13:46Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15779" length="30"/>
+        <text>2-hydroxy-3-methoxybenzylidene</text>
+      </annotation>
+      <annotation id="267">
+        <infon key="score">0.9820941</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:14:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15823" length="1"/>
+        <text>4</text>
+      </annotation>
+      <annotation id="268">
+        <infon key="score">0.9549013</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:14:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15826" length="1"/>
+        <text>6</text>
+      </annotation>
+      <annotation id="269">
+        <infon key="score">0.97834396</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:14:36Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15832" length="1"/>
+        <text>8</text>
+      </annotation>
+      <annotation id="270">
+        <infon key="score">0.9932685</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:15:00Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15878" length="10"/>
+        <text>vRNP assay</text>
+      </annotation>
+      <annotation id="271">
+        <infon key="score">0.9910517</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:14:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15895" length="4"/>
+        <text>CC50</text>
+      </annotation>
+      <annotation id="272">
+        <infon key="score">0.95631367</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:14:52Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15925" length="17"/>
+        <text>selectivity index</text>
+      </annotation>
+      <annotation id="273">
+        <infon key="score">0.98919183</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:14:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15953" length="4"/>
+        <text>CC50</text>
+      </annotation>
+      <annotation id="274">
+        <infon key="score">0.9908751</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:56:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15961" length="4"/>
+        <text>EC50</text>
+      </annotation>
+      <annotation id="275">
+        <infon key="score">0.99630535</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:14:38Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16003" length="2"/>
+        <text>18</text>
+      </annotation>
+      <annotation id="276">
+        <infon key="score">0.99594265</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:14:40Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16010" length="2"/>
+        <text>19</text>
+      </annotation>
+      <annotation id="277">
+        <infon key="score">0.998738</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:15:13Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16027" length="24"/>
+        <text>2,3-dihydroxybenzylidene</text>
+      </annotation>
+      <annotation id="278">
+        <infon key="score">0.9988715</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:15:17Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16055" length="30"/>
+        <text>2-hydroxy-3-methoxybenzylidene</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>16192</offset>
+      <text>Some N-acylhydrazone compounds were devoid of activity in the enzymatic assay, yet showed good to moderate efficacy in cell culture (e.g. 14 and 19, having EC50 values of 2.2 and 7.1 μM, respectively). For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay. On the other hand, this difference was 8-fold or more for 7, 10, 14, 22, 25 and 28. Some N-acylhydrazone compounds showed good to moderate efficacy in the vRNP assay (e.g. 14 and 19, having EC50 values of 2.3 and 5.7 μM, respectively), yet were devoid of activity in the enzymatic assay. This observation suggests that they may inhibit the viral polymerase in an endonuclease-independent manner. To achieve a clear insight into the antiviral profile of the N-acylhydrazones, specific mechanistic experiments are currently ongoing in our laboratory, in which we are analyzing in full depth their effects on virus entry, polymerase-dependent RNA synthesis or the late stage (maturation and release) of the virus replication cycle.</text>
+      <annotation id="279">
+        <infon key="score">0.9978528</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16197" length="15"/>
+        <text>N-acylhydrazone</text>
+      </annotation>
+      <annotation id="280">
+        <infon key="score">0.99858165</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:55:31Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16254" length="15"/>
+        <text>enzymatic assay</text>
+      </annotation>
+      <annotation id="281">
+        <infon key="score">0.9975471</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:15:27Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16330" length="2"/>
+        <text>14</text>
+      </annotation>
+      <annotation id="282">
+        <infon key="score">0.99714905</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:15:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16337" length="2"/>
+        <text>19</text>
+      </annotation>
+      <annotation id="283">
+        <infon key="score">0.99172443</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:56:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16348" length="4"/>
+        <text>EC50</text>
+      </annotation>
+      <annotation id="284">
+        <infon key="score">0.9981059</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:16:11Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16433" length="1"/>
+        <text>9</text>
+      </annotation>
+      <annotation id="285">
+        <infon key="score">0.9972927</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:16:14Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16436" length="2"/>
+        <text>11</text>
+      </annotation>
+      <annotation id="286">
+        <infon key="score">0.9962166</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:16:16Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16440" length="2"/>
+        <text>13</text>
+      </annotation>
+      <annotation id="287">
+        <infon key="score">0.9952614</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:16:18Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16444" length="5"/>
+        <text>15–21</text>
+      </annotation>
+      <annotation id="288">
+        <infon key="score">0.9978259</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:16:21Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16451" length="2"/>
+        <text>23</text>
+      </annotation>
+      <annotation id="289">
+        <infon key="score">0.99684983</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:16:23Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16455" length="2"/>
+        <text>24</text>
+      </annotation>
+      <annotation id="290">
+        <infon key="score">0.9975376</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:16:25Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16462" length="2"/>
+        <text>26</text>
+      </annotation>
+      <annotation id="641">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:15:43Z</infon>
+        <infon key="error:misaligned:641">16505-&gt;16506</infon>
+        <location offset="16506" length="17"/>
+        <text>cell-based assays</text>
+      </annotation>
+      <annotation id="291">
+        <infon key="score">0.9952685</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:56:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16535" length="4"/>
+        <text>EC50</text>
+      </annotation>
+      <annotation id="292">
+        <infon key="score">0.99638575</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:15:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16563" length="10"/>
+        <text>vRNP assay</text>
+      </annotation>
+      <annotation id="293">
+        <infon key="score">0.9971117</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:38:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16613" length="4"/>
+        <text>EC90</text>
+      </annotation>
+      <annotation id="294">
+        <infon key="score">0.9901541</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:15:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16632" length="17"/>
+        <text>virus yield assay</text>
+      </annotation>
+      <annotation id="295">
+        <infon key="score">0.9980354</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:16:27Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16709" length="1"/>
+        <text>7</text>
+      </annotation>
+      <annotation id="296">
+        <infon key="score">0.9968274</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:16:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16712" length="2"/>
+        <text>10</text>
+      </annotation>
+      <annotation id="297">
+        <infon key="score">0.99701536</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:16:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16716" length="2"/>
+        <text>14</text>
+      </annotation>
+      <annotation id="298">
+        <infon key="score">0.9983103</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:16:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16720" length="2"/>
+        <text>22</text>
+      </annotation>
+      <annotation id="299">
+        <infon key="score">0.9979874</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:16:37Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16724" length="2"/>
+        <text>25</text>
+      </annotation>
+      <annotation id="300">
+        <infon key="score">0.9980386</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:16:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16731" length="2"/>
+        <text>28</text>
+      </annotation>
+      <annotation id="301">
+        <infon key="score">0.99742246</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16740" length="15"/>
+        <text>N-acylhydrazone</text>
+      </annotation>
+      <annotation id="302">
+        <infon key="score">0.99691087</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:15:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16806" length="10"/>
+        <text>vRNP assay</text>
+      </annotation>
+      <annotation id="303">
+        <infon key="score">0.99795216</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:16:43Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16823" length="2"/>
+        <text>14</text>
+      </annotation>
+      <annotation id="304">
+        <infon key="score">0.9975484</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:16:08Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16830" length="2"/>
+        <text>19</text>
+      </annotation>
+      <annotation id="305">
+        <infon key="score">0.9897635</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:56:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16841" length="4"/>
+        <text>EC50</text>
+      </annotation>
+      <annotation id="306">
+        <infon key="score">0.9986216</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:55:31Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16922" length="15"/>
+        <text>enzymatic assay</text>
+      </annotation>
+      <annotation id="307">
+        <infon key="score">0.9979644</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:47:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16991" length="5"/>
+        <text>viral</text>
+      </annotation>
+      <annotation id="308">
+        <infon key="score">0.99281305</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:49:34Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16997" length="10"/>
+        <text>polymerase</text>
+      </annotation>
+      <annotation id="521">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:46Z</infon>
+        <location offset="17014" length="12"/>
+        <text>endonuclease</text>
+      </annotation>
+      <annotation id="309">
+        <infon key="score">0.99821377</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:46Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="17108" length="16"/>
+        <text>N-acylhydrazones</text>
+      </annotation>
+      <annotation id="310">
+        <infon key="score">0.9980268</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17257" length="5"/>
+        <text>virus</text>
+      </annotation>
+      <annotation id="311">
+        <infon key="score">0.98708135</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:49:34Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17270" length="10"/>
+        <text>polymerase</text>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="updated_at">2023-09-20T15:50:22Z</infon>
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+        <text>RNA</text>
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+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17355" length="5"/>
+        <text>virus</text>
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+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
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+      <text>Docking studies</text>
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+        <infon key="annotator">cleaner0</infon>
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+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>17404</offset>
+      <text>In order to explore the possible binding mode of the synthesized compounds, docking simulations by GOLD program were performed by using the structural coordinates (PDB code 4AWM) for the PA-Nter endonuclease in complex with EGCG. Considering that the position of the side-chains of some residues changes depending on which pocket the ligand is occupying, we superimposed some X-ray structures of complexes between PA-Nter endonuclease and known active ligands. It was observed that the side-chain of amino acid Tyr24 shows greater movement than the other residues and for this reason we considered it as a flexible residue during the docking procedure.</text>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>GOLD program</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="updated_at">2023-09-20T15:45:13Z</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>X-ray structures</text>
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+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:17:43Z</infon>
+        <location offset="18038" length="17"/>
+        <text>docking procedure</text>
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+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>18057</offset>
+      <text>First, test docking calculations, using EGCG, L-742,001 and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one (Fig. 1), were carried out to compare experimental and predicted binding modes and validate docking procedure. Their best docking poses agreed well with the experimental binding modes (rmsd values of 0.8, 1.2 and 0.7, respectively).</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:17:39Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18064" length="25"/>
+        <text>test docking calculations</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:53:50Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18097" length="4"/>
+        <text>EGCG</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:18:18Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18103" length="9"/>
+        <text>L-742,001</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:18:23Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18117" length="59"/>
+        <text>2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one</text>
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+        <infon key="score">0.97238517</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:17:43Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>docking procedure</text>
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+      <annotation id="329">
+        <infon key="score">0.9985305</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:18:28Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18362" length="4"/>
+        <text>rmsd</text>
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+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>18410</offset>
+      <text>Next, docking of several N-acylhydrazones was performed and this generated a number of possible binding conformations, highlighting that the active site cavity of the PA endonuclease is quite spacious, as already demonstrated by crystallographic studies, and confirming the ability of this scaffold to chelate the two M2+ ions in different ways (Mode A-C in Fig. 4).</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:43:59Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>docking</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:46Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18435" length="16"/>
+        <text>N-acylhydrazones</text>
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+        <infon key="score">0.99903184</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:17:59Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>active site cavity</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18577" length="2"/>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18580" length="12"/>
+        <text>endonuclease</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:44:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18639" length="24"/>
+        <text>crystallographic studies</text>
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+      <annotation id="336">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:18:09Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18728" length="3"/>
+        <text>M2+</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>18777</offset>
+      <text>Figure 5 displays the first (panel A) and second (panel B) GOLD cluster docked solutions for compound 23. These two complex structures represent the largest clusters with similar fitness values (59.20 and 58.65, respectively). In both cases, 23 appears able to coordinate the two M2+ ions in the active site through the three contiguous OH groups (Fig. 5). In addition, 23 was predicted to form two hydrogen bonding interactions, i.e. with the catalytic Lys134 on the one side and Glu26 on the other side. Furthermore, in these two different binding modes, 23 forms π–π interactions with the aromatic ring of Tyr24, in a fashion similar to that described for other endonuclease inhibitors, i.e. EGCG and L-742,001.</text>
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:34:00Z</infon>
+        <location offset="18836" length="19"/>
+        <text>GOLD cluster docked</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:08Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18879" length="2"/>
+        <text>23</text>
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+      <annotation id="338">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:18:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>structures</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:10Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19038" length="10"/>
+        <text>coordinate</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:05Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <text>M2+</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19073" length="11"/>
+        <text>active site</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:12Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="19147" length="2"/>
+        <text>23</text>
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+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19176" length="29"/>
+        <text>hydrogen bonding interactions</text>
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+      <annotation id="345">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:20:31Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>catalytic</text>
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+      <annotation id="346">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19231" length="6"/>
+        <text>Lys134</text>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>Glu26</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:14Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="19334" length="2"/>
+        <text>23</text>
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+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:18Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19343" length="16"/>
+        <text>π–π interactions</text>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:17:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19386" length="5"/>
+        <text>Tyr24</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:46Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>endonuclease</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:53:50Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="19472" length="4"/>
+        <text>EGCG</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:18:19Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="19481" length="9"/>
+        <text>L-742,001</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>19496</offset>
+      <text>The best docked conformation for compound 15 (Fig. 6, fitness value 68.56), which has an activity slightly lower than 23, reveals a different role for the gallic moiety. The ligand seems to form two hydrogen bonding interactions with Tyr130 as well as a cation–π interaction with Lys134. Tyr130 lies in a pocket that also contains Arg124, a residue that was proposed to have a crucial role in binding of the RNA substrate. Compound 15 appears further stabilized by hydrogen bonding interactions between two hydroxyl groups and Arg82 and Asp108. In this case, chelation of the two M2+ ions is carried out by involving the imine group (mode A in Fig. 4).</text>
+      <annotation id="354">
+        <infon key="score">0.9544191</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:38Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="19538" length="2"/>
+        <text>15</text>
+      </annotation>
+      <annotation id="355">
+        <infon key="score">0.83134425</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:44:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19550" length="13"/>
+        <text>fitness value</text>
+      </annotation>
+      <annotation id="356">
+        <infon key="score">0.9965567</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19695" length="29"/>
+        <text>hydrogen bonding interactions</text>
+      </annotation>
+      <annotation id="357">
+        <infon key="score">0.9994832</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:20:09Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19730" length="6"/>
+        <text>Tyr130</text>
+      </annotation>
+      <annotation id="358">
+        <infon key="score">0.99628747</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19750" length="20"/>
+        <text>cation–π interaction</text>
+      </annotation>
+      <annotation id="359">
+        <infon key="score">0.99945813</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19776" length="6"/>
+        <text>Lys134</text>
+      </annotation>
+      <annotation id="360">
+        <infon key="score">0.99948764</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:20:09Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19784" length="6"/>
+        <text>Tyr130</text>
+      </annotation>
+      <annotation id="361">
+        <infon key="score">0.99882525</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:43:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19801" length="6"/>
+        <text>pocket</text>
+      </annotation>
+      <annotation id="362">
+        <infon key="score">0.99945635</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:20:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19827" length="6"/>
+        <text>Arg124</text>
+      </annotation>
+      <annotation id="363">
+        <infon key="score">0.9987018</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:50:22Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="19904" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="364">
+        <infon key="score">0.94156957</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:20:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="19928" length="2"/>
+        <text>15</text>
+      </annotation>
+      <annotation id="365">
+        <infon key="score">0.9967854</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19961" length="29"/>
+        <text>hydrogen bonding interactions</text>
+      </annotation>
+      <annotation id="366">
+        <infon key="score">0.9993685</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:20:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20023" length="5"/>
+        <text>Arg82</text>
+      </annotation>
+      <annotation id="367">
+        <infon key="score">0.9993944</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:51Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20033" length="6"/>
+        <text>Asp108</text>
+      </annotation>
+      <annotation id="368">
+        <infon key="score">0.8217899</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:48Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20055" length="9"/>
+        <text>chelation</text>
+      </annotation>
+      <annotation id="369">
+        <infon key="score">0.98686737</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:20:00Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20076" length="3"/>
+        <text>M2+</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>20152</offset>
+      <text>It is important to highlight that compounds 23 and 15, although in different ways, both are able to chelate the metal cofactors and to establish interactions with highly conserved aminoacids (Tyr24, Glu26, Arg124, Tyr130 and Lys134) that are very important for both endonuclease activity and transcription in vitro. The crucial role of such interactions is underlined by the differences in activity between 15 (IC50 = 9.0 μM) and 19 (&gt;500 μM): their coordinating features are similar, since both coordinate to the divalent metal ion through the phenolic oxygen, the iminic nitrogen and the carbonylic oxygen (mode A in Fig. 4), but the biological activity could be related to their different ability to engage interactions with the protein environment.</text>
+      <annotation id="370">
+        <infon key="score">0.9976574</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:20:36Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20196" length="2"/>
+        <text>23</text>
+      </annotation>
+      <annotation id="371">
+        <infon key="score">0.99680054</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:20:38Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20203" length="2"/>
+        <text>15</text>
+      </annotation>
+      <annotation id="372">
+        <infon key="score">0.9988625</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:21:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20315" length="16"/>
+        <text>highly conserved</text>
+      </annotation>
+      <annotation id="373">
+        <infon key="score">0.999537</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:17:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20344" length="5"/>
+        <text>Tyr24</text>
+      </annotation>
+      <annotation id="374">
+        <infon key="score">0.99951553</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20351" length="5"/>
+        <text>Glu26</text>
+      </annotation>
+      <annotation id="375">
+        <infon key="score">0.9995223</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:20:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20358" length="6"/>
+        <text>Arg124</text>
+      </annotation>
+      <annotation id="376">
+        <infon key="score">0.99954116</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:20:09Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20366" length="6"/>
+        <text>Tyr130</text>
+      </annotation>
+      <annotation id="377">
+        <infon key="score">0.99951816</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20377" length="6"/>
+        <text>Lys134</text>
+      </annotation>
+      <annotation id="378">
+        <infon key="score">0.9048646</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20418" length="12"/>
+        <text>endonuclease</text>
+      </annotation>
+      <annotation id="379">
+        <infon key="score">0.9975988</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:20:41Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20559" length="2"/>
+        <text>15</text>
+      </annotation>
+      <annotation id="380">
+        <infon key="score">0.9984628</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20563" length="4"/>
+        <text>IC50</text>
+      </annotation>
+      <annotation id="381">
+        <infon key="score">0.997436</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:20:43Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20582" length="2"/>
+        <text>19</text>
+      </annotation>
+      <annotation id="382">
+        <infon key="score">0.72198266</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:51:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20648" length="10"/>
+        <text>coordinate</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>20915</offset>
+      <text>Crystallographic Studies</text>
+      <annotation id="383">
+        <infon key="score">0.9979657</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:21:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20915" length="24"/>
+        <text>Crystallographic Studies</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>20940</offset>
+      <text>Attempts were made to co-crystallize PA-Nter with 15, 20, 21 and 23 in one to four molar excess. While crystals appeared and diffracted well, upon data processing, no or very little electron density for the inhibitors was observed. Attempts to soak apo crystals in crystallization solution containing 5 mM inhibitor overnight also did not result in substantial electron density for the inhibitor. As a last resort, dry powder of the inhibitor was sprinkled over the crystallization drop containing apo crystals and left over night. This experiment was successful for compound 23, the crystals diffracted to 2.15 Å and diffraction data were collected (PDB ID 5EGA). The refined structure shows unambiguous electron density for the inhibitor (Table S1 and Fig. 7). The complex structure confirms one of the two binding modes predicted by the docking simulations (Fig. 5, panel B). The galloyl moiety chelates the manganese ions, while the trihydroxyphenyl group stacks against the Tyr24 side chain. It is interesting to note that two of these hydroxyl groups are in position to form hydrogen bonds with the side chain of Glu26 and Lys34 (Fig. 7). These interactions suggest that other functional groups, e.g. halogens, could be used in place of the hydroxyl groups for better interactions with Glu26 and Lys34 side chains, and the inhibitory potency of these compounds could be further improved.</text>
+      <annotation id="384">
+        <infon key="score">0.99876714</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:21:18Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20962" length="14"/>
+        <text>co-crystallize</text>
+      </annotation>
+      <annotation id="545">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="20977" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="566">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="20980" length="4"/>
+        <text>Nter</text>
+      </annotation>
+      <annotation id="385">
+        <infon key="score">0.9974802</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:22:17Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20990" length="2"/>
+        <text>15</text>
+      </annotation>
+      <annotation id="386">
+        <infon key="score">0.9937837</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:22:20Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20994" length="2"/>
+        <text>20</text>
+      </annotation>
+      <annotation id="387">
+        <infon key="score">0.99413764</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:22:22Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20998" length="2"/>
+        <text>21</text>
+      </annotation>
+      <annotation id="388">
+        <infon key="score">0.9973648</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:22:25Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="21005" length="2"/>
+        <text>23</text>
+      </annotation>
+      <annotation id="389">
+        <infon key="score">0.98446065</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:21:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21043" length="8"/>
+        <text>crystals</text>
+      </annotation>
+      <annotation id="390">
+        <infon key="score">0.99861944</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:22:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21122" length="16"/>
+        <text>electron density</text>
+      </annotation>
+      <annotation id="391">
+        <infon key="score">0.9992843</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:21:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21189" length="3"/>
+        <text>apo</text>
+      </annotation>
+      <annotation id="392">
+        <infon key="score">0.9942742</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:21:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21193" length="8"/>
+        <text>crystals</text>
+      </annotation>
+      <annotation id="393">
+        <infon key="score">0.9986007</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:22:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21301" length="16"/>
+        <text>electron density</text>
+      </annotation>
+      <annotation id="394">
+        <infon key="score">0.9993243</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:21:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21438" length="3"/>
+        <text>apo</text>
+      </annotation>
+      <annotation id="395">
+        <infon key="score">0.99404055</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:21:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21442" length="8"/>
+        <text>crystals</text>
+      </annotation>
+      <annotation id="396">
+        <infon key="score">0.99720263</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:22:27Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="21516" length="2"/>
+        <text>23</text>
+      </annotation>
+      <annotation id="397">
+        <infon key="score">0.9950954</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:21:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21524" length="8"/>
+        <text>crystals</text>
+      </annotation>
+      <annotation id="398">
+        <infon key="score">0.9976955</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:44:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21617" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="399">
+        <infon key="score">0.9985931</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:22:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21645" length="16"/>
+        <text>electron density</text>
+      </annotation>
+      <annotation id="400">
+        <infon key="score">0.71468544</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:21:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21707" length="17"/>
+        <text>complex structure</text>
+      </annotation>
+      <annotation id="401">
+        <infon key="score">0.99876976</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:44:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21780" length="19"/>
+        <text>docking simulations</text>
+      </annotation>
+      <annotation id="402">
+        <infon key="score">0.998395</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:08Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="21851" length="9"/>
+        <text>manganese</text>
+      </annotation>
+      <annotation id="403">
+        <infon key="score">0.999305</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:17:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21919" length="5"/>
+        <text>Tyr24</text>
+      </annotation>
+      <annotation id="404">
+        <infon key="score">0.9967308</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:08:11Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22021" length="14"/>
+        <text>hydrogen bonds</text>
+      </annotation>
+      <annotation id="405">
+        <infon key="score">0.999268</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22059" length="5"/>
+        <text>Glu26</text>
+      </annotation>
+      <annotation id="406">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:35:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22069" length="5"/>
+        <text>Lys34</text>
+      </annotation>
+      <annotation id="407">
+        <infon key="score">0.9992293</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22232" length="5"/>
+        <text>Glu26</text>
+      </annotation>
+      <annotation id="408">
+        <infon key="score">0.9991905</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:35:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22242" length="5"/>
+        <text>Lys34</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">CONCL</infon>
+      <infon key="type">title_1</infon>
+      <offset>22335</offset>
+      <text>Conclusions</text>
+    </passage>
+    <passage>
+      <infon key="section_type">CONCL</infon>
+      <infon key="type">paragraph</infon>
+      <offset>22347</offset>
+      <text>The development of new agents for the treatment of influenza infection that exert their action by inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is a strategy that recently is gaining a lot of interest. The results here presented add the N-acylhydrazone scaffold to the library of the chelating molecules with potent antiviral activity (EC90 &lt; 5 μM, virus yield assay in influenza virus-infected MDCK cells). The structure of the N-acylhydrazone 23 co-crystallized with PA-Nter is important not only because confirms that the polyhydroxypheyl group efficiently coordinates two metal ions in the active site of the enzyme, but also because highlights the importance of the (flexible) inhibitor backbone in order to engage effective interactions with crucial aminoacids of the protein. Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase could represent another example, after carbonic anhydrase, histone deacetylase, and HIV-1 integrase, of metal binding as a successful strategy in drug design.</text>
+      <annotation id="409">
+        <infon key="score">0.9472973</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22398" length="9"/>
+        <text>influenza</text>
+      </annotation>
+      <annotation id="522">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:47Z</infon>
+        <location offset="22463" length="12"/>
+        <text>endonuclease</text>
+      </annotation>
+      <annotation id="410">
+        <infon key="score">0.9804986</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22488" length="9"/>
+        <text>influenza</text>
+      </annotation>
+      <annotation id="411">
+        <infon key="score">0.785363</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:23:09Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22498" length="28"/>
+        <text>RNA-dependent RNA polymerase</text>
+      </annotation>
+      <annotation id="412">
+        <infon key="score">0.999037</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="22620" length="15"/>
+        <text>N-acylhydrazone</text>
+      </annotation>
+      <annotation id="413">
+        <infon key="score">0.97529185</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:38:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22719" length="4"/>
+        <text>EC90</text>
+      </annotation>
+      <annotation id="642">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:15:59Z</infon>
+        <location offset="22732" length="17"/>
+        <text>virus yield assay</text>
+      </annotation>
+      <annotation id="414">
+        <infon key="score">0.9345518</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22753" length="9"/>
+        <text>influenza</text>
+      </annotation>
+      <annotation id="597">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <location offset="22763" length="5"/>
+        <text>virus</text>
+      </annotation>
+      <annotation id="415">
+        <infon key="score">0.99737966</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:23:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22795" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="416">
+        <infon key="score">0.99909306</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="22812" length="15"/>
+        <text>N-acylhydrazone</text>
+      </annotation>
+      <annotation id="417">
+        <infon key="score">0.99650013</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:23:23Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="22828" length="2"/>
+        <text>23</text>
+      </annotation>
+      <annotation id="418">
+        <infon key="score">0.9978605</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:44:18Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22831" length="15"/>
+        <text>co-crystallized</text>
+      </annotation>
+      <annotation id="546">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="22852" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="567">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="22855" length="4"/>
+        <text>Nter</text>
+      </annotation>
+      <annotation id="419">
+        <infon key="score">0.59459054</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:43:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22943" length="11"/>
+        <text>coordinates</text>
+      </annotation>
+      <annotation id="420">
+        <infon key="score">0.9235175</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:23:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="22959" length="5"/>
+        <text>metal</text>
+      </annotation>
+      <annotation id="421">
+        <infon key="score">0.9990668</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22977" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="523">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:47Z</infon>
+        <location offset="23184" length="12"/>
+        <text>endonuclease</text>
+      </annotation>
+      <annotation id="422">
+        <infon key="score">0.9762899</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23209" length="9"/>
+        <text>influenza</text>
+      </annotation>
+      <annotation id="423">
+        <infon key="score">0.89355963</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:23:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23219" length="28"/>
+        <text>RNA-dependent RNA polymerase</text>
+      </annotation>
+      <annotation id="424">
+        <infon key="score">0.99830073</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:23:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23287" length="18"/>
+        <text>carbonic anhydrase</text>
+      </annotation>
+      <annotation id="425">
+        <infon key="score">0.9986998</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:23:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23307" length="19"/>
+        <text>histone deacetylase</text>
+      </annotation>
+      <annotation id="426">
+        <infon key="score">0.8409801</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:34Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23332" length="5"/>
+        <text>HIV-1</text>
+      </annotation>
+      <annotation id="427">
+        <infon key="score">0.9830672</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:47Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23338" length="9"/>
+        <text>integrase</text>
+      </annotation>
+      <annotation id="428">
+        <infon key="score">0.98587555</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:43:20Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="23352" length="5"/>
+        <text>metal</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_1</infon>
+      <offset>23412</offset>
+      <text>Experimental Section</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>23433</offset>
+      <text>Materials and methods. Chemistry</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>23466</offset>
+      <text>All reagents of commercial quality were purchased from Sigma-Aldrich and used without further purification. The purity of the compounds was determined by elemental analysis and verified to be ≥95% for all synthesized molecules. NMR spectra were recorded at 25 °C on a Bruker Avance 400 FT spectrophotometer. The attenuate total reflectance IR spectra were recorded by means of a Nicolet-Nexus (Thermo Fisher) spectrophotometer by using a diamond crystal plate in the range of 4000–400 cm−1. Elemental analyses were performed by using a FlashEA 1112 series CHNS/O analyzer (Thermo Fisher) with gas-chromatographic separation. Electrospray mass spectral analyses (ESI-MS) were performed with an electrospray ionization (ESI) time-of-flight Micromass 4LCZ spectrometer. MS spectra were acquired in positive EI mode by means of a direct exposure probe mounting on the tip of a Re-filament with a DSQII Thermo Fisher apparatus, equipped with a single quadrupole analyzer. UV–Vis spectra were recorded on an Evolution 260 Bio Thermo spectrophotometer by using cells of 1 cm path length. UV-vis absorption spectra of 19 and 23 were registered using a ca. 10−5 M solution in methanol. Each metal/ligand system was studied by titrating a 2.8 ml sample of the ligand solution with a methanolic solution of Mg(CH3COO)2; 8–12 spectra of samples with M:L molar ratio ranging from 0 to 6 were measured.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>24870</offset>
+      <text>Synthesis of the ligands (general procedure)</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>24915</offset>
+      <text>All the N-acylhydrazones were prepared in a manner similar to reported procedures. Briefly, to a solution of the aldehyde in absolute ethanol or toluene, an equimolar amount of the hydrazide dissolved in the same solvent was added. The mixture was refluxed for 6 hours, cooled at room temperature and concentrated in vacuum. The resulting precipitate was filtered off, washed with cold ethanol and dried in vacuum.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>25330</offset>
+      <text>3,4,5-trihydroxybenzohydrazide (28) and 3,4,5-trimethoxybenzohydrazide (29) were obtained by reaction of the corresponding methyl esters with hydrazine monohydrate. Hydrazine was added to an ethanol suspension of the ester and stirred at room temperature until the solute completely dissolved. Reaction mixture was then refluxed overnight. On concentrating the solution, a precipitate was observed, which was filtered and washed with cold ethanol. Chemical characterization of 1–29 and of Mg(HL)2 4H2O is collected in the Supplementary Information.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>25881</offset>
+      <text>Computational Studies</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>25903</offset>
+      <text>The crystal structure of PA-Nter endonuclease in complex with EGCG was retrieved from the RCSB Protein Data Bank (entry code 4AWM). The ligand and water molecules were discarded and the hydrogens were added to the protein by Discovery Studio 2.5. The charge on the metal ions was set as +2. EGCG, L-742,001, and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one structures were extracted from their X-ray complexes (PDB IDs 4AWM, 4W9S and 4E5H respectively). The other ligand structures were constructed using Discovery Studio 2.5.5 (Accelrys, Discovery Studio) and energy minimized using the Smart Minimizer protocol (1000 steps) which combines the Steepest Descent and the Conjugate Gradient methods.</text>
+      <annotation id="655">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:37:10Z</infon>
+        <location offset="26050" length="5"/>
+        <text>water</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>26618</offset>
+      <text>The minimized ligands were docked in their corresponding proteins by means of GOLD Suite 5.0.1. The region of interest used by the GOLD program was defined in order to contain the residues within 15 Å from the original position of the ligand in the X-ray structure. The side-chain of residue Tyr24 was allowed to rotate according to the internal rotamer libraries in GOLD Suite 5.0.1. GoldScore was chosen as fitness function. The standard default settings were used in all calculations and the ligands were submitted to 100 genetic algorithm runs. The “allow early termination” command was deactivated. Results differing by less than 0.75 Å in ligand-all atom rmsd, were clustered together. The best GOLD-calculated conformation was used both for analysis and representation.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>27401</offset>
+      <text>Plasmid-based endonuclease assay</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>27434</offset>
+      <text>This enzymatic assay was performed according to a previously published method. One microgram of recombinant PA-Nter (residues 1–217 from the PA protein of influenza virus strain A/X-31) was incubated with 1 μg (16.7 nM) of single-stranded circular DNA plasmid M13mp18 (Bayou Biolabs, Metairie, Louisiana) in the presence of the test compounds and at a final volume of 25 μL. The assay buffer contained 50 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM β-mercaptoethanol and 1 mM MnCl2. The reaction was incubated at 37 °C for 2 h and then stopped by heat inactivation (80 °C, 20 min), followed by visualization of the endonucleolytic digestion of the plasmid by gel electrophoresis on a 1% agarose gel with ethidium bromide staining. The amount of remaining intact plasmid was quantified by ImageQuant TL software (GE Healthcare, Diegem, Belgium). The percentage inhibition of PA endonuclease activity was plotted against the compound concentration on a semi-logarithmic plot, using GraphPad Prism software (GraphPad Software, La Jolla, CA). The 50% inhibitory concentrations (IC50) were obtained by nonlinear least-squares regression analysis of the results from three independent experiments. 2,4-Dioxo-4-phenylbutanoic acid (DPBA; Interchim, Montluçon, France) was included as the reference compound.</text>
+      <annotation id="661">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:39:37Z</infon>
+        <location offset="27746" length="11"/>
+        <text>presence of</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>28740</offset>
+      <text>Cells and media</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>28756</offset>
+      <text>MDCK cells (a kind gift from M. Matrosovich, Marburg, Germany) and HEK293T cells (purchased from Thermo Fisher Scientific, Waltham, MA) were cultivated in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, and 0.075% sodium bicarbonate. During virus experiments, the MDCK cells were maintained in MDCK infection medium, consisting of Ultra MDCK medium (Lonza, Basel, Switzerland) supplemented with 0.0225% sodium bicarbonate, 2 mM L-glutamine, and 2 μg/ml tosyl phenylalanyl chloromethyl ketone-treated trypsin (Sigma-Aldrich, St. Louis, MO). The cells were incubated in a humidified atmosphere containing 5% CO2.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>29417</offset>
+      <text>vRNP reconstitution assay</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>29443</offset>
+      <text>The procedure to determine the inhibitory effect of the compounds on influenza virus vRNPs reconstituted in HEK293T cells, is described in full detail elsewhere. Briefly, the four relevant plasmids (i.e. the expression plasmids for PB1, PB2, PA and NP) were combined with the fluc reporter plasmid, and co-transfected into HEK293T cells using Lipofectamin 2000 (Invitrogen, Life Technologies, Gent, Belgium). After incubation at 37 °C for 24 h in the presence of serial dilutions of the test compounds, the ONE-Glo luciferase assay system (Promega, Madison, WI) was used to determine luciferase activity. EC50 was defined as the compound concentration causing 50% reduction in the vRNP-driven firefly luciferase signal, as compared to cells receiving medium instead of compound. These EC50 values were calculated by interpolation assuming a semi-log dose-response effect using GraphPad Prism software. In parallel, compound cytotoxic activity was determined in untransfected HEK293T cells which had been incubated with serial dilutions of the compounds for 24 h, using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay; Promega). These spectrophotometric data were used to calculate the 50% cytotoxic concentration (CC50), i.e. the concentration reducing cell viability by 50%, as compared to wells receiving medium instead of compound. Ribavirin (Virazole; ICN Pharmaceuticals, Costa Mesa, CA) was included as the reference compound.</text>
+      <annotation id="662">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:39:37Z</infon>
+        <location offset="29894" length="11"/>
+        <text>presence of</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>31011</offset>
+      <text>Virus yield assay</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>31029</offset>
+      <text>We previously published in full detail the virus yield assay to determine the anti-influenza virus activity in MDCK cell cultures. Briefly, one day prior to infection, MDCK cells were seeded into 96-well plates at 25,000 cells per well. At day 0, serial dilutions of the test compounds were added, immediately followed by infection with influenza A/PR/8/34 virus. After 24 h incubation at 35 °C, the virus amount in the supernatants was estimated by determining the viral genome copy number in a one-step quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay (CellsDirect One-Step qRT-PCR kit; Invitrogen), with influenza virus M1-specific primers and probe. The compound concentration values causing a 2-log10 (EC99) and a 1-log10 (EC90) reduction in viral RNA (vRNA) copy number at 24 h p.i., as compared to the virus control receiving no compound, were calculated by interpolation from data of at least three experiments. In parallel, the CC50 values after 24 h incubation with compounds were determined in uninfected MDCK cells, using the spectrophotometric MTS cell viability assay described above, respectively. Ribavirin was included as the reference compound.</text>
+      <annotation id="657">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:38:12Z</infon>
+        <location offset="31774" length="4"/>
+        <text>EC99</text>
+      </annotation>
+      <annotation id="659">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:38:22Z</infon>
+        <location offset="31795" length="4"/>
+        <text>EC90</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>32233</offset>
+      <text>Crystallographic analysis</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>32259</offset>
+      <text>A PAN construct (PANΔLoop) with a loop (residues 51–72) deleted and replaced with GGS from A/California/04/2009 H1N1 strain was used for the crystallographic studies. The details of cloning, over-expression and purification are described elsewhere. Briefly, the gene was cloned into pET52b vector and transformed into BL21 (DE3) cells, and the protein was expressed in LB medium overnight at 18 °C after induction at an OD600 ~0.8 with 0.2 mM isopropyl-β-thiogalactopyranoside (IPTG). The protein was purified from cell lysates by HisTrap affinity chromatography and the 10xHis tag was removed by digestion with thrombin. The protein was further purified by gel filtration using a Superdex 75 size-exclusion chromatography column in 20 mM Tris pH 8.0, 150 mM NaCl and 1 mM TCEP. The protein was concentrated to 10–12 mg/ml for crystallization. Crystals were grown in 0.2 M MgCl2, 2 mM MnCl2, 0.1 M Tris pH 8.5, 30% (w/v) PEG 4000 using the hanging drop method. For determination of the protein-inhibitor complex structure, the powder of the inhibitor was sprinkled on a 2 μl drop of a 1:1 ratio mixture of protein solution and well solution, on a cover slide hanging over 500 μl well solution, and left overnight. Next day, the crystals were cryo-protected using well solution supplemented with 25% ethylene glycol and flash frozen in liquid nitrogen. The data were collected at the 22-ID beam line maintained by Southeast Regional Collaborative Access Team (SERCAT) at the Advanced Photon Source, Argonne National Laboratory. The data were indexed, integrated and scaled using the HKL2000 suite of programs. Phase determination, structure refinement and model building were completed using Phaser, Refmac and Coot (part of the CCP4 package). The apo structure of PANΔLoop (PDB ID: 5DES) was used as starting model for molecular replacement. The details of the data collection and refinement statistics are given in Table S1.</text>
+      <annotation id="651">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:35:15Z</infon>
+        <location offset="32276" length="8"/>
+        <text>PANΔLoop</text>
+      </annotation>
+      <annotation id="652">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:35:15Z</infon>
+        <location offset="34023" length="8"/>
+        <text>PANΔLoop</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_1</infon>
+      <offset>34198</offset>
+      <text>Additional Information</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>34221</offset>
+      <text>How to cite this article: Carcelli, M. et al. N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes. Sci. Rep. 6, 31500; doi: 10.1038/srep31500 (2016).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
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+      <offset>34416</offset>
+      <text>Supplementary Material</text>
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+      <infon key="lpage">3674</infon>
+      <infon key="name_0">surname:Song;given-names:M. S.</infon>
+      <infon key="pub-id_pmid">26976575</infon>
+      <infon key="section_type">REF</infon>
+      <infon key="source">PNAS</infon>
+      <infon key="type">ref</infon>
+      <infon key="volume">113</infon>
+      <infon key="year">2016</infon>
+      <offset>39650</offset>
+      <text>Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor</text>
+    </passage>
+    <passage>
+      <infon key="fpage">307</infon>
+      <infon key="lpage">326</infon>
+      <infon key="name_0">surname:Otwinowski;given-names:Z.</infon>
+      <infon key="name_1">surname:Minor;given-names:W.</infon>
+      <infon key="name_2">surname:Sweet;given-names:R. M.</infon>
+      <infon key="section_type">REF</infon>
+      <infon key="source">In Methods in Enzymology, Volume 276: Macromolecular Crystallography, part A</infon>
+      <infon key="type">ref</infon>
+      <infon key="year">1997</infon>
+      <offset>39752</offset>
+      <text>Processing of X-ray diffraction data collected in oscillation mode</text>
+    </passage>
+    <passage>
+      <infon key="fpage">235</infon>
+      <infon key="lpage">42</infon>
+      <infon key="name_0">surname:Winn;given-names:M. D.</infon>
+      <infon key="pub-id_pmid">21460441</infon>
+      <infon key="section_type">REF</infon>
+      <infon key="source">Acta Crystallogr. D Biol Crystallogr</infon>
+      <infon key="type">ref</infon>
+      <infon key="volume">67</infon>
+      <infon key="year">2011</infon>
+      <offset>39819</offset>
+      <text>Overview of the CCP4 suite and current developments</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>39871</offset>
+      <text>Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. M.C., D.R., A.G. and M.S. drug design and chemical synthesis; L.D.L. docking studies; G.K. and S.W.W. crystallographic studies; A.S. and L.N. biological studies.</text>
+    </passage>
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+      <infon key="file">srep31500-f1.jpg</infon>
+      <infon key="id">f1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>40191</offset>
+      <text>Chemical structures of some prototype inhibitors of influenza virus endonuclease.</text>
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+      <infon key="type">fig_caption</infon>
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+      <text>Inhibitor activity in enzymatic assays (IC50, μM) as reported in: aref., bref., cref., dref..</text>
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+      <text>First (A) and second (B) GOLD cluster docked solutions of compound 23 (orange and cyan, respectively) in complex with PA endonuclease. Key residues of the pocket are presented using PyMOL [ http://www.pymol.org] and LIGPLUS [Laskowski, R. A.; Swindells, M. B. Journal of chemical information and modeling
+2011,
+51, 2778]. Hydrogen bonds are illustrated by dotted lines, while the divalent metal ions are shown as purple spheres. Schematic drawings of the interactions of the first (C) and second (D) GOLD cluster docked solutions generated using LIGPLUS. Dashed lines are hydrogen bonds and ‘eyelashes’ show residues involved in hydrophobic interactions.</text>
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+      <infon key="type">fig_caption</infon>
+      <offset>41286</offset>
+      <text>(A) Binding mode of compound 15 (orange) in complex with PA endonuclease. Key residues of the pocket are presented using PyMOL [ http://www.pymol.org] and LIGPLUS [Laskowski, R. A.; Swindells, M. B. Journal of chemical information and modeling
+2011,
+51, 2778]. Hydrogen bonds are illustrated by dotted lines while the divalent metal ions are shown as purple spheres. (B) Schematic drawing of the interactions of compound 15 generated using LIGPLUS. Dashed lines are hydrogen bonds and ‘eyelashes’ show residues involved in hydrophobic interactions.</text>
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+      <text>Active site residues are shown in sticks with green carbons, manganese atoms are shown as purple spheres and water molecules as red spheres. Compound 23 is shown in sticks with yellow carbons. Protein secondary structure is shown as ribbons in salmon color. 2Fo-Fc electron density map contoured at 1σ is shown as blue mesh. Hydrogen bonds and metal coordination are shown with dotted lines. The H-bond distances from the side chain carboxyl group of Glu26 to p-OH and m-OH of the trihydroxyphenyl group of the inhibitor are 2.7 Å and 3.0 Å, respectively. The H-bond distance from the side chain of Lys34 to p-OH of the trihydroxyphenyl group is 3.6 Å. The H-bond distance to the water molecule from m-OH of the galloyl moiety is 3.0 Å, which in turn is H-bonded to the side chain of Tyr130 with a distance of 2.7 Å. Crystal structure has been deposited in the RCSB Protein Data Bank with PDB ID: 5EGA.</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:45:00Z</infon>
+        <location offset="41902" length="11"/>
+        <text>Active site</text>
+      </annotation>
+      <annotation id="464">
+        <infon key="score">0.999</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:52:08Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="41963" length="9"/>
+        <text>manganese</text>
+      </annotation>
+      <annotation id="465">
+        <infon key="score">0.99719954</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:37:10Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="42011" length="5"/>
+        <text>water</text>
+      </annotation>
+      <annotation id="466">
+        <infon key="score">0.99787354</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:43:26Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="42052" length="2"/>
+        <text>23</text>
+      </annotation>
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+        <infon key="score">0.9711051</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:35:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="42160" length="27"/>
+        <text>2Fo-Fc electron density map</text>
+      </annotation>
+      <annotation id="468">
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+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:08:12Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="42227" length="14"/>
+        <text>Hydrogen bonds</text>
+      </annotation>
+      <annotation id="653">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:36:30Z</infon>
+        <location offset="42246" length="18"/>
+        <text>metal coordination</text>
+      </annotation>
+      <annotation id="469">
+        <infon key="score">0.9292416</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:35:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="42298" length="6"/>
+        <text>H-bond</text>
+      </annotation>
+      <annotation id="470">
+        <infon key="score">0.9993598</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:19:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="42353" length="5"/>
+        <text>Glu26</text>
+      </annotation>
+      <annotation id="471">
+        <infon key="score">0.7898533</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:35:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="42462" length="6"/>
+        <text>H-bond</text>
+      </annotation>
+      <annotation id="472">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:35:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="42501" length="5"/>
+        <text>Lys34</text>
+      </annotation>
+      <annotation id="473">
+        <infon key="score">0.9379614</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:35:56Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="42559" length="6"/>
+        <text>H-bond</text>
+      </annotation>
+      <annotation id="474">
+        <infon key="score">0.99886</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:37:09Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="42582" length="5"/>
+        <text>water</text>
+      </annotation>
+      <annotation id="475">
+        <infon key="score">0.9967211</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:35:38Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="42656" length="8"/>
+        <text>H-bonded</text>
+      </annotation>
+      <annotation id="476">
+        <infon key="score">0.9994579</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:20:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="42686" length="6"/>
+        <text>Tyr130</text>
+      </annotation>
+      <annotation id="477">
+        <infon key="score">0.73657626</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:52Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="42719" length="17"/>
+        <text>Crystal structure</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">t1.xml</infon>
+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_title_caption</infon>
+      <offset>42811</offset>
+      <text>Inhibitory activity of the N-acylhydrazones 1–27 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays.</text>
+      <annotation id="478">
+        <infon key="score">0.9986432</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:44:46Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="42838" length="16"/>
+        <text>N-acylhydrazones</text>
+      </annotation>
+      <annotation id="479">
+        <infon key="score">0.99474984</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:37:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="42855" length="4"/>
+        <text>1–27</text>
+      </annotation>
+      <annotation id="480">
+        <infon key="score">0.99926764</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:12:16Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="42864" length="9"/>
+        <text>hydrazide</text>
+      </annotation>
+      <annotation id="481">
+        <infon key="score">0.9974287</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:37:06Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="42874" length="2"/>
+        <text>28</text>
+      </annotation>
+      <annotation id="482">
+        <infon key="score">0.9988385</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:55:31Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="42884" length="15"/>
+        <text>enzymatic assay</text>
+      </annotation>
+      <annotation id="579">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:44Z</infon>
+        <location offset="42905" length="9"/>
+        <text>influenza</text>
+      </annotation>
+      <annotation id="599">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <location offset="42915" length="5"/>
+        <text>virus</text>
+      </annotation>
+      <annotation id="547">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <location offset="42921" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="568">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="42924" length="4"/>
+        <text>Nter</text>
+      </annotation>
+      <annotation id="483">
+        <infon key="score">0.99725777</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:41:47Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="42929" length="12"/>
+        <text>endonuclease</text>
+      </annotation>
+      <annotation id="654">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:36:59Z</infon>
+        <location offset="42949" length="31"/>
+        <text>cellular influenza virus assays</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">t1.xml</infon>
+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table</infon>
+      <infon key="xml">&lt;?xml version="1.0" encoding="UTF-8"?&gt;
+&lt;table frame="hsides" rules="groups" border="1"&gt;&lt;colgroup&gt;&lt;col align="left"/&gt;&lt;col align="center"/&gt;&lt;col align="center"/&gt;&lt;col align="center"/&gt;&lt;col align="center"/&gt;&lt;col align="center"/&gt;&lt;col align="center"/&gt;&lt;col align="center"/&gt;&lt;/colgroup&gt;&lt;thead valign="bottom"&gt;&lt;tr&gt;&lt;th rowspan="3" align="left" valign="top" charoff="50"&gt;Compound&lt;/th&gt;&lt;th rowspan="2" align="center" valign="top" charoff="50"&gt;Enzyme assay with PA-Nter&lt;xref ref-type="fn" rid="t1-fn1"&gt;a&lt;/xref&gt;&lt;/th&gt;&lt;th colspan="4" align="center" valign="top" charoff="50"&gt;Virus yield assay in influenza virus-infected MDCK cells&lt;xref ref-type="fn" rid="t1-fn2"&gt;b&lt;/xref&gt;&lt;/th&gt;&lt;th colspan="2" align="center" valign="top" charoff="50"&gt;vRNP reconstitution assay in HEK293T cells&lt;xref ref-type="fn" rid="t1-fn3"&gt;c&lt;/xref&gt;&lt;/th&gt;&lt;/tr&gt;&lt;tr&gt;&lt;th colspan="2" align="center" valign="top" charoff="50"&gt;Antiviral activity&lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;Cytotoxicity&lt;/th&gt;&lt;th rowspan="2" align="center" valign="top" charoff="50"&gt;SI&lt;xref ref-type="fn" rid="t1-fn4"&gt;d&lt;/xref&gt;&lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;Activity&lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;Cytotoxicity&lt;/th&gt;&lt;/tr&gt;&lt;tr&gt;&lt;th align="center" valign="top" charoff="50"&gt;IC&lt;sub&gt;50&lt;/sub&gt;&lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;EC&lt;sub&gt;99&lt;/sub&gt;&lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;EC&lt;sub&gt;90&lt;/sub&gt;&lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;CC&lt;sub&gt;50&lt;/sub&gt;&lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;EC&lt;sub&gt;50&lt;/sub&gt;&lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;CC&lt;sub&gt;50&lt;/sub&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign="top"&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(1)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;24&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;xref ref-type="fn" rid="t1-fn6"&gt;f&lt;/xref&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;107&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(2)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;500&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;100&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(3)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;500&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;5.9&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;48&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(4)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;500&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;6.3&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;33&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(5)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;67&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;25&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;25&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;≥146&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;2.6&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;10&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(6)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;500&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;50&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;50&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;15&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;14&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(7)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;54&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;172&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;100&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;2.0&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;3.2&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;8.9&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(8)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;500&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;12.5&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;12.5&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;1.9&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;15&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(9)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;34&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;16&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;5.3&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;38&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;5.5&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(10)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;68&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;14&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;8.5&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;111&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;13&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;0.40&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;132&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(11)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;45&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;30&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;12&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;17&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;5.6&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(12)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;500&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;12.5&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;12.5&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;20&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;39&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(13)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;69&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;71&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;34&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;5.9&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;6.3&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(14)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;500&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;63&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;37&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;5.4&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;2.3&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(15)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;8.9&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;18&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;7.5&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;≥172&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;≥23&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;14&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(16)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;454&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;67&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;28&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;7.1&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;5.2&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(17)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;482&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;21&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;8.1&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;25&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;7.1&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(18)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;83&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;6.2&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;2.2&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;91&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;3.3&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(19)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;500&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;53&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;26&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;7.7&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;5.7&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(20)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;18&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;35&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;11&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;18&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;2.2&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(21)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;13&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;8.3&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;3.6&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;56&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;2.5&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(22)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;75&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;7.4&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;3.4&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;59&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;0.42&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(23)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;8.7&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;11&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;3.5&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;57&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;3.1&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(24)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;131&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;58&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;26&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;7.7&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;25&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(25)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;40&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;132&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;70&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;2.9&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;4.1&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(26)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;30&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;36&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;13&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;15&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;5.5&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(27)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;36&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;21&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;bold&gt;(28)&lt;/bold&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;40&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;158&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;85&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;2.4&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;7.2&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;DPBA&lt;xref ref-type="fn" rid="t1-fn5"&gt;e&lt;/xref&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;5.3&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;Ribavirin&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;ND&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;13&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;8.5&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;24&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;9.4&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon>
+      <offset>42984</offset>
+      <text>Compound	Enzyme assay with PA-Ntera	Virus yield assay in influenza virus-infected MDCK cellsb	vRNP reconstitution assay in HEK293T cellsc	 	Antiviral activity	Cytotoxicity	SId	Activity	Cytotoxicity	 	IC50	EC99	EC90	CC50	EC50	CC50	 	(1)	24	NDf	ND	ND	 	107	&gt;200	 	(2)	&gt;500	ND	ND	ND	 	&gt;100	&gt;200	 	(3)	&gt;500	ND	ND	&gt;200	 	5.9	48	 	(4)	&gt;500	ND	ND	&gt;200	 	6.3	33	 	(5)	67	&gt;25	&gt;25	≥146	 	2.6	10	 	(6)	&gt;500	&gt;50	&gt;50	&gt;200	 	15	14	 	(7)	54	172	100	&gt;200	&gt;2.0	3.2	8.9	 	(8)	&gt;500	&gt;12.5	&gt;12.5	&gt;200	 	1.9	15	 	(9)	34	16	5.3	&gt;200	&gt;38	5.5	&gt;200	 	(10)	68	14	8.5	111	&gt;13	0.40	132	 	(11)	45	30	12	&gt;200	&gt;17	5.6	&gt;200	 	(12)	&gt;500	&gt;12.5	&gt;12.5	&gt;200	 	20	39	 	(13)	69	71	34	&gt;200	&gt;5.9	6.3	&gt;200	 	(14)	&gt;500	63	37	&gt;200	&gt;5.4	2.3	&gt;200	 	(15)	8.9	18	7.5	≥172	≥23	14	&gt;200	 	(16)	454	67	28	&gt;200	&gt;7.1	5.2	&gt;200	 	(17)	482	21	8.1	&gt;200	&gt;25	7.1	&gt;200	 	(18)	83	6.2	2.2	&gt;200	&gt;91	3.3	&gt;200	 	(19)	&gt;500	53	26	&gt;200	&gt;7.7	5.7	&gt;200	 	(20)	18	35	11	&gt;200	&gt;18	2.2	&gt;200	 	(21)	13	8.3	3.6	&gt;200	&gt;56	2.5	&gt;200	 	(22)	75	7.4	3.4	&gt;200	&gt;59	0.42	&gt;200	 	(23)	8.7	11	3.5	&gt;200	&gt;57	3.1	&gt;200	 	(24)	131	58	26	&gt;200	&gt;7.7	25	&gt;200	 	(25)	40	132	70	&gt;200	&gt;2.9	4.1	&gt;200	 	(26)	30	36	13	&gt;200	&gt;15	5.5	&gt;200	 	(27)	36	ND	ND	ND	 	21	&gt;200	 	(28)	40	158	85	&gt;200	&gt;2.4	7.2	&gt;200	 	DPBAe	5.3	ND	ND	ND	 	ND	ND	 	Ribavirin	ND	13	8.5	&gt;200	&gt;24	9.4	&gt;200	 	</text>
+      <annotation id="484">
+        <infon key="score">0.9970269</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:44:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="42984" length="21"/>
+        <text>Compound	Enzyme assay</text>
+      </annotation>
+      <annotation id="485">
+        <infon key="score">0.6912155</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="43011" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="486">
+        <infon key="score">0.96390253</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:15:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="43020" length="17"/>
+        <text>Virus yield assay</text>
+      </annotation>
+      <annotation id="580">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:43:44Z</infon>
+        <location offset="43041" length="9"/>
+        <text>influenza</text>
+      </annotation>
+      <annotation id="600">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <location offset="43051" length="5"/>
+        <text>virus</text>
+      </annotation>
+      <annotation id="487">
+        <infon key="score">0.9968073</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:44:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="43078" length="25"/>
+        <text>vRNP reconstitution assay</text>
+      </annotation>
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+        <infon key="score">0.6241912</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="43184" length="4"/>
+        <text>IC50</text>
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+      <annotation id="656">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:38:12Z</infon>
+        <location offset="43189" length="4"/>
+        <text>EC99</text>
+      </annotation>
+      <annotation id="658">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:38:22Z</infon>
+        <location offset="43194" length="4"/>
+        <text>EC90</text>
+      </annotation>
+      <annotation id="639">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:14:48Z</infon>
+        <location offset="43199" length="4"/>
+        <text>CC50</text>
+      </annotation>
+      <annotation id="620">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:56:18Z</infon>
+        <location offset="43204" length="4"/>
+        <text>EC50</text>
+      </annotation>
+      <annotation id="640">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:14:48Z</infon>
+        <location offset="43209" length="4"/>
+        <text>CC50</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">t1.xml</infon>
+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_footnote</infon>
+      <offset>44265</offset>
+      <text>aRecombinant PA-Nter was incubated with the ssDNA plasmid substrate, a Mn2+-containing buffer and test compounds. Cleavage of the substrate was assessed after 2 hr incubation. The IC50 represents the compound concentration (in μM) required to obtain 50% inhibition of cleavage, calculated by nonlinear least-squares regression analysis (using GraphPad Prism software) of the results from 2–4 independent experiments.</text>
+      <annotation id="489">
+        <infon key="score">0.66438216</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="44278" length="2"/>
+        <text>PA</text>
+      </annotation>
+      <annotation id="569">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:42:28Z</infon>
+        <location offset="44281" length="4"/>
+        <text>Nter</text>
+      </annotation>
+      <annotation id="490">
+        <infon key="score">0.96477795</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:38:56Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="44290" length="9"/>
+        <text>incubated</text>
+      </annotation>
+      <annotation id="491">
+        <infon key="score">0.97936475</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:38:47Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="44309" length="5"/>
+        <text>ssDNA</text>
+      </annotation>
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+        <infon key="score">0.8582004</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:38:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="44336" length="4"/>
+        <text>Mn2+</text>
+      </annotation>
+      <annotation id="493">
+        <infon key="score">0.9971878</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:02:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44445" length="4"/>
+        <text>IC50</text>
+      </annotation>
+      <annotation id="494">
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:38:53Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="44557" length="43"/>
+        <text>nonlinear least-squares regression analysis</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">t1.xml</infon>
+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_footnote</infon>
+      <offset>44687</offset>
+      <text>bMDCK cells were infected with influenza A virus (strain A/PR/8/34) and incubated with the compounds during 24 h. The virus yield in the supernatant was assessed by real-time qPCR. The EC99 and EC90 values represent the compound concentrations (in μM) producing a 2-log10 or 1-log10 reduction in virus titer, respectively, determined in 2–3 independent experiments. The cytotoxicity, assessed in uninfected MDCK cells, was expressed as the CC50 value (50% cytotoxic concentration, determined with the MTS cell viability assay, in μM).</text>
+      <annotation id="660">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:39:14Z</infon>
+        <location offset="44718" length="11"/>
+        <text>influenza A</text>
+      </annotation>
+      <annotation id="495">
+        <infon key="score">0.74896485</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44730" length="5"/>
+        <text>virus</text>
+      </annotation>
+      <annotation id="601">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <location offset="44805" length="5"/>
+        <text>virus</text>
+      </annotation>
+      <annotation id="496">
+        <infon key="score">0.99877644</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:39:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="44852" length="14"/>
+        <text>real-time qPCR</text>
+      </annotation>
+      <annotation id="497">
+        <infon key="score">0.9965879</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:38:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44872" length="4"/>
+        <text>EC99</text>
+      </annotation>
+      <annotation id="498">
+        <infon key="score">0.9963899</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:38:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44881" length="4"/>
+        <text>EC90</text>
+      </annotation>
+      <annotation id="602">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:46:16Z</infon>
+        <location offset="44983" length="5"/>
+        <text>virus</text>
+      </annotation>
+      <annotation id="499">
+        <infon key="score">0.9893111</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:14:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45127" length="4"/>
+        <text>CC50</text>
+      </annotation>
+      <annotation id="500">
+        <infon key="score">0.99815226</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:39:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="45188" length="24"/>
+        <text>MTS cell viability assay</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">t1.xml</infon>
+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_footnote</infon>
+      <offset>45226</offset>
+      <text>cHEK293T cells were co-transfected with the four vRNP-reconstituting plasmids and the luciferase reporter plasmid in the presence of the test compounds. The EC50 represents the compound concentration (in μM) producing 50% reduction in vRNP-driven firefly reporter signal, estimated at 24 h after transfection. The EC50 value was derived from data from 2–4 independent experiments, by nonlinear least-squares regression analysis (using GraphPad Prism software). The CC50 (in μM), i.e. the 50% cytotoxic concentration, was determined in untransfected HEK293T cells by MTS cell viability assay.</text>
+      <annotation id="501">
+        <infon key="score">0.9986248</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:39:27Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="45246" length="14"/>
+        <text>co-transfected</text>
+      </annotation>
+      <annotation id="502">
+        <infon key="score">0.99769396</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:39:31Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="45275" length="4"/>
+        <text>vRNP</text>
+      </annotation>
+      <annotation id="503">
+        <infon key="score">0.93733823</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:39:37Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45347" length="11"/>
+        <text>presence of</text>
+      </annotation>
+      <annotation id="504">
+        <infon key="score">0.98840135</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:56:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45383" length="4"/>
+        <text>EC50</text>
+      </annotation>
+      <annotation id="505">
+        <infon key="score">0.99732447</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:39:33Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="45461" length="4"/>
+        <text>vRNP</text>
+      </annotation>
+      <annotation id="506">
+        <infon key="score">0.99140984</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:56:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45540" length="4"/>
+        <text>EC50</text>
+      </annotation>
+      <annotation id="507">
+        <infon key="score">0.99891144</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:44:29Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="45610" length="43"/>
+        <text>nonlinear least-squares regression analysis</text>
+      </annotation>
+      <annotation id="508">
+        <infon key="score">0.983597</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:14:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45691" length="4"/>
+        <text>CC50</text>
+      </annotation>
+      <annotation id="509">
+        <infon key="score">0.9987122</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:39:41Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="45792" length="24"/>
+        <text>MTS cell viability assay</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">t1.xml</infon>
+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_footnote</infon>
+      <offset>45824</offset>
+      <text>dSI, selectivity index, defined as the ratio between the CC50 and EC90.</text>
+      <annotation id="510">
+        <infon key="score">0.9969549</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:39:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45824" length="3"/>
+        <text>dSI</text>
+      </annotation>
+      <annotation id="511">
+        <infon key="score">0.99642277</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:14:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45829" length="17"/>
+        <text>selectivity index</text>
+      </annotation>
+      <annotation id="512">
+        <infon key="score">0.99740654</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:14:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45881" length="4"/>
+        <text>CC50</text>
+      </annotation>
+      <annotation id="513">
+        <infon key="score">0.9969945</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:38:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45890" length="4"/>
+        <text>EC90</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">t1.xml</infon>
+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_footnote</infon>
+      <offset>45896</offset>
+      <text>eDPBA, 2,4-dioxo-4-phenylbutanoic acid.</text>
+      <annotation id="514">
+        <infon key="score">0.99927</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:39:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="45896" length="5"/>
+        <text>eDPBA</text>
+      </annotation>
+      <annotation id="515">
+        <infon key="score">0.9991686</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T16:39:55Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="45903" length="31"/>
+        <text>2,4-dioxo-4-phenylbutanoic acid</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">t1.xml</infon>
+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_footnote</infon>
+      <offset>45936</offset>
+      <text>fND, not determined.</text>
+    </passage>
+  </document>
+</collection>
diff --git a/annotated_BioC_XML/PMC4981400_ann.xml b/annotated_BioC_XML/PMC4981400_ann.xml
new file mode 100644
index 0000000000000000000000000000000000000000..8b014d7ec52cb6fdae13d511c8b41c368919f391
--- /dev/null
+++ b/annotated_BioC_XML/PMC4981400_ann.xml
@@ -0,0 +1,6049 @@
+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection>
+  <source>PMC</source>
+  <date>20201217</date>
+  <key>pmc.key</key>
+  <document>
+    <id>4981400</id>
+    <infon key="license">CC BY</infon>
+    <infon key="tt_curatable">no</infon>
+    <infon key="tt_version">2</infon>
+    <infon key="tt_round">2</infon>
+    <passage>
+      <infon key="alt-title">Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA</infon>
+      <infon key="article-id_doi">10.1371/journal.pone.0160694</infon>
+      <infon key="article-id_pmc">4981400</infon>
+      <infon key="article-id_pmid">27513867</infon>
+      <infon key="article-id_publisher-id">PONE-D-16-20928</infon>
+      <infon key="elocation-id">e0160694</infon>
+      <infon key="issue">8</infon>
+      <infon key="license">This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</infon>
+      <infon key="name_0">surname:Zhang;given-names:Yinglu</infon>
+      <infon key="name_1">surname:Rataj;given-names:Katarzyna</infon>
+      <infon key="name_2">surname:Simpson;given-names:Gordon G.</infon>
+      <infon key="name_3">surname:Tong;given-names:Liang</infon>
+      <infon key="name_4">surname:Candela;given-names:Hector</infon>
+      <infon key="name_5">surname:Tong;given-names:Liang</infon>
+      <infon key="name_6">surname:Tong;given-names:Liang</infon>
+      <infon key="name_7">surname:Simpson;given-names:Gordon G.</infon>
+      <infon key="name_8">surname:Simpson;given-names:Gordon G.</infon>
+      <infon key="notes">All relevant data are within the paper and its Supporting Information files.</infon>
+      <infon key="section_type">TITLE</infon>
+      <infon key="title">Data Availability</infon>
+      <infon key="type">front</infon>
+      <infon key="volume">11</infon>
+      <infon key="year">2016</infon>
+      <offset>0</offset>
+      <text>Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA</text>
+      <annotation id="1">
+        <infon key="score">0.9986647</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:48:35Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="0" length="17"/>
+        <text>Crystal Structure</text>
+      </annotation>
+      <annotation id="486">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:48:59Z</infon>
+        <location offset="25" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="2">
+        <infon key="score">0.99860686</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44" length="11"/>
+        <text>Arabidopsis</text>
+      </annotation>
+      <annotation id="3">
+        <infon key="score">0.99752444</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="56" length="19"/>
+        <text>Flowering Regulator</text>
+      </annotation>
+      <annotation id="4">
+        <infon key="score">0.9987639</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="76" length="3"/>
+        <text>FPA</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">ABSTRACT</infon>
+      <infon key="type">abstract</infon>
+      <offset>80</offset>
+      <text>The Arabidopsis protein FPA controls flowering time by regulating the alternative 3′-end processing of the FLOWERING LOCUS (FLC) antisense RNA. FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain. The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower. We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution. The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them. Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs. Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.</text>
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+        <text>Arabidopsis</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>FPA</text>
+      </annotation>
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+        <infon key="identifier">GENE:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:50:06Z</infon>
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+        <text>FLOWERING LOCUS</text>
+      </annotation>
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+        <infon key="type">gene</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:50:19Z</infon>
+        <infon key="identifier">GENE:</infon>
+        <location offset="204" length="3"/>
+        <text>FLC</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:50:24Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="209" length="13"/>
+        <text>antisense RNA</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="224" length="3"/>
+        <text>FPA</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:50:49Z</infon>
+        <location offset="243" length="10"/>
+        <text>split ends</text>
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+      <annotation id="520">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:50:59Z</infon>
+        <location offset="255" length="4"/>
+        <text>SPEN</text>
+      </annotation>
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+        <infon key="score">0.9992418</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:51:19Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="306" length="22"/>
+        <text>RNA recognition motifs</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:51:23Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="330" length="4"/>
+        <text>RRMs</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:51:48Z</infon>
+        <location offset="342" length="36"/>
+        <text>SPEN paralog and ortholog C-terminal</text>
+      </annotation>
+      <annotation id="12">
+        <infon key="score">0.999008</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="380" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="13">
+        <infon key="score">0.9993174</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="398" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="14">
+        <infon key="score">0.99878335</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:05:44Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="413" length="16"/>
+        <text>highly conserved</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="436" length="3"/>
+        <text>FPA</text>
+      </annotation>
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+        <infon key="score">0.99871314</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:51:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="452" length="6"/>
+        <text>plants</text>
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+      <annotation id="521">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:50:59Z</infon>
+        <location offset="506" length="4"/>
+        <text>SPEN</text>
+      </annotation>
+      <annotation id="17">
+        <infon key="score">0.9810288</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:48:36Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="558" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="18">
+        <infon key="score">0.99751747</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:40Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="579" length="20"/>
+        <text>Arabidopsis thaliana</text>
+      </annotation>
+      <annotation id="19">
+        <infon key="score">0.99925774</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="600" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="20">
+        <infon key="score">0.99943274</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="604" length="4"/>
+        <text>SPOC</text>
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+      <annotation id="21">
+        <infon key="score">0.9977823</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="649" length="9"/>
+        <text>structure</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="685" length="4"/>
+        <text>SPOC</text>
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+      <annotation id="23">
+        <infon key="score">0.9987803</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="700" length="5"/>
+        <text>human</text>
+      </annotation>
+      <annotation id="24">
+        <infon key="score">0.99765545</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:16Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="706" length="39"/>
+        <text>SMRT/HDAC1 Associated Repressor Protein</text>
+      </annotation>
+      <annotation id="25">
+        <infon key="score">0.9991811</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:19Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="747" length="5"/>
+        <text>SHARP</text>
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+      <annotation id="26">
+        <infon key="score">0.998875</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="832" length="32"/>
+        <text>Structural and sequence analyses</text>
+      </annotation>
+      <annotation id="27">
+        <infon key="score">0.99889666</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="876" length="13"/>
+        <text>surface patch</text>
+      </annotation>
+      <annotation id="28">
+        <infon key="score">0.9987212</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:24Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="898" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="29">
+        <infon key="score">0.9985349</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="914" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="30">
+        <infon key="score">0.9982534</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="920" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="526">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:37Z</infon>
+        <location offset="934" length="9"/>
+        <text>Mutations</text>
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+      <annotation id="31">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:40Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="968" length="13"/>
+        <text>surface patch</text>
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+      <annotation id="32">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="998" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="33">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="1040" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="34">
+        <infon key="score">0.9990607</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="1084" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="35">
+        <infon key="score">0.8297148</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:50Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1102" length="3"/>
+        <text>RNA</text>
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+      <annotation id="36">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="1147" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="37">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="1151" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="38">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="1253" length="5"/>
+        <text>SHARP</text>
+      </annotation>
+      <annotation id="39">
+        <infon key="score">0.99941254</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="1259" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">title_1</infon>
+      <offset>1277</offset>
+      <text>Introduction</text>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>1290</offset>
+      <text>Eukaryotic messenger RNAs (mRNAs) are made as precursors through transcription by RNA polymerase II (Pol II), and these primary transcripts undergo extensive processing, including 3′-end cleavage and polyadenylation. In addition, alternative 3′-end cleavage and polyadenylation is an essential and ubiquitous process in eukaryotes. Misregulation of (alternative) 3′-end processing can lead to various genetic defects, cancer and other diseases. There is currently great interest in understanding the molecular mechanisms and functional impacts of alternative 3′-end processing.</text>
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+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1290" length="10"/>
+        <text>Eukaryotic</text>
+      </annotation>
+      <annotation id="41">
+        <infon key="score">0.99718124</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:53:01Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1301" length="14"/>
+        <text>messenger RNAs</text>
+      </annotation>
+      <annotation id="42">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:53:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1317" length="5"/>
+        <text>mRNAs</text>
+      </annotation>
+      <annotation id="43">
+        <infon key="score">0.93546987</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:53:07Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="1372" length="17"/>
+        <text>RNA polymerase II</text>
+      </annotation>
+      <annotation id="44">
+        <infon key="score">0.9645325</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:53:11Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="1391" length="6"/>
+        <text>Pol II</text>
+      </annotation>
+      <annotation id="45">
+        <infon key="score">0.99861825</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T14:43:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1610" length="10"/>
+        <text>eukaryotes</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>1876</offset>
+      <text>Recently, the split ends (SPEN) family of proteins was identified as RNA binding proteins that regulate alternative 3′-end cleavage and polyadenylation. They are characterized by possessing N-terminal RNA recognition motifs (RRMs) and a conserved SPEN paralog and ortholog C-terminal (SPOC) domain (Fig 1A). The SPOC domain is believed to mediate protein-protein interactions and has diverse functions among SPEN family proteins, but the molecular mechanism of these functions is not well understood.</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:53:35Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1890" length="10"/>
+        <text>split ends</text>
+      </annotation>
+      <annotation id="47">
+        <infon key="score">0.97204024</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:50:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1902" length="4"/>
+        <text>SPEN</text>
+      </annotation>
+      <annotation id="48">
+        <infon key="score">0.9987454</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:54:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1945" length="20"/>
+        <text>RNA binding proteins</text>
+      </annotation>
+      <annotation id="49">
+        <infon key="score">0.99936414</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:51:19Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2077" length="22"/>
+        <text>RNA recognition motifs</text>
+      </annotation>
+      <annotation id="50">
+        <infon key="score">0.99952304</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:51:24Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2101" length="4"/>
+        <text>RRMs</text>
+      </annotation>
+      <annotation id="51">
+        <infon key="score">0.99919945</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:54:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2113" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="527">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:53:58Z</infon>
+        <location offset="2123" length="36"/>
+        <text>SPEN paralog and ortholog C-terminal</text>
+      </annotation>
+      <annotation id="52">
+        <infon key="score">0.9990139</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2161" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="53">
+        <infon key="score">0.9994504</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2188" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="522">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:50:59Z</infon>
+        <location offset="2284" length="4"/>
+        <text>SPEN</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">pone.0160694.g001.jpg</infon>
+      <infon key="id">pone.0160694.g001</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>2379</offset>
+      <text>Sequence conservation of SPOC domains.</text>
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+        <infon key="score">0.91995025</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:55:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2379" length="21"/>
+        <text>Sequence conservation</text>
+      </annotation>
+      <annotation id="487">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="2404" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">pone.0160694.g001.jpg</infon>
+      <infon key="id">pone.0160694.g001</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>2418</offset>
+      <text>(A). Domain organization of A. thaliana FPA. (B). Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP. Residues in surface patch 1 are indicated with the orange dots, and those in surface patch 2 with the green dots. The secondary structure elements in the structure of FPA SPOC are labeled. Residues that are strictly conserved among the five proteins are shown in white with a red background, and those that are mostly conserved in red.</text>
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:54:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2446" length="11"/>
+        <text>A. thaliana</text>
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+      <annotation id="56">
+        <infon key="score">0.9989416</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2458" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="57">
+        <infon key="score">0.9985844</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:55:04Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2468" length="18"/>
+        <text>Sequence alignment</text>
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+      <annotation id="488">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="2494" length="4"/>
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+      <annotation id="58">
+        <infon key="score">0.9974239</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:41Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2510" length="20"/>
+        <text>Arabidopsis thaliana</text>
+      </annotation>
+      <annotation id="59">
+        <infon key="score">0.9992167</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2531" length="3"/>
+        <text>FPA</text>
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+      <annotation id="60">
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2536" length="5"/>
+        <text>human</text>
+      </annotation>
+      <annotation id="61">
+        <infon key="score">0.99920624</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:55:21Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2542" length="5"/>
+        <text>RBM15</text>
+      </annotation>
+      <annotation id="62">
+        <infon key="score">0.7792917</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:55:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2549" length="10"/>
+        <text>Drosophila</text>
+      </annotation>
+      <annotation id="63">
+        <infon key="score">0.9992182</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:50:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2560" length="4"/>
+        <text>SPEN</text>
+      </annotation>
+      <annotation id="64">
+        <infon key="score">0.9206429</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:55:45Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2566" length="5"/>
+        <text>mouse</text>
+      </annotation>
+      <annotation id="65">
+        <infon key="score">0.9989303</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T14:43:50Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2572" length="4"/>
+        <text>MINT</text>
+      </annotation>
+      <annotation id="66">
+        <infon key="score">0.99851686</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2582" length="5"/>
+        <text>human</text>
+      </annotation>
+      <annotation id="67">
+        <infon key="score">0.9991665</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2588" length="5"/>
+        <text>SHARP</text>
+      </annotation>
+      <annotation id="68">
+        <infon key="score">0.93170136</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:56:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2607" length="15"/>
+        <text>surface patch 1</text>
+      </annotation>
+      <annotation id="69">
+        <infon key="score">0.93146116</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:56:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2672" length="15"/>
+        <text>surface patch 2</text>
+      </annotation>
+      <annotation id="70">
+        <infon key="score">0.9454224</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:56:09Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2749" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="71">
+        <infon key="score">0.99068165</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2762" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="72">
+        <infon key="score">0.5914585</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2766" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="73">
+        <infon key="score">0.9987582</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:56:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2802" length="18"/>
+        <text>strictly conserved</text>
+      </annotation>
+      <annotation id="74">
+        <infon key="score">0.9979994</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:56:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2906" length="16"/>
+        <text>mostly conserved</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>2931</offset>
+      <text>FPA, a SPEN family protein in Arabidopsis thaliana and other plants, was found to regulate the 3′-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene. FPA promotes the 3′-end processing of class I FLC antisense RNAs, which includes the proximal polyadenylation site. This is associated with histone demethylase activity and down-regulation of FLC transcription. However, the functional mechanism of this complex is still not clear.</text>
+      <annotation id="75">
+        <infon key="score">0.9988631</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2931" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="523">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:50:59Z</infon>
+        <location offset="2938" length="4"/>
+        <text>SPEN</text>
+      </annotation>
+      <annotation id="76">
+        <infon key="score">0.9964304</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:41Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2961" length="20"/>
+        <text>Arabidopsis thaliana</text>
+      </annotation>
+      <annotation id="77">
+        <infon key="score">0.9980305</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:51:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2992" length="6"/>
+        <text>plants</text>
+      </annotation>
+      <annotation id="78">
+        <infon key="score">0.9736698</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:56:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3081" length="14"/>
+        <text>antisense RNAs</text>
+      </annotation>
+      <annotation id="79">
+        <infon key="score">0.5571705</infon>
+        <infon key="type">gene</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:56:36Z</infon>
+        <infon key="identifier">GENE:</infon>
+        <location offset="3099" length="15"/>
+        <text>FLOWERING LOCUS</text>
+      </annotation>
+      <annotation id="80">
+        <infon key="score">0.5104294</infon>
+        <infon key="type">gene</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:56:39Z</infon>
+        <infon key="identifier">GENE:</infon>
+        <location offset="3116" length="3"/>
+        <text>FLC</text>
+      </annotation>
+      <annotation id="81">
+        <infon key="score">0.9989016</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3150" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="82">
+        <infon key="score">0.54643095</infon>
+        <infon key="type">gene</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:56:39Z</infon>
+        <infon key="identifier">GENE:</infon>
+        <location offset="3196" length="3"/>
+        <text>FLC</text>
+      </annotation>
+      <annotation id="83">
+        <infon key="score">0.9708284</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:56:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3200" length="14"/>
+        <text>antisense RNAs</text>
+      </annotation>
+      <annotation id="84">
+        <infon key="score">0.99701446</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:56:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="3244" length="20"/>
+        <text>polyadenylation site</text>
+      </annotation>
+      <annotation id="85">
+        <infon key="score">0.97197783</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:56:53Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3290" length="19"/>
+        <text>histone demethylase</text>
+      </annotation>
+      <annotation id="528">
+        <infon key="type">gene</infon>
+        <infon key="identifier">GENE:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:56:39Z</infon>
+        <location offset="3342" length="3"/>
+        <text>FLC</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>3435</offset>
+      <text>Although a SPOC domain is found in all the SPEN family proteins, its sequence conservation is rather low. For example, the sequence identity between the SPOC domains of A. thaliana FPA and human SMRT/HDAC1 Associated Repressor Protein (SHARP) is only 19% (Fig 1B). Currently, the SHARP SPOC domain is the only one with structural information.</text>
+      <annotation id="489">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="3446" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="524">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:50:59Z</infon>
+        <location offset="3478" length="4"/>
+        <text>SPEN</text>
+      </annotation>
+      <annotation id="490">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="3588" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="86">
+        <infon key="score">0.9984439</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:55:00Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3604" length="11"/>
+        <text>A. thaliana</text>
+      </annotation>
+      <annotation id="87">
+        <infon key="score">0.9993006</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3616" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="88">
+        <infon key="score">0.9989404</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3624" length="5"/>
+        <text>human</text>
+      </annotation>
+      <annotation id="89">
+        <infon key="score">0.995815</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:57:23Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3630" length="39"/>
+        <text>SMRT/HDAC1 Associated Repressor Protein</text>
+      </annotation>
+      <annotation id="90">
+        <infon key="score">0.9992428</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3671" length="5"/>
+        <text>SHARP</text>
+      </annotation>
+      <annotation id="91">
+        <infon key="score">0.99915516</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3715" length="5"/>
+        <text>SHARP</text>
+      </annotation>
+      <annotation id="491">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="3721" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>3778</offset>
+      <text>As a first step toward understanding the molecular basis for the regulation of alternative 3′-end processing and flowering by FPA, we have determined the crystal structure of the SPOC domain of A. thaliana FPA at 2.7 Å resolution. The overall structure is similar to that of the SHARP SPOC domain, although there are also substantial conformational differences between them. The structure reveals a surface patch that is conserved among FPA homologs.</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3904" length="3"/>
+        <text>FPA</text>
+      </annotation>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:48:36Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3932" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="3957" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="95">
+        <infon key="score">0.99831086</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:55:00Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3972" length="11"/>
+        <text>A. thaliana</text>
+      </annotation>
+      <annotation id="96">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3984" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="97">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:57:37Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>structure</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="4057" length="5"/>
+        <text>SHARP</text>
+      </annotation>
+      <annotation id="492">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="4063" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="99">
+        <infon key="score">0.9983583</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:57:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4157" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="100">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:45Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>surface patch</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:57:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>conserved</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="4215" length="3"/>
+        <text>FPA</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_1</infon>
+      <offset>4232</offset>
+      <text>Results and Discussion</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>4255</offset>
+      <text>Structure of FPA SPOC domain</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4255" length="9"/>
+        <text>Structure</text>
+      </annotation>
+      <annotation id="104">
+        <infon key="score">0.99878544</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="4268" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="105">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4272" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>4284</offset>
+      <text>The crystal structure of the SPOC domain of A. thaliana FPA has been determined at 2.7 Å resolution using the selenomethionyl single-wavelength anomalous dispersion method. The expression construct contained residues 433–565 of FPA, but only residues 439–460 and 465–565 are ordered in the crystal. The atomic model has good agreement with the X-ray diffraction data and the expected bond lengths, bond angles and other geometric parameters (Table 1). All the residues are located in the favored regions of the Ramachandran plot (data not shown). The structure has been deposited in the Protein Data Bank, with accession code 5KXF.</text>
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+        <infon key="updated_at">2023-09-20T12:48:36Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>crystal structure</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>SPOC</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>A. thaliana</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>FPA</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <text>selenomethionyl single-wavelength anomalous dispersion method</text>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>433–565</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="4512" length="3"/>
+        <text>FPA</text>
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+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:57:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4535" length="7"/>
+        <text>439–460</text>
+      </annotation>
+      <annotation id="114">
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+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:57:52Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4547" length="7"/>
+        <text>465–565</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:57:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>crystal</text>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>atomic model</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:57:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4628" length="22"/>
+        <text>X-ray diffraction data</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4795" length="17"/>
+        <text>Ramachandran plot</text>
+      </annotation>
+      <annotation id="119">
+        <infon key="score">0.98965245</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4835" length="9"/>
+        <text>structure</text>
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+    </passage>
+    <passage>
+      <infon key="file">pone.0160694.t001.xml</infon>
+      <infon key="id">pone.0160694.t001</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_title_caption</infon>
+      <offset>4923</offset>
+      <text>Summary of crystallographic information.</text>
+    </passage>
+    <passage>
+      <infon key="file">pone.0160694.t001.xml</infon>
+      <infon key="id">pone.0160694.t001</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table</infon>
+      <infon key="xml">&lt;?xml version="1.0" encoding="UTF-8"?&gt;
+&lt;table frame="hsides" rules="groups"&gt;&lt;colgroup span="1"&gt;&lt;col align="left" valign="middle" span="1"/&gt;&lt;col align="left" valign="middle" span="1"/&gt;&lt;/colgroup&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;Resolution range (Å)&lt;xref ref-type="table-fn" rid="t001fn001"&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align="center" rowspan="1" colspan="1"&gt;50–2.7 (2.8–2.7)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;Number of observations&lt;/td&gt;&lt;td align="center" rowspan="1" colspan="1"&gt;78,008&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;merge&lt;/sub&gt; (%)&lt;/td&gt;&lt;td align="center" rowspan="1" colspan="1"&gt;10.5 (45.3)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;I/σI&lt;/td&gt;&lt;td align="center" rowspan="1" colspan="1"&gt;24.1 (6.3)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;Redundancy&lt;/td&gt;&lt;td align="center" rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;Completeness (%)&lt;/td&gt;&lt;td align="center" rowspan="1" colspan="1"&gt;100 (100)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;&lt;italic&gt;R&lt;/italic&gt; factor (%)&lt;/td&gt;&lt;td align="center" rowspan="1" colspan="1"&gt;19.2 (25.0)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;Free &lt;italic&gt;R&lt;/italic&gt; factor (%)&lt;/td&gt;&lt;td align="center" rowspan="1" colspan="1"&gt;25.4 (35.4)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;Rms deviation in bond lengths (Å)&lt;/td&gt;&lt;td align="center" rowspan="1" colspan="1"&gt;0.017&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;Rms deviation in bond angles (°)&lt;/td&gt;&lt;td align="center" rowspan="1" colspan="1"&gt;1.9&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon>
+      <offset>4964</offset>
+      <text>Resolution range (Å)1	50–2.7 (2.8–2.7)	 	Number of observations	78,008	 	Rmerge (%)	10.5 (45.3)	 	I/σI	24.1 (6.3)	 	Redundancy		 	Completeness (%)	100 (100)	 	R factor (%)	19.2 (25.0)	 	Free R factor (%)	25.4 (35.4)	 	Rms deviation in bond lengths (Å)	0.017	 	Rms deviation in bond angles (°)	1.9	 	</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5123" length="8"/>
+        <text>R factor</text>
+      </annotation>
+      <annotation id="121">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5150" length="13"/>
+        <text>Free R factor</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">pone.0160694.t001.xml</infon>
+      <infon key="id">pone.0160694.t001</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_footnote</infon>
+      <offset>5272</offset>
+      <text>1The numbers in parentheses are for the highest resolution shell.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>5338</offset>
+      <text>The crystal structure of the FPA SPOC domain contains a seven-stranded, mostly anti-parallel β-barrel (β1-β7) and three helices (αA-αC) (Fig 2A). Only two of the neighboring strands, β1 and β3, are parallel to each other. Helix αB covers one end of the barrel, while helices αA and αC are located next to each other at one side of the barrel (Fig 2B). The other end of the β-barrel is covered by the loop connecting strands β2 and β3, which contains the disordered 461–464 segment. The center of the barrel is filled with hydrophobic side chains and is not accessible to the solvent.</text>
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+        <text>crystal structure</text>
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+        <infon key="identifier">PR:</infon>
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+        <text>FPA</text>
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+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
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+        <text>SPOC</text>
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+        <infon key="identifier">SO:</infon>
+        <location offset="5394" length="45"/>
+        <text>seven-stranded, mostly anti-parallel β-barrel</text>
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+        <infon key="identifier">SO:</infon>
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+        <text>β1-β7</text>
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+        <infon key="identifier">SO:</infon>
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+        <text>helices</text>
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+        <infon key="identifier">SO:</infon>
+        <location offset="5467" length="5"/>
+        <text>αA-αC</text>
+      </annotation>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5512" length="7"/>
+        <text>strands</text>
+      </annotation>
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+        <infon key="identifier">SO:</infon>
+        <location offset="5521" length="2"/>
+        <text>β1</text>
+      </annotation>
+      <annotation id="130">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5528" length="2"/>
+        <text>β3</text>
+      </annotation>
+      <annotation id="131">
+        <infon key="score">0.9983072</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5560" length="5"/>
+        <text>Helix</text>
+      </annotation>
+      <annotation id="132">
+        <infon key="score">0.99947184</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:03Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5566" length="2"/>
+        <text>αB</text>
+      </annotation>
+      <annotation id="133">
+        <infon key="score">0.9992513</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:06Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5591" length="6"/>
+        <text>barrel</text>
+      </annotation>
+      <annotation id="134">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:23Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5605" length="7"/>
+        <text>helices</text>
+      </annotation>
+      <annotation id="135">
+        <infon key="score">0.99948055</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5613" length="2"/>
+        <text>αA</text>
+      </annotation>
+      <annotation id="136">
+        <infon key="score">0.99945194</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:29Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5620" length="2"/>
+        <text>αC</text>
+      </annotation>
+      <annotation id="137">
+        <infon key="score">0.99912864</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5673" length="6"/>
+        <text>barrel</text>
+      </annotation>
+      <annotation id="138">
+        <infon key="score">0.99915344</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5711" length="8"/>
+        <text>β-barrel</text>
+      </annotation>
+      <annotation id="139">
+        <infon key="score">0.9990128</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:39Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5738" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="140">
+        <infon key="score">0.9920982</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5754" length="7"/>
+        <text>strands</text>
+      </annotation>
+      <annotation id="141">
+        <infon key="score">0.999331</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T14:42:19Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5762" length="2"/>
+        <text>β2</text>
+      </annotation>
+      <annotation id="142">
+        <infon key="score">0.9992975</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5769" length="2"/>
+        <text>β3</text>
+      </annotation>
+      <annotation id="143">
+        <infon key="score">0.9989806</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:30Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5792" length="10"/>
+        <text>disordered</text>
+      </annotation>
+      <annotation id="144">
+        <infon key="score">0.99722177</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:27Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5803" length="7"/>
+        <text>461–464</text>
+      </annotation>
+      <annotation id="145">
+        <infon key="score">0.99890804</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5838" length="6"/>
+        <text>barrel</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">pone.0160694.g002.jpg</infon>
+      <infon key="id">pone.0160694.g002</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>5966</offset>
+      <text>Crystal structure of the SPOC domain of A. thaliana FPA.</text>
+      <annotation id="146">
+        <infon key="score">0.99874413</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:48:36Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5966" length="17"/>
+        <text>Crystal structure</text>
+      </annotation>
+      <annotation id="494">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="5991" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="147">
+        <infon key="score">0.9980335</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:55:00Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6006" length="11"/>
+        <text>A. thaliana</text>
+      </annotation>
+      <annotation id="148">
+        <infon key="score">0.98107886</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6018" length="3"/>
+        <text>FPA</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">pone.0160694.g002.jpg</infon>
+      <infon key="id">pone.0160694.g002</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>6023</offset>
+      <text>(A). Schematic drawing of the structure of FPA SPOC domain, colored from blue at the N terminus to red at the C terminus. The view is from the side of the β-barrel. The disordered segment (residues 460–465) is indicated with the dotted line. (B). Structure of the FPA SPOC domain, viewed from the end of the β-barrel, after 90° rotation around the horizontal axis from panel A. All structure figures were produced with PyMOL (www.pymol.org).</text>
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+        <infon key="score">0.9700408</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:00:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6053" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="150">
+        <infon key="score">0.984616</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6066" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="151">
+        <infon key="score">0.99939215</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6070" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="152">
+        <infon key="score">0.999288</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6178" length="8"/>
+        <text>β-barrel</text>
+      </annotation>
+      <annotation id="153">
+        <infon key="score">0.9964395</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:05:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6192" length="10"/>
+        <text>disordered</text>
+      </annotation>
+      <annotation id="154">
+        <infon key="score">0.9978326</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:00:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6221" length="7"/>
+        <text>460–465</text>
+      </annotation>
+      <annotation id="155">
+        <infon key="score">0.99287826</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:00:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6270" length="9"/>
+        <text>Structure</text>
+      </annotation>
+      <annotation id="156">
+        <infon key="score">0.99104744</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6287" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="157">
+        <infon key="score">0.9992855</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6291" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="158">
+        <infon key="score">0.9992698</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6331" length="8"/>
+        <text>β-barrel</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>6471</offset>
+      <text>Comparisons to structural homologs of the SPOC domain</text>
+      <annotation id="534">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:00:30Z</infon>
+        <location offset="6471" length="34"/>
+        <text>Comparisons to structural homologs</text>
+      </annotation>
+      <annotation id="495">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="6513" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>6525</offset>
+      <text>Only five structural homologs of the FPA SPOC domain were found in the Protein Data Bank with the DaliLite server, suggesting that the SPOC domain structure is relatively unique. The top hit is the SPOC domain of human SHARP (Fig 3A), with a Z score of 12.3. The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D). The next structural homolog has a Z score of 3.0.</text>
+      <annotation id="159">
+        <infon key="score">0.7332179</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6562" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="160">
+        <infon key="score">0.999361</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6566" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="161">
+        <infon key="score">0.9983363</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:00:40Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6623" length="15"/>
+        <text>DaliLite server</text>
+      </annotation>
+      <annotation id="162">
+        <infon key="score">0.9990138</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6660" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="163">
+        <infon key="score">0.99605185</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:00:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6672" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="164">
+        <infon key="score">0.9993979</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6723" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="165">
+        <infon key="score">0.9984281</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6738" length="5"/>
+        <text>human</text>
+      </annotation>
+      <annotation id="166">
+        <infon key="score">0.9990225</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6744" length="5"/>
+        <text>SHARP</text>
+      </annotation>
+      <annotation id="167">
+        <infon key="score">0.9980933</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:07:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6767" length="7"/>
+        <text>Z score</text>
+      </annotation>
+      <annotation id="530">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:35Z</infon>
+        <location offset="6831" length="8"/>
+        <text>β-barrel</text>
+      </annotation>
+      <annotation id="168">
+        <infon key="score">0.9989506</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:01:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6863" length="4"/>
+        <text>Ku70</text>
+      </annotation>
+      <annotation id="169">
+        <infon key="score">0.9988921</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:01:08Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6872" length="4"/>
+        <text>Ku80</text>
+      </annotation>
+      <annotation id="170">
+        <infon key="score">0.997887</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:00:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6878" length="7"/>
+        <text>Z score</text>
+      </annotation>
+      <annotation id="171">
+        <infon key="score">0.9908906</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:01:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6918" length="20"/>
+        <text>chromodomain protein</text>
+      </annotation>
+      <annotation id="172">
+        <infon key="score">0.99925596</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:01:04Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6939" length="4"/>
+        <text>Chp1</text>
+      </annotation>
+      <annotation id="173">
+        <infon key="score">0.99778926</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:00:51Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6945" length="7"/>
+        <text>Z score</text>
+      </annotation>
+      <annotation id="174">
+        <infon key="score">0.99922377</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:00:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6977" length="28"/>
+        <text>activator interacting domain</text>
+      </annotation>
+      <annotation id="175">
+        <infon key="score">0.9984598</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:00:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7007" length="4"/>
+        <text>ACID</text>
+      </annotation>
+      <annotation id="176">
+        <infon key="score">0.9993298</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:01:15Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7020" length="5"/>
+        <text>Med25</text>
+      </annotation>
+      <annotation id="177">
+        <infon key="score">0.99764967</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:00:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>Z score</text>
+      </annotation>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>Z score</text>
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+    </passage>
+    <passage>
+      <infon key="file">pone.0160694.g003.jpg</infon>
+      <infon key="id">pone.0160694.g003</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
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+      <text>Structural homologs of the FPA SPOC domain.</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>FPA</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="7166" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">pone.0160694.g003.jpg</infon>
+      <infon key="id">pone.0160694.g003</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>7179</offset>
+      <text>(A). Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray). The bound position of a doubly-phosphorylated peptide from SMRT is shown in magenta. (B). Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 β-barrel domain (gray). Ku80 contains a homologous domain (green), which forms a hetero-dimer with that in Ku70. The two domains, and inserted segments on them, mediate the binding of dsDNA (orange). The red rectangle highlights the region of contact between the two β-barrel domains. (C). Overlay of the structures of the FPA SPOC domain (cyan) and the homologous domain in Chp1 (gray). The binding partner of Chp1, Tas3, is shown in green. The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (D). Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray).</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="updated_at">2023-09-20T13:02:28Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">experimental_method</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">protein</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="7808" length="4"/>
+        <text>Chp1</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:01:05Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7844" length="4"/>
+        <text>Chp1</text>
+      </annotation>
+      <annotation id="203">
+        <infon key="score">0.999226</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:09:03Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7850" length="4"/>
+        <text>Tas3</text>
+      </annotation>
+      <annotation id="204">
+        <infon key="score">0.99869466</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:03:08Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7932" length="12"/>
+        <text>binding site</text>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:07:36Z</infon>
+        <location offset="7952" length="5"/>
+        <text>SMART</text>
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+      <annotation id="205">
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+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:10:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7958" length="14"/>
+        <text>phosphopeptide</text>
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+      <annotation id="206">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7976" length="5"/>
+        <text>SHARP</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="7982" length="4"/>
+        <text>SPOC</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:40Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8003" length="4"/>
+        <text>loop</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:09:03Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8011" length="4"/>
+        <text>Tas3</text>
+      </annotation>
+      <annotation id="209">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:01:39Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8038" length="7"/>
+        <text>Overlay</text>
+      </annotation>
+      <annotation id="210">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:07:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>structures</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8071" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="212">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8075" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="213">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:01:16Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8102" length="5"/>
+        <text>Med25</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:00:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8108" length="4"/>
+        <text>ACID</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>8127</offset>
+      <text>SHARP is a transcriptional co-repressor in the nuclear receptor and Notch/RBP-Jκ signaling pathways. The SPOC domain of SHARP interacts directly with silencing mediator for retinoid and thyroid receptor (SMRT), nuclear receptor co-repressor (N-CoR), HDAC, and other components to represses transcription. While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the β-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A). In addition, the SHARP SPOC domain has three extra helices. One of them covers the other end of the β-barrel, and the other two shield an additional surface of the side of the β-barrel from solvent. A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands β1 and β3 (Fig 3A). Such a binding mode probably would not be possible in FPA, as the peptide would clash with the β1-β2 loop.</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8127" length="5"/>
+        <text>SHARP</text>
+      </annotation>
+      <annotation id="216">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:03:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8138" length="28"/>
+        <text>transcriptional co-repressor</text>
+      </annotation>
+      <annotation id="536">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:03:28Z</infon>
+        <location offset="8174" length="16"/>
+        <text>nuclear receptor</text>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:03:37Z</infon>
+        <location offset="8195" length="5"/>
+        <text>Notch</text>
+      </annotation>
+      <annotation id="217">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:04:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8201" length="6"/>
+        <text>RBP-Jκ</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="8232" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="218">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8247" length="5"/>
+        <text>SHARP</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:03:54Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8277" length="52"/>
+        <text>silencing mediator for retinoid and thyroid receptor</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:03:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8331" length="4"/>
+        <text>SMRT</text>
+      </annotation>
+      <annotation id="221">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:04:12Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8338" length="29"/>
+        <text>nuclear receptor co-repressor</text>
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+      <annotation id="541">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:04:31Z</infon>
+        <location offset="8369" length="5"/>
+        <text>N-CoR</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:04:39Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8377" length="4"/>
+        <text>HDAC</text>
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+      <annotation id="223">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:03:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8450" length="9"/>
+        <text>structure</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8467" length="3"/>
+        <text>FPA</text>
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+      <annotation id="500">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="8471" length="4"/>
+        <text>SPOC</text>
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+      <annotation id="225">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8509" length="5"/>
+        <text>SHARP</text>
+      </annotation>
+      <annotation id="501">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="8515" length="4"/>
+        <text>SPOC</text>
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+      <annotation id="226">
+        <infon key="score">0.99924237</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:05:04Z</infon>
+        <infon key="identifier">SO:</infon>
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+      <annotation id="227">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:23Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8605" length="7"/>
+        <text>helices</text>
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+      <annotation id="228">
+        <infon key="score">0.99715745</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:05:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8630" length="5"/>
+        <text>loops</text>
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+      <annotation id="229">
+        <infon key="score">0.9987274</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8714" length="5"/>
+        <text>SHARP</text>
+      </annotation>
+      <annotation id="502">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="8720" length="4"/>
+        <text>SPOC</text>
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+      <annotation id="230">
+        <infon key="score">0.9960328</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:23Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8748" length="7"/>
+        <text>helices</text>
+      </annotation>
+      <annotation id="231">
+        <infon key="score">0.999305</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8797" length="8"/>
+        <text>β-barrel</text>
+      </annotation>
+      <annotation id="232">
+        <infon key="score">0.9993205</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8873" length="8"/>
+        <text>β-barrel</text>
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+      <annotation id="233">
+        <infon key="score">0.99733895</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:05:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8898" length="21"/>
+        <text>doubly-phosphorylated</text>
+      </annotation>
+      <annotation id="545">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:06:45Z</infon>
+        <location offset="8920" length="7"/>
+        <text>peptide</text>
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+      <annotation id="234">
+        <infon key="score">0.99883896</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:03:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8933" length="4"/>
+        <text>SMRT</text>
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+      <annotation id="542">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:06:15Z</infon>
+        <location offset="8941" length="8"/>
+        <text>bound to</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8966" length="6"/>
+        <text>barrel</text>
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+      <annotation id="236">
+        <infon key="score">0.99895597</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8979" length="7"/>
+        <text>strands</text>
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+      <annotation id="237">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T14:42:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8987" length="2"/>
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+      <annotation id="238">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8994" length="2"/>
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+        <infon key="score">0.9890232</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
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+      <annotation id="546">
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:06:45Z</infon>
+        <location offset="9073" length="7"/>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:05:37Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9102" length="10"/>
+        <text>β1-β2 loop</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>9139</offset>
+      <text>The Ku70-Ku80 hetero-dimer is involved in DNA double-strand break repair and the β-barrel domain contributes to DNA binding. In fact, the β-barrel domains of Ku70 and Ku80 form a hetero-dimer, primarily through interactions between the loops connecting the third and fourth strands of the barrel (Fig 3B). The open ends of the two β-barrels face the DNA binding sites, and contact the phosphodiester backbone of the dsDNA. In addition, a long insert connecting strands β2 and β3 in the two domains form an arch-like structure, encircling the dsDNA.</text>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:07:11Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="9143" length="9"/>
+        <text>Ku70-Ku80</text>
+      </annotation>
+      <annotation id="543">
+        <infon key="type">oligomeric_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:06:22Z</infon>
+        <location offset="9153" length="12"/>
+        <text>hetero-dimer</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:35Z</infon>
+        <location offset="9220" length="8"/>
+        <text>β-barrel</text>
+      </annotation>
+      <annotation id="242">
+        <infon key="score">0.81347704</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:07:24Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9251" length="3"/>
+        <text>DNA</text>
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+      <annotation id="533">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:35Z</infon>
+        <location offset="9277" length="8"/>
+        <text>β-barrel</text>
+      </annotation>
+      <annotation id="243">
+        <infon key="score">0.9989766</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:01:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9297" length="4"/>
+        <text>Ku70</text>
+      </annotation>
+      <annotation id="244">
+        <infon key="score">0.99886876</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:01:08Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9306" length="4"/>
+        <text>Ku80</text>
+      </annotation>
+      <annotation id="544">
+        <infon key="type">oligomeric_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:06:22Z</infon>
+        <location offset="9318" length="12"/>
+        <text>hetero-dimer</text>
+      </annotation>
+      <annotation id="245">
+        <infon key="score">0.9985916</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:05:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9375" length="5"/>
+        <text>loops</text>
+      </annotation>
+      <annotation id="246">
+        <infon key="score">0.9872525</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:08:03Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9396" length="24"/>
+        <text>third and fourth strands</text>
+      </annotation>
+      <annotation id="247">
+        <infon key="score">0.99917185</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9428" length="6"/>
+        <text>barrel</text>
+      </annotation>
+      <annotation id="248">
+        <infon key="score">0.9990589</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T14:42:37Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9470" length="9"/>
+        <text>β-barrels</text>
+      </annotation>
+      <annotation id="249">
+        <infon key="score">0.9989037</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:08:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9489" length="17"/>
+        <text>DNA binding sites</text>
+      </annotation>
+      <annotation id="250">
+        <infon key="score">0.99702954</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:06:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9555" length="5"/>
+        <text>dsDNA</text>
+      </annotation>
+      <annotation id="549">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:08:26Z</infon>
+        <location offset="9577" length="11"/>
+        <text>long insert</text>
+      </annotation>
+      <annotation id="251">
+        <infon key="score">0.9117398</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9600" length="7"/>
+        <text>strands</text>
+      </annotation>
+      <annotation id="252">
+        <infon key="score">0.9974274</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T14:42:42Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9608" length="2"/>
+        <text>β2</text>
+      </annotation>
+      <annotation id="253">
+        <infon key="score">0.99721956</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9615" length="2"/>
+        <text>β3</text>
+      </annotation>
+      <annotation id="254">
+        <infon key="score">0.99237406</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:08:32Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9645" length="19"/>
+        <text>arch-like structure</text>
+      </annotation>
+      <annotation id="255">
+        <infon key="score">0.9949091</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:06:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9681" length="5"/>
+        <text>dsDNA</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>9703</offset>
+      <text>Chp1 is a subunit of the RNA-induced initiation of transcriptional gene silencing (RITS) complex. The partner of Chp1, Tas3, is bound between the barrel domain and the second domain of Chp1, and the linker between the two domains is also crucial for this interaction (Fig 3C). It is probably unlikely that the β-barrel itself is sufficient to bind Tas3. Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).</text>
+      <annotation id="256">
+        <infon key="score">0.9983724</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:01:05Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9703" length="4"/>
+        <text>Chp1</text>
+      </annotation>
+      <annotation id="257">
+        <infon key="score">0.74675</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:08:55Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="9728" length="56"/>
+        <text>RNA-induced initiation of transcriptional gene silencing</text>
+      </annotation>
+      <annotation id="258">
+        <infon key="score">0.95602566</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:08:58Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="9786" length="4"/>
+        <text>RITS</text>
+      </annotation>
+      <annotation id="259">
+        <infon key="score">0.99872905</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:01:05Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9816" length="4"/>
+        <text>Chp1</text>
+      </annotation>
+      <annotation id="260">
+        <infon key="score">0.9989574</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:09:02Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9822" length="4"/>
+        <text>Tas3</text>
+      </annotation>
+      <annotation id="261">
+        <infon key="score">0.99926513</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:09:12Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9849" length="13"/>
+        <text>barrel domain</text>
+      </annotation>
+      <annotation id="262">
+        <infon key="score">0.9989255</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T14:42:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9871" length="13"/>
+        <text>second domain</text>
+      </annotation>
+      <annotation id="263">
+        <infon key="score">0.99880016</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:01:05Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9888" length="4"/>
+        <text>Chp1</text>
+      </annotation>
+      <annotation id="264">
+        <infon key="score">0.9988242</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:09:18Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9902" length="6"/>
+        <text>linker</text>
+      </annotation>
+      <annotation id="265">
+        <infon key="score">0.999301</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10013" length="8"/>
+        <text>β-barrel</text>
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+      <annotation id="266">
+        <infon key="score">0.99884015</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:09:03Z</infon>
+        <infon key="identifier">PR:</infon>
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+      </annotation>
+      <annotation id="267">
+        <infon key="score">0.9993482</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:40Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10074" length="4"/>
+        <text>loop</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:09:03Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10082" length="4"/>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:09:23Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10096" length="6"/>
+        <text>strand</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:55Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <infon key="score">0.99932253</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:09:38Z</infon>
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+      <annotation id="538">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:03:59Z</infon>
+        <location offset="10200" length="4"/>
+        <text>SMRT</text>
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+      <annotation id="547">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:06:45Z</infon>
+        <location offset="10205" length="7"/>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:20Z</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="10235" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>10263</offset>
+      <text>Mediator is a coactivator complex that promotes transcription by Pol II. The Med25 subunit ACID is the target of the potent activator VP16 of the herpes simplex virus. The structure of ACID contains a helix at the C-terminus as well as an extended β1-β2 loop. Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands β1 and β3, that is used by the SHARP and Tas3 SPOC domains for binding their partners.</text>
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+        <infon key="type">protein_type</infon>
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+        <infon key="updated_at">2023-09-20T14:43:45Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10263" length="8"/>
+        <text>Mediator</text>
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+        <infon key="updated_at">2023-09-20T12:53:11Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="10328" length="6"/>
+        <text>Pol II</text>
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+        <infon key="updated_at">2023-09-20T13:01:16Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10340" length="5"/>
+        <text>Med25</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:00:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10354" length="4"/>
+        <text>ACID</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:12:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10397" length="4"/>
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:11:35Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10409" length="20"/>
+        <text>herpes simplex virus</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:12:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10435" length="9"/>
+        <text>structure</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:00:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10448" length="4"/>
+        <text>ACID</text>
+      </annotation>
+      <annotation id="282">
+        <infon key="score">0.98497874</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:12:06Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10464" length="5"/>
+        <text>helix</text>
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+      <annotation id="283">
+        <infon key="score">0.99923664</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:05:37Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10511" length="10"/>
+        <text>β1-β2 loop</text>
+      </annotation>
+      <annotation id="284">
+        <infon key="score">0.99889576</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:09:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10540" length="12"/>
+        <text>binding site</text>
+      </annotation>
+      <annotation id="285">
+        <infon key="score">0.9980599</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:12:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10557" length="4"/>
+        <text>VP16</text>
+      </annotation>
+      <annotation id="286">
+        <infon key="score">0.99654675</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10598" length="13"/>
+        <text>surface patch</text>
+      </annotation>
+      <annotation id="287">
+        <infon key="score">0.9965989</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10618" length="7"/>
+        <text>strands</text>
+      </annotation>
+      <annotation id="288">
+        <infon key="score">0.998982</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:12:11Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10626" length="2"/>
+        <text>β1</text>
+      </annotation>
+      <annotation id="289">
+        <infon key="score">0.99914646</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10633" length="2"/>
+        <text>β3</text>
+      </annotation>
+      <annotation id="290">
+        <infon key="score">0.9992305</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10657" length="5"/>
+        <text>SHARP</text>
+      </annotation>
+      <annotation id="291">
+        <infon key="score">0.9992337</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:09:03Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10667" length="4"/>
+        <text>Tas3</text>
+      </annotation>
+      <annotation id="504">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="10672" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>10725</offset>
+      <text>A conserved surface patch in the FPA SPOC domain</text>
+      <annotation id="292">
+        <infon key="score">0.93878067</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:12:35Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10727" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="293">
+        <infon key="score">0.9084494</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10737" length="13"/>
+        <text>surface patch</text>
+      </annotation>
+      <annotation id="294">
+        <infon key="score">0.9349592</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10758" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="505">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="10762" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>10774</offset>
+      <text>An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A). This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B). The first surface patch is electropositive in nature (Fig 4C), and residues Arg477 and Tyr515 are also conserved in the SHARP SPOC domain (Fig 1B). In fact, one of the phosphorylated residues of the SMRT peptide interacts with this surface patch (Fig 3A), suggesting that the FPA SPOC domain might also interact with a phosphorylated segment here. In comparison, the second surface patch is more hydrophobic in nature (Fig 4C).</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="10793" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="295">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10823" length="13"/>
+        <text>surface patch</text>
+      </annotation>
+      <annotation id="296">
+        <infon key="score">0.9886966</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10842" length="7"/>
+        <text>strands</text>
+      </annotation>
+      <annotation id="297">
+        <infon key="score">0.9989058</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:13:17Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10850" length="2"/>
+        <text>β1</text>
+      </annotation>
+      <annotation id="298">
+        <infon key="score">0.9990095</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10854" length="2"/>
+        <text>β3</text>
+      </annotation>
+      <annotation id="299">
+        <infon key="score">0.9991211</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:13:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10858" length="2"/>
+        <text>β5</text>
+      </annotation>
+      <annotation id="300">
+        <infon key="score">0.9990693</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:13:24Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10865" length="2"/>
+        <text>β6</text>
+      </annotation>
+      <annotation id="301">
+        <infon key="score">0.99898845</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:06:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10876" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="302">
+        <infon key="score">0.9987618</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10892" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="303">
+        <infon key="score">0.9980325</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10898" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="304">
+        <infon key="score">0.99669635</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10926" length="13"/>
+        <text>surface patch</text>
+      </annotation>
+      <annotation id="550">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:13:55Z</infon>
+        <location offset="10963" length="11"/>
+        <text>sub-patches</text>
+      </annotation>
+      <annotation id="305">
+        <infon key="score">0.9995813</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:14:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10990" length="6"/>
+        <text>Lys447</text>
+      </annotation>
+      <annotation id="306">
+        <infon key="score">0.9467047</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:13:29Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11001" length="6"/>
+        <text>strand</text>
+      </annotation>
+      <annotation id="307">
+        <infon key="score">0.99921155</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:13:31Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11008" length="2"/>
+        <text>β1</text>
+      </annotation>
+      <annotation id="308">
+        <infon key="score">0.9995802</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:14:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11013" length="6"/>
+        <text>Arg477</text>
+      </annotation>
+      <annotation id="309">
+        <infon key="score">0.99930096</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11021" length="2"/>
+        <text>β3</text>
+      </annotation>
+      <annotation id="310">
+        <infon key="score">0.9995828</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:14:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11026" length="6"/>
+        <text>Tyr515</text>
+      </annotation>
+      <annotation id="311">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:59:03Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11034" length="2"/>
+        <text>αB</text>
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+      <annotation id="312">
+        <infon key="score">0.9995828</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:14:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11042" length="6"/>
+        <text>Arg521</text>
+      </annotation>
+      <annotation id="313">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:14:26Z</infon>
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+      </annotation>
+      <annotation id="551">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:14:48Z</infon>
+        <location offset="11061" length="9"/>
+        <text>sub-patch</text>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:14:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11085" length="6"/>
+        <text>His486</text>
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+      <annotation id="315">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:14:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11093" length="2"/>
+        <text>αA</text>
+      </annotation>
+      <annotation id="316">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:15:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11098" length="6"/>
+        <text>Thr478</text>
+      </annotation>
+      <annotation id="317">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:58:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11106" length="2"/>
+        <text>β3</text>
+      </annotation>
+      <annotation id="318">
+        <infon key="score">0.99960214</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:15:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11111" length="6"/>
+        <text>Val524</text>
+      </annotation>
+      <annotation id="319">
+        <infon key="score">0.99926275</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:15:11Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11119" length="2"/>
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+      </annotation>
+      <annotation id="320">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:15:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11127" length="6"/>
+        <text>Phe534</text>
+      </annotation>
+      <annotation id="321">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:15:21Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11135" length="2"/>
+        <text>β6</text>
+      </annotation>
+      <annotation id="552">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:14:48Z</infon>
+        <location offset="11152" length="9"/>
+        <text>sub-patch</text>
+      </annotation>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:15:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11176" length="19"/>
+        <text>first surface patch</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:15:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11199" length="15"/>
+        <text>electropositive</text>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:14:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11248" length="6"/>
+        <text>Arg477</text>
+      </annotation>
+      <annotation id="325">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:14:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11259" length="6"/>
+        <text>Tyr515</text>
+      </annotation>
+      <annotation id="326">
+        <infon key="score">0.99863356</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:16:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11275" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="327">
+        <infon key="score">0.9354888</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11292" length="5"/>
+        <text>SHARP</text>
+      </annotation>
+      <annotation id="507">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="11298" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="328">
+        <infon key="score">0.9981578</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:15:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11340" length="14"/>
+        <text>phosphorylated</text>
+      </annotation>
+      <annotation id="539">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:03:59Z</infon>
+        <location offset="11371" length="4"/>
+        <text>SMRT</text>
+      </annotation>
+      <annotation id="548">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:06:45Z</infon>
+        <location offset="11376" length="7"/>
+        <text>peptide</text>
+      </annotation>
+      <annotation id="329">
+        <infon key="score">0.997118</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11404" length="13"/>
+        <text>surface patch</text>
+      </annotation>
+      <annotation id="330">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11448" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="508">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="11452" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="331">
+        <infon key="score">0.9987723</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:15:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11491" length="14"/>
+        <text>phosphorylated</text>
+      </annotation>
+      <annotation id="332">
+        <infon key="score">0.867387</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:16:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11539" length="20"/>
+        <text>second surface patch</text>
+      </annotation>
+      <annotation id="333">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:16:27Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11568" length="11"/>
+        <text>hydrophobic</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">pone.0160694.g004.jpg</infon>
+      <infon key="id">pone.0160694.g004</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>11638</offset>
+      <text>A conserved surface patch of FPA SPOC domain.</text>
+      <annotation id="334">
+        <infon key="score">0.92189</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:16:44Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11640" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="335">
+        <infon key="score">0.9779961</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11650" length="13"/>
+        <text>surface patch</text>
+      </annotation>
+      <annotation id="336">
+        <infon key="score">0.70229733</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11667" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="509">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="11671" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">pone.0160694.g004.jpg</infon>
+      <infon key="id">pone.0160694.g004</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>11684</offset>
+      <text>(A). Two views of the molecular surface of FPA SPOC domain colored based on sequence conservation among plant FPA homologs. Purple: most conserved; cyan: least conserved. (B). Residues in the conserved surface patch of FPA SPOC domain. The side chains of the residues are shown in stick models, colored orange in the first sub-patch and green in the second. (C). Molecular surface of FPA SPOC domain colored based on electrostatic potential. Blue: positively charged; red: negatively charged.</text>
+      <annotation id="337">
+        <infon key="score">0.99155676</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11727" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="338">
+        <infon key="score">0.9992993</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11731" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="339">
+        <infon key="score">0.99874276</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11788" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="340">
+        <infon key="score">0.99192095</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11794" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="341">
+        <infon key="score">0.9024767</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:17:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11876" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="342">
+        <infon key="score">0.9458814</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11886" length="13"/>
+        <text>surface patch</text>
+      </annotation>
+      <annotation id="343">
+        <infon key="score">0.9972939</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11903" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="510">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="11907" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="553">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:17:41Z</infon>
+        <location offset="12001" length="15"/>
+        <text>first sub-patch</text>
+      </annotation>
+      <annotation id="344">
+        <infon key="score">0.9975604</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12068" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="345">
+        <infon key="score">0.9992748</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12072" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>12177</offset>
+      <text>Testing the requirement of specific conserved amino acids for FPA functions</text>
+      <annotation id="346">
+        <infon key="score">0.9983504</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12239" length="3"/>
+        <text>FPA</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>12253</offset>
+      <text>We next examined the potential impact of the conserved surface patch on FPA function in vivo. We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain (Fig 1B) and were found to be functionally important. The mutations were introduced into a transgene designed to express FPA from its native control elements (promoter, introns and 3′ UTR). The resulting transgenes were then stably transformed into an fpa-8 mutant background so that the impact of the mutations on FPA function could be assessed. Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig). We examined independent transgenic lines expressing each R477A and Y515A mutation. In each case, we confirmed that detectable levels of FPA protein expression were restored close to wild-type levels in protein blot analyses using antibodies that specifically recognize FPA (S2 Fig).</text>
+      <annotation id="347">
+        <infon key="score">0.9957634</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:17:56Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12298" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="348">
+        <infon key="score">0.99864554</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12308" length="13"/>
+        <text>surface patch</text>
+      </annotation>
+      <annotation id="349">
+        <infon key="score">0.9974778</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="updated_at">2023-09-20T13:20:34Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>protein blot</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="13391" length="3"/>
+        <text>FPA</text>
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+      <infon key="type">paragraph</infon>
+      <offset>13409</offset>
+      <text>We then examined the impact of the surface patch mutations on FPA’s function in controlling RNA 3′-end formation by determining whether the mutant proteins functioned in FPA autoregulation and the repression of FLC expression. FPA autoregulates its expression by promoting cleavage and polyadenylation within intron 1 of its own pre-mRNA, resulting in a truncated transcript that does not encode functional protein. We used RNA gel blot analyses to reveal that in each of three independent transgenic lines for each single mutant, rescue of proximally polyadenylated FPA pre-mRNA can be detected (Fig 5A and 5B). We therefore conclude that neither of these mutations disrupted the ability of FPA to promote RNA 3′-end formation in its own transcript.</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13444" length="13"/>
+        <text>surface patch</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>mutations</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="13471" length="3"/>
+        <text>FPA</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:21:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="13579" length="3"/>
+        <text>FPA</text>
+      </annotation>
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+        <infon key="type">gene</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:56:39Z</infon>
+        <infon key="identifier">GENE:</infon>
+        <location offset="13620" length="3"/>
+        <text>FLC</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <text>FPA</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:19:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13738" length="8"/>
+        <text>pre-mRNA</text>
+      </annotation>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:29:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="13833" length="21"/>
+        <text>RNA gel blot analyses</text>
+      </annotation>
+      <annotation id="390">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:21:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13932" length="6"/>
+        <text>mutant</text>
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+      <annotation id="391">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="13976" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="392">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:19:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13980" length="8"/>
+        <text>pre-mRNA</text>
+      </annotation>
+      <annotation id="393">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="14101" length="3"/>
+        <text>FPA</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">pone.0160694.g005.jpg</infon>
+      <infon key="id">pone.0160694.g005</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>14166</offset>
+      <text>Impact of individual FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA.</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="14187" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="512">
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="14191" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="395">
+        <infon key="score">0.48791844</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:08:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14203" length="9"/>
+        <text>mutations</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="14247" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="397">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:19:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14251" length="8"/>
+        <text>pre-mRNA</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">pone.0160694.g005.jpg</infon>
+      <infon key="id">pone.0160694.g005</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>14261</offset>
+      <text>RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A
+(A), or FPA::FPA Y515A
+(B) using poly(A)+ purified mRNAs. A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs. RNA size (kb) marker (Ambion). TUBULIN was detected as an internal control. Proximally and distally polyadenylated FPA transcripts are marked with arrows. The ratio of distal:proximal polyadenylated forms is given under each lane. (C,D) Impact of individual FPA SPOC domain mutations on FLC transcript levels. qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A
+(C), FPA::FPA Y515A
+(D) plants. Transcript levels were normalized to the control UBC. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.</text>
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+      <text>We next examined whether the corresponding mutations disrupted the ability of FPA to control FLC expression. We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).</text>
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+        <infon key="annotator">cleaner0</infon>
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+    </passage>
+    <passage>
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+      <infon key="type">paragraph</infon>
+      <offset>15573</offset>
+      <text>Since each surface patch mutation appeared to be insufficient to disrupt FPA functions on its own, we combined both mutations into the same transgene. We could again confirm that near wild-type levels of FPA protein were expressed from three independent transgenic lines expressing the FPA R477A;Y515A doubly mutated protein in an fpa-8 mutant background (S3 Fig). We found that FPA R477A;Y515A protein functioned like wild-type FPA to restore FPA pre-mRNA proximal polyadenylation (Fig 6A) and FLC expression to wild-type levels (Fig 6B).</text>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="updated_at">2023-09-20T15:08:18Z</infon>
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+        <text>FPA</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:19:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15757" length="9"/>
+        <text>wild-type</text>
+      </annotation>
+      <annotation id="442">
+        <infon key="score">0.9967384</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="15777" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="560">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T14:32:13Z</infon>
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+        <text>FPA R477A;Y515A</text>
+      </annotation>
+      <annotation id="443">
+        <infon key="score">0.98018205</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:07:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15875" length="14"/>
+        <text>doubly mutated</text>
+      </annotation>
+      <annotation id="444">
+        <infon key="score">0.6438746</infon>
+        <infon key="type">gene</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:18:58Z</infon>
+        <infon key="identifier">GENE:</infon>
+        <location offset="15904" length="5"/>
+        <text>fpa-8</text>
+      </annotation>
+      <annotation id="445">
+        <infon key="score">0.9978872</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:21:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15910" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="561">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T14:32:13Z</infon>
+        <location offset="15952" length="15"/>
+        <text>FPA R477A;Y515A</text>
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+      <annotation id="446">
+        <infon key="score">0.99899787</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:19:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15992" length="9"/>
+        <text>wild-type</text>
+      </annotation>
+      <annotation id="447">
+        <infon key="score">0.9988796</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16002" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="448">
+        <infon key="score">0.9158664</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16017" length="3"/>
+        <text>FPA</text>
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+      <annotation id="449">
+        <infon key="score">0.9955418</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:19:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16021" length="8"/>
+        <text>pre-mRNA</text>
+      </annotation>
+      <annotation id="529">
+        <infon key="type">gene</infon>
+        <infon key="identifier">GENE:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:56:39Z</infon>
+        <location offset="16068" length="3"/>
+        <text>FLC</text>
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+      <annotation id="450">
+        <infon key="score">0.9989771</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:19:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16086" length="9"/>
+        <text>wild-type</text>
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+      <infon key="file">pone.0160694.g006.jpg</infon>
+      <infon key="id">pone.0160694.g006</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>16113</offset>
+      <text>Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and FLC expression.</text>
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+        <infon key="score">0.8876505</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16130" length="3"/>
+        <text>FPA</text>
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+      <annotation id="514">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="16134" length="4"/>
+        <text>SPOC</text>
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+        <infon key="score">0.4253289</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:08:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16146" length="9"/>
+        <text>mutations</text>
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+      <annotation id="453">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16190" length="3"/>
+        <text>FPA</text>
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+      <annotation id="454">
+        <infon key="score">0.9954029</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:19:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16194" length="8"/>
+        <text>pre-mRNA</text>
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+      <annotation id="455">
+        <infon key="score">0.6315412</infon>
+        <infon key="type">gene</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:56:39Z</infon>
+        <infon key="identifier">GENE:</infon>
+        <location offset="16207" length="3"/>
+        <text>FLC</text>
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+    <passage>
+      <infon key="file">pone.0160694.g006.jpg</infon>
+      <infon key="id">pone.0160694.g006</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>16223</offset>
+      <text>(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs. Black arrows indicate the proximally and distally polyadenylated FPA mRNAs. A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs. RNA size (kb) marker (Ambion). TUBULIN was detected as an internal control. The ratio of distal:proximal polyadenylated forms is given under each lane. (B). qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, and FPA::FPA R477A;Y515A plants. Transcript levels were normalized to the control UBC. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:08:41Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16227" length="12"/>
+        <text>RNA gel blot</text>
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+        <infon key="score">0.98714185</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:31:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16252" length="2"/>
+        <text>WT</text>
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:55:00Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16255" length="11"/>
+        <text>A. thaliana</text>
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+        <infon key="score">0.5267927</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:51:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16294" length="6"/>
+        <text>plants</text>
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+      <annotation id="460">
+        <infon key="score">0.7329895</infon>
+        <infon key="type">gene</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:18:58Z</infon>
+        <infon key="identifier">GENE:</infon>
+        <location offset="16301" length="5"/>
+        <text>fpa-8</text>
+      </annotation>
+      <annotation id="461">
+        <infon key="score">0.8120683</infon>
+        <infon key="type">gene</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:18:58Z</infon>
+        <infon key="identifier">GENE:</infon>
+        <location offset="16311" length="5"/>
+        <text>fpa-8</text>
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+      <annotation id="462">
+        <infon key="score">0.9203812</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:30:52Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16317" length="7"/>
+        <text>mutants</text>
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+        <infon key="score">0.9772659</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16336" length="3"/>
+        <text>FPA</text>
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+      <annotation id="562">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T14:32:13Z</infon>
+        <location offset="16341" length="15"/>
+        <text>FPA R477A;Y515A</text>
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+      <annotation id="464">
+        <infon key="score">0.99847776</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:53:05Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16381" length="5"/>
+        <text>mRNAs</text>
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+      <annotation id="465">
+        <infon key="score">0.95337856</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16453" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="466">
+        <infon key="score">0.9983594</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:53:05Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16457" length="5"/>
+        <text>mRNAs</text>
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+      <annotation id="467">
+        <infon key="score">0.94853</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16509" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="468">
+        <infon key="score">0.99780947</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T14:31:21Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16513" length="4"/>
+        <text>mRNA</text>
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+      <annotation id="469">
+        <infon key="score">0.9346011</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16537" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="470">
+        <infon key="score">0.99581194</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:53:05Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16550" length="5"/>
+        <text>mRNAs</text>
+      </annotation>
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+        <infon key="score">0.99899334</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T14:30:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16714" length="7"/>
+        <text>qRT-PCR</text>
+      </annotation>
+      <annotation id="564">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T14:34:56Z</infon>
+        <location offset="16756" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="555">
+        <infon key="type">gene</infon>
+        <infon key="identifier">GENE:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:18:58Z</infon>
+        <location offset="16781" length="5"/>
+        <text>fpa-8</text>
+      </annotation>
+      <annotation id="472">
+        <infon key="score">0.9812045</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16792" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="473">
+        <infon key="score">0.94803566</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T14:32:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16797" length="15"/>
+        <text>FPA R477A;Y515A</text>
+      </annotation>
+      <annotation id="474">
+        <infon key="score">0.9977831</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:51:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16813" length="6"/>
+        <text>plants</text>
+      </annotation>
+      <annotation id="565">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T14:35:12Z</infon>
+        <location offset="16875" length="10"/>
+        <text>Histograms</text>
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+        <infon key="score">0.9770333</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:08:45Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16929" length="3"/>
+        <text>PCR</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>16983</offset>
+      <text>Together our findings suggest that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation, or that this combination of mutations is not sufficient to critically disrupt the function of the FPA SPOC domain. Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain.</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17029" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="477">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17073" length="3"/>
+        <text>FPA</text>
+      </annotation>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:05:29Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="17091" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="479">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:08:48Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17141" length="9"/>
+        <text>mutations</text>
+      </annotation>
+      <annotation id="480">
+        <infon key="score">0.60978085</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17211" length="3"/>
+        <text>FPA</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="17215" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="481">
+        <infon key="score">0.69148296</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:08:51Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17252" length="9"/>
+        <text>mutations</text>
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+        <infon key="score">0.99684554</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:52:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17269" length="5"/>
+        <text>SHARP</text>
+      </annotation>
+      <annotation id="483">
+        <infon key="score">0.99948525</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17275" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="484">
+        <infon key="score">0.99606663</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T15:07:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17317" length="16"/>
+        <text>unphosphorylated</text>
+      </annotation>
+      <annotation id="540">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T13:03:59Z</infon>
+        <location offset="17334" length="4"/>
+        <text>SMRT</text>
+      </annotation>
+      <annotation id="566">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T14:35:45Z</infon>
+        <location offset="17339" length="8"/>
+        <text>peptides</text>
+      </annotation>
+      <annotation id="485">
+        <infon key="score">0.79536206</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17430" length="3"/>
+        <text>FPA</text>
+      </annotation>
+      <annotation id="516">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="17434" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+      <annotation id="517">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:49:04Z</infon>
+        <location offset="17501" length="4"/>
+        <text>SPOC</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_1</infon>
+      <offset>17516</offset>
+      <text>Materials and Methods</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>17538</offset>
+      <text>Protein expression and purification</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>17574</offset>
+      <text>The SPOC domain (residue 433–565) of A. thaliana FPA was sub-cloned into the pET28a vector (Novagen). The recombinant protein, with an N-terminal hexa-histidine tag, was over-expressed in E. coli BL21 Star (DE3) cells (Novagen), which were induced with 0.4 mM IPTG and allowed to grow at 20°C for 14–18 h. The soluble protein was purified by nickel-charged immobilized-metal affinity chromatography and gel filtration chromatography. The purified protein was concentrated and stored at –80°C in a buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 10 mM DTT and 5% (v/v) glycerol. The His-tag was not removed for crystallization.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>18213</offset>
+      <text>The selenomethionine labeled SPOC domain was expressed in E. coli B834(DE3) strain using LeMaster media and purified with the same protocol as the native protein.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>18376</offset>
+      <text>Protein crystallization</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>18400</offset>
+      <text>Crystals of the native SPOC domain of FPA were grown at 20°C with the sitting-drop vapor diffusion method. The protein solution was at 30 mg/ml concentration, and the reservoir solution contained 0.2 M MgSO4, and 20% (v/v) PEG 3350. Fully-grown crystals were obtained two days after set-up. Crystals of the selenomethionine labeled SPOC domain were grown using the same condition as the native protein. The crystals were cryo-protected in the crystallization solution supplemented with 20% (v/v) glycerol and flash-frozen in liquid nitrogen for data collection at 100K.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>18971</offset>
+      <text>Data collection and processing</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>19002</offset>
+      <text>A single-wavelength anomalous dispersion (SAD) X-ray diffraction data set on a selenomethionine labeled SPOC domain crystal was collected at the National Synchrotron Light Source (NSLS) beamline X29A using an ADSC Q315r CCD. The diffraction images were processed and scaled with the HKL package. The crystal belongs to space group P65, with unit cell parameters of a = b = 108.2 Å, and c = 34.2 Å.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>19402</offset>
+      <text>Structure determination and refinement</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>19441</offset>
+      <text>The structure of the SPOC domain was solved by the selenomethionyl SAD method with the program SHELX. The phases were used by program PHENIX for automatic model building. Manual model rebuilding was carried out with Coot. The structure refinement was performed with the program PHENIX, with translation, libration, and screw-rotation (TLS) parameters. The data processing and refinement statistics are summarized in Table 1. The Ramachandran plot showed that 95.8% of the residues are located in the most favored regions, and 4.2% are in additional allowed regions.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>20007</offset>
+      <text>Generation of constructs with mutated genomic FPA sequence</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>20066</offset>
+      <text>A series of constructs containing a mutated FPA genomic sequence was prepared based on pGreen I 0029 vector. pGreen I 0029 vector with inserted FPA genomic sequence was prepared. In this vector FPA genomic sequence is flanked by 2620bp of the native sequence upstream to the start codon and 1178bp downstream to the stop codon. The vector contains kanamycin resistance genes for both the bacteria and plant hosts. In order to obtain a series of constructs with mutated FPA genomic sequence, FPA sequence in this construct was modified using site-directed mutagenesis. Primers used to prepare required constructs are listed in S1 Table. After the mutagenesis reaction the presence of only the desired mutations was confirmed by sequencing of the whole FPA genomic sequence and flanking regions.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>20860</offset>
+      <text>Generation of Arabidopsis thaliana transgenic plants</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>20913</offset>
+      <text>All transgenic plants were prepared in fpa-8 mutant background, which is in Col-0 accession. The prepared vectors for Arabidopsis transformations were introduced into electro-competent Agrobacterium tumefaciens cells (C58 CV3101 strain harbouring pSoup vector). The floral dip method was used for plant transformation. Transgenic plants were selected using kanamycin as a selection marker. Presence of the desired mutations in plants was confirmed with specific dCaps markers.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>21390</offset>
+      <text>Plant growth conditions</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>21414</offset>
+      <text>Wild type Col-0 plants used in this study were obtained from the Nottingham Arabidopsis Stock Centre. Seed of fpa-8 and 35S::FPA:YFP were obtained from Professor Caroline Dean. Plants were grown in pots containing Universal Extra general purpose soil. The glasshouse temperature was maintained at 20°C and the 16 hour daylight was provided by high pressure sodium vapour lamps (Philips Powertone SON-T AGRO 400). In order to grow plants in sterile conditions, seeds were first surface sterilized by a 5 min treatment with sterilizing solution (3% v/v sodium hypochlorite, 0.02% v/v Triton X-100), followed by three washes with 0.02% v/v Triton X-100 and one wash with sterile water. The sterile seeds were sown on MS10 media supplemented with 0.8% w/v agar. MS10 medium was also supplemented with specific antibiotics if required. After sowing, the seeds were stratified at 4°C for two days in order to synchronize their germination. Plants were grown in the tissue culture room at the following conditions: temperature 22°C, 16 hours daylight provided by the Master TL-D 36W/840 (Philips) lamps.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>22514</offset>
+      <text>Plant protein analysis</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>22537</offset>
+      <text>Total protein samples were prepared using extraction buffer containing: 40 mM Tris-HCl, pH 6.8; 0.1 mM EDTA, pH 8.0; 8 M urea; 1.43 M β-mercaptoethanol, 7% v/v Complete Protease Inhibitors (Roche) and 5 mM PMSF. Equal volumes of samples were separated on 8% SDS-PAGE. Proteins were transferred onto Protran nitrocellulose transfer membrane (Whatman) using wet Criterion blotter system (BioRad). The transfer was performed at room temperature for two hours at a stable voltage of 70 V. Membrane was blocked in 3% (w/v) Milk in TBS for 1h at room temperature followed by overnight incubation with anti-FPA antibody (dilution 1:100 in 3% (w/v) Milk in TBS). After washes the membrane was incubated for 75 min with goat anti-rabbit antibody (Thermo Scientific) (1:3000 dilution in 3% (w/v) Milk in TBS). Protein was detected using SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific). Blots were re-probed following treatment with low pH solution (25mM glycine-HCl, pH 2, 1% (w/v) SDS) followed by blocking for 1h at room temperature in 3% (w/v) Milk in TBS. The membrane was incubated overnight with anti-TUBB2A, tubulin, beta 2A antibody (ARP40177_P050 Aviva systems biology; (dilution 1:1000 in 3% (w/v) Milk in TBS). After washes the membrane was incubated for 75 min with goat anti-rabbit antibody (Thermo Scientific) [1:3000 dilution in 3% (w/v) Milk in TBS]. Signal was detected using SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>24024</offset>
+      <text>RNA gel blot analysis and RT-qPCR</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>24058</offset>
+      <text>RNA gel blot analysis and RT-qPCR method performed as previously described.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">title_1</infon>
+      <offset>24134</offset>
+      <text>Supporting Information</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REF</infon>
+      <infon key="type">title</infon>
+      <offset>24157</offset>
+      <text>References</text>
+    </passage>
+    <passage>
+      <infon key="fpage">688</infon>
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+      <offset>24168</offset>
+      <text>Pre-mRNA processing reaches back to transcription and ahead to translation</text>
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+    </passage>
+    <passage>
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+      <infon key="type">ref</infon>
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+      <offset>24776</offset>
+      <text>Split end family RNA binding proteins: novel tumor suppressors coupling transcriptional regulation with RNA processing</text>
+    </passage>
+    <passage>
+      <infon key="fpage">91</infon>
+      <infon key="name_0">surname:Sanchez-Pulido;given-names:L</infon>
+      <infon key="name_1">surname:Rojas;given-names:AM</infon>
+      <infon key="name_2">surname:van Wely;given-names:KH</infon>
+      <infon key="name_3">surname:Martinez-A;given-names:C</infon>
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+      <offset>24895</offset>
+      <text>SPOC: a widely distributed domain associated with cancer, apoptosis and transcription</text>
+    </passage>
+    <passage>
+      <infon key="fpage">6742</infon>
+      <infon key="lpage">6752</infon>
+      <infon key="name_0">surname:Arieti;given-names:F</infon>
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+      <infon key="section_type">REF</infon>
+      <infon key="source">Nucl Acids Res</infon>
+      <infon key="type">ref</infon>
+      <infon key="volume">42</infon>
+      <infon key="year">2014</infon>
+      <offset>24981</offset>
+      <text>The crystal structure of the Split End protein SHARP adds a new layer of complexity to proteins containing RNA recognition motifs</text>
+    </passage>
+    <passage>
+      <infon key="fpage">1427</infon>
+      <infon key="lpage">1436</infon>
+      <infon key="name_0">surname:Schomburg;given-names:FM</infon>
+      <infon key="name_1">surname:Patton;given-names:DA</infon>
+      <infon key="name_2">surname:Meinke;given-names:DW</infon>
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+      <infon key="pub-id_pmid">11402170</infon>
+      <infon key="section_type">REF</infon>
+      <infon key="source">Plant Cell</infon>
+      <infon key="type">ref</infon>
+      <infon key="volume">13</infon>
+      <infon key="year">2001</infon>
+      <offset>25111</offset>
+      <text>FPA, a gene involved in floral induction in Arabidopsis, encodes a protein containing RNA-recognition motifs</text>
+    </passage>
+    <passage>
+      <infon key="fpage">203</infon>
+      <infon key="lpage">213</infon>
+      <infon key="name_0">surname:Hornyik;given-names:C</infon>
+      <infon key="name_1">surname:Terzi;given-names:LC</infon>
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+      <infon key="pub-id_doi">10.1016/j.devcel.2009.12.009</infon>
+      <infon key="pub-id_pmid">20079695</infon>
+      <infon key="section_type">REF</infon>
+      <infon key="source">Dev Cell</infon>
+      <infon key="type">ref</infon>
+      <infon key="volume">18</infon>
+      <infon key="year">2010</infon>
+      <offset>25220</offset>
+      <text>The spen family protein FPA controls alternative cleavage and polyadenylation of RNA</text>
+    </passage>
+    <passage>
+      <infon key="fpage">1077</infon>
+      <infon key="lpage">1081</infon>
+      <infon key="name_0">surname:Hornyik;given-names:C</infon>
+      <infon key="name_1">surname:Duc;given-names:C</infon>
+      <infon key="name_2">surname:Rataj;given-names:K</infon>
+      <infon key="name_3">surname:Terzi;given-names:LC</infon>
+      <infon key="name_4">surname:Simpson;given-names:GG</infon>
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+      <infon key="pub-id_pmid">20659007</infon>
+      <infon key="section_type">REF</infon>
+      <infon key="source">Biochem Soc Trans</infon>
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+      <text>N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.</text>
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+        <infon key="score">0.9378259</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="96" length="22"/>
+        <text>N-terminal acetylation</text>
+      </annotation>
+      <annotation id="6">
+        <infon key="score">0.9623558</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:09Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="120" length="14"/>
+        <text>Nt-acetylation</text>
+      </annotation>
+      <annotation id="7">
+        <infon key="score">0.99854547</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="152" length="29"/>
+        <text>N-terminal acetyltransferases</text>
+      </annotation>
+      <annotation id="8">
+        <infon key="score">0.99888486</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="183" length="4"/>
+        <text>NATs</text>
+      </annotation>
+      <annotation id="985">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:45:09Z</infon>
+        <location offset="241" length="7"/>
+        <text>peptide</text>
+      </annotation>
+      <annotation id="9">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="257" length="5"/>
+        <text>Naa60</text>
+      </annotation>
+      <annotation id="10">
+        <infon key="score">0.99875224</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:55:36Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="275" length="4"/>
+        <text>NatF</text>
+      </annotation>
+      <annotation id="11">
+        <infon key="score">0.99907637</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:27Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="306" length="3"/>
+        <text>NAT</text>
+      </annotation>
+      <annotation id="12">
+        <infon key="score">0.97256976</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="324" length="24"/>
+        <text>multicellular eukaryotes</text>
+      </annotation>
+      <annotation id="13">
+        <infon key="score">0.96585155</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="430" length="14"/>
+        <text>Nt-acetylation</text>
+      </annotation>
+      <annotation id="14">
+        <infon key="score">0.9983275</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="492" length="27"/>
+        <text>lysine Nε-acetyltransferase</text>
+      </annotation>
+      <annotation id="15">
+        <infon key="score">0.99864644</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="521" length="3"/>
+        <text>KAT</text>
+      </annotation>
+      <annotation id="16">
+        <infon key="score">0.99024945</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:48:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="551" length="11"/>
+        <text>acetylation</text>
+      </annotation>
+      <annotation id="17">
+        <infon key="score">0.99478674</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:13Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="566" length="6"/>
+        <text>lysine</text>
+      </annotation>
+      <annotation id="18">
+        <infon key="score">0.9975256</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="602" length="18"/>
+        <text>crystal structures</text>
+      </annotation>
+      <annotation id="19">
+        <infon key="score">0.99812895</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="624" length="5"/>
+        <text>human</text>
+      </annotation>
+      <annotation id="20">
+        <infon key="score">0.9994475</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="630" length="5"/>
+        <text>Naa60</text>
+      </annotation>
+      <annotation id="21">
+        <infon key="score">0.99933773</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="637" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="22">
+        <infon key="score">0.99842304</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="645" length="15"/>
+        <text>in complex with</text>
+      </annotation>
+      <annotation id="23">
+        <infon key="score">0.99915624</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="661" length="17"/>
+        <text>Acetyl-Coenzyme A</text>
+      </annotation>
+      <annotation id="24">
+        <infon key="score">0.9992083</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:03Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="680" length="6"/>
+        <text>Ac-CoA</text>
+      </annotation>
+      <annotation id="25">
+        <infon key="score">0.99916434</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:09Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="691" length="10"/>
+        <text>Coenzyme A</text>
+      </annotation>
+      <annotation id="26">
+        <infon key="score">0.999223</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:14Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="703" length="3"/>
+        <text>CoA</text>
+      </annotation>
+      <annotation id="27">
+        <infon key="score">0.99931455</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="713" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="967">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:48Z</infon>
+        <location offset="740" length="17"/>
+        <text>amphipathic helix</text>
+      </annotation>
+      <annotation id="28">
+        <infon key="score">0.99922186</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:29:42Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="772" length="11"/>
+        <text>GNAT domain</text>
+      </annotation>
+      <annotation id="29">
+        <infon key="score">0.9993635</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="829" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="30">
+        <infon key="score">0.99896705</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="845" length="13"/>
+        <text>β7-β8 hairpin</text>
+      </annotation>
+      <annotation id="31">
+        <infon key="score">0.9745685</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="898" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="32">
+        <infon key="score">0.89123774</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:24:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="905" length="5"/>
+        <text>1-242</text>
+      </annotation>
+      <annotation id="1042">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:32:03Z</infon>
+        <location offset="916" length="13"/>
+        <text>hNaa60(1-199)</text>
+      </annotation>
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+        <infon key="score">0.9984098</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="930" length="18"/>
+        <text>crystal structures</text>
+      </annotation>
+      <annotation id="34">
+        <infon key="score">0.99716777</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="994" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="932">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:13:42Z</infon>
+        <location offset="1035" length="8"/>
+        <text>coenzyme</text>
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+        <infon key="score">0.98665667</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:35:25Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1150" length="45"/>
+        <text>structural comparison and biochemical studies</text>
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+      <annotation id="36">
+        <infon key="score">0.9976251</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1211" length="6"/>
+        <text>Tyr 97</text>
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+      <annotation id="37">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:48:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1222" length="7"/>
+        <text>His 138</text>
+      </annotation>
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+        <infon key="score">0.9988678</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:26:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1281" length="13"/>
+        <text>non-conserved</text>
+      </annotation>
+      <annotation id="39">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:50Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="1295" length="15"/>
+        <text>β3-β4 long loop</text>
+      </annotation>
+      <annotation id="40">
+        <infon key="score">0.9993338</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="1345" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>1386</offset>
+      <text>Acetylation is one of the most ubiquitous modifications that plays a vital role in many biological processes, such as transcriptional regulation, protein-protein interaction, enzyme activity, protein stability, antibiotic resistance, biological rhythm and so on. Protein acetylation can be grouped into lysine Nε-acetylation and peptide N-terminal acetylation (Nt-acetylation). Generally, Nε-acetylation refers to the transfer of an acetyl group from an acetyl coenzyme A (Ac-CoA) to the ε-amino group of lysine. This kind of modification is catalyzed by lysine acetyltransferases (KATs), some of which are named histone acetyltransferases (HATs) because early studies focused mostly on the post-transcriptional acetylation of histones.</text>
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+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:48:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1386" length="11"/>
+        <text>Acetylation</text>
+      </annotation>
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+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:48:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1657" length="11"/>
+        <text>acetylation</text>
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+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:24:40Z</infon>
+        <location offset="1689" length="21"/>
+        <text>lysine Nε-acetylation</text>
+      </annotation>
+      <annotation id="986">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:45:09Z</infon>
+        <location offset="1715" length="7"/>
+        <text>peptide</text>
+      </annotation>
+      <annotation id="894">
+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:03Z</infon>
+        <location offset="1723" length="22"/>
+        <text>N-terminal acetylation</text>
+      </annotation>
+      <annotation id="43">
+        <infon key="score">0.9702212</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1747" length="14"/>
+        <text>Nt-acetylation</text>
+      </annotation>
+      <annotation id="44">
+        <infon key="score">0.97078913</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:48:53Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1775" length="14"/>
+        <text>Nε-acetylation</text>
+      </annotation>
+      <annotation id="1021">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:19:06Z</infon>
+        <location offset="1819" length="6"/>
+        <text>acetyl</text>
+      </annotation>
+      <annotation id="45">
+        <infon key="score">0.9988362</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:13:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1840" length="17"/>
+        <text>acetyl coenzyme A</text>
+      </annotation>
+      <annotation id="46">
+        <infon key="score">0.9988877</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1859" length="6"/>
+        <text>Ac-CoA</text>
+      </annotation>
+      <annotation id="47">
+        <infon key="score">0.9945634</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:24:44Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="1891" length="6"/>
+        <text>lysine</text>
+      </annotation>
+      <annotation id="48">
+        <infon key="score">0.9981992</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:49:17Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1941" length="25"/>
+        <text>lysine acetyltransferases</text>
+      </annotation>
+      <annotation id="49">
+        <infon key="score">0.9988122</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:49:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1968" length="4"/>
+        <text>KATs</text>
+      </annotation>
+      <annotation id="50">
+        <infon key="score">0.9982982</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:49:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1999" length="26"/>
+        <text>histone acetyltransferases</text>
+      </annotation>
+      <annotation id="51">
+        <infon key="score">0.9989518</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:49:33Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2027" length="4"/>
+        <text>HATs</text>
+      </annotation>
+      <annotation id="52">
+        <infon key="score">0.97209585</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:48:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2098" length="11"/>
+        <text>acetylation</text>
+      </annotation>
+      <annotation id="53">
+        <infon key="score">0.66079366</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:49:40Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2113" length="8"/>
+        <text>histones</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>2141</offset>
+      <text>Despite the prominent accomplishments in the field regarding Nε-acetylation by KATs for over 50 years, the significance of the more evolutionarily conserved Nt-acetylation is still inconclusive. Nt-acetylation is an abundant and evolutionarily conserved modification occurring in bacteria, archaea and eukaryotes. It is estimated that about 80–90% of soluble human proteins and 50–70% of yeast proteins are subjected to Nt-acetylation, where an acetyl moiety is transferred from Ac-CoA to the α-amino group of the first residue. Recently Nt-acetylome expands the Nt-acetylation to transmembrane proteins. Unlike Nε-acetylation that can be eliminated by deacetylases, Nt-acetylation is considered irreversible since no corresponding deacetylase is found to date. Although Nt-acetylation has been regarded as a co-translational modification traditionally, there is evidence that post-translational Nt-acetylation exists. During the past decades, a large number of Nt-acetylome researches have shed light on the functional roles of Nt-acetylation, including protein degradation, subcellular localization, protein-protein interaction, protein-membrane interaction, plant development, stress-response and protein stability.</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:48:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2202" length="14"/>
+        <text>Nε-acetylation</text>
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+      <annotation id="55">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:49:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2220" length="4"/>
+        <text>KATs</text>
+      </annotation>
+      <annotation id="56">
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+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2298" length="14"/>
+        <text>Nt-acetylation</text>
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+      <annotation id="57">
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+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2336" length="14"/>
+        <text>Nt-acetylation</text>
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+      <annotation id="58">
+        <infon key="score">0.9987232</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:24:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2421" length="8"/>
+        <text>bacteria</text>
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+      <annotation id="59">
+        <infon key="score">0.9986106</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:24:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2431" length="7"/>
+        <text>archaea</text>
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+      <annotation id="60">
+        <infon key="score">0.9983525</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:24:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2443" length="10"/>
+        <text>eukaryotes</text>
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+      <annotation id="61">
+        <infon key="score">0.9985355</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2500" length="5"/>
+        <text>human</text>
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+      <annotation id="62">
+        <infon key="score">0.9988913</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:50:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2529" length="5"/>
+        <text>yeast</text>
+      </annotation>
+      <annotation id="63">
+        <infon key="score">0.97266906</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2561" length="14"/>
+        <text>Nt-acetylation</text>
+      </annotation>
+      <annotation id="64">
+        <infon key="score">0.7109769</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:19:06Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2586" length="6"/>
+        <text>acetyl</text>
+      </annotation>
+      <annotation id="65">
+        <infon key="score">0.99906653</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2620" length="6"/>
+        <text>Ac-CoA</text>
+      </annotation>
+      <annotation id="66">
+        <infon key="score">0.96215796</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2704" length="14"/>
+        <text>Nt-acetylation</text>
+      </annotation>
+      <annotation id="67">
+        <infon key="score">0.9716828</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:48:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2753" length="14"/>
+        <text>Nε-acetylation</text>
+      </annotation>
+      <annotation id="68">
+        <infon key="score">0.99580246</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:50:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2794" length="12"/>
+        <text>deacetylases</text>
+      </annotation>
+      <annotation id="69">
+        <infon key="score">0.97022635</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2808" length="14"/>
+        <text>Nt-acetylation</text>
+      </annotation>
+      <annotation id="70">
+        <infon key="score">0.90664417</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:26:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2837" length="12"/>
+        <text>irreversible</text>
+      </annotation>
+      <annotation id="71">
+        <infon key="score">0.9765928</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:50:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2873" length="11"/>
+        <text>deacetylase</text>
+      </annotation>
+      <annotation id="72">
+        <infon key="score">0.9757602</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2912" length="14"/>
+        <text>Nt-acetylation</text>
+      </annotation>
+      <annotation id="73">
+        <infon key="score">0.9744993</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3037" length="14"/>
+        <text>Nt-acetylation</text>
+      </annotation>
+      <annotation id="74">
+        <infon key="score">0.9702752</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3170" length="14"/>
+        <text>Nt-acetylation</text>
+      </annotation>
+      <annotation id="75">
+        <infon key="score">0.99510866</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:51:24Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3302" length="5"/>
+        <text>plant</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>3376</offset>
+      <text>The Nt-acetylation is carried out by N-terminal acetyltransferases (NATs) that belong to the GNAT superfamily. To date, six NATs (NatA/B/C/D/E/F) have been identified in eukaryotes. About 40 percent of Nt-acetylation of soluble proteins in cells is catalyzed by NatA complex which is composed of the catalytic subunit Naa10p and the auxiliary subunit Naa15p. NatE was found to physically interact with the NatA complex without any observation of impact on NatA-activity. Two other multimeric complexes of NATs are NatB and NatC which contain the catalytic subunits Naa20 and Naa30 and the auxiliary subunits Naa25 and Naa35/Naa38, respectively. Furthermore, only the catalytic subunits Naa40 and Naa60 were found for NatD and NatF, respectively. Besides Nt-acetylation, accumulating reports have proposed Nε-acetylation carried out by NATs.</text>
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+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3380" length="14"/>
+        <text>Nt-acetylation</text>
+      </annotation>
+      <annotation id="77">
+        <infon key="score">0.99842566</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3413" length="29"/>
+        <text>N-terminal acetyltransferases</text>
+      </annotation>
+      <annotation id="78">
+        <infon key="score">0.9991702</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3444" length="4"/>
+        <text>NATs</text>
+      </annotation>
+      <annotation id="79">
+        <infon key="score">0.9988775</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:51:39Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3469" length="16"/>
+        <text>GNAT superfamily</text>
+      </annotation>
+      <annotation id="80">
+        <infon key="score">0.99918216</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3500" length="4"/>
+        <text>NATs</text>
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+        <infon key="score">0.9953088</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:54:46Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="3506" length="4"/>
+        <text>NatA</text>
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+      <annotation id="82">
+        <infon key="score">0.9346398</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:59:09Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="3511" length="1"/>
+        <text>B</text>
+      </annotation>
+      <annotation id="913">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:59:27Z</infon>
+        <location offset="3513" length="1"/>
+        <text>C</text>
+      </annotation>
+      <annotation id="83">
+        <infon key="score">0.8967032</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:59:37Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="3515" length="1"/>
+        <text>D</text>
+      </annotation>
+      <annotation id="914">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:59:50Z</infon>
+        <location offset="3517" length="1"/>
+        <text>E</text>
+      </annotation>
+      <annotation id="84">
+        <infon key="score">0.5994063</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:02:21Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="3519" length="1"/>
+        <text>F</text>
+      </annotation>
+      <annotation id="85">
+        <infon key="score">0.99849737</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:24:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3546" length="10"/>
+        <text>eukaryotes</text>
+      </annotation>
+      <annotation id="86">
+        <infon key="score">0.96252584</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3578" length="14"/>
+        <text>Nt-acetylation</text>
+      </annotation>
+      <annotation id="87">
+        <infon key="score">0.9793975</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:54:46Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="3638" length="4"/>
+        <text>NatA</text>
+      </annotation>
+      <annotation id="88">
+        <infon key="score">0.99895537</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:52:15Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3694" length="6"/>
+        <text>Naa10p</text>
+      </annotation>
+      <annotation id="89">
+        <infon key="score">0.99897397</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:52:20Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3727" length="6"/>
+        <text>Naa15p</text>
+      </annotation>
+      <annotation id="90">
+        <infon key="score">0.9929033</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:00:05Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="3735" length="4"/>
+        <text>NatE</text>
+      </annotation>
+      <annotation id="91">
+        <infon key="score">0.5679324</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:54:45Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="3782" length="4"/>
+        <text>NatA</text>
+      </annotation>
+      <annotation id="92">
+        <infon key="score">0.9276306</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:54:46Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="3832" length="4"/>
+        <text>NatA</text>
+      </annotation>
+      <annotation id="93">
+        <infon key="score">0.99913484</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3881" length="4"/>
+        <text>NATs</text>
+      </annotation>
+      <annotation id="94">
+        <infon key="score">0.9946814</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:54:37Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="3890" length="4"/>
+        <text>NatB</text>
+      </annotation>
+      <annotation id="95">
+        <infon key="score">0.9961499</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:54:58Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="3899" length="4"/>
+        <text>NatC</text>
+      </annotation>
+      <annotation id="96">
+        <infon key="score">0.9987263</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:53:02Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3941" length="5"/>
+        <text>Naa20</text>
+      </annotation>
+      <annotation id="97">
+        <infon key="score">0.9987482</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:53:08Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3951" length="5"/>
+        <text>Naa30</text>
+      </annotation>
+      <annotation id="98">
+        <infon key="score">0.99873227</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:53:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3984" length="5"/>
+        <text>Naa25</text>
+      </annotation>
+      <annotation id="99">
+        <infon key="score">0.998733</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:53:19Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3994" length="5"/>
+        <text>Naa35</text>
+      </annotation>
+      <annotation id="100">
+        <infon key="score">0.99870276</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:53:23Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4000" length="5"/>
+        <text>Naa38</text>
+      </annotation>
+      <annotation id="101">
+        <infon key="score">0.9986804</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:53:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4062" length="5"/>
+        <text>Naa40</text>
+      </annotation>
+      <annotation id="102">
+        <infon key="score">0.9985952</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4072" length="5"/>
+        <text>Naa60</text>
+      </annotation>
+      <annotation id="103">
+        <infon key="score">0.9984458</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:55:17Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="4093" length="4"/>
+        <text>NatD</text>
+      </annotation>
+      <annotation id="104">
+        <infon key="score">0.9985499</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:55:35Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="4102" length="4"/>
+        <text>NatF</text>
+      </annotation>
+      <annotation id="105">
+        <infon key="score">0.96562463</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4130" length="14"/>
+        <text>Nt-acetylation</text>
+      </annotation>
+      <annotation id="106">
+        <infon key="score">0.9625132</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:48:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4181" length="14"/>
+        <text>Nε-acetylation</text>
+      </annotation>
+      <annotation id="107">
+        <infon key="score">0.99909365</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4211" length="4"/>
+        <text>NATs</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>4223</offset>
+      <text>There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family. Unlike other NATs that are highly conserved among lower and higher eukaryotes, NatF only exists in higher eukaryotes. Subsequent researches indicated that NatF displays its catalytic ability with both Nt-acetylation and lysine Nε-acetylation. As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91. Another important feature of NatF is that this protein is anchored on the Golgi apparatus through its C-terminal membrane-integrating region and takes part in the maintaining of Golgi integrity. With its unique intracellular organellar localization and substrate selectivity, NatF appears to provide more evolutionary information among the NAT family members.</text>
+      <annotation id="108">
+        <infon key="score">0.9552281</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4276" length="14"/>
+        <text>Nt-acetylation</text>
+      </annotation>
+      <annotation id="109">
+        <infon key="score">0.9985532</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:50:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4299" length="5"/>
+        <text>yeast</text>
+      </annotation>
+      <annotation id="110">
+        <infon key="score">0.9977108</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4309" length="5"/>
+        <text>human</text>
+      </annotation>
+      <annotation id="111">
+        <infon key="score">0.9987839</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:55:36Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="4370" length="4"/>
+        <text>NatF</text>
+      </annotation>
+      <annotation id="112">
+        <infon key="score">0.99850816</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:02:41Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4389" length="28"/>
+        <text>N-terminal acetyltransferase</text>
+      </annotation>
+      <annotation id="113">
+        <infon key="score">0.99880755</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:55:36Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="4446" length="4"/>
+        <text>NatF</text>
+      </annotation>
+      <annotation id="114">
+        <infon key="score">0.6020324</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4463" length="5"/>
+        <text>NAA60</text>
+      </annotation>
+      <annotation id="915">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:03:06Z</infon>
+        <location offset="4487" length="42"/>
+        <text>Histone acetyltransferase type B protein 4</text>
+      </annotation>
+      <annotation id="115">
+        <infon key="score">0.99775237</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:03:18Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4531" length="4"/>
+        <text>HAT4</text>
+      </annotation>
+      <annotation id="116">
+        <infon key="score">0.99884844</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4538" length="5"/>
+        <text>Naa60</text>
+      </annotation>
+      <annotation id="916">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:03:53Z</infon>
+        <location offset="4547" length="22"/>
+        <text>N-acetyltransferase 15</text>
+      </annotation>
+      <annotation id="117">
+        <infon key="score">0.9983645</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:03:57Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4571" length="5"/>
+        <text>NAT15</text>
+      </annotation>
+      <annotation id="897">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:28Z</infon>
+        <location offset="4609" length="3"/>
+        <text>NAT</text>
+      </annotation>
+      <annotation id="118">
+        <infon key="score">0.999131</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4634" length="4"/>
+        <text>NATs</text>
+      </annotation>
+      <annotation id="119">
+        <infon key="score">0.99893486</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:26:56Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4648" length="16"/>
+        <text>highly conserved</text>
+      </annotation>
+      <annotation id="120">
+        <infon key="score">0.9907494</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:24:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4671" length="5"/>
+        <text>lower</text>
+      </annotation>
+      <annotation id="121">
+        <infon key="score">0.8346692</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:06:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4681" length="17"/>
+        <text>higher eukaryotes</text>
+      </annotation>
+      <annotation id="122">
+        <infon key="score">0.99874425</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:55:36Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="4700" length="4"/>
+        <text>NatF</text>
+      </annotation>
+      <annotation id="123">
+        <infon key="score">0.98239946</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:06:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4720" length="17"/>
+        <text>higher eukaryotes</text>
+      </annotation>
+      <annotation id="124">
+        <infon key="score">0.99875</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:55:36Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="4776" length="4"/>
+        <text>NatF</text>
+      </annotation>
+      <annotation id="125">
+        <infon key="score">0.9238295</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4822" length="14"/>
+        <text>Nt-acetylation</text>
+      </annotation>
+      <annotation id="919">
+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:07:04Z</infon>
+        <location offset="4841" length="21"/>
+        <text>lysine Nε-acetylation</text>
+      </annotation>
+      <annotation id="126">
+        <infon key="score">0.9984901</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:02:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4870" length="28"/>
+        <text>N-terminal acetyltransferase</text>
+      </annotation>
+      <annotation id="127">
+        <infon key="score">0.99684405</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:55:36Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="4900" length="4"/>
+        <text>NatF</text>
+      </annotation>
+      <annotation id="128">
+        <infon key="score">0.9915748</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:48:11Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4931" length="11"/>
+        <text>acetylation</text>
+      </annotation>
+      <annotation id="933">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:14:47Z</infon>
+        <location offset="5051" length="8"/>
+        <text>Met-Lys-</text>
+      </annotation>
+      <annotation id="934">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:15:08Z</infon>
+        <location offset="5061" length="8"/>
+        <text>Met-Val-</text>
+      </annotation>
+      <annotation id="935">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:15:27Z</infon>
+        <location offset="5071" length="8"/>
+        <text>Met-Ala-</text>
+      </annotation>
+      <annotation id="936">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:15:45Z</infon>
+        <location offset="5084" length="8"/>
+        <text>Met-Met-</text>
+      </annotation>
+      <annotation id="129">
+        <infon key="score">0.9971757</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:54:58Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="5157" length="4"/>
+        <text>NatC</text>
+      </annotation>
+      <annotation id="130">
+        <infon key="score">0.99452823</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:00:06Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="5166" length="4"/>
+        <text>NatE</text>
+      </annotation>
+      <annotation id="131">
+        <infon key="score">0.9983417</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:55:36Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="5191" length="4"/>
+        <text>NatF</text>
+      </annotation>
+      <annotation id="132">
+        <infon key="score">0.99512225</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:08:12Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5206" length="24"/>
+        <text>lysine acetyltransferase</text>
+      </annotation>
+      <annotation id="917">
+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:05:36Z</infon>
+        <location offset="5254" length="18"/>
+        <text>lysine acetylation</text>
+      </annotation>
+      <annotation id="133">
+        <infon key="score">0.98459953</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:08:43Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5281" length="7"/>
+        <text>histone</text>
+      </annotation>
+      <annotation id="134">
+        <infon key="score">0.9713598</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:09:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5289" length="2"/>
+        <text>H4</text>
+      </annotation>
+      <annotation id="926">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:11:31Z</infon>
+        <location offset="5303" length="2"/>
+        <text>H4</text>
+      </annotation>
+      <annotation id="927">
+        <infon key="type">residue_name_number</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:11:44Z</infon>
+        <location offset="5305" length="3"/>
+        <text>K20</text>
+      </annotation>
+      <annotation id="928">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:12:04Z</infon>
+        <location offset="5310" length="2"/>
+        <text>H4</text>
+      </annotation>
+      <annotation id="929">
+        <infon key="type">residue_name_number</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:12:28Z</infon>
+        <location offset="5312" length="3"/>
+        <text>K79</text>
+      </annotation>
+      <annotation id="930">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:12:49Z</infon>
+        <location offset="5320" length="2"/>
+        <text>H4</text>
+      </annotation>
+      <annotation id="931">
+        <infon key="type">residue_name_number</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:13:05Z</infon>
+        <location offset="5322" length="3"/>
+        <text>K91</text>
+      </annotation>
+      <annotation id="135">
+        <infon key="score">0.9989195</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:55:36Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="5356" length="4"/>
+        <text>NatF</text>
+      </annotation>
+      <annotation id="136">
+        <infon key="score">0.9942046</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:29:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5440" length="27"/>
+        <text>membrane-integrating region</text>
+      </annotation>
+      <annotation id="137">
+        <infon key="score">0.9988495</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:55:36Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="5603" length="4"/>
+        <text>NatF</text>
+      </annotation>
+      <annotation id="898">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:28Z</infon>
+        <location offset="5667" length="3"/>
+        <text>NAT</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>5697</offset>
+      <text>It was recently found that NatF facilitates nucleosomes assembly and that NAA60 knockdown in MCF7-cell inhibits cell proliferation, sensitizes cells to DNA damage and induces cell apoptosis. In Drosophila cells, NAA60 knockdown induces chromosomal segregation defects during anaphase including lagging chromosomes and chromosomal bridges. Much recent attention has also been focused on the requirement of NatF for regulation of organellar structure. In HeLa cells, NAA60 knockdown causes Golgi apparatus fragmentation which can be rescued by overexpression Naa60. The systematic investigation of publicly available microarray data showed that NATs share distinct tissue-specific expression patterns in Drosophila and NatF shows a higher expression level in central nervous system of Drosophila.</text>
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+        <infon key="score">0.939754</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:55:36Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="5724" length="4"/>
+        <text>NatF</text>
+      </annotation>
+      <annotation id="139">
+        <infon key="score">0.9885904</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:22:55Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="5741" length="11"/>
+        <text>nucleosomes</text>
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+      <annotation id="140">
+        <infon key="score">0.89290154</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="5771" length="5"/>
+        <text>NAA60</text>
+      </annotation>
+      <annotation id="141">
+        <infon key="score">0.9981092</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:13:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5891" length="10"/>
+        <text>Drosophila</text>
+      </annotation>
+      <annotation id="142">
+        <infon key="score">0.7164299</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="5909" length="5"/>
+        <text>NAA60</text>
+      </annotation>
+      <annotation id="143">
+        <infon key="score">0.9589997</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:55:36Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="6102" length="4"/>
+        <text>NatF</text>
+      </annotation>
+      <annotation id="144">
+        <infon key="score">0.79295474</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6162" length="5"/>
+        <text>NAA60</text>
+      </annotation>
+      <annotation id="145">
+        <infon key="score">0.9425036</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:13:29Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6239" length="14"/>
+        <text>overexpression</text>
+      </annotation>
+      <annotation id="146">
+        <infon key="score">0.99772257</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6254" length="5"/>
+        <text>Naa60</text>
+      </annotation>
+      <annotation id="147">
+        <infon key="score">0.9991636</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6340" length="4"/>
+        <text>NATs</text>
+      </annotation>
+      <annotation id="148">
+        <infon key="score">0.99801564</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:13:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6399" length="10"/>
+        <text>Drosophila</text>
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+      <annotation id="149">
+        <infon key="score">0.9858074</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:55:36Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="6414" length="4"/>
+        <text>NatF</text>
+      </annotation>
+      <annotation id="150">
+        <infon key="score">0.99814355</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:13:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6480" length="10"/>
+        <text>Drosophila</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>6492</offset>
+      <text>In this study, we solved the structures of human Naa60 (NatF) in complex with coenzyme. The hNaa60 protein contains a unique amphipathic α-helix (α5) following its GNAT domain that might account for the Golgi localization of this protein. Crystal structures showed that the β7-β8 hairpin rotated about 50 degrees upon removing the C-terminal region of the protein and this movement substantially changed the geometry of the substrate-binding pocket. Remarkably, we find that Phe 34 may participate in the proper positioning of the coenzyme for the transfer reaction to occur. Further structure comparison and biochemical studies also identified other key structural elements essential for the enzyme activity of Naa60.</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:35:30Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>solved</text>
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+        <infon key="score">0.94823563</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:25:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>structures</text>
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6535" length="5"/>
+        <text>human</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6541" length="5"/>
+        <text>Naa60</text>
+      </annotation>
+      <annotation id="155">
+        <infon key="score">0.99935895</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:55:36Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="6548" length="4"/>
+        <text>NatF</text>
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+      <annotation id="156">
+        <infon key="score">0.9983024</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6554" length="15"/>
+        <text>in complex with</text>
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+        <infon key="score">0.9977319</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:13:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="6570" length="8"/>
+        <text>coenzyme</text>
+      </annotation>
+      <annotation id="158">
+        <infon key="score">0.9993267</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6584" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="1038">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:27:15Z</infon>
+        <location offset="6617" length="19"/>
+        <text>amphipathic α-helix</text>
+      </annotation>
+      <annotation id="159">
+        <infon key="score">0.9994553</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:29:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6638" length="2"/>
+        <text>α5</text>
+      </annotation>
+      <annotation id="160">
+        <infon key="score">0.99896514</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:29:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6656" length="11"/>
+        <text>GNAT domain</text>
+      </annotation>
+      <annotation id="161">
+        <infon key="score">0.99477154</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6731" length="18"/>
+        <text>Crystal structures</text>
+      </annotation>
+      <annotation id="162">
+        <infon key="score">0.99920386</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:46Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6766" length="13"/>
+        <text>β7-β8 hairpin</text>
+      </annotation>
+      <annotation id="1048">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:36:24Z</infon>
+        <location offset="6823" length="17"/>
+        <text>C-terminal region</text>
+      </annotation>
+      <annotation id="163">
+        <infon key="score">0.99904025</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:39:15Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6916" length="24"/>
+        <text>substrate-binding pocket</text>
+      </annotation>
+      <annotation id="164">
+        <infon key="score">0.9975388</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6967" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="165">
+        <infon key="score">0.87373805</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:13:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7023" length="8"/>
+        <text>coenzyme</text>
+      </annotation>
+      <annotation id="166">
+        <infon key="score">0.99868906</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:35:36Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7076" length="20"/>
+        <text>structure comparison</text>
+      </annotation>
+      <annotation id="167">
+        <infon key="score">0.9906101</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:15:55Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7101" length="19"/>
+        <text>biochemical studies</text>
+      </annotation>
+      <annotation id="168">
+        <infon key="score">0.99938595</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7204" length="5"/>
+        <text>Naa60</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_1</infon>
+      <offset>7225</offset>
+      <text>Results</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>7233</offset>
+      <text>Overall structure of hNaa60</text>
+      <annotation id="169">
+        <infon key="score">0.9977296</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:16:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7241" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="170">
+        <infon key="score">0.99925464</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7254" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>7261</offset>
+      <text>In the effort to prepare the protein for structural studies, we tried a large number of hNaa60 constructs but all failed due to heavy precipitation or aggregation. Sequence alignment of Naa60 from different species revealed a Glu-Glu-Arg (EER) versus Val-Val-Pro (VVP) sequence difference near the N-terminus of the protein in Xenopus Laevis versus Homo sapiens (Fig. 1A). Considering that terminal residues may lack higher-order structure and hydrophobic residues in this region may expose to solvent and hence cause protein aggregation, we mutated residues 4–6 from VVP to EER for the purpose of improving solubility of this protein. According to previous studies, this N-terminal region should not interfere with hNaa60’s Golgi localization. We tried many hNaa60 constructs with the three-residues mutation but only the truncated variant 1-199 and the full-length protein behaved well. We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C). Hereafter, all deletions or point mutants of hNaa60 we describe here are with the EER mutation. The crystal structures of hNaa60(1-242)/Ac-CoA and hNaa60(1-199)/CoA were determined by molecular replacement and refined to 1.38 Å and 1.60 Å resolution, respectively (Table 1). The electron density maps were of sufficient quality to trace residues 1-211 of hNaa60(1-242) and residues 5-199 of hNaa60(1-199).</text>
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+        <infon key="score">0.99925226</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7349" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="172">
+        <infon key="score">0.99790084</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:35:39Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7425" length="18"/>
+        <text>Sequence alignment</text>
+      </annotation>
+      <annotation id="173">
+        <infon key="score">0.9993876</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7447" length="5"/>
+        <text>Naa60</text>
+      </annotation>
+      <annotation id="939">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:18:40Z</infon>
+        <location offset="7487" length="11"/>
+        <text>Glu-Glu-Arg</text>
+      </annotation>
+      <annotation id="174">
+        <infon key="score">0.50987583</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:18:49Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7500" length="3"/>
+        <text>EER</text>
+      </annotation>
+      <annotation id="940">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:19:16Z</infon>
+        <location offset="7512" length="11"/>
+        <text>Val-Val-Pro</text>
+      </annotation>
+      <annotation id="175">
+        <infon key="score">0.8827366</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:19:25Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7525" length="3"/>
+        <text>VVP</text>
+      </annotation>
+      <annotation id="176">
+        <infon key="score">0.99861956</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:20:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7588" length="14"/>
+        <text>Xenopus Laevis</text>
+      </annotation>
+      <annotation id="177">
+        <infon key="score">0.9984832</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:20:07Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7610" length="12"/>
+        <text>Homo sapiens</text>
+      </annotation>
+      <annotation id="178">
+        <infon key="score">0.9906036</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:35:43Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7803" length="7"/>
+        <text>mutated</text>
+      </annotation>
+      <annotation id="179">
+        <infon key="score">0.9951412</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:32:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7820" length="3"/>
+        <text>4–6</text>
+      </annotation>
+      <annotation id="938">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:18:21Z</infon>
+        <location offset="7829" length="10"/>
+        <text>VVP to EER</text>
+      </annotation>
+      <annotation id="180">
+        <infon key="score">0.9993231</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7977" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="181">
+        <infon key="score">0.99922884</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8020" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="182">
+        <infon key="score">0.8594266</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:06Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8062" length="8"/>
+        <text>mutation</text>
+      </annotation>
+      <annotation id="183">
+        <infon key="score">0.99651885</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:20:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+      <annotation id="184">
+        <infon key="score">0.905786</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:20:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8102" length="5"/>
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+        <infon key="score">0.99910736</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:20:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8116" length="11"/>
+        <text>full-length</text>
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+      <annotation id="186">
+        <infon key="score">0.9986326</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:25:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:20:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8181" length="9"/>
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+        <infon key="type">residue_range</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:20:54Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:14Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8221" length="3"/>
+        <text>CoA</text>
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+      <annotation id="190">
+        <infon key="score">0.99869686</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:25:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8270" length="7"/>
+        <text>crystal</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:20:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8285" length="11"/>
+        <text>full-length</text>
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+        <infon key="score">0.9829355</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:24:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8324" length="5"/>
+        <text>1-242</text>
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+        <infon key="score">0.9957215</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8331" length="15"/>
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+        <infon key="score">0.9988327</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8347" length="6"/>
+        <text>Ac-CoA</text>
+      </annotation>
+      <annotation id="1010">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:38Z</infon>
+        <location offset="8401" length="7"/>
+        <text>mutants</text>
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+      <annotation id="195">
+        <infon key="score">0.99930966</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8412" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="196">
+        <infon key="score">0.6943366</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:19:33Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8449" length="3"/>
+        <text>EER</text>
+      </annotation>
+      <annotation id="942">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:07Z</infon>
+        <location offset="8453" length="8"/>
+        <text>mutation</text>
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+      <annotation id="197">
+        <infon key="score">0.99876124</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8467" length="18"/>
+        <text>crystal structures</text>
+      </annotation>
+      <annotation id="944">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:34Z</infon>
+        <location offset="8489" length="20"/>
+        <text>hNaa60(1-242)/Ac-CoA</text>
+      </annotation>
+      <annotation id="951">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:53Z</infon>
+        <location offset="8514" length="17"/>
+        <text>hNaa60(1-199)/CoA</text>
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+      <annotation id="198">
+        <infon key="score">0.9986177</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:17:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8551" length="21"/>
+        <text>molecular replacement</text>
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+      <annotation id="199">
+        <infon key="score">0.9987355</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:45:31Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8646" length="21"/>
+        <text>electron density maps</text>
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+      <annotation id="200">
+        <infon key="score">0.9942498</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:32:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8713" length="5"/>
+        <text>1-211</text>
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+        <infon key="score">0.8964569</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8722" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="937">
+        <infon key="type">residue_range</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:16:33Z</infon>
+        <location offset="8729" length="5"/>
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+      <annotation id="202">
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+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:32:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8749" length="5"/>
+        <text>5-199</text>
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+      <annotation id="1043">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:32:35Z</infon>
+        <location offset="8758" length="13"/>
+        <text>hNaa60(1-199)</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>8783</offset>
+      <text>The structure of hNaa60 protein contains a central domain exhibiting a classic GCN5-related N-acetyltransferase (GNAT) folding, along with the extended N- and C-terminal regions (Fig. 1B,C). The central domain includes nine β strands (β1-β9) and four α-helixes (α1-α4) and is highly similar to the known hNaa50p and other reported NATs (Fig. 1D). However, in hNaa60, there is an extra 20-residue loop between β3 and β4 that forms a small subdomain with well-defined 3D structure (Fig. 1B–D). Furthermore, the β7-β8 strands form an approximately antiparallel β-hairpin structure remarkably different from that in hNaa50p (Fig. 1D). The N- and C-terminal regions form helical structures (α0 and α5) stretching out from the central GCN5-domain (Fig. 1C).</text>
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+        <infon key="score">0.9957825</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:22:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8787" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="204">
+        <infon key="score">0.9993191</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8800" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="205">
+        <infon key="score">0.99772197</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:22:27Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8826" length="14"/>
+        <text>central domain</text>
+      </annotation>
+      <annotation id="206">
+        <infon key="score">0.9719784</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:22:09Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8862" length="32"/>
+        <text>GCN5-related N-acetyltransferase</text>
+      </annotation>
+      <annotation id="207">
+        <infon key="score">0.9992061</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:22:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8896" length="4"/>
+        <text>GNAT</text>
+      </annotation>
+      <annotation id="208">
+        <infon key="score">0.7512091</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:26:37Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8926" length="8"/>
+        <text>extended</text>
+      </annotation>
+      <annotation id="209">
+        <infon key="score">0.9954179</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:26:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8935" length="25"/>
+        <text>N- and C-terminal regions</text>
+      </annotation>
+      <annotation id="210">
+        <infon key="score">0.9985126</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:22:27Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8978" length="14"/>
+        <text>central domain</text>
+      </annotation>
+      <annotation id="211">
+        <infon key="score">0.9941509</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:22:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9007" length="9"/>
+        <text>β strands</text>
+      </annotation>
+      <annotation id="212">
+        <infon key="score">0.9976589</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:30:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9018" length="5"/>
+        <text>β1-β9</text>
+      </annotation>
+      <annotation id="213">
+        <infon key="score">0.98797673</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:26:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9034" length="9"/>
+        <text>α-helixes</text>
+      </annotation>
+      <annotation id="214">
+        <infon key="score">0.99689</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:26:03Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9045" length="5"/>
+        <text>α1-α4</text>
+      </annotation>
+      <annotation id="215">
+        <infon key="score">0.99236286</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:26:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9059" length="14"/>
+        <text>highly similar</text>
+      </annotation>
+      <annotation id="216">
+        <infon key="score">0.99932754</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9087" length="7"/>
+        <text>hNaa50p</text>
+      </annotation>
+      <annotation id="217">
+        <infon key="score">0.99929893</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9114" length="4"/>
+        <text>NATs</text>
+      </annotation>
+      <annotation id="218">
+        <infon key="score">0.9993673</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9142" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="219">
+        <infon key="score">0.9921711</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:25:51Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9162" length="21"/>
+        <text>extra 20-residue loop</text>
+      </annotation>
+      <annotation id="220">
+        <infon key="score">0.999292</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:25:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9192" length="2"/>
+        <text>β3</text>
+      </annotation>
+      <annotation id="221">
+        <infon key="score">0.9991374</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:25:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9199" length="2"/>
+        <text>β4</text>
+      </annotation>
+      <annotation id="222">
+        <infon key="score">0.9988525</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:26:28Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9215" length="15"/>
+        <text>small subdomain</text>
+      </annotation>
+      <annotation id="223">
+        <infon key="score">0.99903136</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:30:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9292" length="13"/>
+        <text>β7-β8 strands</text>
+      </annotation>
+      <annotation id="224">
+        <infon key="score">0.9855807</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:22:43Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9314" length="46"/>
+        <text>approximately antiparallel β-hairpin structure</text>
+      </annotation>
+      <annotation id="225">
+        <infon key="score">0.99932003</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:49Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9395" length="7"/>
+        <text>hNaa50p</text>
+      </annotation>
+      <annotation id="226">
+        <infon key="score">0.9703472</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:26:08Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9418" length="25"/>
+        <text>N- and C-terminal regions</text>
+      </annotation>
+      <annotation id="227">
+        <infon key="score">0.8584138</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:26:14Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9449" length="18"/>
+        <text>helical structures</text>
+      </annotation>
+      <annotation id="228">
+        <infon key="score">0.99945825</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:25:49Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9469" length="2"/>
+        <text>α0</text>
+      </annotation>
+      <annotation id="229">
+        <infon key="score">0.9993788</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:25:46Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9476" length="2"/>
+        <text>α5</text>
+      </annotation>
+      <annotation id="230">
+        <infon key="score">0.99567384</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:25:42Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9512" length="11"/>
+        <text>GCN5-domain</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>9579</offset>
+      <text>Interestingly, we found that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) (Figure S1), indicating that residues 200–242 may have some auto-inhibitory effect on the activity of the enzyme. However, since this region was not visible in the hNaa60(1-242) crystal structure, we do not yet understand how this happens. Another possibility is that since hNaa60 is localized on Golgi apparatus, the observed low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies. For the convenience of studying the kinetics of mutants, the mutagenesis studies described hereafter were all based on hNaa60 (1-199).</text>
+      <annotation id="231">
+        <infon key="score">0.9958061</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9634" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="232">
+        <infon key="score">0.993637</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:24:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9641" length="5"/>
+        <text>1-242</text>
+      </annotation>
+      <annotation id="965">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:24:44Z</infon>
+        <location offset="9675" length="13"/>
+        <text>hNaa60(1-199)</text>
+      </annotation>
+      <annotation id="233">
+        <infon key="score">0.99601203</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:33:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9727" length="7"/>
+        <text>200–242</text>
+      </annotation>
+      <annotation id="903">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <location offset="9853" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="959">
+        <infon key="type">residue_range</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:24:08Z</infon>
+        <location offset="9860" length="5"/>
+        <text>1-242</text>
+      </annotation>
+      <annotation id="234">
+        <infon key="score">0.99869287</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:33:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9867" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="235">
+        <infon key="score">0.99932134</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9963" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="236">
+        <infon key="score">0.99910516</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:20:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10036" length="11"/>
+        <text>full-length</text>
+      </annotation>
+      <annotation id="237">
+        <infon key="score">0.9993279</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10048" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="1011">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:38Z</infon>
+        <location offset="10189" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="966">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:25:37Z</infon>
+        <location offset="10202" length="19"/>
+        <text>mutagenesis studies</text>
+      </annotation>
+      <annotation id="1044">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:33:03Z</infon>
+        <location offset="10260" length="14"/>
+        <text>hNaa60 (1-199)</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>10278</offset>
+      <text>An amphipathic α-helix in the C-terminal region may contribute to Golgi localization of hNaa60</text>
+      <annotation id="1039">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:27:40Z</infon>
+        <location offset="10281" length="19"/>
+        <text>amphipathic α-helix</text>
+      </annotation>
+      <annotation id="238">
+        <infon key="score">0.996947</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:30:08Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10308" length="17"/>
+        <text>C-terminal region</text>
+      </annotation>
+      <annotation id="239">
+        <infon key="score">0.9990615</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10366" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>10377</offset>
+      <text>There is one hNaa60 molecule in the asymmetric unit in the hNaa60(1-242)/Ac-CoA structure. The C-terminal region extended from the GCN5-domain forms an amphipathic helix (α5) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between α5-helix and a hydrophobic groove between the N-terminal β1 and β3 strands of the neighbor molecule (Fig. 2A). The C-terminal extension following α5-helix forms a β-turn that wraps around and interacts with the neighbor protein molecule through hydrophobic interactions, too. In the hNaa60(1-199)/CoA structure, a part of the α5-helix is deleted due to truncation of the C-terminal region (Fig. 1B). Interestingly, the remaining residues in α5-helix still form an amphipathic helix although the hydrophobic interaction with the N-terminal hydrophobic groove of a neighbor molecule is abolished and the helix is largely exposed in solvent due to different crystal packing (Fig. 2B).</text>
+      <annotation id="240">
+        <infon key="score">0.9991192</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10390" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="241">
+        <infon key="score">0.9910477</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:35Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="10436" length="20"/>
+        <text>hNaa60(1-242)/Ac-CoA</text>
+      </annotation>
+      <annotation id="242">
+        <infon key="score">0.9924954</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:25:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10457" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="243">
+        <infon key="score">0.99688613</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:30:12Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10472" length="17"/>
+        <text>C-terminal region</text>
+      </annotation>
+      <annotation id="244">
+        <infon key="score">0.99803805</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:25:43Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10508" length="11"/>
+        <text>GCN5-domain</text>
+      </annotation>
+      <annotation id="245">
+        <infon key="score">0.9984503</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:11Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10529" length="17"/>
+        <text>amphipathic helix</text>
+      </annotation>
+      <annotation id="246">
+        <infon key="score">0.99945563</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:30:16Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10548" length="2"/>
+        <text>α5</text>
+      </annotation>
+      <annotation id="247">
+        <infon key="score">0.9969907</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:26:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10620" length="24"/>
+        <text>hydrophobic interactions</text>
+      </annotation>
+      <annotation id="248">
+        <infon key="score">0.9992375</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:02Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10653" length="8"/>
+        <text>α5-helix</text>
+      </annotation>
+      <annotation id="249">
+        <infon key="score">0.99887705</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:22Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10668" length="18"/>
+        <text>hydrophobic groove</text>
+      </annotation>
+      <annotation id="250">
+        <infon key="score">0.99939835</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:14Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10710" length="2"/>
+        <text>β1</text>
+      </annotation>
+      <annotation id="251">
+        <infon key="score">0.99835706</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:17Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10717" length="10"/>
+        <text>β3 strands</text>
+      </annotation>
+      <annotation id="252">
+        <infon key="score">0.9985872</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:30:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10768" length="20"/>
+        <text>C-terminal extension</text>
+      </annotation>
+      <annotation id="253">
+        <infon key="score">0.9992624</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:03Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10799" length="8"/>
+        <text>α5-helix</text>
+      </annotation>
+      <annotation id="254">
+        <infon key="score">0.99928707</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:30:23Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10816" length="6"/>
+        <text>β-turn</text>
+      </annotation>
+      <annotation id="255">
+        <infon key="score">0.9970007</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:26:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10898" length="24"/>
+        <text>hydrophobic interactions</text>
+      </annotation>
+      <annotation id="256">
+        <infon key="score">0.9390323</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:54Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="10936" length="17"/>
+        <text>hNaa60(1-199)/CoA</text>
+      </annotation>
+      <annotation id="257">
+        <infon key="score">0.99651355</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:25:24Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10954" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="258">
+        <infon key="score">0.99932104</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:03Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10979" length="8"/>
+        <text>α5-helix</text>
+      </annotation>
+      <annotation id="259">
+        <infon key="score">0.9868099</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:30:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11024" length="17"/>
+        <text>C-terminal region</text>
+      </annotation>
+      <annotation id="260">
+        <infon key="score">0.9992686</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:03Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11094" length="8"/>
+        <text>α5-helix</text>
+      </annotation>
+      <annotation id="968">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:48Z</infon>
+        <location offset="11117" length="17"/>
+        <text>amphipathic helix</text>
+      </annotation>
+      <annotation id="261">
+        <infon key="score">0.9966192</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11148" length="23"/>
+        <text>hydrophobic interaction</text>
+      </annotation>
+      <annotation id="262">
+        <infon key="score">0.9988097</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:22Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11192" length="18"/>
+        <text>hydrophobic groove</text>
+      </annotation>
+      <annotation id="263">
+        <infon key="score">0.9987142</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:42Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11255" length="5"/>
+        <text>helix</text>
+      </annotation>
+      <annotation id="980">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:44:17Z</infon>
+        <location offset="11308" length="15"/>
+        <text>crystal packing</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>11364</offset>
+      <text>A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.</text>
+      <annotation id="264">
+        <infon key="score">0.99668574</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:28:24Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11403" length="7"/>
+        <text>182–216</text>
+      </annotation>
+      <annotation id="265">
+        <infon key="score">0.9992968</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11449" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="266">
+        <infon key="score">0.99788886</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:25:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11483" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="267">
+        <infon key="score">0.8807922</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:28:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11498" length="15"/>
+        <text>solvent-exposed</text>
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+        <text>amphipathic helix</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <location offset="11959" length="6"/>
+        <text>PB1-F2</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
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+      <text>The β7-β8 hairpin showed alternative conformations in the hNaa60 crystal structures</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12040" length="18"/>
+        <text>crystal structures</text>
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+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>12065</offset>
+      <text>Superposition of hNaa60(1-242)/Ac-CoA, hNaa60(1-199)/CoA and hNaa50/CoA/peptide (PDB 3TFY) revealed considerable difference in the β7-β8 hairpin region despite the overall stability and similarity of the GNAT domain (Fig. 1D). In hNaa60(1-242), the β7-β8 hairpin is located in close proximity to the α1-α2 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (β6-β7 loop). Upon removing the C-terminal region of hNaa60, we observed that hNaa60 (1-199) molecules pack in a different way involving the β7-β8 hairpin in the crystal, leading to about 50 degree rotation of the hairpin which moves away from the α1-α2 loop (Figs 1D and 2C).</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>Superposition</text>
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+        <infon key="updated_at">2023-09-20T10:21:35Z</infon>
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+        <infon key="updated_at">2023-09-20T10:21:54Z</infon>
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+        <text>hNaa60(1-199)/CoA</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:29:54Z</infon>
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+        <text>hNaa50/CoA/peptide</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:46Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <text>GNAT domain</text>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
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+        <text>hNaa60</text>
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:24:08Z</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:46Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>β7-β8 hairpin</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:30:06Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12365" length="10"/>
+        <text>α1-α2 loop</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:30:46Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>substrate binding site</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12437" length="6"/>
+        <text>hNaa50</text>
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+      <annotation id="290">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:30:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12477" length="8"/>
+        <text>flexible</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <text>loop</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:30:44Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12505" length="10"/>
+        <text>β6-β7 loop</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:35:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12523" length="8"/>
+        <text>removing</text>
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+      <annotation id="1047">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:36:03Z</infon>
+        <location offset="12536" length="17"/>
+        <text>C-terminal region</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="12557" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="295">
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+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:30:22Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>hNaa60 (1-199)</text>
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+      <annotation id="296">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:46Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>β7-β8 hairpin</text>
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+      <annotation id="297">
+        <infon key="score">0.9906501</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:30:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12666" length="7"/>
+        <text>crystal</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:31:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12718" length="7"/>
+        <text>hairpin</text>
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+      <annotation id="299">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:30:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12752" length="10"/>
+        <text>α1-α2 loop</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>12821</offset>
+      <text>This conformational change substantially altered the geometry of the substrate binding site, which could potentially change the way in which the substrate accesses the active site of the enzyme. In hNaa60(1-242), the β7-β8 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only. KAT activity of hNaa60 toward histone H4 has been noted in previous study, and our enzyme kinetic data also indicated that hNaa60 can acetylate H3-H4 tetramer in vitro (Figure S3). Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4). We also conducted liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis on a synthetic peptide (NH2-MKGKEEKEGGAR-COOH) after treatment with hNaa60(1-199), and the data confirmed that both the N-terminal α-amine and lysine side-chain ε-amine were robustly acetylated after the treatment (Table S1). Despite these observations, the mechanism for this alternative activity remains unknown. Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site. Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D). Interestingly, in the hNaa60(1-199) crystal structure, the displaced β7-β8 hairpin opened a second way for the substrate to access the active center that would readily accommodate the binding of the H4 peptide (Fig. 2E), thus implied a potential explanation for KAT activity of this enzyme from a structural biological view. However, since hNaa60(1-242) and hNaa60(1-199) were crystallized in different crystal forms, the observed conformational change of the β7-β8 hairpin may simply be an artifact related to the different crystal packing. Whether the KAT substrates bind to the β7-β8 hairpin displaced conformation of the enzyme needs to be verified by further structural and functional studies.</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="updated_at">2023-09-20T09:46:45Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:24:08Z</infon>
+        <location offset="13026" length="5"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:46Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>β7-β8 hairpin</text>
+      </annotation>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:33:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13063" length="11"/>
+        <text>active site</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="13112" length="6"/>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:34:00Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <infon key="updated_at">2023-09-20T10:34:22Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>tunnel</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <text>Ac-CoA</text>
+      </annotation>
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+        <text>active center</text>
+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <text>H4</text>
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+      <annotation id="989">
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:45:09Z</infon>
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+        <text>peptide</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:54Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>KAT</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:43:57Z</infon>
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+        <text>crystallized</text>
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+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:43:36Z</infon>
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+        <text>crystal forms</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:46Z</infon>
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+        <text>β7-β8 hairpin</text>
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:44:16Z</infon>
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+        <text>crystal packing</text>
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+      <annotation id="902">
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:54Z</infon>
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+      <annotation id="911">
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:46Z</infon>
+        <location offset="15307" length="13"/>
+        <text>β7-β8 hairpin</text>
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+      <annotation id="983">
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:44:28Z</infon>
+        <location offset="15390" length="33"/>
+        <text>structural and functional studies</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>15485</offset>
+      <text>Phe 34 facilitates proper positioning of the cofactor for acetyl-transfer</text>
+      <annotation id="357">
+        <infon key="score">0.9979638</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15485" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="1022">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:19:06Z</infon>
+        <location offset="15543" length="6"/>
+        <text>acetyl</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>15559</offset>
+      <text>The electron density of Phe 34 side-chain is well defined in the hNaa60(1-242)/Ac-CoA structure, but becomes invisible in the hNaa60(1-199)/CoA structure, indicating displacement of the Phe 34 side-chain in the latter (Fig. 3A,B). A solvent-derived malonate molecule is found beside Phe 34 and the ethanethioate moiety of Ac-CoA in the high-resolution hNaa60(1-242)/Ac-CoA structure (Fig. 3A). Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A). Interestingly, in the structure of hNaa60(1-199)/CoA, the terminal thiol of CoA adopts alternative conformations. One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the α1-α2 loop and away from the substrate amine (Fig. 3B). To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15563" length="16"/>
+        <text>electron density</text>
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+        <text>Phe 34</text>
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+      <annotation id="946">
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:35Z</infon>
+        <location offset="15624" length="20"/>
+        <text>hNaa60(1-242)/Ac-CoA</text>
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+        <infon key="score">0.99336183</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:27Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>structure</text>
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+      <annotation id="953">
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+        <infon key="updated_at">2023-09-20T10:21:54Z</infon>
+        <location offset="15685" length="17"/>
+        <text>hNaa60(1-199)/CoA</text>
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+      <annotation id="361">
+        <infon key="score">0.9961402</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:25Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15703" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="362">
+        <infon key="score">0.9921007</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15745" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="363">
+        <infon key="score">0.9992324</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:40:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15808" length="8"/>
+        <text>malonate</text>
+      </annotation>
+      <annotation id="364">
+        <infon key="score">0.9924878</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15842" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="365">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:40:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15857" length="13"/>
+        <text>ethanethioate</text>
+      </annotation>
+      <annotation id="366">
+        <infon key="score">0.9987211</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15881" length="6"/>
+        <text>Ac-CoA</text>
+      </annotation>
+      <annotation id="367">
+        <infon key="score">0.90510833</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:35Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="15911" length="20"/>
+        <text>hNaa60(1-242)/Ac-CoA</text>
+      </annotation>
+      <annotation id="368">
+        <infon key="score">0.99532664</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15932" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="369">
+        <infon key="score">0.99804014</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:29:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15953" length="13"/>
+        <text>Superposition</text>
+      </annotation>
+      <annotation id="370">
+        <infon key="score">0.99360776</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15975" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="371">
+        <infon key="score">0.9954337</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:36:25Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="15996" length="19"/>
+        <text>hNaa50p/CoA/peptide</text>
+      </annotation>
+      <annotation id="372">
+        <infon key="score">0.99910945</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:40:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16031" length="8"/>
+        <text>malonate</text>
+      </annotation>
+      <annotation id="373">
+        <infon key="score">0.9969879</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:40:25Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="16081" length="10"/>
+        <text>methionine</text>
+      </annotation>
+      <annotation id="990">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:45:09Z</infon>
+        <location offset="16109" length="7"/>
+        <text>peptide</text>
+      </annotation>
+      <annotation id="374">
+        <infon key="score">0.99384165</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16129" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="375">
+        <infon key="score">0.9994134</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="16139" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="376">
+        <infon key="score">0.9929946</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16163" length="6"/>
+        <text>Phe 27</text>
+      </annotation>
+      <annotation id="377">
+        <infon key="score">0.9994166</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16173" length="6"/>
+        <text>hNaa50</text>
+      </annotation>
+      <annotation id="378">
+        <infon key="score">0.99719894</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16213" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="954">
+        <infon key="type">complex_assembly</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:54Z</infon>
+        <location offset="16226" length="17"/>
+        <text>hNaa60(1-199)/CoA</text>
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+        <infon key="score">0.9990219</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16267" length="3"/>
+        <text>CoA</text>
+      </annotation>
+      <annotation id="380">
+        <infon key="score">0.52795213</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:23:22Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16338" length="5"/>
+        <text>amine</text>
+      </annotation>
+      <annotation id="381">
+        <infon key="score">0.9921836</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:39:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16365" length="12"/>
+        <text>superimposed</text>
+      </annotation>
+      <annotation id="382">
+        <infon key="score">0.9987024</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:38:27Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="16378" length="18"/>
+        <text>hNaa50/CoA/peptide</text>
+      </annotation>
+      <annotation id="383">
+        <infon key="score">0.984336</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:31Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16397" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="384">
+        <infon key="score">0.9991757</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:40:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16433" length="13"/>
+        <text>ethanethioate</text>
+      </annotation>
+      <annotation id="385">
+        <infon key="score">0.9990859</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16450" length="6"/>
+        <text>Ac-CoA</text>
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+      <annotation id="386">
+        <infon key="score">0.99643755</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:35Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16464" length="9"/>
+        <text>structure</text>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:35Z</infon>
+        <location offset="16477" length="20"/>
+        <text>hNaa60(1-242)/Ac-CoA</text>
+      </annotation>
+      <annotation id="387">
+        <infon key="score">0.9990897</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:30:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="16528" length="10"/>
+        <text>α1-α2 loop</text>
+      </annotation>
+      <annotation id="388">
+        <infon key="score">0.9987115</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16621" length="16"/>
+        <text>electron density</text>
+      </annotation>
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+        <infon key="score">0.99693334</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16714" length="16"/>
+        <text>electron density</text>
+      </annotation>
+      <annotation id="390">
+        <infon key="score">0.99336606</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16762" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="391">
+        <infon key="score">0.9901281</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:36:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16773" length="6"/>
+        <text>solved</text>
+      </annotation>
+      <annotation id="392">
+        <infon key="score">0.9979323</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:33:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16784" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="973">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:38:45Z</infon>
+        <location offset="16805" length="22"/>
+        <text>hNaa60(1-199) F34A/CoA</text>
+      </annotation>
+      <annotation id="393">
+        <infon key="score">0.9976439</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16833" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="394">
+        <infon key="score">0.9936772</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:43:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16851" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="955">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:54Z</infon>
+        <location offset="16879" length="17"/>
+        <text>hNaa60(1-199)/CoA</text>
+      </annotation>
+      <annotation id="395">
+        <infon key="score">0.99862635</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16931" length="16"/>
+        <text>electron density</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>17026</offset>
+      <text>Phe 27 in hNaa50p (equivalent to Phe 34 in hNaa60) has been implicated to facilitate the binding of N-terminal methionine of the substrate peptide through hydrophobic interaction. However, in the hNaa60/Ac-CoA structure, a hydrophilic malonate molecule is found at the same location where the N-terminal methionine should bind as is indicated by the superposition (Fig. 3A), suggesting that Phe 34 may accommodate binding of hydrophilic substrate, too. Moreover, orientation of Phe 34 side-chain seems to be co-related to positioning of the terminus of the co-enzyme and important for placing it at a location in close proximity to the substrate amine. We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme. Indeed, our enzyme kinetic data showed that hNaa60(1-199) F34A mutant showed no detectable activity (Fig. 5A). In order to rule out the possibility that the observed loss of activity may be related to bad folding of the mutant protein, we studied the circular dichroism (CD) spectrum of the protein (Fig. 5B) and determined its crystal structure (Fig. 3C). Both studies proved that the F34A mutant protein is well-folded. Many studies have addressed the crucial effect of α1-α2 loop on catalysis, showing that some residues located in this area are involved in the binding of substrates. We propose that Phe 34 may play a dual role both in interacting with the peptide substrate (recognition) and in positioning of the ethanethioate moiety of Ac-CoA to the right location to facilitate acetyl-transfer.</text>
+      <annotation id="396">
+        <infon key="score">0.99686694</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17026" length="6"/>
+        <text>Phe 27</text>
+      </annotation>
+      <annotation id="397">
+        <infon key="score">0.9991411</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:49Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17036" length="7"/>
+        <text>hNaa50p</text>
+      </annotation>
+      <annotation id="398">
+        <infon key="score">0.9948791</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17059" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="399">
+        <infon key="score">0.9993376</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:46Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17069" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="400">
+        <infon key="score">0.997621</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:24:49Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17137" length="10"/>
+        <text>methionine</text>
+      </annotation>
+      <annotation id="991">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:45:09Z</infon>
+        <location offset="17165" length="7"/>
+        <text>peptide</text>
+      </annotation>
+      <annotation id="401">
+        <infon key="score">0.996629</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:29Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17181" length="23"/>
+        <text>hydrophobic interaction</text>
+      </annotation>
+      <annotation id="402">
+        <infon key="score">0.9990492</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:41:12Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="17222" length="13"/>
+        <text>hNaa60/Ac-CoA</text>
+      </annotation>
+      <annotation id="403">
+        <infon key="score">0.99804497</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:25:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17236" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="404">
+        <infon key="score">0.9988341</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:40:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="17261" length="8"/>
+        <text>malonate</text>
+      </annotation>
+      <annotation id="405">
+        <infon key="score">0.9970541</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:24:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17330" length="10"/>
+        <text>methionine</text>
+      </annotation>
+      <annotation id="406">
+        <infon key="score">0.9985031</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:29:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17376" length="13"/>
+        <text>superposition</text>
+      </annotation>
+      <annotation id="407">
+        <infon key="score">0.9941642</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17417" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="408">
+        <infon key="score">0.9908397</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17504" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="409">
+        <infon key="score">0.99226713</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17702" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="410">
+        <infon key="score">0.9970092</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:41:23Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17776" length="3"/>
+        <text>Met</text>
+      </annotation>
+      <annotation id="411">
+        <infon key="score">0.9900765</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:36:35Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17792" length="6"/>
+        <text>mutate</text>
+      </annotation>
+      <annotation id="412">
+        <infon key="score">0.99656135</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:41:19Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17807" length="3"/>
+        <text>Phe</text>
+      </annotation>
+      <annotation id="413">
+        <infon key="score">0.9962882</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:41:21Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17814" length="3"/>
+        <text>Ala</text>
+      </annotation>
+      <annotation id="414">
+        <infon key="score">0.9943061</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17884" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="415">
+        <infon key="score">0.99901795</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:40:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="17936" length="13"/>
+        <text>ethanethioate</text>
+      </annotation>
+      <annotation id="416">
+        <infon key="score">0.99916965</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="17960" length="6"/>
+        <text>Ac-CoA</text>
+      </annotation>
+      <annotation id="417">
+        <infon key="score">0.9942807</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:07Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17972" length="8"/>
+        <text>mutation</text>
+      </annotation>
+      <annotation id="418">
+        <infon key="score">0.99212605</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:25:44Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18051" length="19"/>
+        <text>enzyme kinetic data</text>
+      </annotation>
+      <annotation id="419">
+        <infon key="score">0.77918214</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:42:51Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18083" length="13"/>
+        <text>hNaa60(1-199)</text>
+      </annotation>
+      <annotation id="420">
+        <infon key="score">0.99832374</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:42:55Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18097" length="4"/>
+        <text>F34A</text>
+      </annotation>
+      <annotation id="421">
+        <infon key="score">0.9989729</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:43:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18102" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="422">
+        <infon key="score">0.9986871</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:43:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18259" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="423">
+        <infon key="score">0.8455858</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:42:29Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18290" length="18"/>
+        <text>circular dichroism</text>
+      </annotation>
+      <annotation id="424">
+        <infon key="score">0.57051194</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:42:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18310" length="2"/>
+        <text>CD</text>
+      </annotation>
+      <annotation id="425">
+        <infon key="score">0.9649881</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:42:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18314" length="8"/>
+        <text>spectrum</text>
+      </annotation>
+      <annotation id="426">
+        <infon key="score">0.99833393</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:33:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18367" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="427">
+        <infon key="score">0.9987686</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:42:56Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18425" length="4"/>
+        <text>F34A</text>
+      </annotation>
+      <annotation id="428">
+        <infon key="score">0.9987796</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:43:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18430" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="974">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:42:06Z</infon>
+        <location offset="18448" length="11"/>
+        <text>well-folded</text>
+      </annotation>
+      <annotation id="429">
+        <infon key="score">0.99931383</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:30:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18511" length="10"/>
+        <text>α1-α2 loop</text>
+      </annotation>
+      <annotation id="430">
+        <infon key="score">0.9952917</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18643" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="992">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:45:09Z</infon>
+        <location offset="18700" length="7"/>
+        <text>peptide</text>
+      </annotation>
+      <annotation id="431">
+        <infon key="score">0.9990681</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:40:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18758" length="13"/>
+        <text>ethanethioate</text>
+      </annotation>
+      <annotation id="432">
+        <infon key="score">0.99910814</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18782" length="6"/>
+        <text>Ac-CoA</text>
+      </annotation>
+      <annotation id="1023">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:19:06Z</infon>
+        <location offset="18825" length="6"/>
+        <text>acetyl</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>18850</offset>
+      <text>Structural basis for hNaa60 substrate binding</text>
+      <annotation id="433">
+        <infon key="score">0.99913543</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:46Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18871" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>18896</offset>
+      <text>Several studies have demonstrated that the substrate specificities of hNaa60 and hNaa50 are highly overlapped. The structure of hNaa50p/CoA/peptide provides detailed information about the position of substrate N-terminal residues in the active site of hNaa50. Comparing the active site of hNaa60(1-242)/Ac-CoA with hNaa50p/CoA/peptide revealed that key catalytic and substrate binding residues are highly conserved in both proteins (Fig. 4A). With respect to catalysis, hNaa50p has been shown to employ residues Tyr 73 and His 112 to abstract proton from the α-amino group from the substrate’s first residue through a well-ordered water. A well-ordered water was also found between Tyr 97 and His 138 in hNaa60 (1-199)/CoA and hNaa60 (1-242)/Ac-CoA (Fig. 4B). To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B). Purity of all proteins were also analyzed by SDS-PAGE (Figure S5). As show in Fig. 5A, the mutants Y97A, Y97F, H138A and H138F abolished the activity of hNaa60. In contrast, to mutate the nearby solvent exposed residue Glu 37 to Ala (E37A) has little impact on the activity of hNaa60 (Figs 4B and 5A). In conclusion, the structural and functional studies indicate that hNaa60 applies the same two base mechanism through Tyr 97, His 138 and a well-ordered water as was described for hNaa50.</text>
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+        <infon key="updated_at">2023-09-20T10:49:15Z</infon>
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+        <text>kinetic assays</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="updated_at">2023-09-20T10:49:04Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:48:52Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:48:57Z</infon>
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+        <infon key="updated_at">2023-09-20T12:37:26Z</infon>
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+        <infon key="updated_at">2023-09-20T12:36:57Z</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="20269" length="6"/>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="updated_at">2023-09-20T10:47:55Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20355" length="5"/>
+        <text>water</text>
+      </annotation>
+      <annotation id="481">
+        <infon key="score">0.99939847</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="20382" length="6"/>
+        <text>hNaa50</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>20394</offset>
+      <text>The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A). Residues Tyr 38, Asn 143 and Tyr 165 are located around the malonate and interact with it through direct hydrogen bonds or water bridge (Fig. 4C). Although malonate is negatively charged, which is different from that of lysine ε-amine or peptide N-terminal amine, similar hydrophilic interactions may take place when substrate amine presents in the same position, since Tyr 38, Asn 143 and Tyr 165 are not positively or negatively charged. In agreement with this hypothesis, it was found that the Y38A, N143A and Y165A mutants all showed remarkably reduced activities as compared to WT, implying that these residues may be critical for substrate binding (Figs 4C and 5A).</text>
+      <annotation id="482">
+        <infon key="score">0.99926704</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:40:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20398" length="8"/>
+        <text>malonate</text>
+      </annotation>
+      <annotation id="483">
+        <infon key="score">0.99800014</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:35Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="20432" length="20"/>
+        <text>hNaa60(1-242)/Ac-CoA</text>
+      </annotation>
+      <annotation id="484">
+        <infon key="score">0.9984876</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:33:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20453" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="485">
+        <infon key="score">0.9993979</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:46Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="20526" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="486">
+        <infon key="score">0.9989399</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:34:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="20560" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="487">
+        <infon key="score">0.9947962</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:50:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="20600" length="3"/>
+        <text>Met</text>
+      </annotation>
+      <annotation id="993">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:45:09Z</infon>
+        <location offset="20621" length="7"/>
+        <text>peptide</text>
+      </annotation>
+      <annotation id="488">
+        <infon key="score">0.9987845</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:29:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20636" length="13"/>
+        <text>superposition</text>
+      </annotation>
+      <annotation id="489">
+        <infon key="score">0.999078</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:49:41Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="20659" length="19"/>
+        <text>hNaa50p/CoA/peptide</text>
+      </annotation>
+      <annotation id="490">
+        <infon key="score">0.9886729</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:50:30Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20679" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="491">
+        <infon key="score">0.9944216</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:50:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20709" length="6"/>
+        <text>Tyr 38</text>
+      </annotation>
+      <annotation id="492">
+        <infon key="score">0.99544126</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:50:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20717" length="7"/>
+        <text>Asn 143</text>
+      </annotation>
+      <annotation id="493">
+        <infon key="score">0.99556684</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:50:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20729" length="7"/>
+        <text>Tyr 165</text>
+      </annotation>
+      <annotation id="494">
+        <infon key="score">0.9989592</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:40:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20760" length="8"/>
+        <text>malonate</text>
+      </annotation>
+      <annotation id="495">
+        <infon key="score">0.99632376</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:49:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20805" length="14"/>
+        <text>hydrogen bonds</text>
+      </annotation>
+      <annotation id="496">
+        <infon key="score">0.9881353</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:49:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20823" length="12"/>
+        <text>water bridge</text>
+      </annotation>
+      <annotation id="497">
+        <infon key="score">0.9986852</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:40:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20856" length="8"/>
+        <text>malonate</text>
+      </annotation>
+      <annotation id="498">
+        <infon key="score">0.9957231</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:50:06Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="20920" length="6"/>
+        <text>lysine</text>
+      </annotation>
+      <annotation id="994">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:45:09Z</infon>
+        <location offset="20938" length="7"/>
+        <text>peptide</text>
+      </annotation>
+      <annotation id="499">
+        <infon key="score">0.9969008</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:49:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20972" length="24"/>
+        <text>hydrophilic interactions</text>
+      </annotation>
+      <annotation id="500">
+        <infon key="score">0.99480635</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:50:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21070" length="6"/>
+        <text>Tyr 38</text>
+      </annotation>
+      <annotation id="501">
+        <infon key="score">0.9955844</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:50:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21078" length="7"/>
+        <text>Asn 143</text>
+      </annotation>
+      <annotation id="502">
+        <infon key="score">0.9956566</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:50:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21090" length="7"/>
+        <text>Tyr 165</text>
+      </annotation>
+      <annotation id="503">
+        <infon key="score">0.99890196</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:34:31Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21197" length="4"/>
+        <text>Y38A</text>
+      </annotation>
+      <annotation id="504">
+        <infon key="score">0.99893147</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:34:34Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21203" length="5"/>
+        <text>N143A</text>
+      </annotation>
+      <annotation id="505">
+        <infon key="score">0.99892247</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:34:36Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21213" length="5"/>
+        <text>Y165A</text>
+      </annotation>
+      <annotation id="506">
+        <infon key="score">0.9975339</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21219" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="507">
+        <infon key="score">0.99915206</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:50:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21283" length="2"/>
+        <text>WT</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>21378</offset>
+      <text>The β3-β4 loop participates in the regulation of hNaa60-activity</text>
+      <annotation id="508">
+        <infon key="score">0.9993284</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:50:36Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="21382" length="10"/>
+        <text>β3-β4 loop</text>
+      </annotation>
+      <annotation id="509">
+        <infon key="score">0.9993001</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:46Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="21427" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>21449</offset>
+      <text>Residues between β3 and β4 of hNaa60 form a unique 20-residue long loop (residues 73–92) that is a short turn in many other NAT members (Fig. 1D). Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain. We therefore used mass spectrometry to analyze if Lys 79 was acetylated in our bacterially purified proteins, and observed no modification on this residue (Figure S6). To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively. Interestingly, both K79R and K79Q mutants led to an increase in the catalytic activity of hNaa60, while K79A mutant led to modest decrease of the activity (Fig. 5A). These data indicate that the acetylation of Lys 79 is not required for optimal catalytic activity of hNaa60 in vitro.</text>
+      <annotation id="510">
+        <infon key="score">0.99939644</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:00:24Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="21466" length="2"/>
+        <text>β3</text>
+      </annotation>
+      <annotation id="511">
+        <infon key="score">0.9992778</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:00:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="21473" length="2"/>
+        <text>β4</text>
+      </annotation>
+      <annotation id="512">
+        <infon key="score">0.99935776</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:46Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="21479" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="1014">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:00:43Z</infon>
+        <location offset="21500" length="20"/>
+        <text>20-residue long loop</text>
+      </annotation>
+      <annotation id="513">
+        <infon key="score">0.99748373</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:00:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21531" length="5"/>
+        <text>73–92</text>
+      </annotation>
+      <annotation id="514">
+        <infon key="score">0.9959029</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:30:32Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="21548" length="10"/>
+        <text>short turn</text>
+      </annotation>
+      <annotation id="899">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:28Z</infon>
+        <location offset="21573" length="3"/>
+        <text>NAT</text>
+      </annotation>
+      <annotation id="515">
+        <infon key="score">0.97910434</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:55:34Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21626" length="16"/>
+        <text>auto-acetylation</text>
+      </annotation>
+      <annotation id="1008">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:55:49Z</infon>
+        <location offset="21646" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="1009">
+        <infon key="type">residue_name_number</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:02Z</infon>
+        <location offset="21652" length="3"/>
+        <text>K79</text>
+      </annotation>
+      <annotation id="516">
+        <infon key="score">0.9993579</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:46Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="21688" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="517">
+        <infon key="score">0.99005854</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21738" length="6"/>
+        <text>Lys 79</text>
+      </annotation>
+      <annotation id="518">
+        <infon key="score">0.9959693</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21748" length="10"/>
+        <text>acetylated</text>
+      </annotation>
+      <annotation id="519">
+        <infon key="score">0.9988948</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21766" length="18"/>
+        <text>crystal structures</text>
+      </annotation>
+      <annotation id="520">
+        <infon key="score">0.9986427</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21812" length="16"/>
+        <text>electron density</text>
+      </annotation>
+      <annotation id="521">
+        <infon key="score">0.9889725</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21832" length="6"/>
+        <text>Lys 79</text>
+      </annotation>
+      <annotation id="522">
+        <infon key="score">0.9986272</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:32:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21869" length="17"/>
+        <text>mass spectrometry</text>
+      </annotation>
+      <annotation id="523">
+        <infon key="score">0.98724186</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21901" length="6"/>
+        <text>Lys 79</text>
+      </annotation>
+      <annotation id="524">
+        <infon key="score">0.9929476</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21912" length="10"/>
+        <text>acetylated</text>
+      </annotation>
+      <annotation id="1012">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:57:12Z</infon>
+        <location offset="22043" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="1013">
+        <infon key="type">residue_name_number</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:57:25Z</infon>
+        <location offset="22049" length="3"/>
+        <text>K79</text>
+      </annotation>
+      <annotation id="525">
+        <infon key="score">0.9744764</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22053" length="16"/>
+        <text>auto-acetylation</text>
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+      <annotation id="526">
+        <infon key="score">0.9990439</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22098" length="4"/>
+        <text>K79R</text>
+      </annotation>
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+        <infon key="score">0.99908864</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:25Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22107" length="4"/>
+        <text>K79Q</text>
+      </annotation>
+      <annotation id="528">
+        <infon key="score">0.98770905</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:37Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22112" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="529">
+        <infon key="score">0.99886703</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22136" length="13"/>
+        <text>un-acetylated</text>
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+      <annotation id="530">
+        <infon key="score">0.9990865</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:46Z</infon>
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+        <infon key="type">residue_name_number</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+      <annotation id="532">
+        <infon key="score">0.99891984</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:38Z</infon>
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+      <annotation id="535">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:46Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="22285" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="536">
+        <infon key="score">0.9991049</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:30Z</infon>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:48:11Z</infon>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>Lys 79</text>
+      </annotation>
+      <annotation id="540">
+        <infon key="score">0.9993723</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:46Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="22462" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>22485</offset>
+      <text>It is noted that the β3-β4 loop of hNaa60 acts like a door leaf to partly cover the substrate-binding pathway. We hence hypothesize that the β3-β4 loop may interfere with the access of the peptide substrates and that the solvent-exposing Lys 79 may play a potential role to remove the door leaf when it hovers in solvent (Fig. 4D). Acidic residues Glu 80, Asp 81 and Asp 83 interact with His 138, His 159 and His 158 to maintain the conformation of the β3-β4 loop, thus contribute to control the substrate binding (Fig. 4D). To verify this hypothesis, we mutated Glu 80, Asp 81 and Asp 83 to Ala respectively. In line with our hypothesis, E80A, D81A and D83A mutants exhibit at least 2-fold increase in hNaa60-activity (Fig. 5A). Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">protein</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="22569" length="25"/>
+        <text>substrate-binding pathway</text>
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+        <infon key="type">structure_element</infon>
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+        <infon key="updated_at">2023-09-20T10:50:37Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22626" length="10"/>
+        <text>β3-β4 loop</text>
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+      <annotation id="995">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:45:09Z</infon>
+        <location offset="22674" length="7"/>
+        <text>peptide</text>
+      </annotation>
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+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:37:45Z</infon>
+        <location offset="22706" length="16"/>
+        <text>solvent-exposing</text>
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+        <infon key="type">residue_name_number</infon>
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+        <infon key="updated_at">2023-09-20T10:56:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22723" length="6"/>
+        <text>Lys 79</text>
+      </annotation>
+      <annotation id="546">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:01:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22833" length="6"/>
+        <text>Glu 80</text>
+      </annotation>
+      <annotation id="547">
+        <infon key="score">0.99640024</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:01:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22841" length="6"/>
+        <text>Asp 81</text>
+      </annotation>
+      <annotation id="548">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:01:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22852" length="6"/>
+        <text>Asp 83</text>
+      </annotation>
+      <annotation id="549">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:48:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22873" length="7"/>
+        <text>His 138</text>
+      </annotation>
+      <annotation id="550">
+        <infon key="score">0.99452215</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:01:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22882" length="7"/>
+        <text>His 159</text>
+      </annotation>
+      <annotation id="551">
+        <infon key="score">0.9952251</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:01:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22894" length="7"/>
+        <text>His 158</text>
+      </annotation>
+      <annotation id="552">
+        <infon key="score">0.99881583</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:50:37Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22938" length="10"/>
+        <text>β3-β4 loop</text>
+      </annotation>
+      <annotation id="553">
+        <infon key="score">0.96276665</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:37:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23040" length="7"/>
+        <text>mutated</text>
+      </annotation>
+      <annotation id="554">
+        <infon key="score">0.99578</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:01:35Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23048" length="6"/>
+        <text>Glu 80</text>
+      </annotation>
+      <annotation id="555">
+        <infon key="score">0.996125</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:01:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23056" length="6"/>
+        <text>Asp 81</text>
+      </annotation>
+      <annotation id="556">
+        <infon key="score">0.9965105</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:01:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23067" length="6"/>
+        <text>Asp 83</text>
+      </annotation>
+      <annotation id="557">
+        <infon key="score">0.99566793</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:02:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23077" length="3"/>
+        <text>Ala</text>
+      </annotation>
+      <annotation id="558">
+        <infon key="score">0.99891067</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:02:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23124" length="4"/>
+        <text>E80A</text>
+      </annotation>
+      <annotation id="559">
+        <infon key="score">0.9990312</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:02:18Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23130" length="4"/>
+        <text>D81A</text>
+      </annotation>
+      <annotation id="560">
+        <infon key="score">0.9990434</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:02:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23139" length="4"/>
+        <text>D83A</text>
+      </annotation>
+      <annotation id="561">
+        <infon key="score">0.9980446</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23144" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="562">
+        <infon key="score">0.99934214</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:46Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="23188" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="563">
+        <infon key="score">0.998055</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:02:09Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23234" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="564">
+        <infon key="score">0.99928576</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23260" length="3"/>
+        <text>NAT</text>
+      </annotation>
+      <annotation id="565">
+        <infon key="score">0.99852866</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:02:34Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23269" length="15"/>
+        <text>S. solfataricus</text>
+      </annotation>
+      <annotation id="1015">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:04:25Z</infon>
+        <location offset="23301" length="25"/>
+        <text>10-residue long extension</text>
+      </annotation>
+      <annotation id="566">
+        <infon key="score">0.99925405</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:30:36Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23335" length="2"/>
+        <text>β3</text>
+      </annotation>
+      <annotation id="567">
+        <infon key="score">0.9989618</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:30:39Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23342" length="2"/>
+        <text>β4</text>
+      </annotation>
+      <annotation id="568">
+        <infon key="score">0.9890509</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:04:33Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23354" length="33"/>
+        <text>structure and biochemical studies</text>
+      </annotation>
+      <annotation id="569">
+        <infon key="score">0.9962972</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:30:46Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23404" length="9"/>
+        <text>extension</text>
+      </annotation>
+      <annotation id="570">
+        <infon key="score">0.9946806</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:03:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="23417" length="5"/>
+        <text>SsNat</text>
+      </annotation>
+      <annotation id="571">
+        <infon key="score">0.998391</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:34:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23469" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="572">
+        <infon key="score">0.9979418</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:03:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="23496" length="5"/>
+        <text>SsNat</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">title_1</infon>
+      <offset>23536</offset>
+      <text>Discussion</text>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>23547</offset>
+      <text>Nt-acetylation, which is carried out by the NAT family acetyltransferases, is an ancient and essential modification of proteins. Although many NATs are highly conserved from lower to higher eukaryotes and the substrate bias of them appears to be partially overlapped, there is a significant increase in the overall level of N-terminal acetylation from lower to higher eukaryotes. In this study we provide structural insights into Naa60 found only in multicellular eukaryotes.</text>
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+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23547" length="14"/>
+        <text>Nt-acetylation</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:06:21Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>NAT family acetyltransferases</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23690" length="4"/>
+        <text>NATs</text>
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+        <infon key="score">0.9988533</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:06:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>highly conserved</text>
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+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:06:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23721" length="5"/>
+        <text>lower</text>
+      </annotation>
+      <annotation id="578">
+        <infon key="score">0.94689333</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:06:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>higher eukaryotes</text>
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+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:03Z</infon>
+        <location offset="23871" length="22"/>
+        <text>N-terminal acetylation</text>
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+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:06:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23899" length="5"/>
+        <text>lower</text>
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+      <annotation id="580">
+        <infon key="score">0.97923696</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:06:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23908" length="17"/>
+        <text>higher eukaryotes</text>
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+      <annotation id="581">
+        <infon key="score">0.99926347</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="23977" length="5"/>
+        <text>Naa60</text>
+      </annotation>
+      <annotation id="582">
+        <infon key="score">0.9782407</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23997" length="24"/>
+        <text>multicellular eukaryotes</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>24023</offset>
+      <text>The N-terminus of hNaa60 harbors three hydrophobic residues (VVP) that makes it very difficult to express and purify the protein. This problem was solved by replacing residues 4–6 from VVP to EER that are found in Naa60 from Xenopus Laevis. Since Naa60 from human and from Xenopus Laevis are highly homologous (Fig. 1A), we speculate that these two proteins should have the same biological function. Therefore it is deduced that the VVP to EER replacement on the N-terminus of hNaa60 may not interfere with its function. However, in the hNaa60(1-242) structure the N-terminus adopts an α-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing. Hence it is not clear if the N-terminal end of wild-type hNaa60 is an α-helix, and what roles the hydrophobic residues 4–6 play in structure and function of wild-type hNaa60. In addition to the three-residue mutation (VVP to EER), we also tried many other hNaa60 constructs, but only the full-length protein and the truncated variant 1-199 behaved well. The finding that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) is intriguing. We speculate that low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies or there remains some undiscovered auto-inhibitory regulation in the full-length protein.</text>
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+        <infon key="updated_at">2023-09-20T09:46:46Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24041" length="6"/>
+        <text>hNaa60</text>
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+      <annotation id="584">
+        <infon key="score">0.32769075</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:19:39Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24084" length="3"/>
+        <text>VVP</text>
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+      <annotation id="585">
+        <infon key="score">0.99827373</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:38:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="24180" length="9"/>
+        <text>replacing</text>
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+        <infon key="score">0.99464613</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:33:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24199" length="3"/>
+        <text>4–6</text>
+      </annotation>
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+        <infon key="score">0.39737746</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:19:39Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24208" length="3"/>
+        <text>VVP</text>
+      </annotation>
+      <annotation id="588">
+        <infon key="score">0.3354909</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:19:33Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24215" length="3"/>
+        <text>EER</text>
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+      <annotation id="589">
+        <infon key="score">0.9994191</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24237" length="5"/>
+        <text>Naa60</text>
+      </annotation>
+      <annotation id="590">
+        <infon key="score">0.99843544</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:20:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="24248" length="14"/>
+        <text>Xenopus Laevis</text>
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+      <annotation id="591">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24270" length="5"/>
+        <text>Naa60</text>
+      </annotation>
+      <annotation id="592">
+        <infon key="score">0.9987526</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="24281" length="5"/>
+        <text>human</text>
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+        <infon key="score">0.9984007</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:20:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="24296" length="14"/>
+        <text>Xenopus Laevis</text>
+      </annotation>
+      <annotation id="594">
+        <infon key="score">0.655985</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:28:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24315" length="17"/>
+        <text>highly homologous</text>
+      </annotation>
+      <annotation id="595">
+        <infon key="score">0.6868272</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:05:36Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="24456" length="10"/>
+        <text>VVP to EER</text>
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+      <annotation id="596">
+        <infon key="score">0.99554366</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:05:39Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="24467" length="11"/>
+        <text>replacement</text>
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+      <annotation id="597">
+        <infon key="score">0.9993748</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:46Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24500" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="908">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:46Z</infon>
+        <location offset="24560" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="964">
+        <infon key="type">residue_range</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:24:08Z</infon>
+        <location offset="24567" length="5"/>
+        <text>1-242</text>
+      </annotation>
+      <annotation id="598">
+        <infon key="score">0.9979784</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:07:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24574" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="599">
+        <infon key="score">0.9973535</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:30:51Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24609" length="19"/>
+        <text>α-helical structure</text>
+      </annotation>
+      <annotation id="600">
+        <infon key="score">0.9877457</infon>
+        <infon key="type">residue_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:07:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24670" length="1"/>
+        <text>6</text>
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+      <annotation id="601">
+        <infon key="score">0.9979997</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:07:43Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24675" length="7"/>
+        <text>proline</text>
+      </annotation>
+      <annotation id="602">
+        <infon key="score">0.95973444</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:07:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="24705" length="13"/>
+        <text>hNaa60(1-199)</text>
+      </annotation>
+      <annotation id="603">
+        <infon key="score">0.9979778</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:07:28Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24719" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="604">
+        <infon key="score">0.9946813</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:30:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24763" length="22"/>
+        <text>semi-helical structure</text>
+      </annotation>
+      <annotation id="981">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:44:17Z</infon>
+        <location offset="24820" length="15"/>
+        <text>crystal packing</text>
+      </annotation>
+      <annotation id="605">
+        <infon key="score">0.9991526</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:07:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24884" length="9"/>
+        <text>wild-type</text>
+      </annotation>
+      <annotation id="606">
+        <infon key="score">0.9993598</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:46Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24894" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="607">
+        <infon key="score">0.9992042</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:30:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24907" length="7"/>
+        <text>α-helix</text>
+      </annotation>
+      <annotation id="608">
+        <infon key="score">0.9663139</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:08:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24956" length="3"/>
+        <text>4–6</text>
+      </annotation>
+      <annotation id="609">
+        <infon key="score">0.9991584</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:07:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24994" length="9"/>
+        <text>wild-type</text>
+      </annotation>
+      <annotation id="610">
+        <infon key="score">0.9993875</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:46Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25004" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="611">
+        <infon key="score">0.5227871</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:07Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="25045" length="8"/>
+        <text>mutation</text>
+      </annotation>
+      <annotation id="612">
+        <infon key="score">0.4418587</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:19:39Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25055" length="3"/>
+        <text>VVP</text>
+      </annotation>
+      <annotation id="613">
+        <infon key="score">0.51431394</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:19:33Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25062" length="3"/>
+        <text>EER</text>
+      </annotation>
+      <annotation id="614">
+        <infon key="score">0.99928135</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:46Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25093" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="615">
+        <infon key="score">0.99907476</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:20:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25125" length="11"/>
+        <text>full-length</text>
+      </annotation>
+      <annotation id="616">
+        <infon key="score">0.986394</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:20:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25153" length="9"/>
+        <text>truncated</text>
+      </annotation>
+      <annotation id="617">
+        <infon key="score">0.9944792</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:08:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25171" length="5"/>
+        <text>1-199</text>
+      </annotation>
+      <annotation id="618">
+        <infon key="score">0.959848</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25234" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="619">
+        <infon key="score">0.9730511</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:24:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25241" length="5"/>
+        <text>1-242</text>
+      </annotation>
+      <annotation id="1046">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:34:58Z</infon>
+        <location offset="25275" length="13"/>
+        <text>hNaa60(1-199)</text>
+      </annotation>
+      <annotation id="620">
+        <infon key="score">0.9990978</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:20:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25342" length="11"/>
+        <text>full-length</text>
+      </annotation>
+      <annotation id="621">
+        <infon key="score">0.99933344</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25354" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="622">
+        <infon key="score">0.9991409</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:20:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25515" length="11"/>
+        <text>full-length</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>25544</offset>
+      <text>The hNaa60 protein was proven to be localized on Golgi apparatus. Aksnes and colleagues predicted putative transmembrane domains and two putative sites of S-palmitoylation, by bioinformatics means, to account for Golgi localization of the protein. They then mutated all five cysteine residues of hNaa60’s to serine, including the two putative S-palmitoylation sites. However, these mutations did not abolish Naa60 membrane localization, indicating that S-palmitoylation is unlikely to (solely) account for targeting hNaa60 on Golgi. Furthermore, adding residues 217–242 of hNaa60 (containing residues 217–236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182–216 are important for Golgi localization of hNaa60. We found that residues 190–202 formed an amphipathic helix with an array of hydrophobic residues located on one side. This observation is reminiscent of the protein/membrane interaction through amphipathic helices in the cases of KalSec14, Atg3, PB1-F2 etc. In this model an amphipathic helix can immerse its hydrophobic side into the lipid bilayer through hydrophobic interactions. Therefore we propose that the amphipathic helix α5 may contribute to Golgi localization of hNaa60. This model, though may need further studies, is supported by the Aksnes studies.</text>
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+        <infon key="score">0.9991893</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25548" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="624">
+        <infon key="score">0.99866015</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:10:17Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25651" length="21"/>
+        <text>transmembrane domains</text>
+      </annotation>
+      <annotation id="625">
+        <infon key="score">0.97566384</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:10:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="25699" length="16"/>
+        <text>S-palmitoylation</text>
+      </annotation>
+      <annotation id="626">
+        <infon key="score">0.99236155</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:38:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="25802" length="7"/>
+        <text>mutated</text>
+      </annotation>
+      <annotation id="627">
+        <infon key="score">0.9971693</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:08:41Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25819" length="8"/>
+        <text>cysteine</text>
+      </annotation>
+      <annotation id="628">
+        <infon key="score">0.9992047</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25840" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="629">
+        <infon key="score">0.9971943</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:08:44Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25852" length="6"/>
+        <text>serine</text>
+      </annotation>
+      <annotation id="630">
+        <infon key="score">0.99831057</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:10:32Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25887" length="22"/>
+        <text>S-palmitoylation sites</text>
+      </annotation>
+      <annotation id="631">
+        <infon key="score">0.97516257</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:38:30Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="25926" length="9"/>
+        <text>mutations</text>
+      </annotation>
+      <annotation id="632">
+        <infon key="score">0.99930024</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:44:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25952" length="5"/>
+        <text>Naa60</text>
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+      <annotation id="633">
+        <infon key="score">0.9770059</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:10:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="25997" length="16"/>
+        <text>S-palmitoylation</text>
+      </annotation>
+      <annotation id="634">
+        <infon key="score">0.99929214</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="26060" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="635">
+        <infon key="score">0.9947547</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:38:43Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26090" length="6"/>
+        <text>adding</text>
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+      <annotation id="636">
+        <infon key="score">0.9976034</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:08:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26106" length="7"/>
+        <text>217–242</text>
+      </annotation>
+      <annotation id="637">
+        <infon key="score">0.9992981</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="26117" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="638">
+        <infon key="score">0.9976539</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:10:37Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26145" length="7"/>
+        <text>217–236</text>
+      </annotation>
+      <annotation id="639">
+        <infon key="score">0.997695</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:09:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26174" length="21"/>
+        <text>transmembrane domains</text>
+      </annotation>
+      <annotation id="640">
+        <infon key="score">0.92742413</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:09:25Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26218" length="4"/>
+        <text>eGFP</text>
+      </annotation>
+      <annotation id="641">
+        <infon key="score">0.7607362</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:23:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26293" length="4"/>
+        <text>eGFP</text>
+      </annotation>
+      <annotation id="642">
+        <infon key="score">0.8553622</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:35:19Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26298" length="13"/>
+        <text>hNaa60182-242</text>
+      </annotation>
+      <annotation id="643">
+        <infon key="score">0.9976466</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:10:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26356" length="7"/>
+        <text>182–216</text>
+      </annotation>
+      <annotation id="644">
+        <infon key="score">0.9992848</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="26404" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="645">
+        <infon key="score">0.99767846</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:10:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26435" length="7"/>
+        <text>190–202</text>
+      </annotation>
+      <annotation id="646">
+        <infon key="score">0.97816676</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26453" length="17"/>
+        <text>amphipathic helix</text>
+      </annotation>
+      <annotation id="1016">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:11:02Z</infon>
+        <location offset="26606" length="19"/>
+        <text>amphipathic helices</text>
+      </annotation>
+      <annotation id="647">
+        <infon key="score">0.9991417</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:28:53Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="26642" length="8"/>
+        <text>KalSec14</text>
+      </annotation>
+      <annotation id="648">
+        <infon key="score">0.9991431</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:28:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="26652" length="4"/>
+        <text>Atg3</text>
+      </annotation>
+      <annotation id="649">
+        <infon key="score">0.999237</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:29:04Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="26658" length="6"/>
+        <text>PB1-F2</text>
+      </annotation>
+      <annotation id="969">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:48Z</infon>
+        <location offset="26687" length="17"/>
+        <text>amphipathic helix</text>
+      </annotation>
+      <annotation id="650">
+        <infon key="score">0.99526036</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:26:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26769" length="24"/>
+        <text>hydrophobic interactions</text>
+      </annotation>
+      <annotation id="970">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:48Z</infon>
+        <location offset="26825" length="17"/>
+        <text>amphipathic helix</text>
+      </annotation>
+      <annotation id="651">
+        <infon key="score">0.99937785</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:31:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26843" length="2"/>
+        <text>α5</text>
+      </annotation>
+      <annotation id="652">
+        <infon key="score">0.99927133</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="26886" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>26986</offset>
+      <text>Previous studies indicated that members of NAT family are bi-functional NAT and KAT enzymes. However, known structures of NATs do not well support this hypothesis, since the β6-β7 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the α1-α2 loop, which would be good for the NAT but not KAT activity of the enzyme. Kinetic studies have been conducted to compare the NAT and KAT activity of hNaa50 in vitro, and indicate that the NAT activity of Naa50 is much higher than KAT activity. However, the substrate used in this study for assessing KAT activity was a small peptide which could not really mimic the 3D structure of a folded protein substrate in vivo. Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro. Conformational change of the β7-β8 hairpin (corresponding to the β6-β7 loop of other NATs) is noted in our structures (Figs 1D and 2C), which might provide an explanation to the NAT/KAT dual-activity in a structural biological view, but we were unable to rule out the possibility that the observed conformational change of this hairpin might be an artifact related to crystal packing or truncation of the C-terminal end of the protein. Further studies are therefore needed to reveal the mechanism for the KAT activity of this enzyme.</text>
+      <annotation id="900">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:28Z</infon>
+        <location offset="27029" length="3"/>
+        <text>NAT</text>
+      </annotation>
+      <annotation id="653">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27058" length="3"/>
+        <text>NAT</text>
+      </annotation>
+      <annotation id="654">
+        <infon key="score">0.9953199</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27066" length="3"/>
+        <text>KAT</text>
+      </annotation>
+      <annotation id="655">
+        <infon key="score">0.99714917</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:11:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27094" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="656">
+        <infon key="score">0.9986947</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27108" length="4"/>
+        <text>NATs</text>
+      </annotation>
+      <annotation id="657">
+        <infon key="score">0.9985568</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:31:12Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27160" length="13"/>
+        <text>β6-β7 hairpin</text>
+      </annotation>
+      <annotation id="658">
+        <infon key="score">0.99843925</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:12:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27174" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="659">
+        <infon key="score">0.9988381</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27190" length="4"/>
+        <text>NATs</text>
+      </annotation>
+      <annotation id="1052">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:39:50Z</infon>
+        <location offset="27229" length="34"/>
+        <text>tunnel-like substrate-binding site</text>
+      </annotation>
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+        <infon key="score">0.99911714</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:30:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27273" length="10"/>
+        <text>α1-α2 loop</text>
+      </annotation>
+      <annotation id="661">
+        <infon key="score">0.995466</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27313" length="3"/>
+        <text>NAT</text>
+      </annotation>
+      <annotation id="662">
+        <infon key="score">0.9779108</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27325" length="3"/>
+        <text>KAT</text>
+      </annotation>
+      <annotation id="663">
+        <infon key="score">0.9656652</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:12:15Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27353" length="15"/>
+        <text>Kinetic studies</text>
+      </annotation>
+      <annotation id="664">
+        <infon key="score">0.9873094</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27404" length="3"/>
+        <text>NAT</text>
+      </annotation>
+      <annotation id="665">
+        <infon key="score">0.96045804</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27412" length="3"/>
+        <text>KAT</text>
+      </annotation>
+      <annotation id="666">
+        <infon key="score">0.9993749</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="27428" length="6"/>
+        <text>hNaa50</text>
+      </annotation>
+      <annotation id="667">
+        <infon key="score">0.97203314</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27467" length="3"/>
+        <text>NAT</text>
+      </annotation>
+      <annotation id="668">
+        <infon key="score">0.99943024</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:21:03Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="27483" length="5"/>
+        <text>Naa50</text>
+      </annotation>
+      <annotation id="669">
+        <infon key="score">0.9692684</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27509" length="3"/>
+        <text>KAT</text>
+      </annotation>
+      <annotation id="670">
+        <infon key="score">0.71763927</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27579" length="3"/>
+        <text>KAT</text>
+      </annotation>
+      <annotation id="996">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:45:09Z</infon>
+        <location offset="27604" length="7"/>
+        <text>peptide</text>
+      </annotation>
+      <annotation id="1040">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:28:21Z</infon>
+        <location offset="27645" length="12"/>
+        <text>3D structure</text>
+      </annotation>
+      <annotation id="671">
+        <infon key="score">0.9810611</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:28:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27663" length="6"/>
+        <text>folded</text>
+      </annotation>
+      <annotation id="672">
+        <infon key="score">0.9983854</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:32:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27701" length="17"/>
+        <text>mass spectrometry</text>
+      </annotation>
+      <annotation id="1041">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:28:39Z</infon>
+        <location offset="27719" length="4"/>
+        <text>data</text>
+      </annotation>
+      <annotation id="673">
+        <infon key="score">0.99387944</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:48:11Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27757" length="11"/>
+        <text>acetylation</text>
+      </annotation>
+      <annotation id="923">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:08:43Z</infon>
+        <location offset="27772" length="7"/>
+        <text>histone</text>
+      </annotation>
+      <annotation id="972">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:32:13Z</infon>
+        <location offset="27780" length="5"/>
+        <text>H3-H4</text>
+      </annotation>
+      <annotation id="674">
+        <infon key="score">0.9655266</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:12:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27786" length="8"/>
+        <text>tetramer</text>
+      </annotation>
+      <annotation id="675">
+        <infon key="score">0.992084</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:24:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27795" length="7"/>
+        <text>lysines</text>
+      </annotation>
+      <annotation id="896">
+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:03Z</infon>
+        <location offset="27812" length="22"/>
+        <text>N-terminal acetylation</text>
+      </annotation>
+      <annotation id="918">
+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:05:37Z</infon>
+        <location offset="27839" length="18"/>
+        <text>lysine acetylation</text>
+      </annotation>
+      <annotation id="997">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:45:09Z</infon>
+        <location offset="27865" length="7"/>
+        <text>peptide</text>
+      </annotation>
+      <annotation id="676">
+        <infon key="score">0.99767715</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:12:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27885" length="14"/>
+        <text>activity assay</text>
+      </annotation>
+      <annotation id="677">
+        <infon key="score">0.97320694</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27920" length="3"/>
+        <text>KAT</text>
+      </annotation>
+      <annotation id="678">
+        <infon key="score">0.9989168</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:46Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27987" length="13"/>
+        <text>β7-β8 hairpin</text>
+      </annotation>
+      <annotation id="679">
+        <infon key="score">0.99842685</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:12:50Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28023" length="10"/>
+        <text>β6-β7 loop</text>
+      </annotation>
+      <annotation id="680">
+        <infon key="score">0.9988734</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="28043" length="4"/>
+        <text>NATs</text>
+      </annotation>
+      <annotation id="681">
+        <infon key="score">0.99845386</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:11:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28065" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="682">
+        <infon key="score">0.99693537</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="28136" length="3"/>
+        <text>NAT</text>
+      </annotation>
+      <annotation id="683">
+        <infon key="score">0.9884763</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="28140" length="3"/>
+        <text>KAT</text>
+      </annotation>
+      <annotation id="684">
+        <infon key="score">0.99828833</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:12:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28286" length="7"/>
+        <text>hairpin</text>
+      </annotation>
+      <annotation id="982">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:44:17Z</infon>
+        <location offset="28326" length="15"/>
+        <text>crystal packing</text>
+      </annotation>
+      <annotation id="685">
+        <infon key="score">0.97712255</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="28463" length="3"/>
+        <text>KAT</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>28518</offset>
+      <text>The relationship between enzyme, co-enzyme and substrates has been documented for several years. In early years, researchers found adjustment of GCN5 histone acetyltransferase structure when it binds CoA molecule. The complexed form of NatA is more suitable for catalytic activation, since the α1-α2 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA). In the structure of hNaa50/CoA/peptide, Phe 27 in the α1-α2 loop appears to make hydrophobic interaction with the N-terminal Met of substrate peptide. However, the hNaa60(1-242)/Ac-CoA crystal structure indicated that its counterpart in hNaa60, Phe 34, could also accommodate the binding of a hydrophilic malonate that occupied the substrate binding site although it maintained the same conformation as that observed in hNaa50. Interestingly, the terminal thiol of CoA adopted alternative conformations in the structure of hNaa60(1-199)/CoA. One was to approach the substrate amine; the other was to approach the α1-α2 loop and away from the substrate amine. Same alternative conformations of CoA were observed in the hNaa60(1-199)(F34A) crystal structure, and our kinetic data showed that the F34A mutation abolished the activity of the enzyme. Taken together, our data indicated that Phe 34 in hNaa60 may play a role in placing co-enzyme at the right location to facilitate the acetyl-transfer. However, these data did not rule out that possibility that Phe 34 may coordinate the binding of the N-terminal Met through hydrophobic interaction as was proposed by previous studies.</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:14:32Z</infon>
+        <location offset="28663" length="30"/>
+        <text>GCN5 histone acetyltransferase</text>
+      </annotation>
+      <annotation id="686">
+        <infon key="score">0.99628496</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:14:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28694" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="687">
+        <infon key="score">0.9992446</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="28718" length="3"/>
+        <text>CoA</text>
+      </annotation>
+      <annotation id="688">
+        <infon key="score">0.9990343</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:28:52Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28736" length="9"/>
+        <text>complexed</text>
+      </annotation>
+      <annotation id="689">
+        <infon key="score">0.996225</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:54:46Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="28754" length="4"/>
+        <text>NatA</text>
+      </annotation>
+      <annotation id="690">
+        <infon key="score">0.9991602</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:30:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28812" length="10"/>
+        <text>α1-α2 loop</text>
+      </annotation>
+      <annotation id="691">
+        <infon key="score">0.9989286</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:34:06Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28890" length="22"/>
+        <text>substrate-binding site</text>
+      </annotation>
+      <annotation id="692">
+        <infon key="score">0.9994199</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:21:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="28940" length="5"/>
+        <text>Naa15</text>
+      </annotation>
+      <annotation id="693">
+        <infon key="score">0.99938476</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:21:17Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="28961" length="5"/>
+        <text>Naa10</text>
+      </annotation>
+      <annotation id="694">
+        <infon key="score">0.74719286</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:28:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28972" length="9"/>
+        <text>catalytic</text>
+      </annotation>
+      <annotation id="695">
+        <infon key="score">0.6046873</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:31:19Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28982" length="7"/>
+        <text>subunit</text>
+      </annotation>
+      <annotation id="696">
+        <infon key="score">0.97488165</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:54:46Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="28993" length="4"/>
+        <text>NatA</text>
+      </annotation>
+      <annotation id="697">
+        <infon key="score">0.99712</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:14:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29007" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="698">
+        <infon key="score">0.99884164</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:15:05Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="29020" length="18"/>
+        <text>hNaa50/CoA/peptide</text>
+      </annotation>
+      <annotation id="699">
+        <infon key="score">0.9969653</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29040" length="6"/>
+        <text>Phe 27</text>
+      </annotation>
+      <annotation id="700">
+        <infon key="score">0.9991973</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:30:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="29054" length="10"/>
+        <text>α1-α2 loop</text>
+      </annotation>
+      <annotation id="701">
+        <infon key="score">0.9969026</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:29Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="29081" length="23"/>
+        <text>hydrophobic interaction</text>
+      </annotation>
+      <annotation id="702">
+        <infon key="score">0.9954798</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:16:34Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="29125" length="3"/>
+        <text>Met</text>
+      </annotation>
+      <annotation id="998">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:45:09Z</infon>
+        <location offset="29142" length="7"/>
+        <text>peptide</text>
+      </annotation>
+      <annotation id="948">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:35Z</infon>
+        <location offset="29164" length="20"/>
+        <text>hNaa60(1-242)/Ac-CoA</text>
+      </annotation>
+      <annotation id="703">
+        <infon key="score">0.99837536</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:33:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29185" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="704">
+        <infon key="score">0.9994266</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29237" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="705">
+        <infon key="score">0.9955821</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29245" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="706">
+        <infon key="score">0.99918216</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:40:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="29305" length="8"/>
+        <text>malonate</text>
+      </annotation>
+      <annotation id="707">
+        <infon key="score">0.9986929</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:39:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="29332" length="22"/>
+        <text>substrate binding site</text>
+      </annotation>
+      <annotation id="708">
+        <infon key="score">0.99944645</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29420" length="6"/>
+        <text>hNaa50</text>
+      </annotation>
+      <annotation id="709">
+        <infon key="score">0.6209677</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:23:27Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="29456" length="5"/>
+        <text>thiol</text>
+      </annotation>
+      <annotation id="710">
+        <infon key="score">0.99915993</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="29465" length="3"/>
+        <text>CoA</text>
+      </annotation>
+      <annotation id="711">
+        <infon key="score">0.99825984</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:25:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29510" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="712">
+        <infon key="score">0.9801244</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:54Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="29523" length="17"/>
+        <text>hNaa60(1-199)/CoA</text>
+      </annotation>
+      <annotation id="713">
+        <infon key="score">0.99917614</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:30:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="29613" length="10"/>
+        <text>α1-α2 loop</text>
+      </annotation>
+      <annotation id="714">
+        <infon key="score">0.9990846</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="29693" length="3"/>
+        <text>CoA</text>
+      </annotation>
+      <annotation id="1018">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:16:09Z</infon>
+        <location offset="29718" length="19"/>
+        <text>hNaa60(1-199)(F34A)</text>
+      </annotation>
+      <annotation id="715">
+        <infon key="score">0.998599</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:33:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29738" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="716">
+        <infon key="score">0.97129583</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:25:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29765" length="12"/>
+        <text>kinetic data</text>
+      </annotation>
+      <annotation id="717">
+        <infon key="score">0.9991026</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:42:56Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="29794" length="4"/>
+        <text>F34A</text>
+      </annotation>
+      <annotation id="943">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:07Z</infon>
+        <location offset="29799" length="8"/>
+        <text>mutation</text>
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+      <annotation id="718">
+        <infon key="score">0.9954623</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29886" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="719">
+        <infon key="score">0.9994444</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29896" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="1020">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:19:05Z</infon>
+        <location offset="29980" length="6"/>
+        <text>acetyl</text>
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+      <annotation id="720">
+        <infon key="score">0.99600923</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="30056" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="721">
+        <infon key="score">0.99463564</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:16:30Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="30108" length="3"/>
+        <text>Met</text>
+      </annotation>
+      <annotation id="722">
+        <infon key="score">0.99646735</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:27:29Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30120" length="23"/>
+        <text>hydrophobic interaction</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>30205</offset>
+      <text>Furthermore, we showed that hNaa60 adopts the classical two base mechanism to catalyze acetyl-transfer. Although sequence identity between hNaa60 and hNaa50 is low, key residues in the active site of both enzymes are highly conserved. This can reasonably explain the high overlapping substrates specificities between hNaa60 and hNaa50. Another structural feature of hNaa60 that distinguishes it from other NATs is the β3-β4 long loop which appears to inhibit the catalytic activity of hNaa60. However, this loop also seems to stabilize the whole hNaa60 structure, because deletion mutations of this region led to protein precipitation and aggregation (Figure S7). A previous study suggested that the auto-acetylation of Lys 79 was important for hNaa60-activity, whereas the point mutation K79R did not decrease the activity of hNaa60 in our study. Meanwhile, no electron density of acetyl group was found on Lys 79 in our structures and mass spectrometry analysis. Hence, it appears that the auto-acetylation of hNaa60 is not an essential modification for its activity for the protein we used here. As for the reason why K79R in Yang’s previous studies reduced the activity of the enzyme, but in our studies it didn’t, we suspect that the stability of this mutant may play some role. K79R is less stable than the wild-type enzyme as was judged by its poorer gel-filtration behavior and tendency to precipitate. In our studies we have paid special attention and carefully handled this protein to ensure that we did get enough of the protein in good condition for kinetic assays. The intracellular environment is more complicated than our in vitro assay and the substrate specificity of hNaa60 most focuses on transmembrane proteins. The interaction between hNaa60 and its substrates may involve the protein-membrane interaction which would further increase the complexity. It is not clear if the structure of hNaa60 is different in vivo or if other potential partner proteins may help to regulate its activity. Nevertheless, our study may be an inspiration for further studies on the functions and regulation of this youngest member of the NAT family.</text>
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+        <infon key="score">0.9992888</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30233" length="6"/>
+        <text>hNaa60</text>
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+      <annotation id="1024">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:19:06Z</infon>
+        <location offset="30292" length="6"/>
+        <text>acetyl</text>
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+      <annotation id="724">
+        <infon key="score">0.9992924</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30344" length="6"/>
+        <text>hNaa60</text>
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+      <annotation id="725">
+        <infon key="score">0.9993185</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30355" length="6"/>
+        <text>hNaa50</text>
+      </annotation>
+      <annotation id="726">
+        <infon key="score">0.9990032</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:34:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="30390" length="11"/>
+        <text>active site</text>
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+        <infon key="score">0.99891543</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:29:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="30422" length="16"/>
+        <text>highly conserved</text>
+      </annotation>
+      <annotation id="728">
+        <infon key="score">0.99930453</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30522" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="729">
+        <infon key="score">0.9993167</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30533" length="6"/>
+        <text>hNaa50</text>
+      </annotation>
+      <annotation id="730">
+        <infon key="score">0.99931</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30571" length="6"/>
+        <text>hNaa60</text>
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+        <infon key="score">0.9991873</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30611" length="4"/>
+        <text>NATs</text>
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+      <annotation id="732">
+        <infon key="score">0.99923515</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:51Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="30623" length="15"/>
+        <text>β3-β4 long loop</text>
+      </annotation>
+      <annotation id="733">
+        <infon key="score">0.9992981</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30690" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="734">
+        <infon key="score">0.9965564</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:18:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="30712" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="735">
+        <infon key="score">0.99934274</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30751" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="736">
+        <infon key="score">0.9930085</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:26:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="30758" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="737">
+        <infon key="score">0.9984491</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:38:48Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30777" length="18"/>
+        <text>deletion mutations</text>
+      </annotation>
+      <annotation id="738">
+        <infon key="score">0.9753645</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:18:11Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30905" length="16"/>
+        <text>auto-acetylation</text>
+      </annotation>
+      <annotation id="739">
+        <infon key="score">0.98703116</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="30925" length="6"/>
+        <text>Lys 79</text>
+      </annotation>
+      <annotation id="740">
+        <infon key="score">0.9992586</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30950" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="741">
+        <infon key="score">0.879584</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:17:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30979" length="14"/>
+        <text>point mutation</text>
+      </annotation>
+      <annotation id="742">
+        <infon key="score">0.99910057</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30994" length="4"/>
+        <text>K79R</text>
+      </annotation>
+      <annotation id="743">
+        <infon key="score">0.99931717</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="31032" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="744">
+        <infon key="score">0.9931169</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:35:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="31067" length="16"/>
+        <text>electron density</text>
+      </annotation>
+      <annotation id="1025">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:19:06Z</infon>
+        <location offset="31087" length="6"/>
+        <text>acetyl</text>
+      </annotation>
+      <annotation id="745">
+        <infon key="score">0.9799822</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="31113" length="6"/>
+        <text>Lys 79</text>
+      </annotation>
+      <annotation id="746">
+        <infon key="score">0.9984842</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:26:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="31127" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="747">
+        <infon key="score">0.9960762</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:32:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="31142" length="17"/>
+        <text>mass spectrometry</text>
+      </annotation>
+      <annotation id="748">
+        <infon key="score">0.97629786</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:18:18Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="31197" length="16"/>
+        <text>auto-acetylation</text>
+      </annotation>
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+        <infon key="score">0.9993073</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="31217" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="750">
+        <infon key="score">0.9990808</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="31326" length="4"/>
+        <text>K79R</text>
+      </annotation>
+      <annotation id="751">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:43:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="31462" length="6"/>
+        <text>mutant</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:56:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="31489" length="4"/>
+        <text>K79R</text>
+      </annotation>
+      <annotation id="753">
+        <infon key="score">0.89987457</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:18:27Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="31502" length="6"/>
+        <text>stable</text>
+      </annotation>
+      <annotation id="754">
+        <infon key="score">0.9989593</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:07:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="31518" length="9"/>
+        <text>wild-type</text>
+      </annotation>
+      <annotation id="1019">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:17:54Z</infon>
+        <location offset="31563" length="14"/>
+        <text>gel-filtration</text>
+      </annotation>
+      <annotation id="1007">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:49:16Z</infon>
+        <location offset="31767" length="14"/>
+        <text>kinetic assays</text>
+      </annotation>
+      <annotation id="755">
+        <infon key="score">0.99931145</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="31890" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="756">
+        <infon key="score">0.99931645</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="31961" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="757">
+        <infon key="score">0.9949426</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:18:35Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="32100" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="758">
+        <infon key="score">0.9992893</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:47Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="32113" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="901">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:28Z</infon>
+        <location offset="32344" length="3"/>
+        <text>NAT</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_1</infon>
+      <offset>32362</offset>
+      <text>Methods</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>32370</offset>
+      <text>Cloning, expression and purification of Homo sapiens Naa60 (hNaa60)</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>32438</offset>
+      <text>The cDNA encoding hNaa60 residues 1–242 (full-length) or residues 1–199 were amplified by PCR and inserted into the pET23a vector, which had been modified to provide an N-terminal 6xHis-tag followed by a tobacco etch virus (TEV) protease cleavage site. The VVP to EER (residues 4–6) mutation and other mutations for functional studies were introduced using the quick change method. The protein was expressed in Escherichia coli BL21 (DE3) or Escherichia coli BL21 (DE3) pLysS at 16 °C for 15 h in the presence of 0.1 mM IPTG. Cells were harvested at 4 °C by centrifugation (4,000 g for 10 min) and resuspended in buffer A containing 20 mM Tris, pH 8.0, 500 mM NaCl, 50 mM imidazole, 10% glycerol, 1 mM protease inhibitor PMSF (Phenylmethylsulfonyl fluoride) and 1 mM Tris (2-carboxyethyl)phosphine (TCEP) hydrochloride. Cells were lysed by sonication and the lysate was cleared by centrifugation (18,000 g at 4 °C for 20 min). Then the supernatant was loaded onto a 5-mL Chelating Sepharose column (GE Healthcare) charged with Ni2+ and washed with buffer B (20 mM Tris, pH 8.0, 500 mM NaCl, 50 mM imidazole, 1% glycerol and 1 mM TCEP). The protein was eluted with buffer C (20 mM Tris, pH 8.0, 500 mM NaCl, 300 mM imidazole, 1% glycerol and 1 mM TCEP). The eluent was digested by His-tagged TEV protease and concentrated by ultrafiltration at the same time. After 3 hours, the concentrated eluent was diluted 10 times with buffer D (20 mM Tris, pH 8.0, 500 mM NaCl, 1% glycerol and 1 mM TCEP) and the diluent was passed through the nickel column once again to remove the His-tagged TEV protease and the un-cleaved His-hNaa60 protein. The flow-through was concentrated to 500 μl and loaded onto a Superose 6 or Superdex 200 10/300 gel-filtration column (GE Healthcare) equilibrated with buffer E (20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP). Fractions containing the protein were collected and concentrated to a final concentration of 10 mg/ml for crystallization or acetyltransferases assays.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>34517</offset>
+      <text>Circular Dichroism (CD) Spectroscopy</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>34554</offset>
+      <text>CD spectra of the proteins were obtained using a Jasco J-810 circular dichroism spectropolarimeter scanning from 190 to 250 nm with a 1 mm quartz cuvette. The wild-type and mutant proteins were examined at 4.5 μM concentration in 20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP at room temperature. All samples were centrifuged at 10,000 g for 5 min before analysis.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>34949</offset>
+      <text>Crystallization, data collection and structure determination</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>35010</offset>
+      <text>The purified hNaa60(1-242), hNaa60(1-199) or F34A(1-199) protein was mixed with acetyl coenzyme A (Ac-CoA) or coenzyme A (CoA) (Sigma), respectively, at a 1:5 molar ratio before crystallization. All crystals were made by the hanging-drop vapor diffusion method. The crystallization reservoir solution for hNaa60(1-242) was 10 mM Tris pH 8.0, 75 mM NaCl, 0.5% glycerol, 3% v/v Tacsimate pH 4.0 (Hampton Research) and 7.5% w/v polyethylene glycol 3350 (PEG 3350), and for hNaa60(1-199) was 0.2 M L-Proline, 0.1 M HEPES pH 7.5, 10% w/v PEG 3350. Crystals of F34A mutation were obtained in 0.2 M Lithium Sulfate monohydrate, 0.1 M Tris pH 8.5, 20% w/v PEG 3350. The crystals were flash-frozen in liquid nitrogen in a cryo-protectant made of the reservoir solution supplemented with 25% glycerol.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>35814</offset>
+      <text>The diffraction data were collected at the Shanghai SSRF BL18U1 beamline or at the Argonne National Laboratory APS ID19 beamline at 100 K. The data were processed with HKL3000. The hNaa60(1-199) structure was determined by molecular replacement with Phaser using a previously reported GNAT family acetyltransferase structure (PDB 2AE6) as the search model. The hNaa60(1-242) structure was solved by molecular replacement using hNaa60(1-199) structure as the search model. To improve the model quality, the programs ARP/wARP in CCP4 or simulated-annealing in CNS were used. Iterative cycles of manual refitting and crystallographic refinement were performed using COOT and Phenix. Ac-CoA/CoA and malonate were modeled into the closely fitting positive Fo-Fc electron density and then included in following refinement cycles. Topology and parameter files for Ac-CoA/CoA and malonate were generated using PRODRG. All figures for the molecular models were prepared using the PyMOL program. Statistics of diffraction data processing and structure refinement are shown in Table 1.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>36891</offset>
+      <text>Acetyltransferase assay</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>36915</offset>
+      <text>Acetyltransferase assay of hNaa60 was conducted as described previously. Briefly, a reaction cocktail containing 100 mM Tris-HCl buffer, pH 8.5, 0.07% alkylated BSA, 0.01% NP-40, 1 mM EDTA, 150 μM Ac-CoA (Sigma) was prepared and varied concentrations of the substrate peptide (0–400 μM) (NH2-MKGKEEKEGGAR-COOH) was added in a 1.5-mL microfuge tube, and then the respective enzyme was added to initiate the reaction with a final assay volume of 100 μL. The reaction was carried out for 20 minutes at 37 °C. Aliquots (40 μL) of the reaction were then removed and quenched with 40 μL of ice-cold isopropanol in individual wells of a 96-well black microplate (Corning), and then mixed with 80 μl of 25 μM 7-diethylamino-3-(49 maleimidylphenyl)-4-methylcoumarin (CPM) (Sigma) in 100 mM Tris-HCl (pH 8.5) and 1% Triton X-100 and allowed to react in darkness for 10 minutes prior to reading. The fluorescence signal was monitored using a Varioskan Flash plate reader (Thermo Scientific) at Exmax = 385 nm and Emmax = 465 nm. Substrate inhibition appeared at high concentrations of substrate peptide prevented our kinetics assays from reaching saturation of the enzyme. Therefore, we determined the value of kcat/Km by fitting our data to the equation: v = (kcat/Km)[ET][S] when the substrate concentration was far less than Km. The assays were done in triplicate. The slope of the line indicates the kcat/Km value of the enzyme (Figure S1).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_1</infon>
+      <offset>38401</offset>
+      <text>Additional Information</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>38424</offset>
+      <text>How to cite this article: Chen, J.-Y. et al. Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase. Sci. Rep. 6, 31425; doi: 10.1038/srep31425 (2016).</text>
+    </passage>
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+      <infon key="type">title_1</infon>
+      <offset>38617</offset>
+      <text>Supplementary Material</text>
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+      <text>Author Contributions J.-Y.C., L.L., C.-L.C., M.-J.L. and X.Y. designed and performed the experiments. K.T. collected diffraction data at APS. C.-H.Y. conceived and instructed the project. All authors are involved in data analysis. J.-Y.C. and C.-H.Y. wrote the manuscript.</text>
+    </passage>
+    <passage>
+      <infon key="file">srep31425-f1.jpg</infon>
+      <infon key="id">f1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>42521</offset>
+      <text>Overall structure of Naa60.</text>
+      <annotation id="759">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:07:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="42529" length="9"/>
+        <text>structure</text>
+      </annotation>
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+        <infon key="updated_at">2023-09-20T09:44:48Z</infon>
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+        <location offset="42542" length="5"/>
+        <text>Naa60</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">srep31425-f1.jpg</infon>
+      <infon key="id">f1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>42549</offset>
+      <text>(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus). Alignment was generated using NPS@ and ESPript.3.0 (http://espript.ibcp.fr/ESPript/ESPript/). Residues 4–6 are highlighted in red box. (B) The structure of hNaa60(1-199)/CoA complex is shown as a yellow cartoon model. The CoA molecule is shown as sticks. (C) The structure of hNaa60(1-242)/Ac-CoA complex is presented as a cartoon model in cyan. The Ac-CoA and malonate molecules are shown as cyan and purple sticks, respectively. The secondary structures are labeled starting with α0. (D) Superposition of hNaa60(1-242) (cyan), hNaa60(1-199) (yellow) and hNaa50 (pink, PDB 3TFY). The Ac-CoA of hNaa60(1-242)/Ac-CoA complex is represented as cyan sticks.</text>
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+        <infon key="identifier">MESH:</infon>
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+        <text>Sequence alignment</text>
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+        <location offset="42575" length="5"/>
+        <text>Naa60</text>
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+        <infon key="updated_at">2023-09-20T09:55:36Z</infon>
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+        <text>NatF</text>
+      </annotation>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="updated_at">2023-09-20T09:47:15Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="updated_at">2023-09-20T09:47:04Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:40:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="43091" length="8"/>
+        <text>malonate</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:29:16Z</infon>
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+        <infon key="updated_at">2023-09-20T10:24:08Z</infon>
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <text>hNaa60(1-242)/Ac-CoA</text>
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+    <passage>
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+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
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+      <text>Amphipathicity of the α5 helix and alternative conformations of the β7-β8 hairpin.</text>
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+        <location offset="43457" length="13"/>
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+      <infon key="section_type">FIG</infon>
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+      <text>(A) The α5 helix of hNaa60(1-242) in one asymmetric unit (slate) interacts with another hNaa60 molecule in a neighboring asymmetric unit (cyan). A close view of the interaction is shown in red box. Side-chains of hydrophobic residues on α5 helix and the neighboring molecule participating in the interaction are shown as yellow and green sticks, respectively. (B) The α5 helix of hNaa60(1-199) in one asymmetric unit (yellow) interacts with another hNaa60 molecule in the neighboring asymmetric units (green). A close view of the interaction is shown in the red box. Side-chains of hydrophobic residues on α5 helix and the neighboring molecule (green) participating in the interaction are shown as yellow and green sticks, respectively. The third molecule (pink) does not directly interact with the α5 helix. (C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E). The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D). The α-amine of the NAT substrate and ε-amine of the KAT substrate (along with the lysine side-chain) subject to acetylation are shown as sticks.</text>
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:10:20Z</infon>
+        <location offset="43862" length="13"/>
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+        <location offset="43931" length="6"/>
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+        <infon key="identifier">SO:</infon>
+        <location offset="44281" length="8"/>
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+        <text>Superposition</text>
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+        <infon key="updated_at">2023-09-20T12:11:01Z</infon>
+        <location offset="44312" length="13"/>
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+        <infon key="identifier">PR:</infon>
+        <location offset="44339" length="6"/>
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+        <location offset="44346" length="5"/>
+        <text>1-242</text>
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+        <infon key="identifier">SO:</infon>
+        <location offset="44397" length="13"/>
+        <text>β7-β8 hairpin</text>
+      </annotation>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:11:43Z</infon>
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+        <location offset="44424" length="10"/>
+        <text>structures</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:29:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="44442" length="13"/>
+        <text>Superposition</text>
+      </annotation>
+      <annotation id="808">
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:34:33Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="44459" length="5"/>
+        <text>Hat1p</text>
+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:09:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="44465" length="2"/>
+        <text>H4</text>
+      </annotation>
+      <annotation id="810">
+        <infon key="score">0.994859</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:48Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="44501" length="6"/>
+        <text>hNaa60</text>
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+        <infon key="type">residue_range</infon>
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+        <infon key="updated_at">2023-09-20T10:24:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44508" length="5"/>
+        <text>1-242</text>
+      </annotation>
+      <annotation id="1030">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:11:20Z</infon>
+        <location offset="44528" length="13"/>
+        <text>hNaa60(1-199)</text>
+      </annotation>
+      <annotation id="924">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:08:43Z</infon>
+        <location offset="44559" length="7"/>
+        <text>histone</text>
+      </annotation>
+      <annotation id="812">
+        <infon key="score">0.9862388</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:09:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="44567" length="2"/>
+        <text>H4</text>
+      </annotation>
+      <annotation id="999">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:45:10Z</infon>
+        <location offset="44570" length="7"/>
+        <text>peptide</text>
+      </annotation>
+      <annotation id="813">
+        <infon key="score">0.9983368</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="44581" length="3"/>
+        <text>KAT</text>
+      </annotation>
+      <annotation id="814">
+        <infon key="score">0.99917674</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:11:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44596" length="8"/>
+        <text>bound to</text>
+      </annotation>
+      <annotation id="815">
+        <infon key="score">0.9994037</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:34:33Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="44605" length="5"/>
+        <text>Hat1p</text>
+      </annotation>
+      <annotation id="1000">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:45:10Z</infon>
+        <location offset="44647" length="7"/>
+        <text>peptide</text>
+      </annotation>
+      <annotation id="816">
+        <infon key="score">0.9989772</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:11:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44655" length="8"/>
+        <text>bound to</text>
+      </annotation>
+      <annotation id="817">
+        <infon key="score">0.99940395</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="44664" length="6"/>
+        <text>hNaa50</text>
+      </annotation>
+      <annotation id="818">
+        <infon key="score">0.9982008</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="44674" length="3"/>
+        <text>NAT</text>
+      </annotation>
+      <annotation id="1036">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:23:52Z</infon>
+        <location offset="44730" length="10"/>
+        <text>Nt-peptide</text>
+      </annotation>
+      <annotation id="819">
+        <infon key="score">0.99784434</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:39:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="44748" length="13"/>
+        <text>superimposing</text>
+      </annotation>
+      <annotation id="820">
+        <infon key="score">0.99932384</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="44762" length="6"/>
+        <text>hNaa50</text>
+      </annotation>
+      <annotation id="821">
+        <infon key="score">0.999337</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:48Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="44794" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="822">
+        <infon key="score">0.9966156</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="44825" length="3"/>
+        <text>NAT</text>
+      </annotation>
+      <annotation id="823">
+        <infon key="score">0.9981242</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:45:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="44858" length="3"/>
+        <text>KAT</text>
+      </annotation>
+      <annotation id="824">
+        <infon key="score">0.9917366</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:11:34Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="44888" length="6"/>
+        <text>lysine</text>
+      </annotation>
+      <annotation id="825">
+        <infon key="score">0.9904163</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:48:11Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="44918" length="11"/>
+        <text>acetylation</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">srep31425-f3.jpg</infon>
+      <infon key="id">f3</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>44987</offset>
+      <text>Electron density map of the active site.</text>
+      <annotation id="826">
+        <infon key="score">0.9986318</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:12:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44987" length="20"/>
+        <text>Electron density map</text>
+      </annotation>
+      <annotation id="827">
+        <infon key="score">0.9990533</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:34:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="45015" length="11"/>
+        <text>active site</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">srep31425-f3.jpg</infon>
+      <infon key="id">f3</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>45028</offset>
+      <text>The 2Fo-Fc maps contoured at 1.0σ are shown for hNaa60(1-242)/Ac-CoA (A), hNaa60(1-199)/CoA (B) and hNaa60(1-199) F34A/CoA (C). The putative substrate peptide binding site is indicated by the peptide (shown as pink sticks) from the hNaa50/CoA/peptide complex structure after superimposing hNaa50 on the hNaa60 structures determined in this study. The black arrow indicates the α-amine of the first Met (M1) (all panels). The purple arrow indicates the acetyl moiety of Ac-CoA (A). The red arrow indicates the alternative conformation of the thiol moiety of the co-enzyme when Phe 34 side-chain is displaced (B) or mutated to Ala (C).</text>
+      <annotation id="828">
+        <infon key="score">0.99838984</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:12:09Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45032" length="11"/>
+        <text>2Fo-Fc maps</text>
+      </annotation>
+      <annotation id="949">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:35Z</infon>
+        <location offset="45076" length="20"/>
+        <text>hNaa60(1-242)/Ac-CoA</text>
+      </annotation>
+      <annotation id="957">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:54Z</infon>
+        <location offset="45102" length="17"/>
+        <text>hNaa60(1-199)/CoA</text>
+      </annotation>
+      <annotation id="1031">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:12:54Z</infon>
+        <location offset="45128" length="22"/>
+        <text>hNaa60(1-199) F34A/CoA</text>
+      </annotation>
+      <annotation id="829">
+        <infon key="score">0.99883485</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:13:11Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="45169" length="30"/>
+        <text>substrate peptide binding site</text>
+      </annotation>
+      <annotation id="830">
+        <infon key="score">0.7129295</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:45:10Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="45220" length="7"/>
+        <text>peptide</text>
+      </annotation>
+      <annotation id="831">
+        <infon key="score">0.99902344</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:13:05Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="45260" length="18"/>
+        <text>hNaa50/CoA/peptide</text>
+      </annotation>
+      <annotation id="832">
+        <infon key="score">0.930311</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:13:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45287" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="833">
+        <infon key="score">0.9982256</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:39:06Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="45303" length="13"/>
+        <text>superimposing</text>
+      </annotation>
+      <annotation id="834">
+        <infon key="score">0.99938524</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="45317" length="6"/>
+        <text>hNaa50</text>
+      </annotation>
+      <annotation id="835">
+        <infon key="score">0.9993223</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:48Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="45331" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="836">
+        <infon key="score">0.9975768</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:26:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45338" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="1032">
+        <infon key="type">residue_name_number</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:13:44Z</infon>
+        <location offset="45420" length="9"/>
+        <text>first Met</text>
+      </annotation>
+      <annotation id="837">
+        <infon key="score">0.99554753</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:13:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45431" length="2"/>
+        <text>M1</text>
+      </annotation>
+      <annotation id="1026">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T11:19:06Z</infon>
+        <location offset="45480" length="6"/>
+        <text>acetyl</text>
+      </annotation>
+      <annotation id="838">
+        <infon key="score">0.9990597</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="45497" length="6"/>
+        <text>Ac-CoA</text>
+      </annotation>
+      <annotation id="839">
+        <infon key="score">0.9792496</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:47:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45604" length="6"/>
+        <text>Phe 34</text>
+      </annotation>
+      <annotation id="840">
+        <infon key="score">0.99492764</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:39:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="45642" length="7"/>
+        <text>mutated</text>
+      </annotation>
+      <annotation id="841">
+        <infon key="score">0.9958605</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:15:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="45653" length="3"/>
+        <text>Ala</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">srep31425-f4.jpg</infon>
+      <infon key="id">f4</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>45670</offset>
+      <text>Structural basis for hNaa60 catalytic activity.</text>
+      <annotation id="842">
+        <infon key="score">0.99925846</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:48Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="45691" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">srep31425-f4.jpg</infon>
+      <infon key="id">f4</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>45718</offset>
+      <text>(A) Superposition of hNaa60 active site (cyan) on that of hNaa50 (pink, PDB 3TFY). Side-chains of key catalytic and substrate-binding residues are highlighted as sticks. The malonate molecule in the hNaa60(1-242)/Ac-CoA structure and the peptide in the hNaa50/CoA/peptide structure are shown as purple and yellow sticks respectively. (B) A close view of the active site of hNaa60. Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks. The water molecules participating in catalysis in the hNaa60 and hNaa50 structures are showed as green and red spheres, separately. (C) The interaction between the malonate molecule and surrounding residues observed in the hNaa60(1-242)/Ac-CoA structure. The yellow dotted lines indicate the hydrogen bonds. (D) A zoomed view of β3-β4 loop of hNaa60. Key residues discussed in the text (cyan), the malonate (purple) and Ac-CoA (gray) are shown as sticks. The yellow dotted lines indicate the salt bridges.</text>
+      <annotation id="843">
+        <infon key="score">0.9982657</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:29:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="45722" length="13"/>
+        <text>Superposition</text>
+      </annotation>
+      <annotation id="844">
+        <infon key="score">0.9993099</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T09:46:48Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="45739" length="6"/>
+        <text>hNaa60</text>
+      </annotation>
+      <annotation id="845">
+        <infon key="score">0.99906766</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:34:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="45746" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="846">
+        <infon key="score">0.9993212</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="45776" length="6"/>
+        <text>hNaa50</text>
+      </annotation>
+      <annotation id="847">
+        <infon key="score">0.9985394</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:18:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="45820" length="40"/>
+        <text>catalytic and substrate-binding residues</text>
+      </annotation>
+      <annotation id="848">
+        <infon key="score">0.99922884</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:40:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="45892" length="8"/>
+        <text>malonate</text>
+      </annotation>
+      <annotation id="849">
+        <infon key="score">0.99856216</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:35Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="45917" length="20"/>
+        <text>hNaa60(1-242)/Ac-CoA</text>
+      </annotation>
+      <annotation id="850">
+        <infon key="score">0.8332482</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:18:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45938" length="9"/>
+        <text>structure</text>
+      </annotation>
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+        <infon key="type">chemical</infon>
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+&lt;table frame="hsides" rules="groups" border="1"&gt;&lt;colgroup&gt;&lt;col align="left"/&gt;&lt;col align="center"/&gt;&lt;col align="center"/&gt;&lt;col align="center"/&gt;&lt;/colgroup&gt;&lt;thead valign="bottom"&gt;&lt;tr&gt;&lt;th align="left" valign="top" charoff="50"&gt;Structure and PDB ID&lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;hNaa60(1-242)/Ac-CoA 5HGZ&lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;hNaa60(1-199)/CoA 5HH0&lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;hNaa60(1-199)F34A/CoA 5HH1&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign="top"&gt;&lt;tr&gt;&lt;td colspan="4" align="left" valign="top" charoff="50"&gt;Data collection&lt;xref ref-type="fn" rid="t1-fn1"&gt;*&lt;/xref&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Space group&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&lt;italic&gt;P2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&lt;italic&gt;P2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&lt;italic&gt;P2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="4" align="left" valign="top" charoff="50"&gt;Cell dimensions&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; &lt;italic&gt;a, b, c&lt;/italic&gt; (Å)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;53.3, 57.4, 68.8&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;67.8, 73.8, 43.2&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;66.7, 74.0, 43.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; α,β,γ (°)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;90.0, 90.0, 90.0&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;90.0, 90.0, 90.0&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;90.0, 90.0, 90.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;Resolution (Å)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;50–1.38 (1.42–1.38)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;50–1.60 (1.66–1.60)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;50–1.80 (1.86–1.80)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;p.i.m.&lt;/sub&gt;(%)&lt;xref ref-type="fn" rid="t1-fn2"&gt;**&lt;/xref&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;3.0 (34.4)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;2.1 (32.5)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;2.6 (47.8)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;italic&gt;I&lt;/italic&gt;/&lt;italic&gt;σ&lt;/italic&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;21.5 (2.0)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;31.8 (2.0)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;28.0 (2.4)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;Completeness (%)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;99.8 (99.1)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;99.6 (98.5)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;99.9 (99.7)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;Redundancy&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;6.9 (5.0)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;6.9 (6.2)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;6.3 (5.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="4" align="left" valign="top" charoff="50"&gt;Refinement&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Resolution (Å)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;25.81–1.38&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;33.55–1.60&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;43.52–1.80&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; No. reflections&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;43660&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;28588&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;20490&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;italic&gt; R&lt;/italic&gt;&lt;sub&gt;work&lt;/sub&gt;/&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;0.182/0.192&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;0.181/0.184&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;0.189/0.209&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="4" align="left" valign="top" charoff="50"&gt;No. atoms&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Protein&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;1717&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;1576&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;1566&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Ligand/ion&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;116&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;96&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;96&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Water&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;289&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;258&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;168&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="4" align="left" valign="top" charoff="50"&gt;&lt;italic&gt;B&lt;/italic&gt;-factors&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Protein&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;23.8&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;32.0&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;37.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Ligand/ion&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;22.2&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;34.6&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;43.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Water&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;35.1&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;46.4&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;49.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="4" align="left" valign="top" charoff="50"&gt;R.m.s. deviations&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Bond lengths (Å)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;0.018&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;0.017&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;0.015&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Bond angles (°)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;1.529&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;1.651&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;1.581&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="4" align="left" valign="top" charoff="50"&gt;Ramachandran Plot&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Favoured region&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;98.98%&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;98.93%&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;98.96%&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Allowed region&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;1.02%&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;1.07%&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;1.04%&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Outliers&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;0.00%&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;0.00%&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;0.00%&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon>
+      <offset>47302</offset>
+      <text>Structure and PDB ID	hNaa60(1-242)/Ac-CoA 5HGZ	hNaa60(1-199)/CoA 5HH0	hNaa60(1-199)F34A/CoA 5HH1	 	Data collection*	 	 Space group	P212121	P21212	P21212	 	Cell dimensions	 	 a, b, c (Å)	53.3, 57.4, 68.8	67.8, 73.8, 43.2	66.7, 74.0, 43.5	 	 α,β,γ (°)	90.0, 90.0, 90.0	90.0, 90.0, 90.0	90.0, 90.0, 90.0	 	Resolution (Å)	50–1.38 (1.42–1.38)	50–1.60 (1.66–1.60)	50–1.80 (1.86–1.80)	 	Rp.i.m.(%)**	3.0 (34.4)	2.1 (32.5)	2.6 (47.8)	 	I/σ	21.5 (2.0)	31.8 (2.0)	28.0 (2.4)	 	Completeness (%)	99.8 (99.1)	99.6 (98.5)	99.9 (99.7)	 	Redundancy	6.9 (5.0)	6.9 (6.2)	6.3 (5.9)	 	Refinement	 	 Resolution (Å)	25.81–1.38	33.55–1.60	43.52–1.80	 	 No. reflections	43660	28588	20490	 	 Rwork/Rfree	0.182/0.192	0.181/0.184	0.189/0.209	 	No. atoms	 	 Protein	1717	1576	1566	 	 Ligand/ion	116	96	96	 	 Water	289	258	168	 	B-factors	 	 Protein	23.8	32.0	37.4	 	 Ligand/ion	22.2	34.6	43.7	 	 Water	35.1	46.4	49.1	 	R.m.s. deviations	 	 Bond lengths (Å)	0.018	0.017	0.015	 	 Bond angles (°)	1.529	1.651	1.581	 	Ramachandran Plot	 	 Favoured region	98.98%	98.93%	98.96%	 	 Allowed region	1.02%	1.07%	1.04%	 	 Outliers	0.00%	0.00%	0.00%	 	</text>
+      <annotation id="950">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:35Z</infon>
+        <location offset="47323" length="20"/>
+        <text>hNaa60(1-242)/Ac-CoA</text>
+      </annotation>
+      <annotation id="958">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:21:54Z</infon>
+        <location offset="47349" length="17"/>
+        <text>hNaa60(1-199)/CoA</text>
+      </annotation>
+      <annotation id="1034">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:02Z</infon>
+        <location offset="47372" length="21"/>
+        <text>hNaa60(1-199)F34A/CoA</text>
+      </annotation>
+      <annotation id="1005">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:47:55Z</infon>
+        <location offset="48082" length="5"/>
+        <text>Water</text>
+      </annotation>
+      <annotation id="1006">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T10:47:55Z</infon>
+        <location offset="48170" length="5"/>
+        <text>Water</text>
+      </annotation>
+      <annotation id="1035">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:22Z</infon>
+        <location offset="48193" length="17"/>
+        <text>R.m.s. deviations</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">t1.xml</infon>
+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_footnote</infon>
+      <offset>48477</offset>
+      <text>*Values in parentheses are for highest-resolution shell. One crystal was used for each data set.</text>
+      <annotation id="891">
+        <infon key="score">0.97377807</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:27Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="48538" length="7"/>
+        <text>crystal</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">t1.xml</infon>
+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_footnote</infon>
+      <offset>48574</offset>
+      <text>**Rp.i.m., a redundancy-independent R factor was used to evaluate the diffraction data quality as was proposed by Evans.</text>
+      <annotation id="892">
+        <infon key="score">0.9949857</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="48610" length="8"/>
+        <text>R factor</text>
+      </annotation>
+      <annotation id="893">
+        <infon key="score">0.9745873</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-20T12:20:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="48644" length="16"/>
+        <text>diffraction data</text>
+      </annotation>
+    </passage>
+  </document>
+</collection>
diff --git a/annotated_BioC_XML/PMC5012862_ann.xml b/annotated_BioC_XML/PMC5012862_ann.xml
new file mode 100644
index 0000000000000000000000000000000000000000..6a68623c5ef431498ab6a46343b84bd59bf3c107
--- /dev/null
+++ b/annotated_BioC_XML/PMC5012862_ann.xml
@@ -0,0 +1,22218 @@
+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection>
+  <source>PMC</source>
+  <date>20230811</date>
+  <key>pmc.key</key>
+  <document>
+    <id>5012862</id>
+    <infon key="license">CC BY</infon>
+    <infon key="tt_curatable">no</infon>
+    <infon key="tt_version">2</infon>
+    <infon key="tt_round">2</infon>
+    <passage>
+      <infon key="article-id_doi">10.7554/eLife.18972</infon>
+      <infon key="article-id_pmc">5012862</infon>
+      <infon key="article-id_pmid">27529188</infon>
+      <infon key="article-id_publisher-id">18972</infon>
+      <infon key="elocation-id">e18972</infon>
+      <infon key="kwd">encapsulin encapsulated ferritin ferritin Rhodospirillum rubrum Other</infon>
+      <infon key="license">This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.</infon>
+      <infon key="name_0">surname:He;given-names:Didi</infon>
+      <infon key="name_1">surname:Hughes;given-names:Sam</infon>
+      <infon key="name_10">surname:Losick;given-names:Richard</infon>
+      <infon key="name_11">surname:He;given-names:Didi</infon>
+      <infon key="name_12">surname:Hughes;given-names:Sam</infon>
+      <infon key="name_13">surname:Altenbach;given-names:Kirsten</infon>
+      <infon key="name_14">surname:Clarke;given-names:David J</infon>
+      <infon key="name_15">surname:Tarrant;given-names:Emma</infon>
+      <infon key="name_16">surname:Waldron;given-names:Kevin J</infon>
+      <infon key="name_17">surname:Clarke;given-names:David J</infon>
+      <infon key="name_18">surname:Marles-Wright;given-names:Jon</infon>
+      <infon key="name_19">surname:Marles-Wright;given-names:Jon</infon>
+      <infon key="name_2">surname:Vanden-Hehir;given-names:Sally</infon>
+      <infon key="name_3">surname:Georgiev;given-names:Atanas</infon>
+      <infon key="name_4">surname:Altenbach;given-names:Kirsten</infon>
+      <infon key="name_5">surname:Tarrant;given-names:Emma</infon>
+      <infon key="name_6">surname:Mackay;given-names:C Logan</infon>
+      <infon key="name_7">surname:Waldron;given-names:Kevin J</infon>
+      <infon key="name_8">surname:Clarke;given-names:David J</infon>
+      <infon key="name_9">surname:Marles-Wright;given-names:Jon</infon>
+      <infon key="section_type">TITLE</infon>
+      <infon key="title">Author Keywords Research Organism</infon>
+      <infon key="type">front</infon>
+      <infon key="volume">5</infon>
+      <infon key="year">2016</infon>
+      <offset>0</offset>
+      <text>Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments</text>
+      <annotation id="1">
+        <infon key="score">0.979727</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:18Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="0" length="27"/>
+        <text>Structural characterization</text>
+      </annotation>
+      <annotation id="1847">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:29:05Z</infon>
+        <location offset="31" length="12"/>
+        <text>encapsulated</text>
+      </annotation>
+      <annotation id="2">
+        <infon key="score">0.98685956</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="44" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+      <annotation id="3">
+        <infon key="score">0.9957735</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:28Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="75" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="4">
+        <infon key="score">0.99892694</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="91" length="9"/>
+        <text>bacterial</text>
+      </annotation>
+      <annotation id="1913">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:47:29Z</infon>
+        <location offset="101" length="16"/>
+        <text>nanocompartments</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">ABSTRACT</infon>
+      <infon key="type">abstract</infon>
+      <offset>118</offset>
+      <text>Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage. We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid. We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface. EncFtn adopts an open decameric structure that is topologically distinct from other ferritins. While EncFtn acts as a ferroxidase, it cannot mineralize iron. Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage. This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins.</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="118" length="9"/>
+        <text>Ferritins</text>
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+      <annotation id="6">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:29Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="175" length="4"/>
+        <text>iron</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:00Z</infon>
+        <location offset="197" length="5"/>
+        <text>shell</text>
+      </annotation>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="269" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="1833">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:04Z</infon>
+        <location offset="315" length="8"/>
+        <text>ferritin</text>
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+      <annotation id="1850">
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+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:25Z</infon>
+        <location offset="366" length="10"/>
+        <text>encapsulin</text>
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+      <annotation id="8">
+        <infon key="score">0.6466904</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:29:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="403" length="12"/>
+        <text>encapsulated</text>
+      </annotation>
+      <annotation id="9">
+        <infon key="score">0.99924374</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:04Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="416" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+      <annotation id="10">
+        <infon key="score">0.9991804</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:35Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="426" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1860">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:31:26Z</infon>
+        <location offset="442" length="18"/>
+        <text>main alpha helices</text>
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+      <annotation id="1861">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:31:44Z</infon>
+        <location offset="482" length="15"/>
+        <text>metal dependent</text>
+      </annotation>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:31:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="515" length="18"/>
+        <text>ferroxidase center</text>
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+      <annotation id="12">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="539" length="15"/>
+        <text>dimer interface</text>
+      </annotation>
+      <annotation id="13">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:36Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="556" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="14">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="573" length="4"/>
+        <text>open</text>
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+      <annotation id="15">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="578" length="9"/>
+        <text>decameric</text>
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+      <annotation id="16">
+        <infon key="score">0.98166347</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:04:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="588" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="17">
+        <infon key="score">0.99918705</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="640" length="9"/>
+        <text>ferritins</text>
+      </annotation>
+      <annotation id="18">
+        <infon key="score">0.9992372</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:36Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="657" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="19">
+        <infon key="score">0.9985642</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:29Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="674" length="11"/>
+        <text>ferroxidase</text>
+      </annotation>
+      <annotation id="20">
+        <infon key="score">0.9986583</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:29Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="708" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="21">
+        <infon key="score">0.94227946</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:26Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="730" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="22">
+        <infon key="score">0.99792576</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="741" length="5"/>
+        <text>shell</text>
+      </annotation>
+      <annotation id="23">
+        <infon key="score">0.99881506</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:29Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="763" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="24">
+        <infon key="score">0.950607</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="776" length="24"/>
+        <text>not enzymatically active</text>
+      </annotation>
+      <annotation id="25">
+        <infon key="score">0.9991411</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:36Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="826" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="26">
+        <infon key="score">0.9428895</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:26Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="859" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="27">
+        <infon key="score">0.9981583</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:29Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="874" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="28">
+        <infon key="score">0.9898727</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:26Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="893" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="2184">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:03:16Z</infon>
+        <location offset="904" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+      <annotation id="29">
+        <infon key="score">0.99831957</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="945" length="8"/>
+        <text>bacteria</text>
+      </annotation>
+      <annotation id="30">
+        <infon key="score">0.99752516</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:28Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="958" length="7"/>
+        <text>archaea</text>
+      </annotation>
+      <annotation id="31">
+        <infon key="score">0.96602964</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:29Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1001" length="4"/>
+        <text>iron</text>
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+      <annotation id="32">
+        <infon key="score">0.99873704</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:29Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1064" length="4"/>
+        <text>iron</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">ABSTRACT</infon>
+      <infon key="type">abstract</infon>
+      <offset>1107</offset>
+      <text>DOI: http://dx.doi.org/10.7554/eLife.18972.001</text>
+    </passage>
+    <passage>
+      <infon key="section_type">ABSTRACT</infon>
+      <infon key="type">abstract_title_1</infon>
+      <offset>1154</offset>
+      <text>eLife digest</text>
+    </passage>
+    <passage>
+      <infon key="section_type">ABSTRACT</infon>
+      <infon key="type">abstract</infon>
+      <offset>1167</offset>
+      <text>Iron is essential for life as it is a key component of many different enzymes that participate in processes such as energy production and metabolism. However, iron can also be highly toxic to cells because it readily reacts with oxygen. This reaction can damage DNA, proteins and the membranes that surround cells.</text>
+      <annotation id="33">
+        <infon key="score">0.99911195</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:29Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1167" length="4"/>
+        <text>Iron</text>
+      </annotation>
+      <annotation id="34">
+        <infon key="score">0.99917394</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:29Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1326" length="4"/>
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+      </annotation>
+      <annotation id="35">
+        <infon key="score">0.9944981</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:35:26Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1396" length="6"/>
+        <text>oxygen</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">ABSTRACT</infon>
+      <infon key="type">abstract</infon>
+      <offset>1482</offset>
+      <text>To balance the cell’s need for iron against its potential damaging effects, organisms have evolved iron storage proteins known as ferritins that form cage-like structures. The ferritins convert iron into a less reactive form that is mineralised and safely stored in the central cavity of the ferritin cage and is available for cells when they need it.</text>
+      <annotation id="36">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:29Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1513" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="37">
+        <infon key="score">0.9981699</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:55Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1581" length="21"/>
+        <text>iron storage proteins</text>
+      </annotation>
+      <annotation id="38">
+        <infon key="score">0.99874544</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1612" length="9"/>
+        <text>ferritins</text>
+      </annotation>
+      <annotation id="39">
+        <infon key="score">0.80043113</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:34:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="1632" length="20"/>
+        <text>cage-like structures</text>
+      </annotation>
+      <annotation id="40">
+        <infon key="score">0.99869776</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1658" length="9"/>
+        <text>ferritins</text>
+      </annotation>
+      <annotation id="41">
+        <infon key="score">0.9990459</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:29Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1676" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="42">
+        <infon key="score">0.9951139</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:34:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="1752" length="14"/>
+        <text>central cavity</text>
+      </annotation>
+      <annotation id="43">
+        <infon key="score">0.9974579</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:04Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1774" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">ABSTRACT</infon>
+      <infon key="type">abstract</infon>
+      <offset>1836</offset>
+      <text>Recently, a new family of ferritins known as encapsulated ferritins have been found in some microorganisms. These ferritins are found in bacterial genomes with a gene that codes for a protein cage called an encapsulin. Although the structure of the encapsulin cage is known to look like the shell of a virus, the structure that the encapsulated ferritin itself forms is not known. It is also not clear how encapsulin and the encapsulated ferritin work together to store iron.</text>
+      <annotation id="44">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1862" length="9"/>
+        <text>ferritins</text>
+      </annotation>
+      <annotation id="45">
+        <infon key="score">0.89042205</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:29:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1881" length="12"/>
+        <text>encapsulated</text>
+      </annotation>
+      <annotation id="46">
+        <infon key="score">0.9987853</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1894" length="9"/>
+        <text>ferritins</text>
+      </annotation>
+      <annotation id="47">
+        <infon key="score">0.9978846</infon>
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+      <text>Biochemical studies revealed that the encapsulated ferritin is able to convert iron into a less reactive form, but it cannot store iron on its own since it does not form a cage. Thus, the encapsulated ferritin needs to be housed within the encapsulin cage to store iron.</text>
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+        <infon key="identifier">CHEBI:</infon>
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+      <offset>2939</offset>
+      <text>Further work is needed to investigate how iron moves into the encapsulin cage to reach the ferritin proteins. Some organisms have both standard ferritin cages and encapsulated ferritins; why this is the case also remains to be discovered.</text>
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+      <text>DOI: http://dx.doi.org/10.7554/eLife.18972.002</text>
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+      <infon key="section_type">INTRO</infon>
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+      <text>Introduction</text>
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+      <text>Encapsulin nanocompartments are a family of proteinaceous metabolic compartments that are widely distributed in bacteria and archaea. They share a common architecture, comprising an icosahedral shell formed by the oligomeric assembly of a protein, encapsulin, that is structurally related to the HK97 bacteriophage capsid protein gp5. Gp5 is known to assemble as a 66 nm diameter icosahedral shell of 420 subunits. In contrast, both the Pyrococcus furiosus and Myxococcus xanthus encapsulin shell-proteins form 32 nm icosahedra with 180 subunits; while the Thermotoga maritima encapsulin is smaller still with a 25 nm, 60-subunit icosahedron. The high structural similarity of the encapsulin shell-proteins to gp5 suggests a common evolutionary origin for these proteins.</text>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="3238" length="10"/>
+        <text>Encapsulin</text>
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+        <infon key="updated_at">2023-09-19T11:47:27Z</infon>
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+        <text>nanocompartments</text>
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+        <text>icosahedral</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">taxonomy_domain</infon>
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+        <infon key="updated_at">2023-09-19T11:36:36Z</infon>
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+        <text>HK97 bacteriophage</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <location offset="3699" length="18"/>
+        <text>Myxococcus xanthus</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:00Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="3795" length="19"/>
+        <text>Thermotoga maritima</text>
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+      <text>The genes encoding encapsulin proteins are found downstream of genes for dye-dependent peroxidase (DyP) family enzymes, or encapsulin-associated ferritins (EncFtn). Enzymes in the DyP family are active against polyphenolic compounds such as azo dyes and lignin breakdown products; although their physiological function and natural substrates are not known. Ferritin family proteins are found in all kingdoms and have a wide range of activities, including ribonucleotide reductase, protecting DNA from oxidative damage, and iron storage. The classical iron storage ferritin nanocages are found in all kingdoms and are essential in eukaryotes; they play a central role in iron homeostasis, where they protect the cell from toxic free Fe2+ by oxidizing it and storing the resulting Fe3+ as ferrihydrite minerals within their central cavity.</text>
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+        <infon key="updated_at">2023-09-19T11:39:21Z</infon>
+        <location offset="4030" length="10"/>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:22:38Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4110" length="3"/>
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+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:40:20Z</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:40:30Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4191" length="10"/>
+        <text>DyP family</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:04Z</infon>
+        <location offset="4368" length="8"/>
+        <text>Ferritin</text>
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+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:40:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4410" length="8"/>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:40:47Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4466" length="24"/>
+        <text>ribonucleotide reductase</text>
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+      <annotation id="118">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:29Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4534" length="4"/>
+        <text>iron</text>
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+      <annotation id="1892">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
+        <location offset="4552" length="9"/>
+        <text>classical</text>
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+      <annotation id="1890">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:41:27Z</infon>
+        <location offset="4562" length="31"/>
+        <text>iron storage ferritin nanocages</text>
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+      <annotation id="119">
+        <infon key="score">0.80968964</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:40:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4611" length="8"/>
+        <text>kingdoms</text>
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+      <annotation id="120">
+        <infon key="score">0.99721307</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:56:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4641" length="10"/>
+        <text>eukaryotes</text>
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+      <annotation id="121">
+        <infon key="score">0.9986492</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:29Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4681" length="4"/>
+        <text>iron</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:41:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4743" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="123">
+        <infon key="score">0.99878216</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:41:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4790" length="4"/>
+        <text>Fe3+</text>
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+      <annotation id="124">
+        <infon key="score">0.99309534</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:23:01Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4798" length="12"/>
+        <text>ferrihydrite</text>
+      </annotation>
+      <annotation id="125">
+        <infon key="score">0.99645233</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:12:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4833" length="14"/>
+        <text>central cavity</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>4849</offset>
+      <text>The encapsulin-associated enzymes are sequestered within the icosahedral shell through interactions between the shell’s inner surface and a short localization sequence (Gly-Ser-Leu-Lys) appended to their C-termini. This motif is well-conserved, and the addition of this sequence to heterologous proteins is sufficient to direct them to the interior of encapsulins.</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:43:35Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4853" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="127">
+        <infon key="score">0.9980197</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:36:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4910" length="11"/>
+        <text>icosahedral</text>
+      </annotation>
+      <annotation id="128">
+        <infon key="score">0.9989114</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4922" length="5"/>
+        <text>shell</text>
+      </annotation>
+      <annotation id="129">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4961" length="5"/>
+        <text>shell</text>
+      </annotation>
+      <annotation id="130">
+        <infon key="score">0.9985676</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:42:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4989" length="27"/>
+        <text>short localization sequence</text>
+      </annotation>
+      <annotation id="131">
+        <infon key="score">0.93535024</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:43:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5018" length="15"/>
+        <text>Gly-Ser-Leu-Lys</text>
+      </annotation>
+      <annotation id="132">
+        <infon key="score">0.89328057</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:59:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5064" length="10"/>
+        <text>This motif</text>
+      </annotation>
+      <annotation id="133">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:43:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5078" length="14"/>
+        <text>well-conserved</text>
+      </annotation>
+      <annotation id="134">
+        <infon key="score">0.90473825</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:22:47Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5201" length="11"/>
+        <text>encapsulins</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>5217</offset>
+      <text>A recent study of the Myxococcus xanthus encapsulin showed that it sequesters a number of different EncFtn proteins and acts as an ‘iron-megastore’ to protect these bacteria from oxidative stress. At 32 nm in diameter, it is much larger than other members of the ferritin superfamily, such as the 12 nm 24-subunit classical ferritin nanocage and the 8 nm 12-subunit Dps (DNA-binding protein from starved cells) complex; and is thus capable of sequestering up to ten times more iron than these ferritins. The primary sequences of EncFtn proteins have Glu-X-X-His metal coordination sites, which are shared features of the ferritin family proteins. Secondary structure prediction identifies two major α-helical regions in these proteins; this is in contrast to other members of the ferritin superfamily, which have four major α-helices (Supplementary file 1). The ‘half-ferritin’ primary sequence of the EncFtn family and their association with encapsulin nanocompartments suggests a distinct biochemical and structural organization to other ferritin family proteins. The Rhodospirillum rubrum EncFtn protein (Rru_A0973) shares 33% protein sequence identity with the M. xanthus (MXAN_4464), 53% with the T. maritima (Tmari_0787), and 29% with the P. furiosus (PF1192) homologues. The GXXH motifs are strictly conserved in each of these species (Supplementary file 1).</text>
+      <annotation id="135">
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:37:09Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5239" length="18"/>
+        <text>Myxococcus xanthus</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:26Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="5258" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:31Z</infon>
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:30Z</infon>
+        <location offset="5349" length="4"/>
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+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:24Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5382" length="8"/>
+        <text>bacteria</text>
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:04Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:51Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5531" length="9"/>
+        <text>classical</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:04Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5541" length="8"/>
+        <text>ferritin</text>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:46:46Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="5550" length="8"/>
+        <text>nanocage</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="5583" length="3"/>
+        <text>Dps</text>
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:46:41Z</infon>
+        <location offset="5588" length="19"/>
+        <text>DNA-binding protein</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <text>iron</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5710" length="9"/>
+        <text>ferritins</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:45:43Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5767" length="11"/>
+        <text>Glu-X-X-His</text>
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+        <infon key="type">site</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="5779" length="24"/>
+        <text>metal coordination sites</text>
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+      <annotation id="1836">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:04Z</infon>
+        <location offset="5838" length="8"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <text>major α-helical regions</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:04Z</infon>
+        <location offset="5997" length="8"/>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:48:00Z</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="updated_at">2023-09-19T11:28:04Z</infon>
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:48:47Z</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:26Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6160" length="10"/>
+        <text>encapsulin</text>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:47:29Z</infon>
+        <infon key="identifier">GO:</infon>
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+        <text>nanocompartments</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:04Z</infon>
+        <location offset="6257" length="8"/>
+        <text>ferritin</text>
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:50:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6287" length="21"/>
+        <text>Rhodospirillum rubrum</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:37Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6309" length="6"/>
+        <text>EncFtn</text>
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+        <infon key="type">gene</infon>
+        <infon key="identifier">GENE:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:49:38Z</infon>
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:50:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6382" length="10"/>
+        <text>M. xanthus</text>
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+      <annotation id="1916">
+        <infon key="type">gene</infon>
+        <infon key="identifier">GENE:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:49:56Z</infon>
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+        <text>MXAN_4464</text>
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+      <annotation id="154">
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:50:38Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6419" length="11"/>
+        <text>T. maritima</text>
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+        <infon key="type">gene</infon>
+        <infon key="identifier">GENE:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:50:14Z</infon>
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:50:45Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6462" length="11"/>
+        <text>P. furiosus</text>
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+        <infon key="type">gene</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:50:55Z</infon>
+        <infon key="identifier">GENE:</infon>
+        <location offset="6475" length="6"/>
+        <text>PF1192</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:51:21Z</infon>
+        <location offset="6499" length="4"/>
+        <text>GXXH</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:51:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6515" length="18"/>
+        <text>strictly conserved</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>6594</offset>
+      <text>Here we investigate the structure and biochemistry of EncFtn in order to understand iron storage within the encapsulin nanocompartment. We have produced recombinant encapsulin (Enc) and EncFtn from the aquatic purple-sulfur bacterium R. rubrum, which serves as a model organism for the study of the control of the bacterial nitrogen fixation machinery, in Escherichia coli. Analysis by transmission electron microscopy (TEM) indicates that their co-expression leads to the production of an icosahedral nanocompartment with encapsulated EncFtn. The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins. We identify a symmetrical iron bound ferroxidase center (FOC) formed between subunits in the decamer and additional metal-binding sites close to the center of the ring and on the outer surface. We also demonstrate the metal-dependent assembly of EncFtn decamers using native PAGE, analytical gel-filtration, and native mass spectrometry. Biochemical assays show that EncFtn is active as a ferroxidase enzyme. Through site-directed mutagenesis we show that the conserved glutamic acid and histidine residues in the FOC influence protein assembly and activity. We use our combined structural and biochemical data to propose a model for the EncFtn-catalyzed sequestration of iron within the encapsulin shell.</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:51:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6618" length="9"/>
+        <text>structure</text>
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+      <annotation id="159">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:37Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6648" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="6678" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="161">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:26Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6702" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="2185">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:03:16Z</infon>
+        <location offset="6713" length="15"/>
+        <text>nanocompartment</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6759" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="163">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:11:26Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6771" length="3"/>
+        <text>Enc</text>
+      </annotation>
+      <annotation id="164">
+        <infon key="score">0.99879503</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:37Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6780" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:56:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6796" length="7"/>
+        <text>aquatic</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:56:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6804" length="23"/>
+        <text>purple-sulfur bacterium</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:22:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6828" length="9"/>
+        <text>R. rubrum</text>
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+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:45Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6908" length="9"/>
+        <text>bacterial</text>
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+      <annotation id="169">
+        <infon key="score">0.9986036</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:52:00Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6950" length="16"/>
+        <text>Escherichia coli</text>
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+      <annotation id="170">
+        <infon key="score">0.9987278</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:52:07Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6980" length="32"/>
+        <text>transmission electron microscopy</text>
+      </annotation>
+      <annotation id="171">
+        <infon key="score">0.9983175</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:52:12Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7014" length="3"/>
+        <text>TEM</text>
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+      <annotation id="1919">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:52:29Z</infon>
+        <location offset="7040" length="13"/>
+        <text>co-expression</text>
+      </annotation>
+      <annotation id="172">
+        <infon key="score">0.99592847</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:36:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7084" length="11"/>
+        <text>icosahedral</text>
+      </annotation>
+      <annotation id="173">
+        <infon key="score">0.9979723</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:56:51Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="7096" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+      <annotation id="1849">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:29:05Z</infon>
+        <location offset="7117" length="12"/>
+        <text>encapsulated</text>
+      </annotation>
+      <annotation id="174">
+        <infon key="score">0.99852824</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:37Z</infon>
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+      <text>(A/B) Negative stain TEM image of recombinant R. rubrum encapsulin and EncFtn-Enc nanocompartments. All samples were imaged at 143,000 x magnification; the scale bar length corresponds to 50 nm. (C) Histogram showing the distribution of nanocompartment diameters. A model Gaussian nonlinear least square function was fitted to the data to obtain a mean diameter of 24.6 nm with a standard deviation of 2.0 nm for encapsulin (grey) and a mean value of 23.9 nm with a standard deviation of 2.2 nm for co-expressed EncFtn and encapsulin (EncFtn-Enc, black).</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:05:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8487" length="9"/>
+        <text>Histogram</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">GO:</infon>
+        <location offset="8525" length="15"/>
+        <text>nanocompartment</text>
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+        <infon key="score">0.99713784</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>Gaussian nonlinear least square function</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.004</text>
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+      <text>Purification of recombinant R. rubrum encapsulin nanocompartments.</text>
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+        <infon key="annotator">cleaner0</infon>
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+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
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+      <text>(A) Recombinantly expressed encapsulin (Enc) and co-expressed EncFtn-Enc were purified by sucrose gradient ultracentrifugation from E. coli B834(DE3) grown in SeMet medium. Samples were resolved by 18% acrylamide SDS-PAGE; the position of the proteins found in the complexes as resolved on the gel are shown with arrows. (B/C) Negative stain TEM image of recombinant encapsulin and EncFtn-Enc nanocompartments. Samples were imaged at 143,000 x magnification, with scale bar shown as 25 nm. Representative encapsulin and EncFtn-Enc complexes are indicated with red arrows.</text>
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+        <text>encapsulin</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="8999" length="3"/>
+        <text>Enc</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:31Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9008" length="12"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <location offset="9021" length="10"/>
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+        <infon key="updated_at">2023-09-19T12:00:57Z</infon>
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+        <infon key="type">species</infon>
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+        <location offset="9091" length="7"/>
+        <text>E. coli</text>
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+      <annotation id="1963">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:16:44Z</infon>
+        <location offset="9118" length="5"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9172" length="8"/>
+        <text>SDS-PAGE</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="9286" length="18"/>
+        <text>Negative stain TEM</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <text>encapsulin</text>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <text>EncFtn-Enc</text>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:47:29Z</infon>
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+      <annotation id="248">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9464" length="10"/>
+        <text>encapsulin</text>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:07Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="9479" length="10"/>
+        <text>EncFtn-Enc</text>
+      </annotation>
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+    <passage>
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+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>9531</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.003</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>9578</offset>
+      <text>We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A). TEM imaging of uranyl acetate-stained samples revealed that, when expressed in isolation, the encapsulin protein forms empty compartments with an average diameter of 24 nm (Figure 1B and Figure 1—figure supplement 1A/C), consistent with the appearance and size of the T. maritima encapsulin. We were not able to resolve any higher-order structures of EncFtn by TEM. Protein purified from co-expression of the encapsulin and EncFtn resulted in 24 nm compartments with regions in the center that exclude stain, consistent with the presence of the EncFtn within the encapsulin shell (Figure 1C and Figure 1—figure supplement 1B/C).</text>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="9602" length="9"/>
+        <text>R. rubrum</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>encapsulin</text>
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+        <infon key="identifier">GO:</infon>
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+        <text>nanocompartments</text>
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>E. coli</text>
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+        <location offset="9675" length="10"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <location offset="9702" length="6"/>
+        <text>EncFtn</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:57Z</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="updated_at">2023-09-19T11:52:14Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>TEM</text>
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+        <infon key="identifier">MESH:</infon>
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+        <text>expressed in isolation</text>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>empty</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:15:29Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>T. maritima</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:37Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10155" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:52:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10165" length="3"/>
+        <text>TEM</text>
+      </annotation>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:19:57Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>co-expression</text>
+      </annotation>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="10213" length="10"/>
+        <text>encapsulin</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <location offset="10378" length="5"/>
+        <text>shell</text>
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+      <infon key="type">title_2</infon>
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+      <text>R. rubrum EncFtn forms a metal-ion stabilized decamer in solution</text>
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+      <infon key="type">fig_title_caption</infon>
+      <offset>10504</offset>
+      <text>Purification of recombinant R. rubrum EncFtnsH.</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <location offset="10542" length="8"/>
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+      <text>(A) Recombinant SeMet-labeled EncFtnsH produced with 1 mM Fe(NH4)2(SO4)2 in the growth medium was purified by nickel affinity chromatography and size-exclusion chromatography using a Superdex 200 16/60 column (GE Healthcare). Chromatogram traces measured at 280 nm and 315 nm are shown with the results from ICP-MS analysis of the iron content of the fractions collected during the experiment. The peak around 73 ml corresponds to a molecular weight of around 130 kDa when compared to calibration standards; this is consistent with a decamer of EncFtnsH. The small peak at 85 ml corresponds to the 13 kDa monomer compared to the standards. Only the decamer peak contains significant amounts of iron as indicated by the ICP-MS analysis. (B) Peak fractions from the gel filtration run were resolved by 15% acrylamide SDS-PAGE and stained with Coomassie blue stain. The bands around 13 kDa and 26 kDa correspond to EncFtnsH, as identified by MALDI peptide mass fingerprinting. The band at 13 kDa is consistent with the monomer mass, while the band at 26 kDa is consistent with a dimer of EncFtnsH. The dimer species only appears in the decamer fractions. (C) SEC-MALLS analysis of EncFtnsH from decamer fractions and monomer fractions allows assignment of an average mass of 132 kDa to decamer fractions and 13 kDa to monomer fractions, consistent with decamer and monomer species (Table 2).</text>
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+        <location offset="10582" length="8"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:17:56Z</infon>
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+        <infon key="score">0.9986612</infon>
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+        <infon key="updated_at">2023-09-19T13:12:51Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="10697" length="29"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:19:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10778" length="12"/>
+        <text>Chromatogram</text>
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+        <infon key="updated_at">2023-09-19T13:13:06Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10860" length="6"/>
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+        <infon key="type">chemical</infon>
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+        <infon key="updated_at">2023-09-19T11:27:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10883" length="4"/>
+        <text>iron</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:19:19Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11097" length="8"/>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">oligomeric_state</infon>
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+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11201" length="7"/>
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+        <infon key="type">chemical</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:07Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11271" length="6"/>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:18Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11316" length="14"/>
+        <text>gel filtration</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:25Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11367" length="8"/>
+        <text>SDS-PAGE</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11464" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="292">
+        <infon key="score">0.99868894</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:20:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11491" length="33"/>
+        <text>MALDI peptide mass fingerprinting</text>
+      </annotation>
+      <annotation id="293">
+        <infon key="score">0.9989442</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11568" length="7"/>
+        <text>monomer</text>
+      </annotation>
+      <annotation id="294">
+        <infon key="score">0.99893254</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:15:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11628" length="5"/>
+        <text>dimer</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11637" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="296">
+        <infon key="score">0.99894065</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:16:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11651" length="5"/>
+        <text>dimer</text>
+      </annotation>
+      <annotation id="297">
+        <infon key="score">0.99875605</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11685" length="7"/>
+        <text>decamer</text>
+      </annotation>
+      <annotation id="298">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11708" length="9"/>
+        <text>SEC-MALLS</text>
+      </annotation>
+      <annotation id="299">
+        <infon key="score">0.99928975</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11730" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="300">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11744" length="7"/>
+        <text>decamer</text>
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+      <annotation id="301">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11766" length="7"/>
+        <text>monomer</text>
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+      <annotation id="302">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11835" length="7"/>
+        <text>decamer</text>
+      </annotation>
+      <annotation id="303">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11867" length="7"/>
+        <text>monomer</text>
+      </annotation>
+      <annotation id="304">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11902" length="7"/>
+        <text>decamer</text>
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+      <annotation id="305">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11914" length="7"/>
+        <text>monomer</text>
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+    <passage>
+      <infon key="file">elife-18972-fig2.jpg</infon>
+      <infon key="id">fig2</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>11944</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.005</text>
+    </passage>
+    <passage>
+      <infon key="file">tbl1.xml</infon>
+      <infon key="id">tbl1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_caption</infon>
+      <offset>11991</offset>
+      <text>Determination of the Fe/EncFtnsH protein ratio by ICP-MS. EncFtnsH was purified as a SeMet derivative from E. coli B834(DE3) cells grown in SeMet medium with 1 mM Fe(NH4)2(SO4)2. Fractions from SEC were collected, acidified and analysed by ICP-MS. EncFtnsH concentration was calculated based on the presence of two SeMet per mature monomer. Samples where the element was undetectable are labelled with n.d. These data were collected from EncFtnsH fractions from a single gel-filtration run.</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:23:07Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <text>Fe</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12015" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:07Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12041" length="6"/>
+        <text>ICP-MS</text>
+      </annotation>
+      <annotation id="309">
+        <infon key="score">0.99813795</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12049" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="310">
+        <infon key="score">0.998733</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:16:43Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12076" length="5"/>
+        <text>SeMet</text>
+      </annotation>
+      <annotation id="1966">
+        <infon key="type">species</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:17:48Z</infon>
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+        <text>E. coli B834(DE3)</text>
+      </annotation>
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+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_caption</infon>
+      <offset>12482</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.006</text>
+    </passage>
+    <passage>
+      <infon key="file">tbl1.xml</infon>
+      <infon key="id">tbl1</infon>
+      <infon key="section_type">TABLE</infon>
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+      <infon key="xml">&lt;?xml version="1.0" encoding="UTF-8"?&gt;
+&lt;table frame="hsides" rules="groups"&gt;&lt;thead&gt;&lt;tr&gt;&lt;th rowspan="2" colspan="1"&gt;Peak&lt;/th&gt;&lt;th rowspan="2" colspan="1"&gt;EncFtn&lt;sub&gt;sH&lt;/sub&gt;
+&lt;break/&gt;retention volume (ml)&lt;/th&gt;&lt;th colspan="4" rowspan="1"&gt;Element concentration (µM)&lt;/th&gt;&lt;th rowspan="2" colspan="1"&gt;Derived EncFtn&lt;sub&gt;sH&lt;/sub&gt;concentration (µM)&lt;/th&gt;&lt;th rowspan="2" colspan="1"&gt;Derived Fe/ &lt;break/&gt;EncFtn&lt;sub&gt;sH&lt;/sub&gt; monomer&lt;/th&gt;&lt;/tr&gt;&lt;tr&gt;&lt;th rowspan="1" colspan="1"&gt;Ca&lt;/th&gt;&lt;th rowspan="1" colspan="1"&gt;Fe&lt;/th&gt;&lt;th rowspan="1" colspan="1"&gt;Zn&lt;/th&gt;&lt;th rowspan="1" colspan="1"&gt;Se&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td rowspan="8" colspan="1"&gt;Decamer&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;66.5&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;6.7&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;24.6&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;12.3&lt;/p&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;0.5&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;68.3&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;28.4&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;124.5&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;62.3&lt;/p&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;0.5&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;70.1&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;2.9&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;93.7&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;2.4&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;301.7&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;150.9&lt;/p&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;0.6&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;71.9&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;6.9&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;120.6&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;3.7&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;379.8&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;189.9&lt;/p&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;0.6&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;73.7&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1.9&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;64.4&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.8&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;240.6&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;120.3&lt;/p&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;0.5&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;75.5&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.9&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;21.1&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;101.7&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;50.8&lt;/p&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;0.4&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;77.3&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;6.2&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;42.6&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;21.3&lt;/p&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;0.3&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;79.1&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.1&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;2.4&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;26.5&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;13.3&lt;/p&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;0.2&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"&gt;80.9&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1.0&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1.5&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;22.3&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;11.2&lt;/p&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;0.1&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"&gt;82.7&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.2&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;29.2&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;14.6&lt;/p&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;n.d&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="5" colspan="1"&gt;Monomer&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;84.5&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.1&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;34.9&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;17.5&lt;/p&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;n.d&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;86.3&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;28.9&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;14.4&lt;/p&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;n.d&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;88.1&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;17.4&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;8.7&lt;/p&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;n.d.&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;89.9&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;5.5&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;2.8&lt;/p&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;n.d.&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;91.7&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;n.d.&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.1&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;0.07&lt;/p&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;p&gt;0.2&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon>
+      <offset>12529</offset>
+      <text>Peak	EncFtnsHretention volume (ml)	Element concentration (µM)	Derived EncFtnsHconcentration (µM)	Derived Fe/ EncFtnsH monomer	 	Ca	Fe	Zn	Se	 	Decamer	66.5	n.d.	6.7	n.d.	24.6	12.3	0.5	 	68.3	n.d.	28.4	n.d	124.5	62.3	0.5	 	70.1	2.9	93.7	2.4	301.7	150.9	0.6	 	71.9	6.9	120.6	3.7	379.8	189.9	0.6	 	73.7	1.9	64.4	0.8	240.6	120.3	0.5	 	75.5	0.9	21.1	n.d.	101.7	50.8	0.4	 	77.3	n.d.	6.2	n.d.	42.6	21.3	0.3	 	79.1	0.1	2.4	n.d.	26.5	13.3	0.2	 		80.9	1.0	1.5	n.d.	22.3	11.2	0.1	 		82.7	n.d.	0.2	n.d.	29.2	14.6	n.d	 	Monomer	84.5	n.d.	0.1	n.d.	34.9	17.5	n.d	 	86.3	n.d.	n.d	n.d.	28.9	14.4	n.d	 	88.1	n.d.	n.d.	n.d.	17.4	8.7	n.d.	 	89.9	n.d.	n.d.	n.d.	5.5	2.8	n.d.	 	91.7	n.d.	n.d.	n.d.	0.1	0.07	0.2	 	</text>
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+      <offset>13228</offset>
+      <text>Estimates of EncFtnsH molecular weight from SEC-MALLS analysis. EncFtnsH was purified from E. coli BL21(DE3) grown in minimal medium (MM) by nickel affinity chromatography and size-exclusion chromatography. Fractions from two peaks (decamer and monomer) were pooled separately (Figure 1C) and analysed by SEC-MALLS using a Superdex 200 10/300 GL column (GE Healthcare) and Viscotek SEC-MALLS instruments (Malvern Instruments) (Figure 2C). The decamer and monomer peaks were both symmetric and monodisperse, allowing the estimation of the molecular weight of the species in these fractions. The molecular weights are quoted to the nearest kDa due to the resolution limit of the instrument. The proteins analyzed by SEC-MALLS came from single protein preparation.</text>
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+        <text>SEC-MALLS</text>
+      </annotation>
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+        <text>EncFtnsH</text>
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+        <location offset="13942" length="9"/>
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+    </passage>
+    <passage>
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+      <infon key="id">tbl2</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_caption</infon>
+      <offset>13997</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.007</text>
+    </passage>
+    <passage>
+      <infon key="file">tbl2.xml</infon>
+      <infon key="id">tbl2</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table</infon>
+      <infon key="xml">&lt;?xml version="1.0" encoding="UTF-8"?&gt;
+&lt;table frame="hsides" rules="groups"&gt;&lt;thead&gt;&lt;tr&gt;&lt;th rowspan="1" colspan="1"&gt;Molecular Weight (kDa)&lt;/th&gt;&lt;th rowspan="1" colspan="1"&gt;Decamer peak&lt;/th&gt;&lt;th rowspan="1" colspan="1"&gt;Monomer peak&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Theoretical&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;133&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;13&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;EncFtn&lt;sub&gt;sH&lt;/sub&gt;-decamer fractions&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;132&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;15&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;EncFtn&lt;sub&gt;sH&lt;/sub&gt;-monomer fractions&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;126&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;13&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon>
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+      <text>Molecular Weight (kDa)	Decamer peak	Monomer peak	 	Theoretical	133	13	 	EncFtnsH-decamer fractions	132	15	 	EncFtnsH-monomer fractions	126	13	 	</text>
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+        <infon key="updated_at">2023-09-19T13:19:19Z</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>Monomer</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:55Z</infon>
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+        <text>EncFtnsH</text>
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+        <location offset="14161" length="7"/>
+        <text>monomer</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>14189</offset>
+      <text>We purified recombinant R. rubrum EncFtn as both the full-length sequence (140 amino acids) and a truncated C-terminal hexahistidine-tagged variant (amino acids 1–96 plus the tag; herein EncFtnsH). In both cases the elution profile from size-exclusion chromatography (SEC) displayed two peaks (Figure 2A). SDS-PAGE analysis of fractions from these peaks showed that the high molecular weight peak was partially resistant to SDS and heat-induced denaturation; in contrast, the low molecular weight peak was consistent with monomeric mass of 13 kDa (Figure 2B). MALDI peptide mass fingerprinting of these bands confirmed the identity of both as EncFtn. Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the SEC fractions showed 100 times more iron in the oligomeric fraction than the monomer (Figure 2A, blue scatter points; Table 1), suggesting that EncFtn oligomerization is associated with iron binding. In order to determine the iron-loading stoichiometry in the EncFtn complex, further ICP-MS experiments were performed using selenomethionine (SeMet)-labelled protein EncFtn (Table 1). In these experiments, we observed sub-stoichiometric metal binding, which is in contrast to the classical ferritins. Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) analysis of samples taken from each peak gave calculated molecular weights consistent with a decamer for the high molecular weight peak and a monomer for the low molecular weight peak (Figure 2C, Table 2).</text>
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+        <location offset="14213" length="9"/>
+        <text>R. rubrum</text>
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+        <text>140 amino acids</text>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>hexahistidine-tagged</text>
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+        <infon key="updated_at">2023-09-19T13:20:44Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>1–96</text>
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+        <infon key="updated_at">2023-09-19T13:19:19Z</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>monomeric</text>
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+        <text>MALDI peptide mass fingerprinting</text>
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+        <infon key="type">protein</infon>
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+        <infon key="updated_at">2023-09-19T11:30:38Z</infon>
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+        <text>EncFtn</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>SEC</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14946" length="4"/>
+        <text>iron</text>
+      </annotation>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>monomer</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <location offset="15054" length="6"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15136" length="4"/>
+        <text>iron</text>
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+        <infon key="identifier">PR:</infon>
+        <location offset="15170" length="6"/>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="15194" length="6"/>
+        <text>ICP-MS</text>
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+      <annotation id="369">
+        <infon key="score">0.9991273</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:23:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15234" length="16"/>
+        <text>selenomethionine</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:16:44Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15252" length="5"/>
+        <text>SeMet</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:38Z</infon>
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+        <location offset="15276" length="6"/>
+        <text>EncFtn</text>
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+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
+        <location offset="15390" length="9"/>
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+        <infon key="score">0.99904937</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:21Z</infon>
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+        <location offset="15400" length="9"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <location offset="15411" length="29"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="15446" length="34"/>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="15482" length="9"/>
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+        <infon key="updated_at">2023-09-19T13:19:19Z</infon>
+        <location offset="15607" length="16"/>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15635" length="7"/>
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:19:19Z</infon>
+        <location offset="15655" length="16"/>
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+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>15702</offset>
+      <text>Effect of metal ions on the oligomeric state of EncFtnsH in solution.</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:55Z</infon>
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+        <location offset="15750" length="8"/>
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+      <infon key="id">fig3s1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
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+      <text>(A/B) EncFtnsH-monomer was incubated with one mole equivalent of various metal salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column. Co2+ and Zn2+ induced the formation of the decameric form of EncFtnsH; while Mn2+, Mg2+ and Fe3+ did not significantly alter the oligomeric state of EncFtnsH.</text>
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+        <infon key="identifier">PR:</infon>
+        <location offset="15779" length="8"/>
+        <text>EncFtnsH</text>
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+      <annotation id="380">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15788" length="7"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:22:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15800" length="9"/>
+        <text>incubated</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15881" length="25"/>
+        <text>analytical gel-filtration</text>
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+      <annotation id="1978">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:21:43Z</infon>
+        <location offset="15946" length="4"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:21:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15955" length="4"/>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15989" length="9"/>
+        <text>decameric</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16007" length="8"/>
+        <text>EncFtnsH</text>
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+      <annotation id="1998">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:28:59Z</infon>
+        <location offset="16023" length="4"/>
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+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:22:10Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:22:13Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16038" length="4"/>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16095" length="8"/>
+        <text>EncFtnsH</text>
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+      <infon key="file">elife-18972-fig3-figsupp1.jpg</infon>
+      <infon key="id">fig3s1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>16105</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.009</text>
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+    <passage>
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+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>16152</offset>
+      <text>PAGE analysis of the effect of metal ions on the oligomeric state of EncFtnsH.</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:22:31Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16152" length="4"/>
+        <text>PAGE</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16221" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig3-figsupp2.jpg</infon>
+      <infon key="id">fig3s2</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>16231</offset>
+      <text>50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE. (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16237" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="391">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16246" length="7"/>
+        <text>monomer</text>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16257" length="7"/>
+        <text>decamer</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:22:38Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16322" length="5"/>
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+      <annotation id="394">
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:22:41Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16328" length="5"/>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:22:44Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16334" length="5"/>
+        <text>Zn2+,</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:22:47Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16340" length="5"/>
+        <text>Mn2+,</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:22:50Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16346" length="5"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:22:52Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16352" length="4"/>
+        <text>Mg2+</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:22:56Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16361" length="5"/>
+        <text>Fe3+,</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16390" length="11"/>
+        <text>Native PAGE</text>
+      </annotation>
+      <annotation id="401">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16412" length="8"/>
+        <text>SDS-PAGE</text>
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+      <annotation id="402">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16430" length="11"/>
+        <text>Native PAGE</text>
+      </annotation>
+      <annotation id="403">
+        <infon key="score">0.99836725</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16454" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="404">
+        <infon key="score">0.99835724</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16463" length="7"/>
+        <text>monomer</text>
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+      <annotation id="405">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16525" length="11"/>
+        <text>Native PAGE</text>
+      </annotation>
+      <annotation id="406">
+        <infon key="score">0.9980385</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16549" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="407">
+        <infon key="score">0.9972319</infon>
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+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.010</text>
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+      <text>Effect of Fe2+ and protein concentration on the oligomeric state of EncFtnsH in solution.</text>
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+      <text>(A) Recombinant EncFtnsH was purified by Gel filtration Superdex 200 chromatography from E. coli BL21(DE3) grown in MM or in MM supplemented with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+). A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH
+in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column (GE Healthcare). Both Fe2+ salts tested induced the formation of decamer indicated by the peak between 1.2 and 1.6 ml. Monomeric and decameric samples of EncFtnsH are shown as controls. Peaks around 0.8 ml were seen as protein aggregation. (C) Analytical gel filtration of EncFtn monomer at different concentrations to illustrate the effect of protein concentration on multimerization. The major peak shows a shift towards a dimer species at high concentration of protein, but the ratio of this peak (1.5–1.8 ml) to the decamer peak (1.2–1.5 ml) does not change when compared to the low concentration sample.</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <location offset="17416" length="25"/>
+        <text>analytical gel-filtration</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="type">oligomeric_state</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">oligomeric_state</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>EncFtnsH</text>
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+        <infon key="identifier">PR:</infon>
+        <location offset="17753" length="6"/>
+        <text>EncFtn</text>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>monomer</text>
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+        <infon key="type">oligomeric_state</infon>
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+        <infon key="updated_at">2023-09-19T18:16:28Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>dimer</text>
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+        <infon key="type">oligomeric_state</infon>
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+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+    <passage>
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+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>18097</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.008</text>
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+      <text>Gel-filtration peak area ratios for EncFtnsH decamer and monomer on addition of different metal ions. EncFtnsH was produced in E. coli BL21(DE3) cultured in MM and MM with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+) and purified by gel-filtration chromatography using an Superdex 200 16/60 column (GE Healthcare). Monomer fractions of EncFtnsH purified from MM were pooled and run in subsequent analytical gel-filtration runs over the course of three days. Samples of EncFtnsH monomer were incubated with one molar equivalent of metal ion salts at room temperature for two hours before analysis by analytical gel filtration chromatography (AGF) using a Superdex 200 10/300 GL column. The area for resulting protein peaks were calculated using the Unicorn software (GE Healthcare); peak ratios were calculated to quantify the propensity of EncFtnsH to multimerize in the presence of the different metal ions. The change in the ratios of monomer to decamer over the three days of experiments may be a consequence of experimental variability, or the propensity of this protein to equilibrate towards decamer over time. The increased decamer: monomer ratio seen in the presence of Fe2+, Co2+, and Zn2+ indicates that these metal ions facilitate multimerization of the EncFtnsH protein, while the other metal ions tested do not appear to induce multimerization. The analytical gel filtration experiment was repeated twice using two independent preparations of protein, of which values calculated from one sample are presented here.</text>
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+http://dx.doi.org/10.7554/eLife.18972.011</text>
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+</infon>
+      <offset>19709</offset>
+      <text>Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	</text>
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+        <infon key="identifier">CHEBI:</infon>
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+        <text>Fe2+</text>
+      </annotation>
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+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:24:28Z</infon>
+        <location offset="19875" length="25"/>
+        <text>Analytical Gel filtration</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <location offset="19906" length="8"/>
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+      </annotation>
+      <annotation id="464">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19915" length="7"/>
+        <text>decamer</text>
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+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <location offset="19949" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="465">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19958" length="7"/>
+        <text>monomer</text>
+      </annotation>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:17:56Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="19991" length="14"/>
+        <text>Fe(NH4)2(SO4)2</text>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <location offset="20006" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20015" length="7"/>
+        <text>monomer</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:23:51Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20039" length="9"/>
+        <text>FeSO4-HCl</text>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <location offset="20049" length="8"/>
+        <text>EncFtnsH</text>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20058" length="7"/>
+        <text>monomer</text>
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+      <annotation id="1981">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:24:28Z</infon>
+        <location offset="20082" length="25"/>
+        <text>Analytical Gel filtration</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <location offset="20113" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20122" length="7"/>
+        <text>monomer</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:23:54Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20155" length="5"/>
+        <text>CoCl2</text>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <location offset="20161" length="8"/>
+        <text>EncFtnsH</text>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20170" length="7"/>
+        <text>monomer</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:23:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20194" length="5"/>
+        <text>MnCl2</text>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <location offset="20200" length="8"/>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>monomer</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:23:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20232" length="5"/>
+        <text>ZnSO4</text>
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+      <annotation id="1948">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <location offset="20238" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="476">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20247" length="7"/>
+        <text>monomer</text>
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+      <annotation id="477">
+        <infon key="score">0.99839336</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:24:01Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20270" length="5"/>
+        <text>FeCl3</text>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <location offset="20276" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="478">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20285" length="7"/>
+        <text>monomer</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:24:28Z</infon>
+        <location offset="20308" length="25"/>
+        <text>Analytical Gel filtration</text>
+      </annotation>
+      <annotation id="1950">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <location offset="20339" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="479">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20348" length="7"/>
+        <text>monomer</text>
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+      <annotation id="480">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:24:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20381" length="5"/>
+        <text>MgSO4</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <location offset="20387" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="481">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20396" length="7"/>
+        <text>monomer</text>
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+      <annotation id="2021">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:34:49Z</infon>
+        <location offset="20419" length="10"/>
+        <text>Ca acetate</text>
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+      <annotation id="1952">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <location offset="20430" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="482">
+        <infon key="score">0.9385806</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20439" length="7"/>
+        <text>monomer</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>20464</offset>
+      <text>We purified EncFtnsH from E. coli grown in MM with or without the addition of 1 mM Fe(NH4)2(SO4)2. The decamer to monomer ratio in the sample purified from cells grown in iron-supplemented media was 4.5, while that from the iron-free media was 0.11, suggesting that iron induces the oligomerization of EncFtnsH in vivo (Figure 3A, Table 3). To test the metal-dependent oligomerization of EncFtnsH in vitro, we incubated the protein with various metal cations and subjected samples to analytical SEC and non-denaturing PAGE. Of the metals tested, only Fe2+, Zn2+ and Co2+ induced the formation of significant amounts of the decamer (Figure 3B, Figure 3—figure supplement 1/2). While Fe2+ induces the multimerization of EncFtnsH, Fe3+ in the form of FeCl3 does not have this effect on the protein, highlighting the apparent preference this protein has for the ferrous form of iron. To determine if the oligomerization of EncFtnsH was concentration dependent we performed analytical SEC at 90 and 700 µM protein concentration (Figure 3C). At the higher concentration, no increase in the decameric form of EncFtn was observed; however, the shift in the major peak from the position of the monomer species indicated a tendency to dimerize at high concentration.</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="20476" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="484">
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:56:09Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20490" length="7"/>
+        <text>E. coli</text>
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+      <annotation id="2013">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:30:27Z</infon>
+        <location offset="20507" length="2"/>
+        <text>MM</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:17:56Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20547" length="14"/>
+        <text>Fe(NH4)2(SO4)2</text>
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+      <annotation id="486">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20567" length="7"/>
+        <text>decamer</text>
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+      <annotation id="487">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20578" length="7"/>
+        <text>monomer</text>
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+      <annotation id="488">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20635" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="489">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:35:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20688" length="9"/>
+        <text>iron-free</text>
+      </annotation>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20730" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="491">
+        <infon key="score">0.9993291</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="20766" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="492">
+        <infon key="score">0.99936074</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="20852" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="493">
+        <infon key="score">0.792685</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:20:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20874" length="9"/>
+        <text>incubated</text>
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+      <annotation id="494">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:35:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20948" length="14"/>
+        <text>analytical SEC</text>
+      </annotation>
+      <annotation id="495">
+        <infon key="score">0.9989158</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:36:00Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20967" length="19"/>
+        <text>non-denaturing PAGE</text>
+      </annotation>
+      <annotation id="496">
+        <infon key="score">0.99724555</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:35:23Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="21015" length="5"/>
+        <text>Fe2+,</text>
+      </annotation>
+      <annotation id="497">
+        <infon key="score">0.9993093</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:35:26Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="21021" length="4"/>
+        <text>Zn2+</text>
+      </annotation>
+      <annotation id="498">
+        <infon key="score">0.9993104</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:35:28Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="21030" length="4"/>
+        <text>Co2+</text>
+      </annotation>
+      <annotation id="499">
+        <infon key="score">0.9988109</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21087" length="7"/>
+        <text>decamer</text>
+      </annotation>
+      <annotation id="500">
+        <infon key="score">0.999256</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:35:31Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="21146" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="501">
+        <infon key="score">0.99936944</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="21182" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="502">
+        <infon key="score">0.99924195</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:35:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="21192" length="4"/>
+        <text>Fe3+</text>
+      </annotation>
+      <annotation id="503">
+        <infon key="score">0.9993135</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:35:37Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="21212" length="5"/>
+        <text>FeCl3</text>
+      </annotation>
+      <annotation id="2161">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:15:46Z</infon>
+        <location offset="21322" length="20"/>
+        <text>ferrous form of iron</text>
+      </annotation>
+      <annotation id="504">
+        <infon key="score">0.99935526</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="21383" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="505">
+        <infon key="score">0.9984896</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:35:48Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21433" length="14"/>
+        <text>analytical SEC</text>
+      </annotation>
+      <annotation id="506">
+        <infon key="score">0.99874544</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21548" length="9"/>
+        <text>decameric</text>
+      </annotation>
+      <annotation id="507">
+        <infon key="score">0.9993456</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:38Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="21566" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="508">
+        <infon key="score">0.998926</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21649" length="7"/>
+        <text>monomer</text>
+      </annotation>
+      <annotation id="509">
+        <infon key="score">0.983941</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:16:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21689" length="8"/>
+        <text>dimerize</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>21725</offset>
+      <text>Crystal structure of EncFtnsH</text>
+      <annotation id="510">
+        <infon key="score">0.99854386</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:36:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21725" length="17"/>
+        <text>Crystal structure</text>
+      </annotation>
+      <annotation id="511">
+        <infon key="score">0.99721366</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="21746" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig4-figsupp1.jpg</infon>
+      <infon key="id">fig4s1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>21755</offset>
+      <text>Electrostatic surface of EncFtnsH.</text>
+      <annotation id="512">
+        <infon key="score">0.98178387</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="21780" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig4-figsupp1.jpg</infon>
+      <infon key="id">fig4s1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>21790</offset>
+      <text>The solvent accessible surface of EncFtnsH is shown, colored by electrostatic potential as calculated using the APBS plugin in PyMOL. Negatively charged regions are colored red and positive regions in blue, neutral regions in grey. (A) View of the surface of the EncFtnsH decamer looking down the central axis. (B) Orthogonal view of (A). (C) Cutaway view of (B) showing the charge distribution within the central cavity.</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="21824" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="514">
+        <infon key="score">0.9770179</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="22053" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="515">
+        <infon key="score">0.99858713</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22062" length="7"/>
+        <text>decamer</text>
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+      <annotation id="516">
+        <infon key="score">0.99757665</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:12:17Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22196" length="14"/>
+        <text>central cavity</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig4-figsupp1.jpg</infon>
+      <infon key="id">fig4s1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>22212</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.013</text>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig4.jpg</infon>
+      <infon key="id">fig4</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>22259</offset>
+      <text>Crystal structure of EncFtnsH.</text>
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+        <infon key="score">0.9986262</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:29:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22259" length="17"/>
+        <text>Crystal structure</text>
+      </annotation>
+      <annotation id="518">
+        <infon key="score">0.9959949</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="22280" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig4.jpg</infon>
+      <infon key="id">fig4</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>22290</offset>
+      <text>(A) Overall architecture of EncFtnsH. Transparent solvent accessible surface view with α-helices shown as tubes and bound metal ions as spheres. Alternating subunits are colored blue and green for clarity. The doughnut-like decamer is 7 nm in diameter and 4.5 nm thick. (B) Monomer of EncFtnsH shown as a secondary structure cartoon. (C/D) Dimer interfaces formed in the decameric ring of EncFtnsH. Subunits are shown as secondary structure cartoons and colored blue and green for clarity. Bound metal ions are shown as orange spheres for Fe3+ and grey and white spheres for Ca2+.</text>
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+        <infon key="score">0.9986072</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="22318" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="520">
+        <infon key="score">0.99932295</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:36:49Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22377" length="9"/>
+        <text>α-helices</text>
+      </annotation>
+      <annotation id="521">
+        <infon key="score">0.98181826</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:35:44Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22447" length="8"/>
+        <text>subunits</text>
+      </annotation>
+      <annotation id="522">
+        <infon key="score">0.98685026</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:16:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22500" length="13"/>
+        <text>doughnut-like</text>
+      </annotation>
+      <annotation id="523">
+        <infon key="score">0.9988224</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22514" length="7"/>
+        <text>decamer</text>
+      </annotation>
+      <annotation id="524">
+        <infon key="score">0.9988906</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22564" length="7"/>
+        <text>Monomer</text>
+      </annotation>
+      <annotation id="525">
+        <infon key="score">0.99908733</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="22575" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="526">
+        <infon key="score">0.99880683</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:36:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22630" length="16"/>
+        <text>Dimer interfaces</text>
+      </annotation>
+      <annotation id="527">
+        <infon key="score">0.9986603</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22661" length="9"/>
+        <text>decameric</text>
+      </annotation>
+      <annotation id="528">
+        <infon key="score">0.99947757</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:00:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22671" length="4"/>
+        <text>ring</text>
+      </annotation>
+      <annotation id="529">
+        <infon key="score">0.99921596</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="22679" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1886">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:35:44Z</infon>
+        <location offset="22689" length="8"/>
+        <text>Subunits</text>
+      </annotation>
+      <annotation id="530">
+        <infon key="score">0.99908435</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:37:00Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="22829" length="4"/>
+        <text>Fe3+</text>
+      </annotation>
+      <annotation id="531">
+        <infon key="score">0.99907744</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:37:03Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="22865" length="4"/>
+        <text>Ca2+</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig4.jpg</infon>
+      <infon key="id">fig4</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>22875</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.012</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>22922</offset>
+      <text>We determined the crystal structure of EncFtnsH by molecular replacement to 2.0 Å resolution (see Table 1 for X-ray data collection and refinement statistics). The crystallographic asymmetric unit contained thirty monomers of EncFtn with visible electron density for residues 7 – 96 in each chain. The protein chains were arranged as three identical annular decamers, each with D5 symmetry. The decamer has a diameter of 7 nm and thickness of 4 nm (Figure 4A). The monomer of EncFtn has an N-terminal 310-helix that precedes two 4 nm long antiparallel α-helices arranged with their long axes at 25° to each other; these helices are followed by a shorter 1.4 nm helix projecting at 70° from α2 (Figure 4B). The C-terminal region of the crystallized construct extends from the outer circumference of the ring, indicating that the encapsulin localization sequence in the full-length protein is on the exterior of the ring and is thus free to interact with its binding site on the encapsulin shell protein.</text>
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+        <infon key="score">0.9976036</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:29:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22940" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="533">
+        <infon key="score">0.99746907</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="22961" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="534">
+        <infon key="score">0.9986429</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:20:35Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22973" length="21"/>
+        <text>molecular replacement</text>
+      </annotation>
+      <annotation id="2198">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:06:07Z</infon>
+        <location offset="23032" length="47"/>
+        <text>X-ray data collection and refinement statistics</text>
+      </annotation>
+      <annotation id="535">
+        <infon key="score">0.9986632</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:16:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23136" length="8"/>
+        <text>monomers</text>
+      </annotation>
+      <annotation id="536">
+        <infon key="score">0.99929357</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:39Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="23148" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="537">
+        <infon key="score">0.99033964</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:05:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23168" length="16"/>
+        <text>electron density</text>
+      </annotation>
+      <annotation id="538">
+        <infon key="score">0.997608</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:38:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23198" length="6"/>
+        <text>7 – 96</text>
+      </annotation>
+      <annotation id="539">
+        <infon key="score">0.67838174</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:16:25Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23272" length="7"/>
+        <text>annular</text>
+      </annotation>
+      <annotation id="540">
+        <infon key="score">0.99786335</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23280" length="8"/>
+        <text>decamers</text>
+      </annotation>
+      <annotation id="541">
+        <infon key="score">0.99868995</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23317" length="7"/>
+        <text>decamer</text>
+      </annotation>
+      <annotation id="542">
+        <infon key="score">0.9987478</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23387" length="7"/>
+        <text>monomer</text>
+      </annotation>
+      <annotation id="543">
+        <infon key="score">0.9993278</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:39Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="23398" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="544">
+        <infon key="score">0.9993064</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:38:22Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23423" length="9"/>
+        <text>310-helix</text>
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+      <annotation id="545">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:38:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23461" length="22"/>
+        <text>antiparallel α-helices</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="23612" length="2"/>
+        <text>α2</text>
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+      <annotation id="2022">
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:39:19Z</infon>
+        <location offset="23632" length="17"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:37:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23750" length="32"/>
+        <text>encapsulin localization sequence</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:20:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>full-length</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:38:46Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>binding site</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <location offset="23910" length="5"/>
+        <text>shell</text>
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+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>23933</offset>
+      <text>The monomer of EncFtnsH forms two distinct dimer interfaces within the decamer (Figure 4 C/D). The first dimer is formed from two monomers arranged antiparallel to each other, with α1 from each monomer interacting along their lengths and α3 interdigitating with α2 and α3 of the partner chain. This interface buries one third of the surface area from each partner and is stabilized by thirty hydrogen bonds and fourteen salt bridges (Figure 4C). The second dimer interface forms an antiparallel four-helix bundle between helices 1 and 2 from each monomer (Figure 4D). This interface is less extensive than the first and is stabilized by twenty-one hydrogen bonds, six salt bridges, and a number of metal ions.</text>
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+        <location offset="23948" length="8"/>
+        <text>EncFtnsH</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="type">oligomeric_state</infon>
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+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24127" length="7"/>
+        <text>monomer</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:00:23Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24171" length="2"/>
+        <text>α3</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:00:26Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:00:29Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24202" length="2"/>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:12:24Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24232" length="9"/>
+        <text>interface</text>
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+      <annotation id="567">
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+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:22:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="24325" length="14"/>
+        <text>hydrogen bonds</text>
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+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:23:01Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>salt bridges</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:02Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>dimer interface</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:00:33Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24415" length="30"/>
+        <text>antiparallel four-helix bundle</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:00:36Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>helices 1 and 2</text>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24480" length="7"/>
+        <text>monomer</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:12:28Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24506" length="9"/>
+        <text>interface</text>
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+      <annotation id="574">
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+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:23:05Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>hydrogen bonds</text>
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+      <annotation id="575">
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+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:23:07Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="24601" length="12"/>
+        <text>salt bridges</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>24659</offset>
+      <text>The arrangement of ten monomers in alternating orientation forms the decamer of EncFtn, which assembles as a pentamer of dimers (Figure 4A). Each monomer lies at 45° relative to the vertical central-axis of the ring, with the N-termini of alternating subunits capping the center of the ring at each end, while the C-termini are arranged around the circumference. The central hole in the ring is 2.5 nm at its widest in the center of the complex, and 1.5 nm at its narrowest point near the outer surface, although it should be noted that a number of residues at the N-terminus are not visible in the crystallographic electron density and these may occupy the central channel. The surface of the decamer has distinct negatively charged patches, both within the central hole and on the outer circumference, which form spokes through the radius of the complex (Figure 4—figure supplement 1).</text>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:16:52Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24682" length="8"/>
+        <text>monomers</text>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24728" length="7"/>
+        <text>decamer</text>
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+      <annotation id="578">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:39Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24739" length="6"/>
+        <text>EncFtn</text>
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+      <annotation id="579">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:39:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24768" length="8"/>
+        <text>pentamer</text>
+      </annotation>
+      <annotation id="580">
+        <infon key="score">0.99871933</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:37:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24780" length="6"/>
+        <text>dimers</text>
+      </annotation>
+      <annotation id="581">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24805" length="7"/>
+        <text>monomer</text>
+      </annotation>
+      <annotation id="582">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:40:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24870" length="4"/>
+        <text>ring</text>
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+      <annotation id="583">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:35:44Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24910" length="8"/>
+        <text>subunits</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:40:13Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24945" length="4"/>
+        <text>ring</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:39:46Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25026" length="12"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="25046" length="4"/>
+        <text>ring</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:06:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25258" length="33"/>
+        <text>crystallographic electron density</text>
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+      <annotation id="588">
+        <infon key="score">0.9828136</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:39:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25317" length="15"/>
+        <text>central channel</text>
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+      <annotation id="589">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25353" length="7"/>
+        <text>decamer</text>
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+      <annotation id="590">
+        <infon key="score">0.99743503</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:39:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25374" length="26"/>
+        <text>negatively charged patches</text>
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+      <annotation id="591">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:39:50Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25418" length="12"/>
+        <text>central hole</text>
+      </annotation>
+      <annotation id="592">
+        <infon key="score">0.96327144</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:39:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25474" length="6"/>
+        <text>spokes</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>25550</offset>
+      <text>EncFtn ferroxidase center</text>
+      <annotation id="1856">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:39Z</infon>
+        <location offset="25550" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1862">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:31:54Z</infon>
+        <location offset="25557" length="18"/>
+        <text>ferroxidase center</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig5-figsupp1.jpg</infon>
+      <infon key="id">fig5s1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>25576</offset>
+      <text>Putative ligand-binding site in EncFtnsH.</text>
+      <annotation id="593">
+        <infon key="score">0.99888396</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:40:51Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25585" length="19"/>
+        <text>ligand-binding site</text>
+      </annotation>
+      <annotation id="594">
+        <infon key="score">0.82397026</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25608" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig5-figsupp1.jpg</infon>
+      <infon key="id">fig5s1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>25618</offset>
+      <text>(A) Wall-eyed stereo view of the dimer interface of EncFtn. Protein chains are shown as sticks, with 2mFo-DFc electron density shown in blue mesh and contoured at 1.5 σ and mFo-DFc shown in green mesh and contoured at 3 σ. (B) Wall-eyed stereo view of putative metal binding site at the external surface of EncFtnsH. Protein chains and electron density maps are shown as in (A).</text>
+      <annotation id="595">
+        <infon key="score">0.9990647</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:02Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25651" length="15"/>
+        <text>dimer interface</text>
+      </annotation>
+      <annotation id="1857">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:39Z</infon>
+        <location offset="25670" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="2023">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:41:22Z</infon>
+        <location offset="25719" length="25"/>
+        <text>2mFo-DFc electron density</text>
+      </annotation>
+      <annotation id="596">
+        <infon key="score">0.75227594</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:05:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25791" length="7"/>
+        <text>mFo-DFc</text>
+      </annotation>
+      <annotation id="597">
+        <infon key="score">0.9990678</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:40:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25879" length="18"/>
+        <text>metal binding site</text>
+      </annotation>
+      <annotation id="1953">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <location offset="25925" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="598">
+        <infon key="score">0.9982582</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:05:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25954" length="21"/>
+        <text>electron density maps</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig5-figsupp1.jpg</infon>
+      <infon key="id">fig5s1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>26005</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.015</text>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig5.jpg</infon>
+      <infon key="id">fig5</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>26052</offset>
+      <text>EncFtnsH metal binding sites.</text>
+      <annotation id="1954">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <location offset="26052" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="599">
+        <infon key="score">0.99899024</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:08:39Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26061" length="19"/>
+        <text>metal binding sites</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig5.jpg</infon>
+      <infon key="id">fig5</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>26082</offset>
+      <text>(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH. Protein residues are shown as sticks with blue and green carbons for the different subunits, iron ions are shown as orange spheres and calcium as grey spheres, and the glycolic acid ligand is shown with yellow carbon atoms coordinated above the di-iron center. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ and the NCS-averaged anomalous difference map is shown as an orange mesh and contoured at 10 σ. (B) Iron coordination within the FOC including residues Glu32, Glu62, His65 and Tyr39 from two chains. Protein and metal ions are shown as in A. Coordination between the protein and iron ions is shown as yellow dashed lines with distances indicated. (C) Coordination of calcium within the dimer interface by four glutamic acid residues (E31 and E34 from two chains). The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.</text>
+      <annotation id="600">
+        <infon key="score">0.9990543</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:08:51Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26115" length="36"/>
+        <text>metal-binding dimerization interface</text>
+      </annotation>
+      <annotation id="601">
+        <infon key="score">0.99433535</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="26155" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="602">
+        <infon key="score">0.62606853</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:35:44Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26248" length="8"/>
+        <text>subunits</text>
+      </annotation>
+      <annotation id="603">
+        <infon key="score">0.9990736</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="26258" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="604">
+        <infon key="score">0.9990526</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:09:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="26300" length="7"/>
+        <text>calcium</text>
+      </annotation>
+      <annotation id="605">
+        <infon key="score">0.9970863</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:24:11Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="26333" length="13"/>
+        <text>glycolic acid</text>
+      </annotation>
+      <annotation id="2024">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:09:23Z</infon>
+        <location offset="26410" length="14"/>
+        <text>di-iron center</text>
+      </annotation>
+      <annotation id="606">
+        <infon key="score">0.99704033</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:09:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26430" length="29"/>
+        <text>2mFo-DFc electron density map</text>
+      </annotation>
+      <annotation id="607">
+        <infon key="score">0.96590286</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:09:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26511" length="37"/>
+        <text>NCS-averaged anomalous difference map</text>
+      </annotation>
+      <annotation id="608">
+        <infon key="score">0.9988978</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="26603" length="4"/>
+        <text>Iron</text>
+      </annotation>
+      <annotation id="2027">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:13Z</infon>
+        <location offset="26608" length="12"/>
+        <text>coordination</text>
+      </annotation>
+      <annotation id="609">
+        <infon key="score">0.48611936</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26632" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="610">
+        <infon key="score">0.9995426</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26655" length="5"/>
+        <text>Glu32</text>
+      </annotation>
+      <annotation id="611">
+        <infon key="score">0.99954283</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:45Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26662" length="5"/>
+        <text>Glu62</text>
+      </annotation>
+      <annotation id="612">
+        <infon key="score">0.9995735</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:24:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26669" length="5"/>
+        <text>His65</text>
+      </annotation>
+      <annotation id="613">
+        <infon key="score">0.99956614</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:51Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26679" length="5"/>
+        <text>Tyr39</text>
+      </annotation>
+      <annotation id="2028">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:13Z</infon>
+        <location offset="26744" length="12"/>
+        <text>Coordination</text>
+      </annotation>
+      <annotation id="614">
+        <infon key="score">0.99900657</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="26781" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="615">
+        <infon key="score">0.8470298</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:12Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26853" length="12"/>
+        <text>Coordination</text>
+      </annotation>
+      <annotation id="616">
+        <infon key="score">0.9982346</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:09:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="26869" length="7"/>
+        <text>calcium</text>
+      </annotation>
+      <annotation id="617">
+        <infon key="score">0.99903464</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:02Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26888" length="15"/>
+        <text>dimer interface</text>
+      </annotation>
+      <annotation id="2026">
+        <infon key="type">residue_name</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:10:55Z</infon>
+        <location offset="26912" length="13"/>
+        <text>glutamic acid</text>
+      </annotation>
+      <annotation id="618">
+        <infon key="score">0.9995981</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26936" length="3"/>
+        <text>E31</text>
+      </annotation>
+      <annotation id="619">
+        <infon key="score">0.9995939</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:12:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26944" length="3"/>
+        <text>E34</text>
+      </annotation>
+      <annotation id="620">
+        <infon key="score">0.9987098</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:09:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="26970" length="7"/>
+        <text>calcium</text>
+      </annotation>
+      <annotation id="621">
+        <infon key="score">0.99776304</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:10:08Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="27012" length="5"/>
+        <text>water</text>
+      </annotation>
+      <annotation id="2029">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:13Z</infon>
+        <location offset="27044" length="12"/>
+        <text>coordination</text>
+      </annotation>
+      <annotation id="622">
+        <infon key="score">0.99873096</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:09:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="27064" length="7"/>
+        <text>calcium</text>
+      </annotation>
+      <annotation id="623">
+        <infon key="score">0.99896955</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:25Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27102" length="23"/>
+        <text>Metal coordination site</text>
+      </annotation>
+      <annotation id="624">
+        <infon key="score">0.9538523</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="27150" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="625">
+        <infon key="score">0.9985858</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:09:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="27168" length="7"/>
+        <text>calcium</text>
+      </annotation>
+      <annotation id="2056">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:19:10Z</infon>
+        <location offset="27185" length="14"/>
+        <text>coordinated by</text>
+      </annotation>
+      <annotation id="626">
+        <infon key="score">0.99950826</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:12:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27209" length="5"/>
+        <text>His57</text>
+      </annotation>
+      <annotation id="627">
+        <infon key="score">0.9995154</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:12:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27216" length="5"/>
+        <text>Glu61</text>
+      </annotation>
+      <annotation id="628">
+        <infon key="score">0.9995223</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:12:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27226" length="5"/>
+        <text>Glu64</text>
+      </annotation>
+      <annotation id="629">
+        <infon key="score">0.3694277</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27259" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="630">
+        <infon key="score">0.99882644</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:16:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27263" length="5"/>
+        <text>dimer</text>
+      </annotation>
+      <annotation id="1921">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <location offset="27353" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="631">
+        <infon key="score">0.99894947</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="27357" length="4"/>
+        <text>iron</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig5.jpg</infon>
+      <infon key="id">fig5</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>27371</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.014</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>27418</offset>
+      <text>The electron density maps of the initial EncFtnsH model displayed significant positive peaks in the mFo-DFc map at the center of the 4-helix bundle dimer (Figure 5—figure supplement 1). Informed by the ICP-MS data indicating the presence of iron in the protein we collected diffraction data at the experimentally determined iron absorption edge (1.74 Å) and calculated an anomalous difference Fourier map using this data. Inspection of this map showed two 10-sigma peaks between residues Glu32, Glu62 and His65 of two adjacent chains, and a statistically smaller 5-sigma peak between residues Glu31 and Glu34 of the two chains. Modeling metal ions into these peaks and refinement of the anomalous scattering parameters allowed us to identify these as two iron ions and a calcium ion respectively (Figure 5A). An additional region of asymmetric electron density near the di-iron binding site in the mFo-DFc map was modeled as glycolic acid, presumably a breakdown product of the PEG 3350 used for crystallization. This di-iron center has an Fe-Fe distance of 3.5 Å, Fe-Glu-O distances between 2.3 and 2.5 Å, and Fe-His-N distances of 2.5 Å (Figure 5B). This coordination geometry is consistent with the di-nuclear ferroxidase center (FOC) found in ferritin. It is interesting to note that although we did not add any additional iron to the crystallization trials, the FOC was fully occupied with iron in the final structure, implying that this site has a very high affinity for iron.</text>
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+        <infon key="score">0.9984922</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:12:37Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27422" length="21"/>
+        <text>electron density maps</text>
+      </annotation>
+      <annotation id="633">
+        <infon key="score">0.9599262</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:57Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="27459" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="2040">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:13:06Z</infon>
+        <location offset="27518" length="11"/>
+        <text>mFo-DFc map</text>
+      </annotation>
+      <annotation id="634">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:13:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27551" length="14"/>
+        <text>4-helix bundle</text>
+      </annotation>
+      <annotation id="635">
+        <infon key="score">0.9987697</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:17:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27566" length="5"/>
+        <text>dimer</text>
+      </annotation>
+      <annotation id="636">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:07Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27620" length="6"/>
+        <text>ICP-MS</text>
+      </annotation>
+      <annotation id="2051">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:18:27Z</infon>
+        <location offset="27647" length="11"/>
+        <text>presence of</text>
+      </annotation>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="27659" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="2041">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:13:46Z</infon>
+        <location offset="27692" length="16"/>
+        <text>diffraction data</text>
+      </annotation>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="27742" length="4"/>
+        <text>iron</text>
+      </annotation>
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+        <infon key="score">0.99854875</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:13:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27790" length="32"/>
+        <text>anomalous difference Fourier map</text>
+      </annotation>
+      <annotation id="640">
+        <infon key="score">0.99651504</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:06:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27859" length="3"/>
+        <text>map</text>
+      </annotation>
+      <annotation id="1989">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:26:00Z</infon>
+        <location offset="27883" length="5"/>
+        <text>peaks</text>
+      </annotation>
+      <annotation id="641">
+        <infon key="score">0.9994722</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27906" length="5"/>
+        <text>Glu32</text>
+      </annotation>
+      <annotation id="642">
+        <infon key="score">0.9994836</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27913" length="5"/>
+        <text>Glu62</text>
+      </annotation>
+      <annotation id="643">
+        <infon key="score">0.9995466</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:24:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27923" length="5"/>
+        <text>His65</text>
+      </annotation>
+      <annotation id="644">
+        <infon key="score">0.99946386</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:14:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28011" length="5"/>
+        <text>Glu31</text>
+      </annotation>
+      <annotation id="645">
+        <infon key="score">0.9994611</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:14:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28021" length="5"/>
+        <text>Glu34</text>
+      </annotation>
+      <annotation id="2199">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:06:39Z</infon>
+        <location offset="28087" length="10"/>
+        <text>refinement</text>
+      </annotation>
+      <annotation id="646">
+        <infon key="score">0.9979587</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:13:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28105" length="31"/>
+        <text>anomalous scattering parameters</text>
+      </annotation>
+      <annotation id="647">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="28173" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="648">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:09:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="28189" length="7"/>
+        <text>calcium</text>
+      </annotation>
+      <annotation id="649">
+        <infon key="score">0.9818403</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:06:28Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28262" length="16"/>
+        <text>electron density</text>
+      </annotation>
+      <annotation id="650">
+        <infon key="score">0.9983818</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:14:29Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28288" length="20"/>
+        <text>di-iron binding site</text>
+      </annotation>
+      <annotation id="2046">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:16:46Z</infon>
+        <location offset="28316" length="11"/>
+        <text>mFo-DFc map</text>
+      </annotation>
+      <annotation id="651">
+        <infon key="score">0.9986642</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:24:23Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="28343" length="13"/>
+        <text>glycolic acid</text>
+      </annotation>
+      <annotation id="652">
+        <infon key="score">0.9276309</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:16:26Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="28396" length="8"/>
+        <text>PEG 3350</text>
+      </annotation>
+      <annotation id="653">
+        <infon key="score">0.9970285</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:14:41Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28436" length="14"/>
+        <text>di-iron center</text>
+      </annotation>
+      <annotation id="2042">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:15:27Z</infon>
+        <location offset="28458" length="14"/>
+        <text>Fe-Fe distance</text>
+      </annotation>
+      <annotation id="2043">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:15:39Z</infon>
+        <location offset="28483" length="18"/>
+        <text>Fe-Glu-O distances</text>
+      </annotation>
+      <annotation id="2044">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:15:50Z</infon>
+        <location offset="28529" length="18"/>
+        <text>Fe-His-N distances</text>
+      </annotation>
+      <annotation id="2030">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:14Z</infon>
+        <location offset="28575" length="12"/>
+        <text>coordination</text>
+      </annotation>
+      <annotation id="654">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:16:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28620" length="29"/>
+        <text>di-nuclear ferroxidase center</text>
+      </annotation>
+      <annotation id="655">
+        <infon key="score">0.99822277</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28651" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="656">
+        <infon key="score">0.98256</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:04Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="28665" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+      <annotation id="657">
+        <infon key="score">0.99911994</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:31Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="28745" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="658">
+        <infon key="score">0.8547493</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:20:39Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="28757" length="22"/>
+        <text>crystallization trials</text>
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+      <annotation id="659">
+        <infon key="score">0.99851245</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28785" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="660">
+        <infon key="score">0.9989792</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:31Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="28813" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="661">
+        <infon key="score">0.99712235</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:06:44Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28831" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="2045">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:16:20Z</infon>
+        <location offset="28882" length="8"/>
+        <text>affinity</text>
+      </annotation>
+      <annotation id="662">
+        <infon key="score">0.99907124</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:31Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="28895" length="4"/>
+        <text>iron</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>28907</offset>
+      <text>The calcium ion coordinated by Glu31 and Glu34 adopts heptacoordinate geometry, with coordination distances of 2.5 Å between the metal ion and carboxylate oxygens of Glu31 and Glu34 (E31/34-site). A number of ordered solvent molecules are also coordinated to this metal ion at a distance of 2.5 Å. This heptacoordinate geometry is common in crystal structures with calcium ions (Figure 5C). While ICP-MS indicated that there were negligible amounts of calcium in the purified protein, the presence of 140 mM calcium acetate in the crystallization mother liquor favors the coordination of calcium at this site. The fact that the protein does not multimerize in solution in the presence of Fe3+ may indicate that these metal binding sites have a lower affinity for the ferric form of iron, which is the product of the ferroxidase reaction. A number of additional metal-ions were present at the outer circumference of at least one decamer in the asymmetric unit (Figure 5D). These ions are coordinated by His57, Glu61 and Glu64 from both chains in the FOC dimer and are 4.5 Å apart; Fe-Glu-O distances are between 2.5 and 3.5 Å and the Fe-His-N distances are 4 and 4.5 Å.</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:09:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="28911" length="7"/>
+        <text>calcium</text>
+      </annotation>
+      <annotation id="2057">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:19:10Z</infon>
+        <location offset="28923" length="14"/>
+        <text>coordinated by</text>
+      </annotation>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:14:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28938" length="5"/>
+        <text>Glu31</text>
+      </annotation>
+      <annotation id="665">
+        <infon key="score">0.9994036</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:14:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28948" length="5"/>
+        <text>Glu34</text>
+      </annotation>
+      <annotation id="666">
+        <infon key="score">0.99332994</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:17:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28961" length="15"/>
+        <text>heptacoordinate</text>
+      </annotation>
+      <annotation id="2031">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:14Z</infon>
+        <location offset="28992" length="12"/>
+        <text>coordination</text>
+      </annotation>
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+      <text>DOI:
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+      <text>(A) Structural alignment of the FOC residues in a dimer of EncFtnsH (green/blue) with a monomer of Pseudo-nitzschia multiseries ferritin (PmFtn) (PDBID: 4ITW) (orange). Iron ions are shown as orange spheres and a single calcium ion as a grey sphere. Residues within the FOC are conserved between EncFtn and ferritin PmFtn, with the exception of residues in the position equivalent to H65’ in the second subunit in the dimer (blue). The site in EncFtn with bound calcium is not present in other family members. (B) Secondary structure of aligned dimeric EncFtnsH and monomeric ferritin highlighting the conserved four-helix bundle. EncFtnsH monomers are shown in green and blue and aligned PmFtn monomer in orange as in A. (C) Cartoon of secondary structure elements in EncFtn dimer and ferritin. In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between α2 and α3 in ferritin PmFtn.</text>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:23:37Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="31926" length="5"/>
+        <text>PmFtn</text>
+      </annotation>
+    </passage>
+    <passage>
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+      <infon key="id">fig6</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>31941</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.016</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>31988</offset>
+      <text>Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins  (Figure 6A). The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65’) (Figure 6A). Structural superimposition of the FOCs of ferritin and EncFtn brings the four-helix bundle of the ferritin fold into close alignment with the EncFtn dimer, showing that the two families of proteins have essentially the same architecture around the di-iron center (Figure 6B). The linker connecting helices 2 and 3 of ferritin is congruent with the start of the C-terminal helix of one EncFtn monomer and the N-terminal 310 helix of the second monomer (Figure 6C).</text>
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+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
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+        <text>EncFtnsH</text>
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+        <location offset="32357" length="8"/>
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+        <infon key="identifier">PR:</infon>
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+        <text>EncFtn</text>
+      </annotation>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">protein_type</infon>
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+      </annotation>
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+        <infon key="type">oligomeric_state</infon>
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+      </annotation>
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+        <infon key="identifier">SO:</infon>
+        <location offset="32784" length="14"/>
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+        <infon key="type">structure_element</infon>
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+        <infon key="updated_at">2023-09-19T18:00:46Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32816" length="6"/>
+        <text>linker</text>
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+        <infon key="identifier">SO:</infon>
+        <location offset="32834" length="15"/>
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+        <location offset="32853" length="8"/>
+        <text>ferritin</text>
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+      <annotation id="792">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:24:30Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32908" length="5"/>
+        <text>helix</text>
+      </annotation>
+      <annotation id="793">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:41Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="32921" length="6"/>
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+      </annotation>
+      <annotation id="794">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="32928" length="7"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:24:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32955" length="9"/>
+        <text>310 helix</text>
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+      <annotation id="796">
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+        <infon key="type">oligomeric_state</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="32979" length="7"/>
+        <text>monomer</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>33004</offset>
+      <text>Mass spectrometry of the EncFtn assembly</text>
+      <annotation id="797">
+        <infon key="score">0.9986815</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:34:47Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="33004" length="17"/>
+        <text>Mass spectrometry</text>
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+      <annotation id="798">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:41Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="33029" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig7-figsupp1.jpg</infon>
+      <infon key="id">fig7s1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>33045</offset>
+      <text>Native IM-MS analysis of the apo-EncFtnsH monomer.</text>
+      <annotation id="799">
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>Native IM-MS</text>
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+        <infon key="identifier">PR:</infon>
+        <location offset="33078" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="802">
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+        <infon key="type">oligomeric_state</infon>
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+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="33087" length="7"/>
+        <text>monomer</text>
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+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig7-figsupp1.jpg</infon>
+      <infon key="id">fig7s1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>33096</offset>
+      <text>(A) Mass spectrum of apo-EncFtnsH acquired from 100 mM ammonium acetate pH 8.0 under native MS conditions. The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1. The +8 to +15 protein charge states have observed CCS between 20–26 nm2, which is significantly higher than the calculated CCS for an EncFtnsH monomer taken from the decameric assembly crystal structure (15.8 nm2). The mobility of the +7 charge state displays broad drift-time distribution with maxima consistent with CCS of 15.9 and 17.9 nm2. Finally, the 6+ charge state of EncFtnsH has mobility consistent with a CCS of 12.3 nm2, indicating a more compact/collapsed structure. It is clear from this data that apo-EncFtnsH exists in several gas phase conformations. The range of charge states occupied by the protein (6+ to 15+) and the range of CCS in which the protein is observed (12.3 nm2 – 26 nm2) are both large. In addition, many of the charge states observed have higher charge than the theoretical maximal charge on spherical globular protein, as determined by the De La Mora relationship (ZR = 0.0778m; for the EncFtnsH monomer ZR = 8.9) Fernandez. As described by Beveridge et al., all these factors are indicative of a disordered protein.</text>
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+        <infon key="updated_at">2023-09-19T18:06:56Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="33100" length="13"/>
+        <text>Mass spectrum</text>
+      </annotation>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>apo</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:58Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="33121" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="2099">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
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+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig7-figsupp2.jpg</infon>
+      <infon key="id">fig7s2</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>35146</offset>
+      <text>The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles). The mass of the ejected-monomer is consistent with apo- EncFtnsH (13.2 kDa), suggesting unfolding of the monomer (and loss of Fe) occurs during ejection from the complex. This observation of asymmetric charge partitioning of the sub-complexes with respect to the mass of the complex is consistent with the 'typical' pathway of dissociation of protein assemblies by CID, as described by. In addition, a third, lower abundance, charge state distribution is observed which overlaps the EncFtn ejected monomer charge state distribution; this region of the spectrum is highlighted in (B). This distribution is consistent with an ejected EncFtnsH dimer (orange circles). Interestingly, closer analysis of the individual charge state of this dimeric CID product shows that this sub-complex exists in three forms – displaying mass consistent with an EncFtnsH dimer binding 0, 1, and 2 Fe ions. This is highlighted in (C), where the 15+ charge state of the EncFtnsH dimer is shown; 3 peaks are observed with m/z 1760.5, 1763.8, and 1767.0 Th – the lowest peak corresponds to neutral masses of 26392.5 Da [predicted EncFtnsH dimer, (C572H884N172O185S2)2; 26388.6 Da]. The two further peaks have a delta-mass of ~+50 Da, consistent with Fe binding. We interpret these observations as partial ‘atypical’ CID fragmentation of the decameric complex – i.e. fragmentation of the initial complex with retention of subunit and ligand interactions. A schematic summary of these results is displayed in (D). We postulate the high stability of this iron-bound dimer sub-complex is due to the metal coordination at the dimer interface, increasing the strength of the dimer interface. Taken together, these observations support our findings that the topology of the decameric EncFtnsH assembly is arranged as a pentamer of dimers, with two Fe ions at each dimer interface.</text>
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+        <infon key="identifier">DUMMY:</infon>
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+http://dx.doi.org/10.7554/eLife.18972.020</text>
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+      <text>Native mass spectrometry and ion mobility analysis of iron loading in EncFtnsH.</text>
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+      <text>All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Native nanoelectrospray ionization (nESI) mass spectrometry of EncFtnsH at varying iron concentrations. A1, nESI spectrum of iron-free EncFtnsH displays a charge state distribution consistent with EncFtnsH monomer (blue circles, 13,194 Da). Addition of 100 µM (A2) and 300 µM (A3) Fe2+ results in the appearance of a second higher molecular weight charge state distribution consistent with a decameric assembly of EncFtnsH (green circles, 132.6 kDa). (B) Ion mobility (IM)-MS of the iron-bound holo-EncFtnsH decamer. Top, Peaks corresponding to the 22+ to 26+ charge states of a homo-decameric assembly of EncFtnsH are observed (132.6 kDa). Top Insert, Analysis of the 24+ charge state of the assembly at m/z 5528.2 Th. The theoretical average m/z of the 24+ charge state with no additional metals bound is marked by a red line (5498.7 Th); the observed m/z of the 24+ charge state indicates that the EncFtnsH assembly binds between 10 (green line, 5521.1 Th) and 15 Fe ions (blue line, 5532.4 Th) per decamer. Bottom, The arrival time distributions (ion mobility data) of all ions in the EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). Bottom right, The arrival time distribution of the 24+ charge state (dashed blue box) has been extracted and plotted. The drift time for this ion is shown (ms), along with the calibrated collision cross section (CCS), Ω (nm2).</text>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="39105" length="1"/>
+        <text>Ω</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig7.jpg</infon>
+      <infon key="id">fig7</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>39130</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.018</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>39177</offset>
+      <text>In order to confirm the assignment of the oligomeric state of EncFtnsH and investigate further the Fe2+-dependent assembly, we used native nano-electrospray ionization (nESI) and ion-mobility mass spectrometry (IM-MS). As described above, by recombinant production of EncFtnsH in minimal media we were able to limit the bioavailability of iron. Native MS analysis of EncFtnsH produced in this way displayed a charge state distribution consistent with an EncFtnsH monomer (blue circles, Figure 7A1) with an average neutral mass of 13,194 Da, in agreement with the predicted mass of the EncFtnsH protein (13,194.53 Da). Under these conditions, no significant higher order assembly was observed and the protein did not have any coordinated metal ions. Titration with Fe2+ directly before native MS analysis resulted in the appearance of a new charge state distribution, consistent with an EncFtnsH decameric assembly (+22 to +26; 132.65 kDa) (Figure 7A2/3). After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa). MS analysis of EncFtnsH after addition of further Fe2+ did not result in iron loading above this stoichiometry. Therefore, the extent of iron binding seen is limited to the FOC and Glu31/34 secondary metal binding site. These data suggest that the decameric assembly of EncFtnsH does not accrue iron in the same manner as classical ferritin, which is able to sequester around 4500 iron ions within its nanocage. Ion mobility analysis of the EncFtnsH decameric assembly, collected with minimal collisional activation, suggested that it consists of a single conformation with a collision cross section (CCS) of 58.2 nm2 (Figure 7B). This observation is in agreement with the calculated CCS of 58.7 nm2derived from our crystal structure of the EncFtnsH decamer. By contrast, IM-MS measurements of the monomeric EncFtnsH at pH 8.0 under the same instrumental conditions revealed that the metal-free protein monomer exists in a wide range of charge states (+6 to +16) and adopts many conformations in the gas phase with collision cross sections ranging from 12 nm2 to 26 nm2 (Figure 7—figure supplement 1). These observations are indicative of an unstructured protein with little secondary or tertiary structure. Thus, IM-MS studies highlight that higher order structure in EncFtnsH is mediated/stabilized by metal binding, an observation that is in agreement with our solution studies. Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure.</text>
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+        <location offset="39239" length="8"/>
+        <text>EncFtnsH</text>
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+        <text>native nano-electrospray ionization</text>
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+        <infon key="identifier">MESH:</infon>
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+        <text>nESI</text>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="39356" length="30"/>
+        <text>ion-mobility mass spectrometry</text>
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+        <text>IM-MS</text>
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+        <text>recombinant production</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>charge state</text>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>monomer</text>
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+        <infon key="identifier">PR:</infon>
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+        <text>EncFtnsH</text>
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+        <infon key="updated_at">2023-09-19T14:35:35Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="39926" length="9"/>
+        <text>Titration</text>
+      </annotation>
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+        <infon key="identifier">CHEBI:</infon>
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+        <text>Fe2+</text>
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+        <text>native MS</text>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="updated_at">2023-09-19T14:26:53Z</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:31Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <text>iron</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <text>Fe</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>decamer</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:18:27Z</infon>
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+        <text>presence of</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <text>irons</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <text>FOC</text>
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+        <infon key="type">bond_interaction</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:13:23Z</infon>
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+        <text>Glu31/34 secondary metal binding site</text>
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+        <infon key="type">oligomeric_state</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="updated_at">2023-09-19T11:46:47Z</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:36:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <location offset="41391" length="7"/>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
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+      <text>We subsequently performed gas phase disassembly of the decameric EncFtnsH using collision-induced dissociation (CID) tandem mass spectrometry. Under the correct CID conditions, protein assemblies can dissociate with retention of subunit and ligand interactions, and thus provide structurally-informative evidence as to the topology of the original assembly; this has been termed ‘atypical’ dissociation. For EncFtnsH, this atypical dissociation pathway was clearly evident; CID of the EncFtnsH decamer resulted in the appearance of a dimeric EncFtnsH subcomplex containing 0, 1, or 2 iron ions (Figure 7—figure supplement 2). In light of the crystal structure, this observation can be rationalized as dissociation of the EncFtnsH decamer by disruption of the non-FOC interface with at least partial retention of the FOC interface and the FOC-Fe. Thus, this observation supports our crystallographic assignment of the overall topology of the EncFtnsH assembly as a pentameric assembly of dimers with two iron ions located at the FOC dimer interface. In addition, this analysis provides evidence that the overall architecture of the complex is consistent in the crystal, solution and gas phases.</text>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="42371" length="24"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <location offset="42796" length="8"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:31Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:29:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="42975" length="8"/>
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+      </annotation>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="42984" length="7"/>
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+      <annotation id="1000">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:38:24Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="43013" length="17"/>
+        <text>non-FOC interface</text>
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+        <infon key="score">0.99905515</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:38:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="43070" length="13"/>
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+      <annotation id="1923">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <location offset="43092" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="2102">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:38:54Z</infon>
+        <location offset="43096" length="2"/>
+        <text>Fe</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="43195" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:19:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="43218" length="10"/>
+        <text>pentameric</text>
+      </annotation>
+      <annotation id="1004">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:37:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="43241" length="6"/>
+        <text>dimers</text>
+      </annotation>
+      <annotation id="1005">
+        <infon key="score">0.99917513</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:31Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="43257" length="4"/>
+        <text>iron</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:39:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="43282" length="19"/>
+        <text>FOC dimer interface</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:39:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="43414" length="7"/>
+        <text>crystal</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>43454</offset>
+      <text>Ferroxidase activity</text>
+      <annotation id="1008">
+        <infon key="score">0.9968352</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:30Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="43454" length="11"/>
+        <text>Ferroxidase</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig8-figsupp1.jpg</infon>
+      <infon key="id">fig8s1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>43475</offset>
+      <text>TEM visualization of iron-loaded bacterial nanocompartments and ferritin.</text>
+      <annotation id="1009">
+        <infon key="score">0.99874395</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:52:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="43475" length="3"/>
+        <text>TEM</text>
+      </annotation>
+      <annotation id="2103">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:39:34Z</infon>
+        <location offset="43496" length="11"/>
+        <text>iron-loaded</text>
+      </annotation>
+      <annotation id="1010">
+        <infon key="score">0.9987717</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:45Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="43508" length="9"/>
+        <text>bacterial</text>
+      </annotation>
+      <annotation id="1011">
+        <infon key="score">0.99808806</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:47:29Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="43518" length="16"/>
+        <text>nanocompartments</text>
+      </annotation>
+      <annotation id="1012">
+        <infon key="score">0.65248877</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="43539" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig8-figsupp1.jpg</infon>
+      <infon key="id">fig8s1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>43549</offset>
+      <text>Decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin, at 8.5 µM, were mixed with 147 µM, 1 mM, 1 mM and 215 µM acidic Fe(NH4)2(SO4)2, respectively. Protein mixtures were incubated at room temperature for 1 hr prior to TEM analysis with or without uranyl acetate stain. (A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.</text>
+      <annotation id="1013">
+        <infon key="score">0.9983839</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="43549" length="9"/>
+        <text>Decameric</text>
+      </annotation>
+      <annotation id="1014">
+        <infon key="score">0.99588805</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="43559" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1015">
+        <infon key="score">0.99626344</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="43569" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1016">
+        <infon key="score">0.721113</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:07Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="43581" length="10"/>
+        <text>EncFtn-Enc</text>
+      </annotation>
+      <annotation id="1017">
+        <infon key="score">0.98189974</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="43596" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+      <annotation id="1967">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:17:56Z</infon>
+        <location offset="43673" length="14"/>
+        <text>Fe(NH4)2(SO4)2</text>
+      </annotation>
+      <annotation id="1018">
+        <infon key="score">0.998538</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:52:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="43773" length="3"/>
+        <text>TEM</text>
+      </annotation>
+      <annotation id="1019">
+        <infon key="score">0.92946935</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:40:05Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="43802" length="14"/>
+        <text>uranyl acetate</text>
+      </annotation>
+      <annotation id="1020">
+        <infon key="score">0.9812164</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="43840" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1021">
+        <infon key="score">0.99050516</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="43850" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1022">
+        <infon key="score">0.8156171</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:07Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="43862" length="10"/>
+        <text>EncFtn-Enc</text>
+      </annotation>
+      <annotation id="1023">
+        <infon key="score">0.9878298</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="43874" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+      <annotation id="1024">
+        <infon key="score">0.9927024</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:39:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="43886" length="11"/>
+        <text>loaded with</text>
+      </annotation>
+      <annotation id="2107">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:40:41Z</infon>
+        <location offset="43898" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1025">
+        <infon key="score">0.719513</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:17:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="44022" length="7"/>
+        <text>Stained</text>
+      </annotation>
+      <annotation id="1026">
+        <infon key="score">0.99073374</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="44030" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1027">
+        <infon key="score">0.9944073</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="44040" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1028">
+        <infon key="score">0.74889445</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:08Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="44052" length="10"/>
+        <text>EncFtn-Enc</text>
+      </annotation>
+      <annotation id="1029">
+        <infon key="score">0.99222344</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44064" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+      <annotation id="1030">
+        <infon key="score">0.99380374</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:39:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44076" length="11"/>
+        <text>loaded with</text>
+      </annotation>
+      <annotation id="2106">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:40:24Z</infon>
+        <location offset="44088" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig8-figsupp1.jpg</infon>
+      <infon key="id">fig8s1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>44180</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.022</text>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig8.jpg</infon>
+      <infon key="id">fig8</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>44227</offset>
+      <text>Spectroscopic evidence for the ferroxidase activity and comparison of iron loading capacity of apoferritin, EncFtnsH, encapsulin, and EncFtn-Enc.</text>
+      <annotation id="1867">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:30Z</infon>
+        <location offset="44258" length="11"/>
+        <text>ferroxidase</text>
+      </annotation>
+      <annotation id="1031">
+        <infon key="score">0.99867576</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="44297" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1032">
+        <infon key="score">0.75865823</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44322" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+      <annotation id="1033">
+        <infon key="score">0.9943082</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="44335" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1034">
+        <infon key="score">0.9900851</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="44345" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1035">
+        <infon key="score">0.9966576</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:08Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="44361" length="10"/>
+        <text>EncFtn-Enc</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig8.jpg</infon>
+      <infon key="id">fig8</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>44373</offset>
+      <text>(A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer. The background oxidation of iron at 20 and 100 μM in enzyme-free controls are shown for reference. (B) Encapsulin and EncFtn-Enc complexes at 10 μM asymmetric unit concentration were incubated with Fe2+ at 20 and 100 μM and progress curves for iron oxidation at A315 were measured in a UV/visible spectrophotometer. Enzyme free controls for background oxidation of Fe2+ are shown for reference. (C) Histogram of the iron loading capacity per biological assembly of EncFtnsH, encapsulin, EncFtn-Enc and apoferritin. The results shown are for three technical replicates and represent the optimal iron loading by the complexes after three hours when incubated with Fe2+.</text>
+      <annotation id="1036">
+        <infon key="score">0.99563134</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44377" length="11"/>
+        <text>Apoferritin</text>
+      </annotation>
+      <annotation id="1037">
+        <infon key="score">0.99856013</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44396" length="7"/>
+        <text>monomer</text>
+      </annotation>
+      <annotation id="1038">
+        <infon key="score">0.99471325</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="44423" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1039">
+        <infon key="score">0.99821883</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44432" length="7"/>
+        <text>decamer</text>
+      </annotation>
+      <annotation id="1040">
+        <infon key="score">0.9977957</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44457" length="7"/>
+        <text>monomer</text>
+      </annotation>
+      <annotation id="1041">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="44486" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1042">
+        <infon key="score">0.9992593</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="44539" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1043">
+        <infon key="score">0.99907815</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:42:17Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="44577" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1044">
+        <infon key="score">0.4217753</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="44586" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1045">
+        <infon key="score">0.754193</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:42:31Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44595" length="15"/>
+        <text>progress curves</text>
+      </annotation>
+      <annotation id="1046">
+        <infon key="score">0.99904156</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:42:20Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="44631" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1047">
+        <infon key="score">0.9989104</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:42:22Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="44639" length="4"/>
+        <text>Fe3+</text>
+      </annotation>
+      <annotation id="1048">
+        <infon key="score">0.9990995</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="44720" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1049">
+        <infon key="score">0.97308445</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="44795" length="10"/>
+        <text>Encapsulin</text>
+      </annotation>
+      <annotation id="1050">
+        <infon key="score">0.97661716</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:08Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="44810" length="10"/>
+        <text>EncFtn-Enc</text>
+      </annotation>
+      <annotation id="1051">
+        <infon key="score">0.5345021</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:21:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="44875" length="9"/>
+        <text>incubated</text>
+      </annotation>
+      <annotation id="1052">
+        <infon key="score">0.999176</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:42:26Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="44890" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1053">
+        <infon key="score">0.74626833</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:42:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44916" length="15"/>
+        <text>progress curves</text>
+      </annotation>
+      <annotation id="1054">
+        <infon key="score">0.9990785</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="44936" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="2108">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:42:11Z</infon>
+        <location offset="44978" length="28"/>
+        <text>UV/visible spectrophotometer</text>
+      </annotation>
+      <annotation id="1055">
+        <infon key="score">0.9991321</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:42:29Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="45057" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1056">
+        <infon key="score">0.99874604</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="45108" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1057">
+        <infon key="score">0.99128133</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="45157" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1058">
+        <infon key="score">0.98507154</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="45167" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1059">
+        <infon key="score">0.94106287</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:08Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="45179" length="10"/>
+        <text>EncFtn-Enc</text>
+      </annotation>
+      <annotation id="1060">
+        <infon key="score">0.99596703</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45194" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+      <annotation id="1061">
+        <infon key="score">0.99891233</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="45286" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1062">
+        <infon key="score">0.99914086</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:42:37Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="45354" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig8.jpg</infon>
+      <infon key="id">fig8</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>45367</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.021</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>45414</offset>
+      <text>In light of the identification of an iron-loaded FOC in the crystal structure of EncFtn and our native mass spectrometry data, we performed ferroxidase and peroxidase assays to demonstrate the catalytic activity of this protein. In addition, we also assayed equine apoferritin, an example of a classical ferritin enzyme, as a positive control. Unlike the Dps family of ferritin-like proteins, EncFtn showed no peroxidase activity when assayed with the substrate ortho-phenylenediamine. The ferroxidase activity of EncFtnsH was measured by recording the progress curve of Fe2+ oxidation to Fe3+ at 315 nm after addition of 20 and 100 µM Fe2+ (2 and 10 times molar ratio Fe2+/FOC). In both experiments the rate of oxidation was faster than background oxidation of Fe2+ by molecular oxygen, and was highest for 100 µM Fe2+ (Figure 8A). These data show that recombinant EncFtnsH acts as an active ferroxidase enzyme. When compared to apoferritin, EncFtnsH oxidized Fe2+ at a slower rate and the reaction did not run to completion over the 1800 s of the experiment. Addition of higher quantities of iron resulted in the formation of a yellow/red precipitate at the end of the reaction. We also performed these assays on purified recombinant encapsulin; which, when assayed alone, did not display ferroxidase activity above background Fe2+ oxidation (Figure 8B). In contrast, complexes of the full EncFtn encapsulin nanocompartment (i.e. the EncFtn-Enc protein complex) displayed ferroxidase activity comparable to apoferritin without the formation of precipitates (Figure 8B).</text>
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:39:36Z</infon>
+        <location offset="45451" length="11"/>
+        <text>iron-loaded</text>
+      </annotation>
+      <annotation id="1063">
+        <infon key="score">0.91124094</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="45463" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1064">
+        <infon key="score">0.99816555</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:29:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45474" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="1065">
+        <infon key="score">0.99925166</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:41Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="45495" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1066">
+        <infon key="score">0.9984116</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:55:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="45510" length="24"/>
+        <text>native mass spectrometry</text>
+      </annotation>
+      <annotation id="1067">
+        <infon key="score">0.99548477</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:43:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="45554" length="33"/>
+        <text>ferroxidase and peroxidase assays</text>
+      </annotation>
+      <annotation id="1068">
+        <infon key="score">0.77726114</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:43:31Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45672" length="6"/>
+        <text>equine</text>
+      </annotation>
+      <annotation id="1069">
+        <infon key="score">0.9988858</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45679" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+      <annotation id="1897">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
+        <location offset="45708" length="9"/>
+        <text>classical</text>
+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="45718" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+      <annotation id="1071">
+        <infon key="score">0.9981369</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:44:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="45769" length="10"/>
+        <text>Dps family</text>
+      </annotation>
+      <annotation id="1072">
+        <infon key="score">0.9988036</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:44:41Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="45783" length="22"/>
+        <text>ferritin-like proteins</text>
+      </annotation>
+      <annotation id="1073">
+        <infon key="score">0.99911267</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:42Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="45807" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1074">
+        <infon key="score">0.99903744</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:43:45Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="45876" length="22"/>
+        <text>ortho-phenylenediamine</text>
+      </annotation>
+      <annotation id="1868">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:30Z</infon>
+        <location offset="45904" length="11"/>
+        <text>ferroxidase</text>
+      </annotation>
+      <annotation id="1075">
+        <infon key="score">0.9989209</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="45928" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1076">
+        <infon key="score">0.8770637</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:44:56Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="45967" length="14"/>
+        <text>progress curve</text>
+      </annotation>
+      <annotation id="1077">
+        <infon key="score">0.99909</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:43:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="45985" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1078">
+        <infon key="score">0.99896085</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:43:51Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="46003" length="4"/>
+        <text>Fe3+</text>
+      </annotation>
+      <annotation id="1079">
+        <infon key="score">0.99907815</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:43:58Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="46050" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1080">
+        <infon key="score">0.9985999</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:43:56Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="46083" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1081">
+        <infon key="score">0.7469009</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="46088" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1082">
+        <infon key="score">0.9989803</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:43:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="46176" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1884">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:35:28Z</infon>
+        <location offset="46194" length="6"/>
+        <text>oxygen</text>
+      </annotation>
+      <annotation id="1083">
+        <infon key="score">0.99907494</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:44:03Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="46229" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1084">
+        <infon key="score">0.99886835</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="46280" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1085">
+        <infon key="score">0.9981989</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:45:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="46300" length="6"/>
+        <text>active</text>
+      </annotation>
+      <annotation id="1086">
+        <infon key="score">0.9988611</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:30Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="46307" length="11"/>
+        <text>ferroxidase</text>
+      </annotation>
+      <annotation id="2162">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:25Z</infon>
+        <location offset="46344" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+      <annotation id="1087">
+        <infon key="score">0.99920136</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="46357" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1088">
+        <infon key="score">0.99899256</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:44:06Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="46375" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1089">
+        <infon key="score">0.998909</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="46508" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1090">
+        <infon key="score">0.99877864</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="46650" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1869">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:30Z</infon>
+        <location offset="46705" length="11"/>
+        <text>ferroxidase</text>
+      </annotation>
+      <annotation id="1091">
+        <infon key="score">0.99899435</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:44:09Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="46743" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1092">
+        <infon key="score">0.9947719</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:44:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="46801" length="4"/>
+        <text>full</text>
+      </annotation>
+      <annotation id="1093">
+        <infon key="score">0.47335646</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:42Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="46806" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1094">
+        <infon key="score">0.79352003</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="46813" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1095">
+        <infon key="score">0.5890131</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:57:02Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="46824" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+      <annotation id="1096">
+        <infon key="score">0.9988458</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:08Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="46850" length="10"/>
+        <text>EncFtn-Enc</text>
+      </annotation>
+      <annotation id="1870">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:30Z</infon>
+        <location offset="46888" length="11"/>
+        <text>ferroxidase</text>
+      </annotation>
+      <annotation id="1097">
+        <infon key="score">0.98846835</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="46923" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>46994</offset>
+      <text>We attributed the precipitates observed in the EncFtnsH ferroxidase assay to the production of insoluble Fe3+ complexes, which led us to propose that EncFtn does not directly store Fe3+ in a mineral form. This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.</text>
+      <annotation id="2109">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:47:33Z</infon>
+        <location offset="47041" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="2110">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:47:45Z</infon>
+        <location offset="47050" length="17"/>
+        <text>ferroxidase assay</text>
+      </annotation>
+      <annotation id="1098">
+        <infon key="score">0.997488</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:45:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="47099" length="4"/>
+        <text>Fe3+</text>
+      </annotation>
+      <annotation id="1099">
+        <infon key="score">0.6162079</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:42Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="47144" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1100">
+        <infon key="score">0.9987997</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:45:38Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="47175" length="4"/>
+        <text>Fe3+</text>
+      </annotation>
+      <annotation id="1101">
+        <infon key="score">0.9978562</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:35:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="47228" length="9"/>
+        <text>native MS</text>
+      </annotation>
+      <annotation id="1102">
+        <infon key="score">0.9989479</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="47273" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1103">
+        <infon key="score">0.99900645</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="47295" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1104">
+        <infon key="score">0.9984871</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="47309" length="9"/>
+        <text>decameric</text>
+      </annotation>
+      <annotation id="1105">
+        <infon key="score">0.9560211</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:42Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="47319" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1106">
+        <infon key="score">0.9984837</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:08:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="47335" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="1107">
+        <infon key="score">0.99901986</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:45:49Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="47382" length="19"/>
+        <text>iron-storage cavity</text>
+      </annotation>
+      <annotation id="1898">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
+        <location offset="47420" length="9"/>
+        <text>classical</text>
+      </annotation>
+      <annotation id="1108">
+        <infon key="score">0.99909246</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="47430" length="9"/>
+        <text>ferritins</text>
+      </annotation>
+      <annotation id="1109">
+        <infon key="score">0.9692733</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:45:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="47444" length="19"/>
+        <text>Dps family proteins</text>
+      </annotation>
+      <annotation id="1110">
+        <infon key="score">0.9987999</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:45:41Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="47501" length="4"/>
+        <text>Fe3+</text>
+      </annotation>
+      <annotation id="1111">
+        <infon key="score">0.98059124</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:57:06Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="47519" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+      <annotation id="1112">
+        <infon key="score">0.95718</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:08:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="47535" length="10"/>
+        <text>structures</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>47549</offset>
+      <text>To analyze the products of these reactions and determine whether the EncFtn and encapsulin were able to store iron in a mineral form, we performed TEM on the reaction mixtures from the ferroxidase assay. The EncFtnsH reaction mixture showed the formation of large, irregular electron-dense precipitates (Figure 8—figure supplement 1A). A similar distribution of particles was observed after addition of Fe2+ to the encapsulin protein (Figure 8—figure supplement 1B). In contrast, addition of Fe2+ to the EncFtn-Enc nanocompartment resulted in small, highly regular, electron dense particles of approximately 5 nm in diameter (Figure 8—figure supplement 1C); we interpret these observations as controlled mineralization of iron within the nanocompartment. Addition of Fe2+ to apoferritin resulted in a mixture of large particles and small (~2 nm) particles consistent with partial mineralization by the ferritin and some background oxidation of the iron (Figure 8—figure supplement 1D). Negative stain TEM of these samples revealed that upon addition of iron, the EncFtnsH protein showed significant aggregation (Figure 8—figure supplement 1F); while the encapsulin, EncFtn-Enc system, and apoferritin are present as distinct nanocompartments without significant protein aggregation (Figure 8—figure supplement 1G–I).</text>
+      <annotation id="1113">
+        <infon key="score">0.9982615</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:42Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="47618" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1114">
+        <infon key="score">0.99852943</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="47629" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1115">
+        <infon key="score">0.9990024</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="47659" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1116">
+        <infon key="score">0.99870753</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:52:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="47696" length="3"/>
+        <text>TEM</text>
+      </annotation>
+      <annotation id="1117">
+        <infon key="score">0.99861383</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:46:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="47734" length="17"/>
+        <text>ferroxidase assay</text>
+      </annotation>
+      <annotation id="1118">
+        <infon key="score">0.9622654</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="47757" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1119">
+        <infon key="score">0.99917233</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:46:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="47952" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1120">
+        <infon key="score">0.9985311</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="47964" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1121">
+        <infon key="score">0.9991504</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:46:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="48041" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1122">
+        <infon key="score">0.99607974</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:08Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="48053" length="10"/>
+        <text>EncFtn-Enc</text>
+      </annotation>
+      <annotation id="1123">
+        <infon key="score">0.9917857</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:57:10Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="48064" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+      <annotation id="1124">
+        <infon key="score">0.9989164</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="48271" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1125">
+        <infon key="score">0.9860162</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:57:14Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="48287" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+      <annotation id="1126">
+        <infon key="score">0.99915993</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:46:29Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="48316" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1127">
+        <infon key="score">0.9991714</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="48324" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+      <annotation id="1128">
+        <infon key="score">0.99494594</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="48451" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+      <annotation id="1129">
+        <infon key="score">0.99889404</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="48497" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1130">
+        <infon key="score">0.99875206</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:59:52Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="48535" length="18"/>
+        <text>Negative stain TEM</text>
+      </annotation>
+      <annotation id="1131">
+        <infon key="score">0.9991665</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="48602" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1132">
+        <infon key="score">0.9977938</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="48612" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1133">
+        <infon key="score">0.9987331</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="48703" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1134">
+        <infon key="score">0.99697083</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:08Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="48715" length="10"/>
+        <text>EncFtn-Enc</text>
+      </annotation>
+      <annotation id="1135">
+        <infon key="score">0.999108</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="48738" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+      <annotation id="1136">
+        <infon key="score">0.97162443</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:47:29Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="48774" length="16"/>
+        <text>nanocompartments</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>48882</offset>
+      <text>Iron storage in encapsulin nanocompartments</text>
+      <annotation id="1137">
+        <infon key="score">0.99887663</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="48882" length="4"/>
+        <text>Iron</text>
+      </annotation>
+      <annotation id="1138">
+        <infon key="score">0.9917041</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="48898" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1139">
+        <infon key="score">0.99208003</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:47:29Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="48909" length="16"/>
+        <text>nanocompartments</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>48926</offset>
+      <text>The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage. To investigate this further, we added Fe2+ at various concentrations to samples of apo-ferritin, EncFtn, isolated encapsulin, and the EncFtn-Enc protein complex, and subjected these samples to a ferrozine assay to quantify the amount of iron associated with the proteins after three hours of incubation. The maximum iron loading capacity of these systems was calculated as the quantity of iron per biological assembly (Figure 8C). In this assay, the EncFtnsH decamer binds a maximum of around 48 iron ions before excess iron induces protein precipitation. The encapsulin shell protein can sequester about 2200 iron ions before significant protein loss occurs, and the reconstituted EncFtn-Enc nanocompartment sequestered about 4150 iron ions. This latter result is significantly more than the apoferritin used in our assay, which sequesters approximately 570 iron ions in this assay (Figure 8C, Table 5).</text>
+      <annotation id="1140">
+        <infon key="score">0.99855113</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:47:07Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="48945" length="17"/>
+        <text>ferroxidase assay</text>
+      </annotation>
+      <annotation id="1141">
+        <infon key="score">0.9556643</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:46:51Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="48967" length="11"/>
+        <text>micrographs</text>
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+      <annotation id="1899">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
+        <location offset="49066" length="9"/>
+        <text>classical</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="49076" length="9"/>
+        <text>ferritins</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:42Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="49108" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1144">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="49119" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1145">
+        <infon key="score">0.9961708</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:42Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="49149" length="6"/>
+        <text>EncFtn</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:30Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="49168" length="11"/>
+        <text>ferroxidase</text>
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+      <annotation id="1147">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="49188" length="10"/>
+        <text>encapsulin</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="49199" length="5"/>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="49247" length="4"/>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:25:08Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="49318" length="4"/>
+        <text>Fe2+</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:25:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="49363" length="3"/>
+        <text>apo</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="49367" length="8"/>
+        <text>ferritin</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:42Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="49377" length="6"/>
+        <text>EncFtn</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="49394" length="10"/>
+        <text>encapsulin</text>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:08Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="49414" length="10"/>
+        <text>EncFtn-Enc</text>
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+      <annotation id="1156">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:21:30Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="49475" length="15"/>
+        <text>ferrozine assay</text>
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+        <infon key="score">0.9990097</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="49517" length="4"/>
+        <text>iron</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="49596" length="4"/>
+        <text>iron</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="49669" length="4"/>
+        <text>iron</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="49730" length="8"/>
+        <text>EncFtnsH</text>
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+      <annotation id="1161">
+        <infon key="score">0.9977925</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="49739" length="7"/>
+        <text>decamer</text>
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+        <infon key="score">0.9991054</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="49776" length="4"/>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="49800" length="4"/>
+        <text>iron</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="49840" length="10"/>
+        <text>encapsulin</text>
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+      <annotation id="1165">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="49851" length="5"/>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="49890" length="4"/>
+        <text>iron</text>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:08Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="49962" length="10"/>
+        <text>EncFtn-Enc</text>
+      </annotation>
+      <annotation id="2186">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:03:16Z</infon>
+        <location offset="49973" length="15"/>
+        <text>nanocompartment</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="50012" length="4"/>
+        <text>iron</text>
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+      <annotation id="1169">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="50073" length="11"/>
+        <text>apoferritin</text>
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+      <annotation id="1170">
+        <infon key="score">0.99898714</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="50139" length="4"/>
+        <text>iron</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>50186</offset>
+      <text>Consideration of the functional oligomeric states of these proteins, where EncFtn is a decamer and encapsulin forms an icosahedral cage, and estimation of the iron loading capacity of these complexes gives insight into the role of the two proteins in iron storage and mineralization. EncFtn decamers bind up to 48 iron ions (Figure 8C), which is significantly higher than the stoichiometry of fifteen metal ions visible in the FOC and E31/34-site of the crystal structure of the EncFtnsH decamer and our MS analysis. The discrepancy between these solution measurements and our MS analysis may indicate that there are additional metal-binding sites on the interior channel and exterior faces of the protein; this is consistent with our identification of a number of weak metal-binding sites at the surface of the protein in the crystal structure (Figure 5D). These observations are consistent with hydrated Fe2+ ions being channeled to the active site from the E31/34-site and the subsequent exit of Fe3+ products on the outer surface, as is seen in other ferritin family proteins. While the isolated encapsulin shell does not display any ferroxidase activity, it binds around 2200 iron ions in our assay (Table 5). This implies that the shell can bind a significant amount of iron on its outer and inner surfaces. While the maximum reported loading capacity of classical ferritins is approximately 4500 iron ions, in our assay system we were only able to load apoferritin with around 570 iron ions. However, the recombinant EncFtn-Enc nanocompartment was able to bind over 4100 iron ions in the same time period, over seven times the amount seen for the apoferritin. We note we do not reach the experimental maximum iron loading for apoferritin and therefore the total iron-loading capacity of our system may be significantly higher than in this experimental system.</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:42Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="50261" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1172">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="50273" length="7"/>
+        <text>decamer</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="50285" length="10"/>
+        <text>encapsulin</text>
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+      <annotation id="1174">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:36:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="50305" length="11"/>
+        <text>icosahedral</text>
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+      <annotation id="1175">
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:01:23Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="50317" length="4"/>
+        <text>cage</text>
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+      <annotation id="1176">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="50345" length="4"/>
+        <text>iron</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="50437" length="4"/>
+        <text>iron</text>
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+      <annotation id="1178">
+        <infon key="score">0.9909506</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:42Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="50470" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1179">
+        <infon key="score">0.9985505</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="50477" length="8"/>
+        <text>decamers</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="50500" length="4"/>
+        <text>iron</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="50613" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1182">
+        <infon key="score">0.998394</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:17:02Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="50621" length="11"/>
+        <text>E31/34-site</text>
+      </annotation>
+      <annotation id="1183">
+        <infon key="score">0.99747866</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:29:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="50640" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
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+        <infon key="score">0.97772205</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="50665" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1185">
+        <infon key="score">0.9986551</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="50674" length="7"/>
+        <text>decamer</text>
+      </annotation>
+      <annotation id="1186">
+        <infon key="score">0.9938304</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:48:44Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="50690" length="2"/>
+        <text>MS</text>
+      </annotation>
+      <annotation id="1187">
+        <infon key="score">0.99414957</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:21:34Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="50733" length="21"/>
+        <text>solution measurements</text>
+      </annotation>
+      <annotation id="1188">
+        <infon key="score">0.9964463</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:48:47Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="50763" length="2"/>
+        <text>MS</text>
+      </annotation>
+      <annotation id="1189">
+        <infon key="score">0.9990403</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:48:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="50814" length="19"/>
+        <text>metal-binding sites</text>
+      </annotation>
+      <annotation id="1190">
+        <infon key="score">0.9957569</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:49:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="50850" length="7"/>
+        <text>channel</text>
+      </annotation>
+      <annotation id="1191">
+        <infon key="score">0.92116725</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:13:29Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="50956" length="19"/>
+        <text>metal-binding sites</text>
+      </annotation>
+      <annotation id="1192">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:29:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="51013" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="1193">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:49:16Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="51092" length="4"/>
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+      </annotation>
+      <annotation id="1194">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:13:34Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="51125" length="11"/>
+        <text>active site</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="51146" length="11"/>
+        <text>E31/34-site</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:54:43Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="51185" length="4"/>
+        <text>Fe3+</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="51241" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+      <annotation id="1198">
+        <infon key="score">0.9989153</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="51286" length="10"/>
+        <text>encapsulin</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="51297" length="5"/>
+        <text>shell</text>
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+      <annotation id="1871">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:30Z</infon>
+        <location offset="51324" length="11"/>
+        <text>ferroxidase</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="51367" length="4"/>
+        <text>iron</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="51423" length="5"/>
+        <text>shell</text>
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+      <annotation id="1202">
+        <infon key="score">0.99907243</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="51462" length="4"/>
+        <text>iron</text>
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+      <annotation id="1900">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
+        <location offset="51547" length="9"/>
+        <text>classical</text>
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+      <annotation id="1203">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="51557" length="9"/>
+        <text>ferritins</text>
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+      <annotation id="1204">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="51589" length="4"/>
+        <text>iron</text>
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+      <annotation id="1205">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="51646" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+      <annotation id="1206">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="51674" length="4"/>
+        <text>iron</text>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:08Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="51710" length="10"/>
+        <text>EncFtn-Enc</text>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:57:20Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="51721" length="15"/>
+        <text>nanocompartment</text>
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+      <annotation id="1209">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="51764" length="4"/>
+        <text>iron</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="51840" length="11"/>
+        <text>apoferritin</text>
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+      <annotation id="1211">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="51902" length="4"/>
+        <text>iron</text>
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+      <annotation id="1212">
+        <infon key="score">0.9990716</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="51919" length="11"/>
+        <text>apoferritin</text>
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+      <annotation id="1213">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="51955" length="4"/>
+        <text>iron</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>52055</offset>
+      <text>Taken together, our data show that EncFtn can catalytically oxidize Fe2+ to Fe3+; however, iron binding in EncFtn is limited to the FOC and several surface metal binding sites. In contrast, the encapsulin protein displays no catalytic activity, but has the ability to bind a considerable amount of iron. Finally, the EncFtn-Enc nanocompartment complex retains the catalytic activity of EncFtn, and sequesters iron within the encapsulin shell at a higher level than the isolated components of the system, and at a significantly higher level than the classical ferritins. Furthermore, our recombinant nanocompartments may not have the physiological subunit stoichiometry, and the iron-loading capacity of native nanocompartments is potentially much higher than the level we have observed.</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="52090" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1215">
+        <infon key="score">0.9990474</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:49:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="52123" length="4"/>
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+      <annotation id="1216">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:49:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="52131" length="4"/>
+        <text>Fe3+</text>
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+        <infon key="score">0.9990871</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="52146" length="4"/>
+        <text>iron</text>
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+      <annotation id="1218">
+        <infon key="score">0.9989825</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="52162" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1219">
+        <infon key="score">0.58354443</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="52187" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1220">
+        <infon key="score">0.97698575</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:08:40Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="52211" length="19"/>
+        <text>metal binding sites</text>
+      </annotation>
+      <annotation id="1221">
+        <infon key="score">0.9950051</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="52249" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1222">
+        <infon key="score">0.9990188</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="52353" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1223">
+        <infon key="score">0.9991837</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:08Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="52372" length="10"/>
+        <text>EncFtn-Enc</text>
+      </annotation>
+      <annotation id="1224">
+        <infon key="score">0.86663836</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:57:24Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="52383" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+      <annotation id="1225">
+        <infon key="score">0.9991554</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="52441" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1226">
+        <infon key="score">0.99898523</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="52464" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1227">
+        <infon key="score">0.75144476</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="52480" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1228">
+        <infon key="score">0.9940952</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="52491" length="5"/>
+        <text>shell</text>
+      </annotation>
+      <annotation id="1901">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
+        <location offset="52604" length="9"/>
+        <text>classical</text>
+      </annotation>
+      <annotation id="1229">
+        <infon key="score">0.9992168</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="52614" length="9"/>
+        <text>ferritins</text>
+      </annotation>
+      <annotation id="1230">
+        <infon key="score">0.99845505</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:47:29Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="52654" length="16"/>
+        <text>nanocompartments</text>
+      </annotation>
+      <annotation id="1231">
+        <infon key="score">0.99830496</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="52733" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1232">
+        <infon key="score">0.8595094</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:56Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="52758" length="6"/>
+        <text>native</text>
+      </annotation>
+      <annotation id="1233">
+        <infon key="score">0.9983076</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:47:29Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="52765" length="16"/>
+        <text>nanocompartments</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>52843</offset>
+      <text>Mutagenesis of the EncFtnsHferroxidase center</text>
+      <annotation id="1234">
+        <infon key="score">0.998384</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:12:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="52843" length="11"/>
+        <text>Mutagenesis</text>
+      </annotation>
+      <annotation id="2111">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:50:35Z</infon>
+        <location offset="52862" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="2112">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:50:48Z</infon>
+        <location offset="52870" length="18"/>
+        <text>ferroxidase center</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig9.jpg</infon>
+      <infon key="id">fig9</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>52889</offset>
+      <text>Purification of recombinant R. rubrum EncFtnsH FOC mutants.</text>
+      <annotation id="1930">
+        <infon key="type">species</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:56:17Z</infon>
+        <location offset="52917" length="9"/>
+        <text>R. rubrum</text>
+      </annotation>
+      <annotation id="1957">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <location offset="52927" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1235">
+        <infon key="score">0.66427743</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="52936" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="2119">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <location offset="52940" length="7"/>
+        <text>mutants</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig9.jpg</infon>
+      <infon key="id">fig9</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>52949</offset>
+      <text>Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex 200 size-exclusion chromatography. (A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.</text>
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+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.023</text>
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+      <infon key="type">paragraph</infon>
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+      <text>To investigate the structural and biochemical role played by the metal binding residues in the di-iron FOC of EncFtnsH we produced alanine mutations in each of these residues: Glu32, Glu62, and His65. These EncFtnsH mutants were produced in E. coli cells grown in MM, both in the absence and presence of additional iron. The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9). For all of the mutants studied, no change in oligomerization state was apparent upon addition of Fe2+ in vitro.</text>
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+        <location offset="53880" length="8"/>
+        <text>EncFtnsH</text>
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+        <infon key="identifier">MESH:</infon>
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+        <text>SEC</text>
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+        <location offset="54187" length="9"/>
+        <text>monomeric</text>
+      </annotation>
+      <annotation id="1279">
+        <infon key="score">0.99932194</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="54205" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="2121">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <location offset="54241" length="7"/>
+        <text>mutants</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:54:56Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="54323" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
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+    <passage>
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+      <infon key="id">fig10</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>54339</offset>
+      <text>Native mass spectrometry of EncFtnsH mutants.</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:55:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="54339" length="24"/>
+        <text>Native mass spectrometry</text>
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+      <annotation id="1282">
+        <infon key="score">0.8879092</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="54367" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1283">
+        <infon key="score">0.6093207</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="54376" length="7"/>
+        <text>mutants</text>
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+      <infon key="id">fig10</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>54385</offset>
+      <text>All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles). (C) E62A EncFtnsH displays charge states consistent with a decamer (green circles). (D) H65A EncFtnsH displays charge states consistent with both monomer (blue circles) and dimer (purple circles).</text>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>spectra</text>
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+        <text>acetate</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="54488" length="9"/>
+        <text>Wild-type</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="54498" length="8"/>
+        <text>EncFtnsH</text>
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+      <annotation id="1288">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="54514" length="10"/>
+        <text>absence of</text>
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+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="54525" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="2133">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:56:17Z</infon>
+        <location offset="54541" length="25"/>
+        <text>charge state distribution</text>
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+      <annotation id="1290">
+        <infon key="score">0.9988834</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="54585" length="7"/>
+        <text>monomer</text>
+      </annotation>
+      <annotation id="1291">
+        <infon key="score">0.99896574</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="54618" length="4"/>
+        <text>E32A</text>
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+      <annotation id="1958">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <location offset="54623" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="2067">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:25:50Z</infon>
+        <location offset="54643" length="13"/>
+        <text>charge states</text>
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+      <annotation id="1292">
+        <infon key="score">0.9988796</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="54675" length="7"/>
+        <text>decamer</text>
+      </annotation>
+      <annotation id="1293">
+        <infon key="score">0.9989023</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="54737" length="7"/>
+        <text>monomer</text>
+      </annotation>
+      <annotation id="1294">
+        <infon key="score">0.99907815</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="54748" length="4"/>
+        <text>E32A</text>
+      </annotation>
+      <annotation id="1295">
+        <infon key="score">0.99743974</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:51:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="54753" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="1296">
+        <infon key="score">0.9990503</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="54797" length="4"/>
+        <text>E62A</text>
+      </annotation>
+      <annotation id="1297">
+        <infon key="score">0.48325944</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="54802" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="2068">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:25:50Z</infon>
+        <location offset="54820" length="13"/>
+        <text>charge states</text>
+      </annotation>
+      <annotation id="1298">
+        <infon key="score">0.9988966</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="54852" length="7"/>
+        <text>decamer</text>
+      </annotation>
+      <annotation id="1299">
+        <infon key="score">0.9989421</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="54881" length="4"/>
+        <text>H65A</text>
+      </annotation>
+      <annotation id="1300">
+        <infon key="score">0.5852406</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="54886" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="2069">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:25:50Z</infon>
+        <location offset="54904" length="13"/>
+        <text>charge states</text>
+      </annotation>
+      <annotation id="1301">
+        <infon key="score">0.9989104</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="54939" length="7"/>
+        <text>monomer</text>
+      </annotation>
+      <annotation id="1302">
+        <infon key="score">0.99890864</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:19:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="54966" length="5"/>
+        <text>dimer</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig10.jpg</infon>
+      <infon key="id">fig10</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>54994</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.024</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>55041</offset>
+      <text>In addition to SEC studies, native mass spectrometry of the apo-EncFtnsH mutants was performed and compared with the wild-type apo-EncFtnsH protein (Figure 10). As described above, the apo-EncFtnsH has a charge state distribution consistent with an unstructured monomer, and decamer formation is only initiated upon addition of ferrous iron. Both the E32A mutant and E62A mutant displayed charge state distributions consistent with decamers, even in the absence of Fe2+. This gas-phase observation is consistent with SEC measurements, which indicate both of these variants were also decamers in solution. Thus it seems that these mutations allow the decamer to form in the absence of iron in the FOC. In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:18:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="55056" length="3"/>
+        <text>SEC</text>
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+      <annotation id="1304">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:55:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="55069" length="24"/>
+        <text>native mass spectrometry</text>
+      </annotation>
+      <annotation id="1305">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:25:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55101" length="3"/>
+        <text>apo</text>
+      </annotation>
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+        <infon key="score">0.99814653</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="55105" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="2122">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <location offset="55114" length="7"/>
+        <text>mutants</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55158" length="9"/>
+        <text>wild-type</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:25:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55168" length="3"/>
+        <text>apo</text>
+      </annotation>
+      <annotation id="1309">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="55172" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:25:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55226" length="3"/>
+        <text>apo</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="55230" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="2089">
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:26:53Z</infon>
+        <location offset="55245" length="12"/>
+        <text>charge state</text>
+      </annotation>
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+        <infon key="score">0.99890757</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:22:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55290" length="12"/>
+        <text>unstructured</text>
+      </annotation>
+      <annotation id="1313">
+        <infon key="score">0.9989256</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55303" length="7"/>
+        <text>monomer</text>
+      </annotation>
+      <annotation id="1314">
+        <infon key="score">0.9987863</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55316" length="7"/>
+        <text>decamer</text>
+      </annotation>
+      <annotation id="1315">
+        <infon key="score">0.9796138</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="55377" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1316">
+        <infon key="score">0.9990503</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="55392" length="4"/>
+        <text>E32A</text>
+      </annotation>
+      <annotation id="1317">
+        <infon key="score">0.99865746</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:51:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55397" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="1318">
+        <infon key="score">0.9990827</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="55408" length="4"/>
+        <text>E62A</text>
+      </annotation>
+      <annotation id="1319">
+        <infon key="score">0.99803156</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:51:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55413" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="2090">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:26:53Z</infon>
+        <location offset="55430" length="12"/>
+        <text>charge state</text>
+      </annotation>
+      <annotation id="1320">
+        <infon key="score">0.9987509</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55473" length="8"/>
+        <text>decamers</text>
+      </annotation>
+      <annotation id="1321">
+        <infon key="score">0.9990344</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:22:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55495" length="10"/>
+        <text>absence of</text>
+      </annotation>
+      <annotation id="1322">
+        <infon key="score">0.9990151</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:56:51Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="55506" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1323">
+        <infon key="score">0.998831</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:18:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="55558" length="3"/>
+        <text>SEC</text>
+      </annotation>
+      <annotation id="1324">
+        <infon key="score">0.9987618</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55624" length="8"/>
+        <text>decamers</text>
+      </annotation>
+      <annotation id="1325">
+        <infon key="score">0.9988433</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55691" length="7"/>
+        <text>decamer</text>
+      </annotation>
+      <annotation id="1326">
+        <infon key="score">0.9988737</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:22:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55714" length="10"/>
+        <text>absence of</text>
+      </annotation>
+      <annotation id="1327">
+        <infon key="score">0.9989624</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="55725" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1924">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <location offset="55737" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="2134">
+        <infon key="type">residue_name</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:57:17Z</infon>
+        <location offset="55761" length="13"/>
+        <text>glutamic acid</text>
+      </annotation>
+      <annotation id="2123">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <location offset="55775" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="1328">
+        <infon key="score">0.9985696</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:21:40Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="55784" length="2"/>
+        <text>MS</text>
+      </annotation>
+      <annotation id="1329">
+        <infon key="score">0.99906534</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="55803" length="4"/>
+        <text>H65A</text>
+      </annotation>
+      <annotation id="1330">
+        <infon key="score">0.99826705</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:51:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55808" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="1331">
+        <infon key="score">0.999105</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55829" length="9"/>
+        <text>wild-type</text>
+      </annotation>
+      <annotation id="1332">
+        <infon key="score">0.9993519</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:25:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55839" length="3"/>
+        <text>apo</text>
+      </annotation>
+      <annotation id="1333">
+        <infon key="score">0.9991072</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="55843" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1334">
+        <infon key="score">0.998919</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55872" length="7"/>
+        <text>monomer</text>
+      </annotation>
+      <annotation id="1335">
+        <infon key="score">0.99894994</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:19:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="55917" length="7"/>
+        <text>dimeric</text>
+      </annotation>
+      <annotation id="1336">
+        <infon key="score">0.9987979</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="55925" length="4"/>
+        <text>H65A</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>55950</offset>
+      <text>We propose that the observed differences in the oligomerization state of the E32A and E62A mutants compared to wild-type are due to the changes in the electrostatic environment within the FOC. At neutral pH the glutamic acid residues are negatively charged, while the histidine residues are predominantly in their uncharged state. In the wild-type (WT) EncFtnsH this leads to electrostatic repulsion between subunits in the absence of iron. Coordination of Fe2+ in this site stabilizes the dimer and reconstitutes the active FOC. The geometric arrangement of Glu32 and Glu62 in the FOC explains their behavior in solution and the gas phase, where they both favor the formation of decamers due to the loss of a repulsive negative charge. The FOC in the H65A mutant is destabilized through the loss of this metal coordinating residue and potential positive charge carrier, thus favoring the monomer in solution and the gas phase.</text>
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+        <infon key="score">0.99901664</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="56027" length="4"/>
+        <text>E32A</text>
+      </annotation>
+      <annotation id="1338">
+        <infon key="score">0.99910337</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="56036" length="4"/>
+        <text>E62A</text>
+      </annotation>
+      <annotation id="1339">
+        <infon key="score">0.9096816</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="56041" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="1340">
+        <infon key="score">0.9989551</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="56061" length="9"/>
+        <text>wild-type</text>
+      </annotation>
+      <annotation id="1341">
+        <infon key="score">0.91614443</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="56138" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1342">
+        <infon key="score">0.9901097</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:57:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="56146" length="10"/>
+        <text>neutral pH</text>
+      </annotation>
+      <annotation id="2135">
+        <infon key="type">residue_name</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:58:05Z</infon>
+        <location offset="56161" length="13"/>
+        <text>glutamic acid</text>
+      </annotation>
+      <annotation id="1343">
+        <infon key="score">0.997472</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:22:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="56218" length="9"/>
+        <text>histidine</text>
+      </annotation>
+      <annotation id="1344">
+        <infon key="score">0.9990551</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="56288" length="9"/>
+        <text>wild-type</text>
+      </annotation>
+      <annotation id="1345">
+        <infon key="score">0.99926955</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:57:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="56299" length="2"/>
+        <text>WT</text>
+      </annotation>
+      <annotation id="1346">
+        <infon key="score">0.9800802</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="56303" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1887">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:35:44Z</infon>
+        <location offset="56358" length="8"/>
+        <text>subunits</text>
+      </annotation>
+      <annotation id="1347">
+        <infon key="score">0.998933</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:22:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="56374" length="10"/>
+        <text>absence of</text>
+      </annotation>
+      <annotation id="1348">
+        <infon key="score">0.9990646</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="56385" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="2035">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:14Z</infon>
+        <location offset="56391" length="12"/>
+        <text>Coordination</text>
+      </annotation>
+      <annotation id="1349">
+        <infon key="score">0.998568</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:54:58Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="56407" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1350">
+        <infon key="score">0.9987746</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:19:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="56440" length="5"/>
+        <text>dimer</text>
+      </annotation>
+      <annotation id="1351">
+        <infon key="score">0.9989641</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:58:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="56468" length="6"/>
+        <text>active</text>
+      </annotation>
+      <annotation id="1352">
+        <infon key="score">0.57369035</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="56475" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1353">
+        <infon key="score">0.99948394</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="56509" length="5"/>
+        <text>Glu32</text>
+      </annotation>
+      <annotation id="1354">
+        <infon key="score">0.9994797</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="56519" length="5"/>
+        <text>Glu62</text>
+      </annotation>
+      <annotation id="1355">
+        <infon key="score">0.90486306</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="56532" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1356">
+        <infon key="score">0.9986803</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="56630" length="8"/>
+        <text>decamers</text>
+      </annotation>
+      <annotation id="1357">
+        <infon key="score">0.9472394</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="56691" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1358">
+        <infon key="score">0.999086</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="56702" length="4"/>
+        <text>H65A</text>
+      </annotation>
+      <annotation id="1359">
+        <infon key="score">0.9983797</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:51:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="56707" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="2094">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:30:46Z</infon>
+        <location offset="56742" length="7"/>
+        <text>loss of</text>
+      </annotation>
+      <annotation id="1360">
+        <infon key="score">0.9975012</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:13:39Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="56755" length="26"/>
+        <text>metal coordinating residue</text>
+      </annotation>
+      <annotation id="1361">
+        <infon key="score">0.99875724</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="56839" length="7"/>
+        <text>monomer</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">tbl4.xml</infon>
+      <infon key="id">tbl4</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_caption</infon>
+      <offset>56879</offset>
+      <text>Data collection and refinement statistics. Statistics for the highest-resolution shell are shown in parentheses. Friedel mates were averaged when calculating reflection numbers and statistics.</text>
+      <annotation id="2136">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:58:53Z</infon>
+        <location offset="56879" length="41"/>
+        <text>Data collection and refinement statistics</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">tbl4.xml</infon>
+      <infon key="id">tbl4</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_caption</infon>
+      <offset>57072</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.025</text>
+    </passage>
+    <passage>
+      <infon key="file">tbl4.xml</infon>
+      <infon key="id">tbl4</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table</infon>
+      <infon key="xml">&lt;?xml version="1.0" encoding="UTF-8"?&gt;
+&lt;table frame="hsides" rules="groups"&gt;&lt;thead&gt;&lt;tr&gt;&lt;th rowspan="1" colspan="1"/&gt;&lt;th rowspan="1" colspan="1"&gt;&lt;styled-content style="color: #000000;"&gt;WT&lt;/styled-content&gt;&lt;/th&gt;&lt;th rowspan="1" colspan="1"&gt;&lt;styled-content style="color: #000000;"&gt;E32A&lt;/styled-content&gt;&lt;/th&gt;&lt;th rowspan="1" colspan="1"&gt;&lt;styled-content style="color: #000000;"&gt;E62A&lt;/styled-content&gt;&lt;/th&gt;&lt;th rowspan="1" colspan="1"&gt;&lt;styled-content style="color: #000000;"&gt;H65A&lt;/styled-content&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Data collection&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Wavelength (Å)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1.74&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1.73&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1.73&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1.74&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Resolution range (Å)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;49.63 - 2.06 &lt;break/&gt;(2.10 - 2.06)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;48.84 - 2.59 &lt;break/&gt;(2.683 - 2.59)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;48.87 - 2.21 &lt;break/&gt;(2.29 - 2.21)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;48.86 - 2.97 &lt;break/&gt;(3.08 - 2.97)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Space group&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;italic&gt;P&lt;/italic&gt; 1 2&lt;sub&gt;1&lt;/sub&gt; 1&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;italic&gt;P&lt;/italic&gt; 1 2&lt;sub&gt;1&lt;/sub&gt; 1&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;italic&gt;P&lt;/italic&gt; 1 2&lt;sub&gt;1&lt;/sub&gt; 1&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;&lt;italic&gt;P&lt;/italic&gt; 1 2&lt;sub&gt;1&lt;/sub&gt; 1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Unit cell (Å) &lt;italic&gt;a&lt;/italic&gt;
+&lt;break/&gt; b &lt;break/&gt;
+&lt;italic&gt; c&lt;/italic&gt;
+&lt;break/&gt; β (°)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;98.18 &lt;break/&gt;120.53 &lt;break/&gt;140.30 &lt;break/&gt;95.36&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;97.78 &lt;break/&gt;120.28 &lt;break/&gt;140.53 &lt;break/&gt;95.41&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;98.09 &lt;break/&gt;120.23 &lt;break/&gt;140.36 &lt;break/&gt;95.50&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;98.03 &lt;break/&gt;120.29 &lt;break/&gt;140.43 &lt;break/&gt;95.39&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Total reflections&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1,264,922 &lt;break/&gt;(41,360)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;405,488 &lt;break/&gt;(36,186)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1,069,345 &lt;break/&gt;(95,716)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;323,853 &lt;break/&gt;(32,120)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Unique reflections&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;197,873 &lt;break/&gt;(8,766)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;100,067 &lt;break/&gt;(9,735)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;162,379 &lt;break/&gt;(15,817)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;66,658 &lt;break/&gt;(6,553)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Multiplicity&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;6.4 (4.7)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;4.1 (3.7)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;6.6 (6.1)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;4.9 (4.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Anomalous multiplicity&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;3.2 (2.6)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;N/A&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;N/A&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;N/A&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Completeness (%)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;99.2 (88.6)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;99.0 (97.0)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;100 (97.0)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;100 (99.0)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Anomalous completeness (%)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;96.7 (77.2)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;N/A&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;N/A&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;N/A&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Mean I/sigma(I)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;10.6 (1.60)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;8.46 (1.79)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;13.74 (1.80)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;8.09 (1.74)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Wilson B-factor&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;26.98&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;40.10&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;33.97&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;52.20&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;R&lt;sub&gt;merge&lt;/sub&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.123 (0.790)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.171 (0.792)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.0979 (1.009)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.177 (0.863)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;R&lt;sub&gt;meas&lt;/sub&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.147 (0.973)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.196 (0.923)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.1064 (1.107)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.199 (0.966)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;CC1/2&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.995 (0.469)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.985 (0.557)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.998 (0.642)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.989 (0.627)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;CC*&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.999 (0.846)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.996 (0.846)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.999 (0.884)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.997 (0.878)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Image DOI&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;10.7488/ds/1342&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;10.7488/ds/1419&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;10.7488/ds/1420&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;10.7488/ds/1421&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Refinement&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;R&lt;sub&gt;work&lt;/sub&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.171 (0.318)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.183 (0.288)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.165 (0.299)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.186 (0.273)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;R&lt;sub&gt;free&lt;/sub&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.206 (0.345)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.225 (0351)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.216 (0.364)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.237 (0.325)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Number of non-hydrogen atoms&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;23,222&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;22,366&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;22,691&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;22,145&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;macromolecules&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;22,276&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;22,019&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;21,965&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;22,066&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;ligands&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;138&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;8&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;24&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;74&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;water&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;808&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;339&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;702&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Protein residues&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;2,703&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;2,686&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;2,675&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;2,700&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;RMS(bonds) (Å)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.012&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.005&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.011&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.002&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;RMS(angles) (°)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1.26&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.58&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1.02&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.40&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Ramachandran favored (%)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;100&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;99&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;100&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;99&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Ramachandran allowed (%)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Ramachandran outliers (%)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Clash score&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1.42&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1.42&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1.79&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.97&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;Average B-factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;33.90&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;42.31&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;41.34&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;47.68&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;macromolecules&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;33.80&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;42.35&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;41.31&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;47.60&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;ligands&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;40.40&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;72.80&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;65.55&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;72.34&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;solvent&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;36.20&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;38.95&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;41.46&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;33.85&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;PDB ID&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;5DA5&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;5L89&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;5L8B&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;5L8G&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon>
+      <offset>57119</offset>
+      <text>	WT	E32A	E62A	H65A	 	Data collection					 	Wavelength (Å)	1.74	1.73	1.73	1.74	 	Resolution range (Å)	49.63 - 2.06 (2.10 - 2.06)	48.84 - 2.59 (2.683 - 2.59)	48.87 - 2.21 (2.29 - 2.21)	48.86 - 2.97 (3.08 - 2.97)	 	Space group	P 1 21 1	P 1 21 1	P 1 21 1	P 1 21 1	 	Unit cell (Å) a b  c β (°)	98.18 120.53 140.30 95.36	97.78 120.28 140.53 95.41	98.09 120.23 140.36 95.50	98.03 120.29 140.43 95.39	 	Total reflections	1,264,922 (41,360)	405,488 (36,186)	1,069,345 (95,716)	323,853 (32,120)	 	Unique reflections	197,873 (8,766)	100,067 (9,735)	162,379 (15,817)	66,658 (6,553)	 	Multiplicity	6.4 (4.7)	4.1 (3.7)	6.6 (6.1)	4.9 (4.9)	 	Anomalous multiplicity	3.2 (2.6)	N/A	N/A	N/A	 	Completeness (%)	99.2 (88.6)	99.0 (97.0)	100 (97.0)	100 (99.0)	 	Anomalous completeness (%)	96.7 (77.2)	N/A	N/A	N/A	 	Mean I/sigma(I)	10.6 (1.60)	8.46 (1.79)	13.74 (1.80)	8.09 (1.74)	 	Wilson B-factor	26.98	40.10	33.97	52.20	 	Rmerge	0.123 (0.790)	0.171 (0.792)	0.0979 (1.009)	0.177 (0.863)	 	Rmeas	0.147 (0.973)	0.196 (0.923)	0.1064 (1.107)	0.199 (0.966)	 	CC1/2	0.995 (0.469)	0.985 (0.557)	0.998 (0.642)	0.989 (0.627)	 	CC*	0.999 (0.846)	0.996 (0.846)	0.999 (0.884)	0.997 (0.878)	 	Image DOI	10.7488/ds/1342	10.7488/ds/1419	10.7488/ds/1420	10.7488/ds/1421	 	Refinement					 	Rwork	0.171 (0.318)	0.183 (0.288)	0.165 (0.299)	0.186 (0.273)	 	Rfree	0.206 (0.345)	0.225 (0351)	0.216 (0.364)	0.237 (0.325)	 	Number of non-hydrogen atoms	23,222	22,366	22,691	22,145	 	macromolecules	22,276	22,019	21,965	22,066	 	ligands	138	8	24	74	 	water	808	339	702	5	 	Protein residues	2,703	2,686	2,675	2,700	 	RMS(bonds) (Å)	0.012	0.005	0.011	0.002	 	RMS(angles) (°)	1.26	0.58	1.02	0.40	 	Ramachandran favored (%)	100	99	100	99	 	Ramachandran allowed (%)	0	1	0	1	 	Ramachandran outliers (%)	0	0	0	0	 	Clash score	1.42	1.42	1.79	0.97	 	Average B-factor (Å2)	33.90	42.31	41.34	47.68	 	macromolecules	33.80	42.35	41.31	47.60	 	ligands	40.40	72.80	65.55	72.34	 	solvent	36.20	38.95	41.46	33.85	 	PDB ID	5DA5	5L89	5L8B	5L8G	 	</text>
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+        <infon key="score">0.8830888</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:57:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="57120" length="2"/>
+        <text>WT</text>
+      </annotation>
+      <annotation id="1363">
+        <infon key="score">0.9837817</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57123" length="4"/>
+        <text>E32A</text>
+      </annotation>
+      <annotation id="1364">
+        <infon key="score">0.991135</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57128" length="4"/>
+        <text>E62A</text>
+      </annotation>
+      <annotation id="1365">
+        <infon key="score">0.9922233</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="57133" length="4"/>
+        <text>H65A</text>
+      </annotation>
+      <annotation id="2025">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:10:09Z</infon>
+        <location offset="58623" length="5"/>
+        <text>water</text>
+      </annotation>
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+    <passage>
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+      <infon key="id">tbl5</infon>
+      <infon key="section_type">TABLE</infon>
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+      <text>Iron loading capacity of EncFtn, encapsulin and ferritin. Protein samples (at 8.5 µM) including decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were mixed with Fe(NH4)2(SO4) (in 0.1% (v/v) HCl) of different concentrations in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl buffer at room temperature for 3 hrs in the air. Protein-Fe mixtures were centrifuged at 13,000 x g to remove precipitated material and desalted prior to the Fe and protein content analysis by ferrozine assay and BCA microplate assay, respectively. Fe to protein ratio was calculated to indicate the Fe binding capacity of the protein. Protein stability was compromised at high iron concentrations; therefore, the highest iron loading with the least protein precipitation was used to derive the maximum iron loading capacity per biological assembly (underlined and highlighted in bold). The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin. Errors are quoted as the standard deviation of three technical repeats in both the ferrozine and BCA microplate assays. The proteins used in Fe loading experiment came from a single preparation.</text>
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+      <offset>60373</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.026</text>
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+      <infon key="xml">&lt;?xml version="1.0" encoding="UTF-8"?&gt;
+&lt;table frame="hsides" rules="groups"&gt;&lt;thead&gt;&lt;tr&gt;&lt;th rowspan="1" colspan="1"&gt;Protein sample&lt;/th&gt;&lt;th rowspan="1" colspan="1"&gt;Fe(NH&lt;sub&gt;4)2&lt;/sub&gt;(SO&lt;sub&gt;4)2&lt;/sub&gt; loading (µM)&lt;/th&gt;&lt;th rowspan="1" colspan="1"&gt;Fe detected by ferrozine assay (µM)&lt;/th&gt;&lt;th rowspan="1" colspan="1"&gt;Protein detected by BCA microplate assay (µM)&lt;/th&gt;&lt;th rowspan="1" colspan="1"&gt;Fe / monomeric protein&lt;/th&gt;&lt;th rowspan="1" colspan="1"&gt;Maximum Fe loading per biological assembly unit&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td rowspan="6" colspan="1"&gt;8.46 µM EncFtn&lt;sub&gt;sH&lt;/sub&gt;-10mer&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;4.73 ± 2.32&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;5.26 ± 0.64&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.90 ± 0.44&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;39.9&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;9.93 ± 1.20&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;5.36 ± 0.69&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1.85 ± 0.22&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;84&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;17.99 ± 2.01&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;4.96 ± 0.04&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;3.63 ± 0.41&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;147&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;21.09 ± 1.94&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;4.44 ± 0.21&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;4.75 ± 0.44&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;48 ± 4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;224&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;28.68 ± 0.30&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;3.73 ± 0.53&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;7.68 ± 0.08&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;301&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;11.27 ± 1.10&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;2.50 ± 0.05&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;4.51 ± 0.44&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="8" colspan="1"&gt;8.50 µM Encapsulin&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;-1.02 ± 0.54&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;8.63 ± 0.17&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;-0.12 ± 0.06&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;224&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;62.24 ± 2.49&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;10.01 ± 0.58&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;6.22 ± 0.35&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;301&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;67.94 ± 3.15&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;8.69 ± 0.42&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;7.81 ± 0.36&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;450&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;107.96 ± 8.88&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;8.50 ± 0.69&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;12.71 ± 1.05&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;700&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;97.51 ± 3.19&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;7.26 ± 0.20&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;13.44 ± 0.44&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;1000&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;308.63 ± 2.06&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;8.42 ± 0.34&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;36.66 ± 0.24&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;2199 ± 15&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;1500&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;57.09 ± 0.90&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1.44 ± 0.21&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;39.77 ± 0.62&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;2000&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;9.2 ± 1.16&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.21 ± 0.14&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;44.73 ± 5.63&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="8" colspan="1"&gt;8.70 µM EncFtn-Enc&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;3.31 ± 1.57&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;6.85 ± 0.07&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.48 ± 0.23&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;224&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;116.27 ± 3.74&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;7.63 ± 0.12&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;15.25 ± 0.49&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;301&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;132.86 ± 4.03&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;6.66 ± 0.31&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;19.96 ± 0.61&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;450&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;220.57 ± 27.33&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;6.12 ± 1.07&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;36.06 ± 4.47&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;700&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;344.03 ± 40.38&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;6.94 ± 0.17&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;49.58 ± 5.82&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;1000&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;496.00 ± 38.48&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;7.19 ± 0.08&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;68.94 ± 5.35&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;4137 ± 321&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;1500&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;569.98 ± 73.63&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;5.73 ± 0.03&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;99.44 ± 12.84&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;2000&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;584.30 ± 28.33&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;4.88 ± 0.22&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;119.62 ± 5.80&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="6" colspan="1"&gt;8.50 µM Apoferritin&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;3.95 ± 2.26&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;9.37 ± 0.24&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;0.42 ± 0.25&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;42.5&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;10.27 ± 1.12&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;8.27 ± 0.30&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;1.24 ± 0.18&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;212.5&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;44.48 ± 2.76&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;7.85 ± 0.77&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;5.67 ± 0.83&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;637.5&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;160.93 ± 4.27&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;6.76 ± 0.81&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;23.79 ± 3.12&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;571 ± 75&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;1275&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;114.92 ± 3.17&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;3.84 ± 0.30&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;29.91 ± 2.95&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"&gt;1700&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;91.40 ± 3.37&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;3.14 ± 0.35&lt;/td&gt;&lt;td rowspan="1" colspan="1"&gt;29.13 ± 3.86&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon>
+      <offset>60420</offset>
+      <text>Protein sample	Fe(NH4)2(SO4)2 loading (µM)	Fe detected by ferrozine assay (µM)	Protein detected by BCA microplate assay (µM)	Fe / monomeric protein	Maximum Fe loading per biological assembly unit	 	8.46 µM EncFtnsH-10mer	0	4.73 ± 2.32	5.26 ± 0.64	0.90 ± 0.44		 	39.9	9.93 ± 1.20	5.36 ± 0.69	1.85 ± 0.22		 	84	17.99 ± 2.01	4.96 ± 0.04	3.63 ± 0.41		 	147	21.09 ± 1.94	4.44 ± 0.21	4.75 ± 0.44	48 ± 4	 	224	28.68 ± 0.30	3.73 ± 0.53	7.68 ± 0.08		 	301	11.27 ± 1.10	2.50 ± 0.05	4.51 ± 0.44		 	8.50 µM Encapsulin	0	-1.02 ± 0.54	8.63 ± 0.17	-0.12 ± 0.06		 	224	62.24 ± 2.49	10.01 ± 0.58	6.22 ± 0.35		 	301	67.94 ± 3.15	8.69 ± 0.42	7.81 ± 0.36		 	450	107.96 ± 8.88	8.50 ± 0.69	12.71 ± 1.05		 	700	97.51 ± 3.19	7.26 ± 0.20	13.44 ± 0.44		 	1000	308.63 ± 2.06	8.42 ± 0.34	36.66 ± 0.24	2199 ± 15	 	1500	57.09 ± 0.90	1.44 ± 0.21	39.77 ± 0.62		 	2000	9.2 ± 1.16	0.21 ± 0.14	44.73 ± 5.63		 	8.70 µM EncFtn-Enc	0	3.31 ± 1.57	6.85 ± 0.07	0.48 ± 0.23		 	224	116.27 ± 3.74	7.63 ± 0.12	15.25 ± 0.49		 	301	132.86 ± 4.03	6.66 ± 0.31	19.96 ± 0.61		 	450	220.57 ± 27.33	6.12 ± 1.07	36.06 ± 4.47		 	700	344.03 ± 40.38	6.94 ± 0.17	49.58 ± 5.82		 	1000	496.00 ± 38.48	7.19 ± 0.08	68.94 ± 5.35	4137 ± 321	 	1500	569.98 ± 73.63	5.73 ± 0.03	99.44 ± 12.84		 	2000	584.30 ± 28.33	4.88 ± 0.22	119.62 ± 5.80		 	8.50 µM Apoferritin	0	3.95 ± 2.26	9.37 ± 0.24	0.42 ± 0.25		 	42.5	10.27 ± 1.12	8.27 ± 0.30	1.24 ± 0.18		 	212.5	44.48 ± 2.76	7.85 ± 0.77	5.67 ± 0.83		 	637.5	160.93 ± 4.27	6.76 ± 0.81	23.79 ± 3.12	571 ± 75	 	1275	114.92 ± 3.17	3.84 ± 0.30	29.91 ± 2.95		 	1700	91.40 ± 3.37	3.14 ± 0.35	29.13 ± 3.86		 	</text>
+      <annotation id="1399">
+        <infon key="score">0.9967003</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:17:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="60435" length="14"/>
+        <text>Fe(NH4)2(SO4)2</text>
+      </annotation>
+      <annotation id="1400">
+        <infon key="score">0.99791986</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:00:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="60463" length="2"/>
+        <text>Fe</text>
+      </annotation>
+      <annotation id="1401">
+        <infon key="score">0.97737515</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:21:45Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="60478" length="15"/>
+        <text>ferrozine assay</text>
+      </annotation>
+      <annotation id="1402">
+        <infon key="score">0.9882868</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:21:48Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="60519" length="20"/>
+        <text>BCA microplate assay</text>
+      </annotation>
+      <annotation id="1403">
+        <infon key="score">0.99756145</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:01:02Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="60545" length="2"/>
+        <text>Fe</text>
+      </annotation>
+      <annotation id="1404">
+        <infon key="score">0.9982145</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:01:10Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="60576" length="2"/>
+        <text>Fe</text>
+      </annotation>
+      <annotation id="1405">
+        <infon key="score">0.7292966</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="60626" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1406">
+        <infon key="score">0.7193685</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:01:27Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="60635" length="5"/>
+        <text>10mer</text>
+      </annotation>
+      <annotation id="1852">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <location offset="60915" length="10"/>
+        <text>Encapsulin</text>
+      </annotation>
+      <annotation id="1931">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:08Z</infon>
+        <location offset="61303" length="10"/>
+        <text>EncFtn-Enc</text>
+      </annotation>
+      <annotation id="2138">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <location offset="61705" length="11"/>
+        <text>Apoferritin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>62115</offset>
+      <text>To understand the impact of the mutants on the organization and metal binding of the FOC, we determined the X-ray crystal structures of each of the EncFtnsH mutants (See Table 4 for data collection and refinement statistics). The crystal packing of all of the mutants in this study is essentially isomorphous to the EncFtnsH structure. All of the mutants display the same decameric arrangement in the crystals as the EncFtnsH structure, and the monomers superimpose with an average RMSDCα of less than 0.2 Å.</text>
+      <annotation id="2124">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <location offset="62147" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="1407">
+        <infon key="score">0.6637988</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="62200" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1408">
+        <infon key="score">0.9985746</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:01:52Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="62223" length="24"/>
+        <text>X-ray crystal structures</text>
+      </annotation>
+      <annotation id="1409">
+        <infon key="score">0.9944206</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="62263" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="2125">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <location offset="62272" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="2126">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <location offset="62375" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="1410">
+        <infon key="score">0.83405113</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="62431" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1411">
+        <infon key="score">0.99745923</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:02:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="62440" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="2127">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <location offset="62462" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="1412">
+        <infon key="score">0.9986298</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="62487" length="9"/>
+        <text>decameric</text>
+      </annotation>
+      <annotation id="1413">
+        <infon key="score">0.9963496</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:01:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="62516" length="8"/>
+        <text>crystals</text>
+      </annotation>
+      <annotation id="1414">
+        <infon key="score">0.7181343</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="62532" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1415">
+        <infon key="score">0.99747676</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:01:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="62541" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="1416">
+        <infon key="score">0.99880767</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:19:37Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="62560" length="8"/>
+        <text>monomers</text>
+      </annotation>
+      <annotation id="1417">
+        <infon key="score">0.9983525</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
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+      <text>(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-H65A. Protein residues are shown as sticks with blue and green carbons for the different subunits. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ. The coordinated calcium ions are shown as a grey spheres with coordination distances in the FOC highlighted with yellow dashed lines. (B) Views of the FOC of the EncFtnsH-H65A mutant. Protein atoms and metal ions shown as in (A).</text>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>64099</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.030</text>
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+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>64146</offset>
+      <text>Comparison of the EncFtnsH FOC mutants vs wild type.</text>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>mutants</text>
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+        <infon key="identifier">DUMMY:</infon>
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+      <text>The structures of the three EncFtnsH mutants were all determined by X-ray crystallography. The E32A, E62A and H65A mutants were crystallized in identical conditions to the wild type. EncFtnsH structure and were essentially isomorphous in terms of their unit cell dimensions. The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue. Of the mutants, only H65A has any coordinated metal ions, which appear to be calcium ions from the crystallization condition. The overall organization of FOC residues is retained in the mutants, with almost no backbone movements. Significant differences center around Tyr39, which moves to coordinate the bound calcium ions in the H65A mutant; and Glu32, which moves away from the metal ions in this structure.</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="64227" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="2128">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <location offset="64236" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="1453">
+        <infon key="score">0.9988324</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:34:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="64267" length="21"/>
+        <text>X-ray crystallography</text>
+      </annotation>
+      <annotation id="1454">
+        <infon key="score">0.99892175</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="64294" length="4"/>
+        <text>E32A</text>
+      </annotation>
+      <annotation id="1455">
+        <infon key="score">0.99892247</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="64300" length="4"/>
+        <text>E62A</text>
+      </annotation>
+      <annotation id="1456">
+        <infon key="score">0.9989938</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="64309" length="4"/>
+        <text>H65A</text>
+      </annotation>
+      <annotation id="1457">
+        <infon key="score">0.59239364</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="64314" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="1458">
+        <infon key="score">0.99857235</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:05:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="64327" length="12"/>
+        <text>crystallized</text>
+      </annotation>
+      <annotation id="1459">
+        <infon key="score">0.99905896</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:05:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="64371" length="9"/>
+        <text>wild type</text>
+      </annotation>
+      <annotation id="1460">
+        <infon key="score">0.6066499</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="64382" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1461">
+        <infon key="score">0.9965065</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:05:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="64391" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="1462">
+        <infon key="score">0.8490344</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="64478" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1463">
+        <infon key="score">0.7875101</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="64498" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="1464">
+        <infon key="score">0.99764305</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:22:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="64510" length="6"/>
+        <text>native</text>
+      </annotation>
+      <annotation id="1465">
+        <infon key="score">0.3265913</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="64517" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1466">
+        <infon key="score">0.99798596</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:05:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="64526" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="2144">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:06:45Z</infon>
+        <location offset="64562" length="11"/>
+        <text>coordinated</text>
+      </annotation>
+      <annotation id="1467">
+        <infon key="score">0.9991374</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:55:06Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="64574" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1468">
+        <infon key="score">0.9991269</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:55:10Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="64593" length="4"/>
+        <text>Ca2+</text>
+      </annotation>
+      <annotation id="1469">
+        <infon key="score">0.9990487</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:05:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="64642" length="9"/>
+        <text>wild type</text>
+      </annotation>
+      <annotation id="1470">
+        <infon key="score">0.998978</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="64659" length="4"/>
+        <text>E32A</text>
+      </annotation>
+      <annotation id="1471">
+        <infon key="score">0.99890566</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="64671" length="4"/>
+        <text>E62A</text>
+      </annotation>
+      <annotation id="1472">
+        <infon key="score">0.998872</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="64684" length="4"/>
+        <text>H65A</text>
+      </annotation>
+      <annotation id="2129">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <location offset="64703" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="1473">
+        <infon key="score">0.9981457</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="64717" length="4"/>
+        <text>H65A</text>
+      </annotation>
+      <annotation id="2143">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:06:30Z</infon>
+        <location offset="64730" length="11"/>
+        <text>coordinated</text>
+      </annotation>
+      <annotation id="1474">
+        <infon key="score">0.9984199</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:09:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="64773" length="7"/>
+        <text>calcium</text>
+      </annotation>
+      <annotation id="1475">
+        <infon key="score">0.820491</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="64850" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1476">
+        <infon key="score">0.6792209</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="64882" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="1477">
+        <infon key="score">0.9995347</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="64964" length="5"/>
+        <text>Tyr39</text>
+      </annotation>
+      <annotation id="2145">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:07:03Z</infon>
+        <location offset="64986" length="10"/>
+        <text>coordinate</text>
+      </annotation>
+      <annotation id="2146">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:07:12Z</infon>
+        <location offset="65001" length="5"/>
+        <text>bound</text>
+      </annotation>
+      <annotation id="1478">
+        <infon key="score">0.9985001</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:09:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="65007" length="7"/>
+        <text>calcium</text>
+      </annotation>
+      <annotation id="1479">
+        <infon key="score">0.9988858</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="65027" length="4"/>
+        <text>H65A</text>
+      </annotation>
+      <annotation id="1480">
+        <infon key="score">0.97360635</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:51:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="65032" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="1481">
+        <infon key="score">0.99951637</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="65044" length="5"/>
+        <text>Glu32</text>
+      </annotation>
+      <annotation id="1482">
+        <infon key="score">0.99818367</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:06:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="65096" length="9"/>
+        <text>structure</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig11.jpg</infon>
+      <infon key="id">fig11</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>65108</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.027</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>65155</offset>
+      <text>Close inspection of the region of the protein around the FOC in each of the mutants highlights their effect on metal binding (Figure 11 and Figure 11—figure supplement 1–3). In the E32A mutant the position of the side chains of the remaining iron coordinating residues in the FOC is essentially unchanged, but the absence of the axial-metal coordinating ligand provided by the Glu32 side chain abrogates metal binding in this site. The Glu31/34-site also lacks metal, with the side chain of Glu31 rotated by 180° at the Cβ in the absence of metal (Figure 11—figure supplement 1). The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11—figure supplement 2). The H65A mutant diverges significantly from the wild type in the position of the residues Glu32 and Tyr39 in the FOC. E32 appears in either the original orientation as the wild type and coordinates Ca2+ in this position, or it is flipped by 180° at the Cβ, moving away from the coordinated calcium ion in the FOC. Tyr39 moves closer to Ca2+ compared to the wild-type and coordinates the calcium ion (Figure 11—figure supplement 3). A single calcium ion is present in the entry site of this mutant; however, Glu31 of one chain is rotated away from the metal ion and is not involved in coordination.</text>
+      <annotation id="1483">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="65212" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="2130">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <location offset="65231" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="1484">
+        <infon key="score">0.9991116</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="65336" length="4"/>
+        <text>E32A</text>
+      </annotation>
+      <annotation id="1485">
+        <infon key="score">0.98977494</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:51:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="65341" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="1486">
+        <infon key="score">0.9979477</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:07:39Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="65397" length="26"/>
+        <text>iron coordinating residues</text>
+      </annotation>
+      <annotation id="1487">
+        <infon key="score">0.9932214</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="65431" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1488">
+        <infon key="score">0.9947363</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:22:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="65469" length="10"/>
+        <text>absence of</text>
+      </annotation>
+      <annotation id="2147">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:08:16Z</infon>
+        <location offset="65496" length="12"/>
+        <text>coordinating</text>
+      </annotation>
+      <annotation id="1489">
+        <infon key="score">0.9994023</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="65532" length="5"/>
+        <text>Glu32</text>
+      </annotation>
+      <annotation id="2148">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:09:25Z</infon>
+        <location offset="65549" length="23"/>
+        <text>abrogates metal binding</text>
+      </annotation>
+      <annotation id="1490">
+        <infon key="score">0.993806</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:13:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="65591" length="13"/>
+        <text>Glu31/34-site</text>
+      </annotation>
+      <annotation id="1491">
+        <infon key="score">0.9983423</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:08:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="65610" length="5"/>
+        <text>lacks</text>
+      </annotation>
+      <annotation id="1492">
+        <infon key="score">0.5333656</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:55:14Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="65616" length="5"/>
+        <text>metal</text>
+      </annotation>
+      <annotation id="1493">
+        <infon key="score">0.99938786</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:14:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="65646" length="5"/>
+        <text>Glu31</text>
+      </annotation>
+      <annotation id="1494">
+        <infon key="score">0.9989635</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:22:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="65685" length="10"/>
+        <text>absence of</text>
+      </annotation>
+      <annotation id="1495">
+        <infon key="score">0.82604885</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
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+      <text>Taken together the results of our data show that these changes to the FOC of EncFtn still permit the formation of the decameric form of the protein. While the proteins all appear decameric in crystals, their solution and gas-phase behavior differs considerably and the mutants no longer show metal-dependent oligomerization. These results highlight the importance of metal coordination in the FOC for the stability and assembly of the EncFtn protein.</text>
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+      <offset>66992</offset>
+      <text>Progress curves recording ferroxidase activity of EncFtnsH mutants. 20 µM wild-type EncFtnsH, E32A, E62A and H65A mutants were mixed with 20 µM or 100 µM acidic Fe(NH4)2(SO4)2, respectively.</text>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="updated_at">2023-09-19T14:53:27Z</infon>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="67106" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="1968">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:17:57Z</infon>
+        <location offset="67153" length="14"/>
+        <text>Fe(NH4)2(SO4)2</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig12-figsupp1.jpg</infon>
+      <infon key="id">fig12s1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>67195</offset>
+      <text>Absorbance at 315 nm was recorded for 1800 s at 25°C as an indication of Fe3+ formation. Protein free samples (dashed and dotted lines) were measured for Fe2+ background oxidation as controls. Assays were performed with three technical repeats. Error bars were showed in shadows behind each curves.</text>
+      <annotation id="1542">
+        <infon key="score">0.99893546</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:10:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="67268" length="4"/>
+        <text>Fe3+</text>
+      </annotation>
+      <annotation id="1543">
+        <infon key="score">0.9990836</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:11:02Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="67349" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig12-figsupp1.jpg</infon>
+      <infon key="id">fig12s1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>67495</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.032</text>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig12.jpg</infon>
+      <infon key="id">fig12</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>67542</offset>
+      <text>Relative ferroxidase activity of EncFtnsH mutants.</text>
+      <annotation id="1873">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:30Z</infon>
+        <location offset="67551" length="11"/>
+        <text>ferroxidase</text>
+      </annotation>
+      <annotation id="1544">
+        <infon key="score">0.87679505</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="67575" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="2131">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <location offset="67584" length="7"/>
+        <text>mutants</text>
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+    <passage>
+      <infon key="file">elife-18972-fig12.jpg</infon>
+      <infon key="id">fig12</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>67593</offset>
+      <text>EncFtnsH, and the mutant forms E32A, E62A and H65A, each at 20 µM, were mixed with 100 µM acidic Fe(NH4)2(SO4)2. Ferroxidase activity of the mutant forms is determined by measuring the absorbance at 315 nm for 1800 s at 25 °C as an indication of Fe3+ formation. The relative ferroxidase activity of mutants is plotted as a proportion of the activity of the wild-type protein using the endpoint measurement of A315. Three technical repeats were performed and the plotted error bars represent the calculated standard deviations. The FOC mutants showed reduced ferroxidase activity to varied extents, among which E62A significantly abrogated the ferroxidase activity.</text>
+      <annotation id="1545">
+        <infon key="score">0.47456783</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="67593" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1546">
+        <infon key="score">0.9963652</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:51:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="67611" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="1547">
+        <infon key="score">0.99853694</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="67624" length="4"/>
+        <text>E32A</text>
+      </annotation>
+      <annotation id="1548">
+        <infon key="score">0.99874085</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="67630" length="4"/>
+        <text>E62A</text>
+      </annotation>
+      <annotation id="1549">
+        <infon key="score">0.9988588</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:27Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="67639" length="4"/>
+        <text>H65A</text>
+      </annotation>
+      <annotation id="1550">
+        <infon key="score">0.9989173</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:17:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="67690" length="14"/>
+        <text>Fe(NH4)2(SO4)2</text>
+      </annotation>
+      <annotation id="1874">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:30Z</infon>
+        <location offset="67706" length="11"/>
+        <text>Ferroxidase</text>
+      </annotation>
+      <annotation id="1551">
+        <infon key="score">0.98657566</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:51:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="67734" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="2154">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:11:58Z</infon>
+        <location offset="67764" length="34"/>
+        <text>measuring the absorbance at 315 nm</text>
+      </annotation>
+      <annotation id="1552">
+        <infon key="score">0.99880314</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:11:28Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="67839" length="4"/>
+        <text>Fe3+</text>
+      </annotation>
+      <annotation id="1875">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:30Z</infon>
+        <location offset="67868" length="11"/>
+        <text>ferroxidase</text>
+      </annotation>
+      <annotation id="2132">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <location offset="67892" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="1553">
+        <infon key="score">0.99907094</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="67950" length="9"/>
+        <text>wild-type</text>
+      </annotation>
+      <annotation id="2153">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:11:43Z</infon>
+        <location offset="67987" length="19"/>
+        <text>measurement of A315</text>
+      </annotation>
+      <annotation id="1554">
+        <infon key="score">0.8451369</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="68124" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1555">
+        <infon key="score">0.95589703</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="68128" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="1876">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:30Z</infon>
+        <location offset="68151" length="11"/>
+        <text>ferroxidase</text>
+      </annotation>
+      <annotation id="1556">
+        <infon key="score">0.9987086</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="68203" length="4"/>
+        <text>E62A</text>
+      </annotation>
+      <annotation id="1877">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:30Z</infon>
+        <location offset="68236" length="11"/>
+        <text>ferroxidase</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig12.jpg</infon>
+      <infon key="id">fig12</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>68266</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.031</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>68313</offset>
+      <text>To address the question of how mutagenesis of the iron coordinating residues affects the enzymatic activity of the EncFtnsH protein we recorded progress curves for the oxidation of Fe2+ to Fe3+ by the different mutants as before. Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%; the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12). Collectively, the effect of mutating these residues in the FOC confirms the importance of the iron coordinating residues for the ferroxidase activity of the EncFtnsH protein.</text>
+      <annotation id="1557">
+        <infon key="score">0.9969042</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:12:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="68344" length="11"/>
+        <text>mutagenesis</text>
+      </annotation>
+      <annotation id="1558">
+        <infon key="score">0.9987772</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:07:40Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="68363" length="26"/>
+        <text>iron coordinating residues</text>
+      </annotation>
+      <annotation id="1559">
+        <infon key="score">0.9993548</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="68428" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1560">
+        <infon key="score">0.9609241</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:12:28Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="68457" length="15"/>
+        <text>progress curves</text>
+      </annotation>
+      <annotation id="1561">
+        <infon key="score">0.99912536</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:12:14Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="68494" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1562">
+        <infon key="score">0.999081</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:12:17Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="68502" length="4"/>
+        <text>Fe3+</text>
+      </annotation>
+      <annotation id="1563">
+        <infon key="score">0.9333361</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:55:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="68524" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="1564">
+        <infon key="score">0.99793833</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:12:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="68543" length="11"/>
+        <text>Mutagenesis</text>
+      </annotation>
+      <annotation id="1565">
+        <infon key="score">0.99888545</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="68558" length="4"/>
+        <text>E32A</text>
+      </annotation>
+      <annotation id="1566">
+        <infon key="score">0.9989735</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:27Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="68567" length="4"/>
+        <text>H65A</text>
+      </annotation>
+      <annotation id="1567">
+        <infon key="score">0.9993606</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="68596" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1568">
+        <infon key="score">0.9990294</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="68627" length="4"/>
+        <text>E62A</text>
+      </annotation>
+      <annotation id="1569">
+        <infon key="score">0.9976634</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:51:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="68632" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="2095">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:30:46Z</infon>
+        <location offset="68693" length="7"/>
+        <text>loss of</text>
+      </annotation>
+      <annotation id="2039">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:11:14Z</infon>
+        <location offset="68714" length="12"/>
+        <text>coordination</text>
+      </annotation>
+      <annotation id="1570">
+        <infon key="score">0.99380463</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:12:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="68752" length="22"/>
+        <text>di-nuclear iron center</text>
+      </annotation>
+      <annotation id="1571">
+        <infon key="score">0.50114024</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="68782" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1572">
+        <infon key="score">0.9930937</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:21:53Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="68827" length="8"/>
+        <text>mutating</text>
+      </annotation>
+      <annotation id="1573">
+        <infon key="score">0.61401594</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="68858" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1574">
+        <infon key="score">0.9987893</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:07:40Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="68893" length="26"/>
+        <text>iron coordinating residues</text>
+      </annotation>
+      <annotation id="1878">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:31Z</infon>
+        <location offset="68928" length="11"/>
+        <text>ferroxidase</text>
+      </annotation>
+      <annotation id="1575">
+        <infon key="score">0.99936014</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="68956" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">title_1</infon>
+      <offset>68975</offset>
+      <text>Discussion</text>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig13.jpg</infon>
+      <infon key="id">fig13</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>68986</offset>
+      <text>Phylogenetic tree of ferritin family proteins.</text>
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+        <infon key="score">0.848318</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:08:51Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="68986" length="17"/>
+        <text>Phylogenetic tree</text>
+      </annotation>
+      <annotation id="1841">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <location offset="69007" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig13.jpg</infon>
+      <infon key="id">fig13</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>69033</offset>
+      <text>The tree was built using the Neighbor-Joining method based on step-wise amino acid sequence alignment of the four-helical bundle portions of ferritin family proteins (Supplementary file 1). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree; the likely root of the tree is indicated by a red arrow. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 2.5). The analysis involved 104 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 262 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 </text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:13:12Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="69062" length="23"/>
+        <text>Neighbor-Joining method</text>
+      </annotation>
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+        <infon key="score">0.99878114</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:13:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="69095" length="39"/>
+        <text>step-wise amino acid sequence alignment</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:13:18Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="69142" length="19"/>
+        <text>four-helical bundle</text>
+      </annotation>
+      <annotation id="1842">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <location offset="69174" length="8"/>
+        <text>ferritin</text>
+      </annotation>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:13:27Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="69426" length="22"/>
+        <text>evolutionary distances</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:13:30Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="69473" length="17"/>
+        <text>p-distance method</text>
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+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig13.jpg</infon>
+      <infon key="id">fig13</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>69870</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.033</text>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>69917</offset>
+      <text>Our study reports on a new class of ferritin-like proteins (EncFtn), which are associated with bacterial encapsulin nanocompartments (Enc). By studying the EncFtn from R. rubrum we demonstrate that iron binding results in assembly of EncFtn decamers, which display a unique annular architecture. Despite a radically different quaternary structure to the classical ferritins, the four-helical bundle scaffold and FOC of EncFtnsH are strikingly similar to ferritin (Figure 6A). A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn). The analysis revealed that the EncFtn and Non-EncFtn proteins form groups distinct from the other clearly delineated groups of ferritins, and represent outliers in the tree (Figure 13). While it is difficult to infer ancestral lineages in protein families, the similarity seen in the active site scaffold of these proteins highlights a shared evolutionary relationship between EncFtn proteins and other members of the ferritin superfamily that has been noted in previous studies (; ). From this analysis, we propose that the four-helical fold of the classical ferritins may have arisen through gene duplication of an ancestor of EncFtn. This gene duplication would result in the C-terminal region of one EncFtn monomer being linked to the N-terminus of another and thus stabilizing the four-helix bundle fold within a single polypeptide chain (Figure 6B). Linking the protein together in this way relaxes the requirement for the maintenance of a symmetrical FOC and thus provides a path to the diversity in active-site residues seen across the ferritin family (Figure 6A, residues Glu95, Gln128 and Glu131 in PmFtn, Supplementary file 1).</text>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <location offset="69953" length="8"/>
+        <text>ferritin</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="69977" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
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+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:45Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="70012" length="9"/>
+        <text>bacterial</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <location offset="70022" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1914">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:47:29Z</infon>
+        <location offset="70033" length="16"/>
+        <text>nanocompartments</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:11:18Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="70051" length="3"/>
+        <text>Enc</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="70073" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1586">
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:56:17Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="70085" length="9"/>
+        <text>R. rubrum</text>
+      </annotation>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="70115" length="4"/>
+        <text>iron</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="70151" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1589">
+        <infon key="score">0.99798334</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="70158" length="8"/>
+        <text>decamers</text>
+      </annotation>
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+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
+        <location offset="70271" length="9"/>
+        <text>classical</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="70281" length="9"/>
+        <text>ferritins</text>
+      </annotation>
+      <annotation id="1591">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:14:14Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="70296" length="28"/>
+        <text>four-helical bundle scaffold</text>
+      </annotation>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="70329" length="3"/>
+        <text>FOC</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="70336" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1594">
+        <infon key="score">0.9987708</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="70371" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+      <annotation id="1595">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:14:18Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="70395" length="32"/>
+        <text>sequence-based phylogenetic tree</text>
+      </annotation>
+      <annotation id="1844">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <location offset="70448" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+      <annotation id="1903">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
+        <location offset="70500" length="9"/>
+        <text>classical</text>
+      </annotation>
+      <annotation id="1596">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="70510" length="9"/>
+        <text>ferritins</text>
+      </annotation>
+      <annotation id="1597">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:14:31Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="70521" length="17"/>
+        <text>bacterioferritins</text>
+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:14:52Z</infon>
+        <location offset="70543" length="3"/>
+        <text>Dps</text>
+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:15:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="70583" length="44"/>
+        <text>encapsulin-associated ferritin-like proteins</text>
+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:15:06Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="70629" length="7"/>
+        <text>EncFtns</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="70684" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:15:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="70705" length="10"/>
+        <text>Non-EncFtn</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="70749" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:15:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="70760" length="10"/>
+        <text>Non-EncFtn</text>
+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="70845" length="9"/>
+        <text>ferritins</text>
+      </annotation>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:16:49Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="71002" length="20"/>
+        <text>active site scaffold</text>
+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:15:50Z</infon>
+        <location offset="71095" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <location offset="71136" length="8"/>
+        <text>ferritin</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
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+        <infon key="updated_at">2023-09-19T15:16:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="71243" length="17"/>
+        <text>four-helical fold</text>
+      </annotation>
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+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
+        <location offset="71268" length="9"/>
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+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="71278" length="9"/>
+        <text>ferritins</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="71347" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1609">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:16:42Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="71397" length="17"/>
+        <text>C-terminal region</text>
+      </annotation>
+      <annotation id="1610">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="71422" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1611">
+        <infon key="score">0.9987987</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:13:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="71429" length="7"/>
+        <text>monomer</text>
+      </annotation>
+      <annotation id="1612">
+        <infon key="score">0.99855626</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:16:36Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="71504" length="22"/>
+        <text>four-helix bundle fold</text>
+      </annotation>
+      <annotation id="1613">
+        <infon key="score">0.49689123</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="71676" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1614">
+        <infon key="score">0.994453</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:16:33Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="71725" length="20"/>
+        <text>active-site residues</text>
+      </annotation>
+      <annotation id="1846">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <location offset="71762" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+      <annotation id="1615">
+        <infon key="score">0.9995757</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:16:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="71799" length="5"/>
+        <text>Glu95</text>
+      </annotation>
+      <annotation id="1616">
+        <infon key="score">0.99958545</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:16:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="71806" length="6"/>
+        <text>Gln128</text>
+      </annotation>
+      <annotation id="1617">
+        <infon key="score">0.99957746</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:16:27Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="71817" length="6"/>
+        <text>Glu131</text>
+      </annotation>
+      <annotation id="1618">
+        <infon key="score">0.9937343</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:20:38Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="71827" length="5"/>
+        <text>PmFtn</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">title_2</infon>
+      <offset>71858</offset>
+      <text>Relationship between ferritin structure and activity</text>
+      <annotation id="1619">
+        <infon key="score">0.98743826</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="71879" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+      <annotation id="1620">
+        <infon key="score">0.55521035</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:08:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="71888" length="9"/>
+        <text>structure</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>71911</offset>
+      <text>The quaternary arrangement of classical ferritins into an octahedral nanocage and Dps into a dodecamer is absolutely required for their function as iron storage compartments. The oxidation and mineralization of iron must be spatially separated from the host cytosol to prevent the formation of damaging hydroxyl radicals in the Fenton and Haber-Weiss reactions. This is achieved in all ferritins by confining the oxidation of iron to the interior of the protein complex, thus achieving sequestration of the Fe3+ mineralization product. A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A). This overlay highlights the fact that the ferroxidase center of EncFtn faces in the opposite direction relative to the classical ferritins and is essentially inside out regarding iron storage space (Figure 6—figure supplement 1B, boxed region). Analysis of each of the single mutations (E32A, E62A and H65A) made in the FOC highlights the importance of the iron-coordinating residues in the catalytic activity of EncFtn. Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC. The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.</text>
+      <annotation id="1905">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
+        <location offset="71941" length="9"/>
+        <text>classical</text>
+      </annotation>
+      <annotation id="1621">
+        <infon key="score">0.99900573</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="71951" length="9"/>
+        <text>ferritins</text>
+      </annotation>
+      <annotation id="1622">
+        <infon key="score">0.99534684</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:17:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="71969" length="10"/>
+        <text>octahedral</text>
+      </annotation>
+      <annotation id="1623">
+        <infon key="score">0.81837994</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:46:47Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="71980" length="8"/>
+        <text>nanocage</text>
+      </annotation>
+      <annotation id="1624">
+        <infon key="score">0.96995956</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:12:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="71993" length="3"/>
+        <text>Dps</text>
+      </annotation>
+      <annotation id="1625">
+        <infon key="score">0.9974191</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:19:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="72004" length="9"/>
+        <text>dodecamer</text>
+      </annotation>
+      <annotation id="1626">
+        <infon key="score">0.9799951</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="72059" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1627">
+        <infon key="score">0.9988739</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="72122" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1628">
+        <infon key="score">0.99887925</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="72297" length="9"/>
+        <text>ferritins</text>
+      </annotation>
+      <annotation id="1629">
+        <infon key="score">0.9988193</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="72337" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1630">
+        <infon key="score">0.9952247</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:18:19Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="72418" length="4"/>
+        <text>Fe3+</text>
+      </annotation>
+      <annotation id="1631">
+        <infon key="score">0.9987408</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:17:09Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="72449" length="20"/>
+        <text>structural alignment</text>
+      </annotation>
+      <annotation id="1632">
+        <infon key="score">0.9852278</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="72477" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1633">
+        <infon key="score">0.99501425</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="72484" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1906">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
+        <location offset="72500" length="9"/>
+        <text>classical</text>
+      </annotation>
+      <annotation id="1634">
+        <infon key="score">0.9988361</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="72510" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+      <annotation id="1635">
+        <infon key="score">0.9954562</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:20:38Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="72519" length="5"/>
+        <text>PmFtn</text>
+      </annotation>
+      <annotation id="1636">
+        <infon key="score">0.99760485</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:17:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="72540" length="12"/>
+        <text>central ring</text>
+      </annotation>
+      <annotation id="1637">
+        <infon key="score">0.9990308</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="72556" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1638">
+        <infon key="score">0.99808574</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="72602" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+      <annotation id="1639">
+        <infon key="score">0.9983908</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="72645" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1640">
+        <infon key="score">0.99829125</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:14:13Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="72680" length="22"/>
+        <text>mineralization surface</text>
+      </annotation>
+      <annotation id="1641">
+        <infon key="score">0.9971945</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="72706" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+      <annotation id="1642">
+        <infon key="score">0.99885035</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:21:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="72753" length="7"/>
+        <text>overlay</text>
+      </annotation>
+      <annotation id="1643">
+        <infon key="score">0.9989034</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:31:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="72790" length="18"/>
+        <text>ferroxidase center</text>
+      </annotation>
+      <annotation id="1644">
+        <infon key="score">0.9981205</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="72812" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1907">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
+        <location offset="72867" length="9"/>
+        <text>classical</text>
+      </annotation>
+      <annotation id="1645">
+        <infon key="score">0.99885356</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="72877" length="9"/>
+        <text>ferritins</text>
+      </annotation>
+      <annotation id="1832">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <location offset="72927" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1646">
+        <infon key="score">0.8510018</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:22:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="73024" length="9"/>
+        <text>mutations</text>
+      </annotation>
+      <annotation id="1647">
+        <infon key="score">0.9988943</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="73035" length="4"/>
+        <text>E32A</text>
+      </annotation>
+      <annotation id="1648">
+        <infon key="score">0.998966</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="73041" length="4"/>
+        <text>E62A</text>
+      </annotation>
+      <annotation id="1649">
+        <infon key="score">0.998933</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:53:27Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="73050" length="4"/>
+        <text>H65A</text>
+      </annotation>
+      <annotation id="1650">
+        <infon key="score">0.8451171</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="73068" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1651">
+        <infon key="score">0.9989415</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:17:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="73105" length="26"/>
+        <text>iron-coordinating residues</text>
+      </annotation>
+      <annotation id="1652">
+        <infon key="score">0.9991235</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="73161" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1653">
+        <infon key="score">0.99879754</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:09:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="73202" length="7"/>
+        <text>calcium</text>
+      </annotation>
+      <annotation id="2058">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:19:10Z</infon>
+        <location offset="73214" length="14"/>
+        <text>coordinated by</text>
+      </annotation>
+      <annotation id="1654">
+        <infon key="score">0.99950826</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:14:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="73229" length="5"/>
+        <text>Glu31</text>
+      </annotation>
+      <annotation id="1655">
+        <infon key="score">0.99950147</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:14:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="73239" length="5"/>
+        <text>Glu34</text>
+      </annotation>
+      <annotation id="1656">
+        <infon key="score">0.99401796</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="73257" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1657">
+        <infon key="score">0.9978822</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:09:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="73266" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="1658">
+        <infon key="score">0.99807453</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:17:46Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="73288" length="10"/>
+        <text>entry site</text>
+      </annotation>
+      <annotation id="1659">
+        <infon key="score">0.99386215</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="73330" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1660">
+        <infon key="score">0.999099</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="73376" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1661">
+        <infon key="score">0.99562365</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="73444" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1662">
+        <infon key="score">0.9986205</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="73517" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1663">
+        <infon key="score">0.99952245</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:12:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="73525" length="5"/>
+        <text>His57</text>
+      </annotation>
+      <annotation id="1664">
+        <infon key="score">0.9995121</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:12:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="73532" length="5"/>
+        <text>Glu61</text>
+      </annotation>
+      <annotation id="1665">
+        <infon key="score">0.9995173</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:12:24Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="73542" length="5"/>
+        <text>Glu64</text>
+      </annotation>
+      <annotation id="1666">
+        <infon key="score">0.9983008</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:17:39Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="73607" length="22"/>
+        <text>mineralization surface</text>
+      </annotation>
+      <annotation id="1667">
+        <infon key="score">0.8620233</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="73641" length="9"/>
+        <text>classical</text>
+      </annotation>
+      <annotation id="1668">
+        <infon key="score">0.99607795</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="73651" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+      <annotation id="1669">
+        <infon key="score">0.8689013</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:46:47Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="73660" length="8"/>
+        <text>nanocage</text>
+      </annotation>
+      <annotation id="1670">
+        <infon key="score">0.9678278</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="73703" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1671">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:17:44Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="73730" length="11"/>
+        <text>exit portal</text>
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+      <annotation id="1672">
+        <infon key="score">0.9972073</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:17:41Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="73746" length="19"/>
+        <text>mineralization site</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>73772</offset>
+      <text>The absolute requirement for the spatial separation of oxidation and mineralization in ferritins suggests that the EncFtn family proteins are not capable of storing iron minerals due to the absence of an enclosed compartment in their structure (Figure 6—figure supplement 1B). Our biochemical characterization of EncFtn supports this hypothesis, indicating that while this protein is capable of oxidizing iron, it does not accrue mineralized iron in an analogous manner to classical ferritins. While EncFtn does not store iron itself, its association with the encapsulin nanocage suggests that mineralization occurs within the cavity of the encapsulin shell. Our ferroxidase assay data on the recombinant EncFtn-Enc nanocompartments, which accrue over 4100 iron ions per complex and form regular nanoparticles, are consistent with the encapsulin protein acting as the store for iron oxidized by the EncFtn enzyme. TEM analysis of the reaction products shows the production of homogeneous iron nanoparticles only in the EncFtn-Enc nanocompartment (Figure 8—figure supplement 1).</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="73859" length="9"/>
+        <text>ferritins</text>
+      </annotation>
+      <annotation id="1859">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:18:33Z</infon>
+        <location offset="73887" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1674">
+        <infon key="score">0.9978358</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="73937" length="4"/>
+        <text>iron</text>
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+      <annotation id="2178">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:22:04Z</infon>
+        <location offset="73962" length="10"/>
+        <text>absence of</text>
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+      <annotation id="1675">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:22:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="74053" length="28"/>
+        <text>biochemical characterization</text>
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+      <annotation id="1676">
+        <infon key="score">0.9992699</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="74085" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1677">
+        <infon key="score">0.99894625</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="74177" length="4"/>
+        <text>iron</text>
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+      <annotation id="1678">
+        <infon key="score">0.9988219</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="74214" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1908">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
+        <location offset="74245" length="9"/>
+        <text>classical</text>
+      </annotation>
+      <annotation id="1679">
+        <infon key="score">0.9991887</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="74255" length="9"/>
+        <text>ferritins</text>
+      </annotation>
+      <annotation id="1680">
+        <infon key="score">0.9990702</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="74272" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1681">
+        <infon key="score">0.9989749</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="74294" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1682">
+        <infon key="score">0.9391922</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="74332" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1683">
+        <infon key="score">0.5729172</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:46:47Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="74343" length="8"/>
+        <text>nanocage</text>
+      </annotation>
+      <annotation id="1684">
+        <infon key="score">0.98972017</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:19:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="74399" length="6"/>
+        <text>cavity</text>
+      </annotation>
+      <annotation id="1685">
+        <infon key="score">0.8813761</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="74413" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1686">
+        <infon key="score">0.9983045</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="74424" length="5"/>
+        <text>shell</text>
+      </annotation>
+      <annotation id="1687">
+        <infon key="score">0.9983613</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:47:08Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="74435" length="17"/>
+        <text>ferroxidase assay</text>
+      </annotation>
+      <annotation id="1688">
+        <infon key="score">0.9979518</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:08Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="74477" length="10"/>
+        <text>EncFtn-Enc</text>
+      </annotation>
+      <annotation id="1689">
+        <infon key="score">0.9949738</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:47:29Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="74488" length="16"/>
+        <text>nanocompartments</text>
+      </annotation>
+      <annotation id="1690">
+        <infon key="score">0.9991097</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="74529" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1691">
+        <infon key="score">0.36210647</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:58:45Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="74568" length="13"/>
+        <text>nanoparticles</text>
+      </annotation>
+      <annotation id="1692">
+        <infon key="score">0.971039</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="74607" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1693">
+        <infon key="score">0.9990445</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="74650" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1694">
+        <infon key="score">0.9991709</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="74671" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1695">
+        <infon key="score">0.9986847</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:52:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="74686" length="3"/>
+        <text>TEM</text>
+      </annotation>
+      <annotation id="1696">
+        <infon key="score">0.99640995</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="74760" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1697">
+        <infon key="score">0.99721414</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:08Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="74791" length="10"/>
+        <text>EncFtn-Enc</text>
+      </annotation>
+      <annotation id="1698">
+        <infon key="score">0.98311573</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:58:50Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="74802" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig14.jpg</infon>
+      <infon key="id">fig14</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>74854</offset>
+      <text>Model of iron oxidation in encapsulin nanocompartments.</text>
+      <annotation id="1699">
+        <infon key="score">0.99905115</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="74863" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1700">
+        <infon key="score">0.9947168</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="74881" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1701">
+        <infon key="score">0.99186254</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:47:29Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="74892" length="16"/>
+        <text>nanocompartments</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig14.jpg</infon>
+      <infon key="id">fig14</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>74910</offset>
+      <text>(A) Model of EncFtnsH docking to the encapsulin shell. A single pentamer of the icosahedral T. maritima encapsulin structure (PDBID: 3DKT) is shown as a blue surface with the encapsulin localization sequence of EncFtn shown as a purple surface. The C-terminal regions of the EncFtn subunits correspond to the position of the localization sequences seen in 3DKT. Alignment of EncFtnsH with 3DKT positions the central channel directly above the pore in the 3DKT pentamer axis (shown as a grey pentagon). (B) Surface view of EncFtn within the encapsulin nanocompartment (grey and blue respectively). The lumen of the encapsulin nanocompartment is considerably larger than the interior of ferritin (shown in orange behind the encapsulin for reference) and thus allows the storage of significantly more iron. The proposed pathway for iron movement through the encapsulin shell and EncFtn FOC is shown with arrows. (C) Model ofiron oxidation within an encapsulin nanocompartment. As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="74923" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="2157">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:19:29Z</infon>
+        <location offset="74932" length="7"/>
+        <text>docking</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="74947" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1704">
+        <infon key="score">0.9856828</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="74958" length="5"/>
+        <text>shell</text>
+      </annotation>
+      <annotation id="1705">
+        <infon key="score">0.99869543</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:39:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="74974" length="8"/>
+        <text>pentamer</text>
+      </annotation>
+      <annotation id="1706">
+        <infon key="score">0.99862695</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:36:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="74990" length="11"/>
+        <text>icosahedral</text>
+      </annotation>
+      <annotation id="1707">
+        <infon key="score">0.9982567</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:50:39Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="75002" length="11"/>
+        <text>T. maritima</text>
+      </annotation>
+      <annotation id="1708">
+        <infon key="score">0.7239029</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="75014" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1709">
+        <infon key="score">0.99784875</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:19:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="75025" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="2196">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:03:59Z</infon>
+        <location offset="75085" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="2197">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:04:11Z</infon>
+        <location offset="75096" length="21"/>
+        <text>localization sequence</text>
+      </annotation>
+      <annotation id="1710">
+        <infon key="score">0.99601483</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="75121" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1711">
+        <infon key="score">0.99636465</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="75185" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1712">
+        <infon key="score">0.9944976</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:35:44Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="75192" length="8"/>
+        <text>subunits</text>
+      </annotation>
+      <annotation id="2181">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:03:44Z</infon>
+        <location offset="75235" length="22"/>
+        <text>localization sequences</text>
+      </annotation>
+      <annotation id="1713">
+        <infon key="score">0.99331784</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:19:47Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="75272" length="9"/>
+        <text>Alignment</text>
+      </annotation>
+      <annotation id="1714">
+        <infon key="score">0.9935847</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="75285" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1715">
+        <infon key="score">0.9828522</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:14:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="75318" length="15"/>
+        <text>central channel</text>
+      </annotation>
+      <annotation id="1716">
+        <infon key="score">0.9973459</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:14:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="75353" length="4"/>
+        <text>pore</text>
+      </annotation>
+      <annotation id="1717">
+        <infon key="score">0.99818194</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:39:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="75370" length="8"/>
+        <text>pentamer</text>
+      </annotation>
+      <annotation id="1718">
+        <infon key="score">0.98649895</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="75432" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1719">
+        <infon key="score">0.80627346</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="75450" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="2182">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:02:59Z</infon>
+        <location offset="75461" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+      <annotation id="1720">
+        <infon key="score">0.8879431</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="75524" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="2183">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:03:15Z</infon>
+        <location offset="75535" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+      <annotation id="1721">
+        <infon key="score">0.9398855</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="75595" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+      <annotation id="1722">
+        <infon key="score">0.48444474</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="75632" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1723">
+        <infon key="score">0.9990338</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="75708" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1724">
+        <infon key="score">0.9990761</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="75739" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1725">
+        <infon key="score">0.7208568</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="75765" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1726">
+        <infon key="score">0.94934714</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="75776" length="5"/>
+        <text>shell</text>
+      </annotation>
+      <annotation id="1727">
+        <infon key="score">0.53844655</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="75786" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1728">
+        <infon key="score">0.8039653</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="75793" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1729">
+        <infon key="score">0.9098615</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="75856" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="2187">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:03:16Z</infon>
+        <location offset="75867" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+      <annotation id="1730">
+        <infon key="score">0.947138</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:43Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="75887" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1731">
+        <infon key="score">0.9990113</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="75918" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1732">
+        <infon key="score">0.9990713</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:19:50Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="75948" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1733">
+        <infon key="score">0.6161996</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="75975" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1734">
+        <infon key="score">0.86503077</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="75986" length="5"/>
+        <text>shell</text>
+      </annotation>
+      <annotation id="1735">
+        <infon key="score">0.9974375</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:19:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="76012" length="18"/>
+        <text>metal binding site</text>
+      </annotation>
+      <annotation id="1736">
+        <infon key="score">0.9860432</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:19:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="76042" length="15"/>
+        <text>central channel</text>
+      </annotation>
+      <annotation id="1737">
+        <infon key="score">0.9866344</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="76061" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1738">
+        <infon key="score">0.9989586</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:20:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="76071" length="10"/>
+        <text>entry site</text>
+      </annotation>
+      <annotation id="1739">
+        <infon key="score">0.7974093</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="76113" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1740">
+        <infon key="score">0.9989891</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:19:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="76132" length="4"/>
+        <text>Fe3+</text>
+      </annotation>
+      <annotation id="1741">
+        <infon key="score">0.88258153</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="76225" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-fig14.jpg</infon>
+      <infon key="id">fig14</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>76242</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.034</text>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>76289</offset>
+      <text>Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A). Thus, it appears that the EncFtn decamer is the physiological state of this protein. This arrangement positions the central ring of EncFtn directly above the pore at the five-fold symmetry axis of the encapsulin shell and highlights a potential route for the entry of iron into the encapsulin and towards the active site of EncFtn. A comparison of the encapsulin nanocompartment and the ferritin nanocage highlights the size differential between the two complexes (Figure 14B) that allows the encapsulin to store significantly more iron. The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment. This arrangement of multiple reaction centers in a single protein assembly is reminiscent of classical ferritins, which has 24 FOCs distributed around the nanocage.</text>
+      <annotation id="1742">
+        <infon key="score">0.99638414</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:22:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="76289" length="7"/>
+        <text>Docking</text>
+      </annotation>
+      <annotation id="1743">
+        <infon key="score">0.99840146</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="76301" length="7"/>
+        <text>decamer</text>
+      </annotation>
+      <annotation id="1744">
+        <infon key="score">0.9968444</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:20:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="76309" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="1745">
+        <infon key="score">0.9991373</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="76322" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1746">
+        <infon key="score">0.9986046</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:39:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="76340" length="8"/>
+        <text>pentamer</text>
+      </annotation>
+      <annotation id="1747">
+        <infon key="score">0.9983079</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:50:39Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="76356" length="11"/>
+        <text>T. maritima</text>
+      </annotation>
+      <annotation id="1748">
+        <infon key="score">0.99425864</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="76368" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="2158">
+        <infon key="type">gene</infon>
+        <infon key="identifier">GENE:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:20:39Z</infon>
+        <location offset="76379" length="10"/>
+        <text>Tmari_0786</text>
+      </annotation>
+      <annotation id="1749">
+        <infon key="score">0.9992826</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:03:27Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="76437" length="21"/>
+        <text>C-terminal extensions</text>
+      </annotation>
+      <annotation id="1750">
+        <infon key="score">0.9988845</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="76466" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1751">
+        <infon key="score">0.99812025</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:20:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="76475" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="1752">
+        <infon key="score">0.87305796</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:04:15Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="76509" length="22"/>
+        <text>localization sequences</text>
+      </annotation>
+      <annotation id="1753">
+        <infon key="score">0.99905574</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:20:45Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="76537" length="8"/>
+        <text>bound to</text>
+      </annotation>
+      <annotation id="1754">
+        <infon key="score">0.9951746</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="76550" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1755">
+        <infon key="score">0.99610835</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="76609" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1756">
+        <infon key="score">0.99860805</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="76616" length="7"/>
+        <text>decamer</text>
+      </annotation>
+      <annotation id="1757">
+        <infon key="score">0.99918133</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:04:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="76699" length="12"/>
+        <text>central ring</text>
+      </annotation>
+      <annotation id="1758">
+        <infon key="score">0.9992908</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="76715" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1759">
+        <infon key="score">0.998437</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:21:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="76741" length="4"/>
+        <text>pore</text>
+      </annotation>
+      <annotation id="1760">
+        <infon key="score">0.9698333</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="76784" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1761">
+        <infon key="score">0.99626005</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="76795" length="5"/>
+        <text>shell</text>
+      </annotation>
+      <annotation id="1762">
+        <infon key="score">0.9989838</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="76851" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1763">
+        <infon key="score">0.9633987</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="76865" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1764">
+        <infon key="score">0.99898756</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:21:02Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="76892" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="1765">
+        <infon key="score">0.9991918</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="76907" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1766">
+        <infon key="score">0.98662937</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="76935" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1767">
+        <infon key="score">0.9967763</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:58:59Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="76946" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+      <annotation id="1768">
+        <infon key="score">0.9652231</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="76970" length="8"/>
+        <text>ferritin</text>
+      </annotation>
+      <annotation id="1769">
+        <infon key="score">0.99120015</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:46:47Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="76979" length="8"/>
+        <text>nanocage</text>
+      </annotation>
+      <annotation id="1770">
+        <infon key="score">0.85045284</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="77076" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1771">
+        <infon key="score">0.999037</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="77115" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="2054">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:18:27Z</infon>
+        <location offset="77125" length="11"/>
+        <text>presence of</text>
+      </annotation>
+      <annotation id="1772">
+        <infon key="score">0.79746157</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:56:23Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="77142" length="4"/>
+        <text>FOCs</text>
+      </annotation>
+      <annotation id="1773">
+        <infon key="score">0.99688536</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T13:14:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="77151" length="8"/>
+        <text>EncFtnsH</text>
+      </annotation>
+      <annotation id="1774">
+        <infon key="score">0.998533</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:53:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="77160" length="7"/>
+        <text>decamer</text>
+      </annotation>
+      <annotation id="1775">
+        <infon key="score">0.9973793</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:36:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="77190" length="11"/>
+        <text>icosahedral</text>
+      </annotation>
+      <annotation id="1776">
+        <infon key="score">0.9507636</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="77202" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1777">
+        <infon key="score">0.99152315</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:46:47Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="77213" length="8"/>
+        <text>nanocage</text>
+      </annotation>
+      <annotation id="1778">
+        <infon key="score">0.9986066</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="77247" length="9"/>
+        <text>decameric</text>
+      </annotation>
+      <annotation id="1779">
+        <infon key="score">0.9985454</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="77257" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1780">
+        <infon key="score">0.9988722</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="77355" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1781">
+        <infon key="score">0.99581426</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:59:04Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="77393" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+      <annotation id="1909">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:44:52Z</infon>
+        <location offset="77503" length="9"/>
+        <text>classical</text>
+      </annotation>
+      <annotation id="1782">
+        <infon key="score">0.99890363</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:28:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="77513" length="9"/>
+        <text>ferritins</text>
+      </annotation>
+      <annotation id="1783">
+        <infon key="score">0.37426853</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:04:29Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="77537" length="4"/>
+        <text>FOCs</text>
+      </annotation>
+      <annotation id="1784">
+        <infon key="score">0.9965545</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:46:47Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="77565" length="8"/>
+        <text>nanocage</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>77576</offset>
+      <text>Our structural data, coupled with biochemical and ICP-MS analysis, suggest a model for the activity of the encapsulin iron-megastore (Figure 14C). The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4—figure supplement 1). The five metal-binding sites on the interior of the ring (Glu31/34-sites) may select for the Fe2+ ion and direct it towards their cognate FOCs. We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by  in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).</text>
+      <annotation id="1785">
+        <infon key="score">0.9957445</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:21:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="77580" length="15"/>
+        <text>structural data</text>
+      </annotation>
+      <annotation id="1786">
+        <infon key="score">0.997255</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:21:25Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="77610" length="22"/>
+        <text>biochemical and ICP-MS</text>
+      </annotation>
+      <annotation id="1787">
+        <infon key="score">0.944305</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="77683" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1788">
+        <infon key="score">0.9900916</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:59:19Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="77694" length="14"/>
+        <text>iron-megastore</text>
+      </annotation>
+      <annotation id="1789">
+        <infon key="score">0.998469</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:29:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="77727" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="1790">
+        <infon key="score">0.99831057</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:50:39Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="77752" length="11"/>
+        <text>T. maritima</text>
+      </annotation>
+      <annotation id="1791">
+        <infon key="score">0.6409168</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="77764" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1792">
+        <infon key="score">0.8687022</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="77775" length="5"/>
+        <text>shell</text>
+      </annotation>
+      <annotation id="1793">
+        <infon key="score">0.9972558</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:21:34Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="77795" length="23"/>
+        <text>negatively charged pore</text>
+      </annotation>
+      <annotation id="1794">
+        <infon key="score">0.9991125</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:56:27Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="77854" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1795">
+        <infon key="score">0.8829711</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="77868" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1796">
+        <infon key="score">0.97599155</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:21:38Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="77922" length="23"/>
+        <text>negatively charged hole</text>
+      </annotation>
+      <annotation id="1797">
+        <infon key="score">0.9972698</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="77953" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1798">
+        <infon key="score">0.9992531</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:04:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="77960" length="4"/>
+        <text>ring</text>
+      </annotation>
+      <annotation id="1799">
+        <infon key="score">0.99906075</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:15:02Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="78006" length="19"/>
+        <text>metal-binding sites</text>
+      </annotation>
+      <annotation id="1800">
+        <infon key="score">0.99935883</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:04:49Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="78049" length="4"/>
+        <text>ring</text>
+      </annotation>
+      <annotation id="2159">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:21:55Z</infon>
+        <location offset="78055" length="14"/>
+        <text>Glu31/34-sites</text>
+      </annotation>
+      <annotation id="1801">
+        <infon key="score">0.9990451</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:21:58Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="78090" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1802">
+        <infon key="score">0.9981737</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:15:06Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="78135" length="4"/>
+        <text>FOCs</text>
+      </annotation>
+      <annotation id="1803">
+        <infon key="score">0.9990736</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:22:11Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="78174" length="4"/>
+        <text>Fe2+</text>
+      </annotation>
+      <annotation id="1804">
+        <infon key="score">0.9990438</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:22:13Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="78182" length="4"/>
+        <text>Fe3+</text>
+      </annotation>
+      <annotation id="1805">
+        <infon key="score">0.9898706</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="78205" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1806">
+        <infon key="score">0.9946569</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="78260" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1807">
+        <infon key="score">0.9892139</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:15:12Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="78274" length="14"/>
+        <text>substrate site</text>
+      </annotation>
+      <annotation id="1808">
+        <infon key="score">0.9989343</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="78303" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1809">
+        <infon key="score">0.9987573</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:15:39Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="78337" length="25"/>
+        <text>weakly coordinating sites</text>
+      </annotation>
+      <annotation id="1810">
+        <infon key="score">0.9995809</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:12:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="78406" length="5"/>
+        <text>His57</text>
+      </annotation>
+      <annotation id="1811">
+        <infon key="score">0.9995735</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:12:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="78413" length="5"/>
+        <text>Glu61</text>
+      </annotation>
+      <annotation id="1812">
+        <infon key="score">0.9995696</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T14:12:24Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="78423" length="5"/>
+        <text>Glu64</text>
+      </annotation>
+      <annotation id="1813">
+        <infon key="score">0.9990777</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:56:31Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="78456" length="12"/>
+        <text>ferrihydrite</text>
+      </annotation>
+      <annotation id="1814">
+        <infon key="score">0.9501573</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="78532" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="2188">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:03:16Z</infon>
+        <location offset="78543" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>78575</offset>
+      <text>Here we describe for the first time the structure and biochemistry of a new class of encapsulin-associated ferritin-like protein and demonstrate that it has an absolute requirement for compartmentalization within an encapsulin nanocage to act as an iron store. Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment.</text>
+      <annotation id="1815">
+        <infon key="score">0.99308926</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:22:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="78615" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="1816">
+        <infon key="score">0.9980296</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:22:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="78660" length="43"/>
+        <text>encapsulin-associated ferritin-like protein</text>
+      </annotation>
+      <annotation id="1817">
+        <infon key="score">0.9901558</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="78791" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1818">
+        <infon key="score">0.9713966</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:46:47Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="78802" length="8"/>
+        <text>nanocage</text>
+      </annotation>
+      <annotation id="1819">
+        <infon key="score">0.9989982</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="78824" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1820">
+        <infon key="score">0.99803776</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T12:00:08Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="78856" length="10"/>
+        <text>EncFtn-Enc</text>
+      </annotation>
+      <annotation id="1821">
+        <infon key="score">0.9541096</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:59:11Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="78867" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+      <annotation id="1822">
+        <infon key="score">0.99910176</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="78939" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1823">
+        <infon key="score">0.97536135</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="78956" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1824">
+        <infon key="score">0.99871886</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:33:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="78967" length="5"/>
+        <text>shell</text>
+      </annotation>
+      <annotation id="1825">
+        <infon key="score">0.99908686</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="78991" length="4"/>
+        <text>iron</text>
+      </annotation>
+      <annotation id="1826">
+        <infon key="score">0.9758664</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:44Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="79013" length="6"/>
+        <text>EncFtn</text>
+      </annotation>
+      <annotation id="1827">
+        <infon key="score">0.9930814</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:54:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="79020" length="3"/>
+        <text>FOC</text>
+      </annotation>
+      <annotation id="1828">
+        <infon key="score">0.99498945</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:30:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="79071" length="10"/>
+        <text>encapsulin</text>
+      </annotation>
+      <annotation id="1829">
+        <infon key="score">0.9556411</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T17:59:24Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="79082" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_1</infon>
+      <offset>79099</offset>
+      <text>Materials and methods</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>79121</offset>
+      <text>Cloning</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>79129</offset>
+      <text>Genes of interest were amplified by PCR using R. rubrum ATCC 11,170 genomic DNA (DSMZ) as the template and KOD Hot Start DNA Polymerase (Novagen). Primers used in this study are listed in Supplementary file 2. PCR products were visualized in 0.8% agarose gel stained with SYBR Safe (Life Technologies, UK). Fragments of interest were purified by gel extraction (Qiagen, UK) before digestion by endonuclease restriction enzymes (Thermo Fisher Scientific, UK) at 37°C for 1 hr, followed by ligation with similarly digested vector pET-28a(+) or pACYCDuet-1 at room temperature for 1 hr. Ligation product was transformed into chemically competent Escherichia coli Top10 cells and screened against 50 ng/μl kanamycin for pET-28a(+) based constructs or 34 ng/μl chloramphenicol for pACYCDuet-1 based constructs. DNA insertion was confirmed through Sanger sequencing (Edinburgh Genomics, The University of Edinburgh, UK). Sequence verified constructs were transformed into E. coli BL21(DE3) or Tuner(DE3) for protein production. Alternatively, plasmids transformed into E. coli B834(DE3) cells were cultured in selenomethionine medium.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>80264</offset>
+      <text>Protein production and purification</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>80300</offset>
+      <text>A single colony of E. coli BL21(DE3) or Tuner(DE3) cells, transformed with protein expression plasmid, was transferred into 10 ml LB medium, or M9 minimal medium (MM), supplemented with appropriate antibiotic, and incubated overnight at 37 °C with 200 rpm shaking. The overnight pre-culture was then inoculated into 1 liter of LB medium and incubated at 37 °C with 200 rpm shaking. Recombinant protein production was induced at OD600= 0.6 by the addition of 1 mM IPTG and the incubation temperature was reduced to 18°C for overnight incubation. Cells were pelleted by centrifugation at 4000 g for 20 min at 4 °C, and resuspended 10-fold (volume per gram of cell pellet) in PBS to wash cells before a second centrifugation step. Cells were resuspended in 10-times (v/w) of appropriate lysis buffer for the purification method used (see details of buffers below) and lysed by sonication on ice, with ten cycles of 30-second burst of sonication at 10 µm amplitude and 30 s of cooling. Cell lysate was clarified by centrifugation at 20,000 x g, 30 min, 4 °C; followed by filtration using a 0.22 µM syringe filter (Millipore, UK).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>81438</offset>
+      <text>Selenomethionine labelled protein was produced by growing a single colony of E. coli B834 (DE3) cells transformed with protein expression plasmids in 100 ml LB medium supplemented with appropriate antibiotic overnight at 37 °C with shaking at 200 rpm. The overnight pre-culture was pelleted by centrifugation 3,000 x g, 4 °C, 15 min and washed twice with M9 minimal medium. The washed cells were transferred to 1 liter of SeMet medium, which contains M9 minimal medium, 40 mg/L of each L-amino acid (without methionine), 40 mg/L selenomethionine, 2 mM MgSO4, 0.4% (w/v) glucose and 1 mM Fe(NH4)2(SO4)2. Cells were incubated at 37 °C with 200 rpm shaking and recombinant protein production was induced at OD600= 0.6 by the addition of 1 mM IPTG and the incubation temperature was reduced to 18 °C for overnight incubation. Cells were harvested and lysed as above.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>82309</offset>
+      <text>His-tagged protein purification</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>82341</offset>
+      <text>Clarified cell lysate was loaded onto a 5 ml HisTrap column (GE Healthcare, UK) pre-equilibrated with HisA buffer (50 mM Tris-HCl, 500 mM NaCl and 50 mM imidazole, pH 8.0). Unbound proteins were washed from the column with HisA buffer. His-tagged proteins were then eluted by a step gradient of 50% HisA buffer and 50% HisB buffer (50 mM Tris-HCl, 500 mM NaCl and 500 mM imidazole, pH 8.0). Fractions containing the protein of interest, as determined by 15% (w/v) acrylamide SDS-PAGE, were pooled before loading onto a gel-filtration column (HiLoad 16/600 Superdex 200, GE Healthcare) equilibrated with GF buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl). Fractions were subjected to 15% SDS-PAGE and those containing the protein of interest were pooled for further analysis.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>83115</offset>
+      <text>Sucrose gradient ultracentrifugation purification</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>83165</offset>
+      <text>Co-expressed encapsulin and EncFtn (EncFtn-Enc) and encapsulin protein were both purified according to the protocol used by M. Sutter. Briefly, EncFtn-Enc or encapsulin was expressed based on pACYCDuet-1 vector. The E. coli cells were grown, induced, harvested and sonicated in a similar way as described above. GF buffer used in this purification contains 50 mM Tris-HCl, pH 8.0, and 150 mM NaCl. To remove RNA contamination, the lysate was supplemented with 50 μg/ml RNase A and rotated at 10 rpm and room temperature for 2 hrs, followed by centrifugation at 34,000 x g and 4 °C for 20 min and filtering through 0.22 µM syringe filter. Proteins were pelleted through 38% (w/v) sucrose cushion by ultracentrifugation at 100,000 x g and 4 °C for 21 hrs. 10% - 50% (w/v) sucrose gradient ultracentrifugation was applied to further separate the proteins at 100,000 x g and 4 °C for 17 hrs. Protein was dialyzed against GF buffer to remove sucrose before being used in chemical assays or TEM.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>84163</offset>
+      <text>Transmission electron microscopy</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>84196</offset>
+      <text>TEM imaging was performed on purified encapsulin, EncFtn, and EncFtn-Enc and apoferritin. Purified protein at 0.1 mg/ml concentration was spotted on glow-discharged 300 mesh carbon-coated copper grids and excess liquid wicked off with filter paper (Whatman, UK). The grids were washed with distilled water and blotted with filter paper three times before staining with 0.2% uranyl acetate, blotting and air-drying. Grids were imaged using a JEM1400 transmission electron microscope and images were collected with a Gatan CCD camera. Images were analyzed using ImageJ (NIH, Bethesda, MD) and size-distribution histograms were plotted using Prism 6 (GraphPad software). To observe iron mineral formation by TEM, protein samples at 8.5 µM concentration including EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were supplemented with acidic Fe(NH4)2(SO4)2 at their maximum iron loading ratio in room temperature for 1 hr. The mixtures were subjected to TEM analysis with or without uranyl acetate staining. TEM experiments without Fe loading were repeated three times, a representative set of images are presented here. Proteins loaded with Fe and imaged by TEM were from single preparation.</text>
+      <annotation id="2163">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <location offset="84273" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+      <annotation id="2164">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <location offset="84993" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>85392</offset>
+      <text>Protein crystallization and X-ray data collection</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>85442</offset>
+      <text>EncFtnsH was purified by anion exchange and Superdex 200 size- exclusion chromatography and concentrated to 10 mg/ml (based on extinction coefficient calculation). Crystallization drops were set up using the hanging drop vapor diffusion method at 292 K. Glass coverslips were set up with 1–2 μl protein mixed with 1 μl well solution (0.14 M calcium acetate and 15% (w/v) PEG 3350) and sealed over 1 ml of well solution. Crystals appeared after 5 days and were harvested from the well using a LithoLoop (Molecular Dimensions Limited, UK), transferred briefly to a cryoprotection solution containing well solution supplemented with 1 mM FeSO4 (in 0.1% (v/v) HCl), 20% (v/v) PEG 200, and subsequently flash cooled in liquid nitrogen. Crystals of the EncFtnsHsingle mutations were produced in the same manner as for the EncFtnsH wild-type protein.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>86295</offset>
+      <text>All crystallographic datasets were collected on the macromolecular crystallography beamlines at Diamond Light Source (Didcot, UK) at 100 K using Pilatus 6M detectors. Diffraction data were integrated and scaled using XDS and symmetry related reflections were merged with Aimless . Data collection statistics are shown in Table 4. The resolution cut-off used for structure determination and refinement was determined based on the CC1/2 criterion proposed by.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>86754</offset>
+      <text>The structure of EncFtnsH was determined by molecular replacement using PDB ID: 3K6C as the search model, modified to match the sequence of the target protein using Chainsaw. A single solution comprising three decamers in the asymmetric unit was found by molecular replacement using Phaser. The initial model was rebuilt using Phenix.autobuild followed by cycles of refinement with Phenix.refine, with manual rebuilding and model inspection in Coot . The final model was refined with isotropic B-factors, torsional NCS restraints, and with anomalous group refinement. The model was validated using MolProbity. Structural superimpositions were calculated using Coot. Crystallographic figures were generated with PyMOL. Multiple sequence alignment of EncFtn and ferritin family proteins was performed using Clustal Omega Sievers and Higgins, 2014 and displayed with Espript 3.0. Model refinement statistics are shown in Table 4. The final models and experimental data are deposited in the PDB and diffraction image files are available at the Edinburgh DataShare repository.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>87828</offset>
+      <text>Horse spleen apoferritin preparation</text>
+      <annotation id="2165">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <location offset="87841" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>87865</offset>
+      <text>Horse spleen apoferritin purchased from Sigma Aldrich (UK) was dissolved in deaerated MOPS buffer (100 mM MOPS, 100 mM NaCl, 3 g/100 ml Na2S2O4 and 0.5 M EDTA, pH 6.5). Protein was dialyzed against 1 liter MOPS buffer in room temperature for two days before buffer exchanging to GF buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl) in a vivaspin column with 5 kDa cut-off (Sartorius, UK) for several times. Fe content of apoferritin was detected using ferrozine assay. Protein concentration was determined using Pierce Microplate BCA Protein Assay Kit. Apoferritin containing less than 0.5 Fe per 24-mer was used in the ferroxidase assay. Apoferritin used in the Fe loading capacity experiment was prepared in the same way with 5–15 Fe per 24-mer.</text>
+      <annotation id="2166">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <location offset="87878" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+      <annotation id="2167">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <location offset="88281" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+      <annotation id="2168">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <location offset="88413" length="11"/>
+        <text>Apoferritin</text>
+      </annotation>
+      <annotation id="2169">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <location offset="88499" length="11"/>
+        <text>Apoferritin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>88612</offset>
+      <text>Ferroxidase assay</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>88630</offset>
+      <text>1 mM and 200 µM Fe(NH4)2(SO4)2 stock solutions were prepared in 0.1% (v/v) HCl anaerobically. Protein solutions with 20 µM FOC were diluted from ~10 mg/ml frozen stock in GF buffer (50 mM Tris-HCl, pH 8.0 and 150 mM NaCl) anaerobically. Ferroxidase activity was initiated by adding 450 μl protein to 50 μl of acidic Fe(NH4)2(SO4)2 at the final concentration of 100 µM and 20 µM in the air, respectively. The ferroxidase activity was measured by monitoring the Fe3+ formation which gives rise to the change of the absorbance at 315 nm. Absorbance at 315 nm was recorded every second over 1800 s using a quartz cuvette in a JASCO V-730 UV/VIS spectrophotometer (JASCO Inc., Easton, MD). In recombinantly coexpressed nanocompartments the ratio of EncFtn to Enc was assumed as 2 to 1, assuming each of the twelve pentameric vertices of the icosahedral encapsulin were occupied with decameric EncFtn. The data are presented as the mean of three technical replicates with error bars indicating one standard deviation from the mean. Proteins used here were from a single preparation.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>89725</offset>
+      <text>Iron loading capacity of ferritins</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>89760</offset>
+      <text>In order to determine the maximum iron loading capacity, around 8.5 µM proteins including decameric EncFtnsH, Encapsulin, EncFtn-Enc and apoferritin were loaded with various amount of acidic Fe(NH4)2(SO4)2 ranging from 0 to 1700 µM. Protein mixtures were incubated in room temperature for 3 hrs before desalting in Zebra spin desalting columns (7 kDa cut-off, Thermo Fisher Scientific, UK) to remove free iron ions. The protein concentration was determined using PierceMicroplate BCA assay kit (Thermo Fisher Scientific). The protein standard curve was plotted according to the manufacturer. The Fe content in the samples was determined using modified ferrozine assay. Briefly speaking, 100 μl protein sample was mixed with 100 μl mixture of equal volume of 1.4 M HCl and 4.5% (w/v) KMnO4 and incubated at 60 °C for 2 hrs. 20 μl of the iron-detection reagent (6.5 mM ferrozine, 6.5 mM neocuproine, 2.5 M ammonium acetate, and 1 M ascorbic acid dissolved in H2O) was added to the cooled tubes. 30 min later, 200 μl of the solution was transferred into a well of 96-well plate and the absorbance at 562 nm was measured on the plate reader Spectramax M5 (Molecular Devices, UK). The standard curve was plotted using various concentrations of FeCl3 (in 10 mM HCl) diluted in the gel-filtration buffer. Three technical repeats were performed for both the ferrozine and microplate BCA assays. Samples analyzed by ICP-MS were prepared in the same way by mixing protein and ferrous ions and desalting. The proteins used in the Fe loading experiment came from a single preparation.</text>
+      <annotation id="2170">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <location offset="89897" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>91345</offset>
+      <text>Peroxidase assay</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>91362</offset>
+      <text>The peroxidase activity of EncFtnsH was determined by measuring the oxidation of ortho-phenylenediamine (OP) by H2O2 . EncFtnsH decameric and monomeric fractions purified from MM were both used in the assay. Ortho-phenylenediamine was prepared as a 92.5 mM stock solution in 50 mM Tris-HCl (pH 8.0). 80, 70, 60, 50, 40, 30, 20 and 10 mM of OP were prepared by diluting the stock solution in the 50 mM Tris-HCl (pH 8.0). 100 μl of each diluted OP was added to a 96-well plate in 3 repeats. 1 μl of 32 µM protein was supplemented into each well to a final concentration of 160 nM, followed by the addition of 2 μl of 30% H2O2. After 15 min shaking in the dark, the reaction was stopped by adding 100 μl of 0.5 M H2SO4. The peroxidase activity was measured by monitoring the absorbance at 490 nm in the SpectraMax M5 Microplate Reader (Molecular Devices).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>92223</offset>
+      <text>ICP-MS analysis</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>92239</offset>
+      <text>Protein samples were diluted 50-fold into a solution of 2.5% HNO3 (Suprapur, Merck, UK) containing 20 µg/L Pt as internal standard. Matrix-matched elemental standards (containing analyte metal concentrations 0 – 1000 µg/L) were prepared by serial dilution from individual metal standard stocks (VWR) with identical solution compositions, including the internal standard. All standards and samples were analyzed by ICP-MS using a Thermo x-series instrument (Thermo Fisher Scientific) operating in collision cell mode (using 3.0 ml min-1 flow of 8% H2 in He as the collision gas). Isotopes 44Ca, 56Fe, 66Zn, 78Se, and 195Pt were monitored using the peak-jump method (100 sweeps, 25–30 ms dwell time on 5 channels per isotope, separated by 0.02 atomic mass units) in triplicate. The protein samples used in ICP-MS came from a single protein preparation.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>93098</offset>
+      <text>Mass spectrometry analysis</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>93125</offset>
+      <text>For native MS analysis, all protein samples were buffer exchanged into 100 mM ammonium acetate (pH 8.0; adjusted with dropwise addition of 1% ammonia solution) using Micro Biospin Chromatography Columns (Bio-Rad, UK) prior to analysis and the resulting protein samples were analyzed at a final concentration of ~5 µM (oligomer concentration). In order to obtain Fe-bound EncFtn, 100 µM or 300 µM of freshly prepared FeCl2 was added to apo-EncFtnsH (monomer peak) immediately prior to buffer exchange into 100 mM ammonium acetate (pH 8.0). Samples were analyzed on a quadrupole ion-mobility time of flight instrument (Synapt G2, Waters Corp., Manchester, UK), equipped with a nanomate nanoelectrospray infusion robot (Advion Biosciences, Ithaca, NY). Instrument parameters were tuned to preserve non-covalent protein complexes. After optimization, typical parameters were: nanoelectrospray voltage 1.54 kV; sample cone 50 V; extractor cone 0 V; trap collision voltage 4 V; source temperature 80°C; and source backing pressure 5.5 mbar. For improved mass resolution the sample cone was raised to 155 V. Ion mobility mass spectrometry (IM-MS) was performed using the travelling-wave mobility cell in the Synapt G2, employing nitrogen as the drift gas. Typically, the IMS wave velocity was set to 300 m/s; wave height to 15 V; and the IMS pressure was 1.8 mbar. All native MS experiments were performed on samples from two independent protein preparations. For collision cross section determination, IM-MS data was calibrated using denatured equine myoglobin and data was analyzed using Driftscope v2.5 and MassLynx v4.1 (Waters Corp., UK). Theoretical collision cross sections (CCS) were calculated from pdb files using IMPACT software v. 0.9.1. In order to obtain information on the topology of the EncFtnsH assembly, gas-phase dissociation of the Fe-associated EncFtnsH complex was achieved by increasing the sample cone and/or trap collision voltage prior to MS analysis.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>95109</offset>
+      <text>SEC-MALLS</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>95119</offset>
+      <text>Size-exclusion chromatography (ÄKTA-Micro; GE Healthcare) coupled to UV, static light scattering and refractive index detection (Viscotec SEC-MALS 20 and Viscotec RI Detector:VE3580; Malvern Instruments, UK) were used to determine the molecular mass of fractions decamer and monomer of EncFtnsH in solution individually. Protein concentration was determined by measurement of absorbance at 280 nm and calculated using the extinction coefficient ε0.1%= 1.462 mg−1 ml-1 cm−1. 100 μl of 1.43 mgml-1 fractions of EncFtnsH decamer and 4.03 mg ml -1 fractions of EncFtnsH monomer were run individually on a Superdex 200 10/300 GL size-exclusion column pre-equilibrated in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl at 22°C with a flow rate of 0.5 ml/min. Light scattering, refractive index (RI) and A280nm were analyzed by a homo-polymer model (OmniSEC software, v 5.1; Malvern Instruments) using the following parameters for fractions of decamer and monomer: the extinction coefficient (dA/dc) at 280 nm was 1.46 AU mg ml−1 and specific refractive index increment (dn/dc) was 0.185 ml g−1. The proteins analyzed by SEC-MALLS came from single protein preparation.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>96289</offset>
+      <text>Metal binding analysis by PAGE</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>96320</offset>
+      <text>Recombinant EncFtnsH fractions at 50 µM concentration were incubated with one molar equivalent of metal ions at room temperature for 2 hrs. Half of each sample was mixed with 5 x native loading buffer (65 mM Tris-HCl, pH 8.5, 20% glycerol and 0.01% bromophenol blue) and run on non-denaturing PAGE gels (10% acrylamide) and run in Tris/glycine buffer, 200 V, 4 °C for 50 min. The remaining samples were left for an additional three hours prior to SDS-PAGE (15% acrylamide) analysis. SDS-PAGE gels were run at room temperature at 200 V, room temperature for 50 min. Gels were stained with Coomassie Brilliant Blue R250 and scanned after de-staining in water. The proteins used in this experiment came from single protein preparation.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>97057</offset>
+      <text>Analytical size-exclusion chromatography</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>97098</offset>
+      <text>For analysis of the multimeric state of EncFtn proteins by analytical size-exclusion gel-filtration chromatography (AGF) 25 μl of 90 µM protein was loaded into Superdex 200 PC 3.2/30 column (GE Healthcare) at 15 °C with GF buffer running at 0.05 ml/min and pressure limit 0.45 MPa. In order to use AGF to determine how metal ions influence the assembly of EncFtnsH, 90 µM EncFtnsH monomer fractions were mixed with equal molar concentrations of metal ion solutions including FeSO4 in 0.1% (v/v) HCl, Fe(NH4)2(SO4)2, FeCl3, CoCl2, calcium acetate (CaAc), ZnSO4 and MnCl2 at room temperature for 2 hrs prior to AGF analysis. Protein samples without metal titration were also analyzed as a control group. Both monomer and decamer fractions of EncFtnsH left at room temperature for 2 hrs, or overnight, were also analysed as controls to show the stability of the protein samples in the absence of additional metal ions. The AGF results have been repeated twice using two independent preparations of protein, of which only one representative trace is presented in the paper.</text>
+      <annotation id="2179">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:22:04Z</infon>
+        <location offset="97980" length="10"/>
+        <text>absence of</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>98177</offset>
+      <text>Accession codes and datasets</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>98206</offset>
+      <text>Coordinates and structure factors for the structures presented in this paper have been deposited in the PDB under the following accession codes: EncFtnsH, 5DA5; EncFtnsH-E32A, 5L89; EncFtnsH-E62A, 5L8B; EncFtnsH-H65A, 5L8G (DOIs for X-ray diffraction image data are shown in Table 4). All MS datasets presented in this paper can be found, in the raw format at http://dx.doi.org/10.7488/ds/1449.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">ACK_FUND</infon>
+      <infon key="type">title_1</infon>
+      <offset>98601</offset>
+      <text>Funding Information</text>
+    </passage>
+    <passage>
+      <infon key="section_type">ACK_FUND</infon>
+      <infon key="type">paragraph</infon>
+      <offset>98621</offset>
+      <text>This paper was supported by the following grants:</text>
+    </passage>
+    <passage>
+      <infon key="section_type">ACK_FUND</infon>
+      <infon key="type">paragraph</infon>
+      <offset>98671</offset>
+      <text> to Didi He.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">ACK_FUND</infon>
+      <infon key="type">paragraph</infon>
+      <offset>98684</offset>
+      <text> to Sam Hughes, Kirsten Altenbach, David J Clarke.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">ACK_FUND</infon>
+      <infon key="type">paragraph</infon>
+      <offset>98735</offset>
+      <text>  to Emma Tarrant, Kevin J Waldron.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">ACK_FUND</infon>
+      <infon key="type">paragraph</infon>
+      <offset>98771</offset>
+      <text>  to David J Clarke, Jon Marles-Wright.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">ACK_FUND</infon>
+      <infon key="type">paragraph</infon>
+      <offset>98811</offset>
+      <text>  to Jon Marles-Wright.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">ACK_FUND</infon>
+      <infon key="type">title_1</infon>
+      <offset>98835</offset>
+      <text>Additional information</text>
+    </passage>
+    <passage>
+      <infon key="section_type">COMP_INT</infon>
+      <infon key="type">title_1</infon>
+      <offset>98858</offset>
+      <text>Competing interests</text>
+    </passage>
+    <passage>
+      <infon key="section_type">COMP_INT</infon>
+      <infon key="type">footnote</infon>
+      <offset>98878</offset>
+      <text>The authors declare that no competing interests exist.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">title_1</infon>
+      <offset>98933</offset>
+      <text>Author contributions</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">footnote</infon>
+      <offset>98954</offset>
+      <text>DH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">footnote</infon>
+      <offset>99073</offset>
+      <text>SH, Acquisition of data, Analysis and interpretation of data.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">footnote</infon>
+      <offset>99135</offset>
+      <text>SV-H, Acquisition of data, Analysis and interpretation of data.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">footnote</infon>
+      <offset>99199</offset>
+      <text>AG, Acquisition of data, Drafting or revising the article.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">footnote</infon>
+      <offset>99258</offset>
+      <text>KA, Acquisition of data, Contributed unpublished essential data or reagents.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">footnote</infon>
+      <offset>99335</offset>
+      <text>ET, Acquisition of data, Analysis and interpretation of data.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">footnote</infon>
+      <offset>99397</offset>
+      <text>CLM, Acquisition of data, Contributed unpublished essential data or reagents.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">footnote</infon>
+      <offset>99475</offset>
+      <text>KJW, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">footnote</infon>
+      <offset>99572</offset>
+      <text>DJC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">footnote</infon>
+      <offset>99692</offset>
+      <text>JM-W, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">title_1</infon>
+      <offset>99813</offset>
+      <text>Additional files</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">title_2</infon>
+      <offset>99830</offset>
+      <text>Major datasets</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">paragraph</infon>
+      <offset>99845</offset>
+      <text>The following datasets were generated:</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REF</infon>
+      <infon key="type">title</infon>
+      <offset>99884</offset>
+      <text>References</text>
+    </passage>
+    <passage>
+      <infon key="fpage">276</infon>
+      <infon key="lpage">278</infon>
+      <infon key="name_0">surname:Aberg;given-names:A</infon>
+      <infon key="name_1">surname:Nordlund;given-names:P</infon>
+      <infon key="name_2">surname:Eklund;given-names:H</infon>
+      <infon key="pub-id_doi">10.1038/361276a0</infon>
+      <infon key="pub-id_pmid">8423856</infon>
+      <infon key="section_type">REF</infon>
+      <infon key="source">Nature</infon>
+      <infon key="type">ref</infon>
+      <infon key="volume">361</infon>
+      <infon key="year">1993</infon>
+      <offset>99895</offset>
+      <text>Unusual clustering of carboxyl side chains in the core of iron-free ribonucleotide reductase</text>
+    </passage>
+    <passage>
+      <infon key="fpage">213</infon>
+      <infon key="lpage">221</infon>
+      <infon key="name_0">surname:Adams;given-names:PD</infon>
+      <infon key="name_1">surname:Afonine;given-names:PV</infon>
+      <infon key="name_10">surname:McCoy;given-names:AJ</infon>
+      <infon key="name_11">surname:Moriarty;given-names:NW</infon>
+      <infon key="name_12">surname:Oeffner;given-names:R</infon>
+      <infon key="name_13">surname:Read;given-names:RJ</infon>
+      <infon key="name_14">surname:Richardson;given-names:DC</infon>
+      <infon key="name_15">surname:Richardson;given-names:JS</infon>
+      <infon key="name_16">surname:Terwilliger;given-names:TC</infon>
+      <infon key="name_17">surname:Zwart;given-names:PH</infon>
+      <infon key="name_2">surname:Bunkóczi;given-names:G</infon>
+      <infon key="name_3">surname:Chen;given-names:VB</infon>
+      <infon key="name_4">surname:Davis;given-names:IW</infon>
+      <infon key="name_5">surname:Echols;given-names:N</infon>
+      <infon key="name_6">surname:Headd;given-names:JJ</infon>
+      <infon key="name_7">surname:Hung;given-names:LW</infon>
+      <infon key="name_8">surname:Kapral;given-names:GJ</infon>
+      <infon key="name_9">surname:Grosse-Kunstleve;given-names:RW</infon>
+      <infon key="pub-id_doi">10.1107/S0907444909052925</infon>
+      <infon key="pub-id_pmid">20124702</infon>
+      <infon key="section_type">REF</infon>
+      <infon key="source">Acta Crystallographica Section D Biological Crystallography</infon>
+      <infon key="type">ref</infon>
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+    <passage>
+      <infon key="fpage">406</infon>
+      <infon key="lpage">425</infon>
+      <infon key="name_0">surname:Saitou;given-names:N</infon>
+      <infon key="name_1">surname:Nei;given-names:M</infon>
+      <infon key="pub-id_pmid">3447015</infon>
+      <infon key="section_type">REF</infon>
+      <infon key="source">Molecular Biology and Evolution</infon>
+      <infon key="type">ref</infon>
+      <infon key="volume">4</infon>
+      <infon key="year">1987</infon>
+      <offset>103868</offset>
+      <text>The neighbor-joining method: a new method for reconstructing phylogenetic trees</text>
+    </passage>
+    <passage>
+      <infon key="fpage">105</infon>
+      <infon key="lpage">116</infon>
+      <infon key="name_0">surname:Sievers;given-names:F</infon>
+      <infon key="name_1">surname:Higgins;given-names:DG</infon>
+      <infon key="pub-id_doi">10.1007/978-1-62703-646-7_6</infon>
+      <infon key="pub-id_pmid">24170397</infon>
+      <infon key="section_type">REF</infon>
+      <infon key="source">Methods in Molecular Biology</infon>
+      <infon key="type">ref</infon>
+      <infon key="volume">1079</infon>
+      <infon key="year">2014</infon>
+      <offset>103948</offset>
+      <text>Clustal Omega, accurate alignment of very large numbers of sequences</text>
+    </passage>
+    <passage>
+      <infon key="fpage">641</infon>
+      <infon key="lpage">643</infon>
+      <infon key="name_0">surname:Stein;given-names:N</infon>
+      <infon key="pub-id_doi">10.1107/S0021889808006985</infon>
+      <infon key="section_type">REF</infon>
+      <infon key="source">Journal of Applied Crystallography</infon>
+      <infon key="type">ref</infon>
+      <infon key="volume">41</infon>
+      <infon key="year">2008</infon>
+      <offset>104017</offset>
+      <text>CHAINSAW : a program for mutating pdb files used as templates in molecular replacement</text>
+    </passage>
+    <passage>
+      <infon key="fpage">939</infon>
+      <infon key="lpage">947</infon>
+      <infon key="name_0">surname:Sutter;given-names:M</infon>
+      <infon key="name_1">surname:Boehringer;given-names:D</infon>
+      <infon key="name_2">surname:Gutmann;given-names:S</infon>
+      <infon key="name_3">surname:Günther;given-names:S</infon>
+      <infon key="name_4">surname:Prangishvili;given-names:D</infon>
+      <infon key="name_5">surname:Loessner;given-names:MJ</infon>
+      <infon key="name_6">surname:Stetter;given-names:KO</infon>
+      <infon key="name_7">surname:Weber-Ban;given-names:E</infon>
+      <infon key="name_8">surname:Ban;given-names:N</infon>
+      <infon key="pub-id_doi">10.1038/nsmb.1473</infon>
+      <infon key="section_type">REF</infon>
+      <infon key="source">Nature Structural &amp; Molecular Biology</infon>
+      <infon key="type">ref</infon>
+      <infon key="volume">15</infon>
+      <infon key="year">2008</infon>
+      <offset>104104</offset>
+      <text>Structural basis of enzyme encapsulation into a bacterial nanocompartment</text>
+      <annotation id="2192">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:03:16Z</infon>
+        <location offset="104162" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">REF</infon>
+      <infon key="type">paragraph</infon>
+      <offset>104178</offset>
+      <text>10.7554/eLife.18972.048</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">title</infon>
+      <offset>104202</offset>
+      <text>Decision letter</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>104218</offset>
+      <text>Losick</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>104225</offset>
+      <text>Richard</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>104233</offset>
+      <text>In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>104517</offset>
+      <text>[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The previous decision letter after peer review is shown below.]</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>104711</offset>
+      <text>Thank you for submitting your work entitled "Structural characterisation of an encapsulated ferritin provides insight into iron storage in bacterial nanocompartments" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Losick as the Senior Editor. Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>105289</offset>
+      <text>In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently, and thus represent an interesting frontier in microbial cell biology. While this study significantly advances our understanding of the structural and biochemical relationship between encapsulins and EncFer, it requires significant revision prior to publication. We do, however, encourage the authors to resubmit when and if they are address the issues raised below.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>105888</offset>
+      <text>Reviewer #1: </text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>105902</offset>
+      <text>1) Methods: What procedures and analyses did the author use to assess whether the iron added to the various ferritin derivatives was protein coated or was simply balls of rust attached to protein fragments? If the latter, it could easily generate reactive oxygen species in air under physiological conditions.</text>
+      <annotation id="1885">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:35:28Z</infon>
+        <location offset="106158" length="6"/>
+        <text>oxygen</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>106212</offset>
+      <text>2) Results:</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>106224</offset>
+      <text>A) Critical data, such as the comparison of maximum amount of iron bound by a monomer in the dodocamer is in the Supplementary information.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>106364</offset>
+      <text>B) The data in Supplementary file 2, shows that the amount of iron bound by an ENCFTN decamer monomer is sub -stoichiometric, ranging from 0.18 to 0.64. In a bona fide ferritin, with ~ 2000 iron atoms/ protein cage (24 subunits), the same parameter is much, much higher.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>106635</offset>
+      <text>Even an experimental situation: 24 subunit (monomer) ferritin with a biomineral prepared experimentally from apoferritin and containing, on average, only 1000 iron atoms/24 subunit cage, the equivalent parameter appears to be 1000/24 = 42. This Fe/protein ratio is 66 times more iron than in the test system described. Moreover, in nature, some ferritin protein cages contain as much as 4500 Fe atoms, several hundred times higher than the test system. Thus the significance of the experimental results in the paper are unclear.</text>
+      <annotation id="2171">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <location offset="106744" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>107164</offset>
+      <text>3) Table 4: Data are shown for three proteins, Encapsulin, Enc-Ftn-10mer, and EncFTN-Enc. Missing are data for the starting material, 24 subunit ferritin or apoferritin (ferritin with the iron removed, by reduction and chelation, as a control.)</text>
+      <annotation id="2172">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <location offset="107321" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>107409</offset>
+      <text>Reviewer #2: </text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>107423</offset>
+      <text>In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently. While the structure of the encapsulin shell has been determined, that of its cargo, the ferritin-like protein (EncFer), has remained elusive. Here, the authors provide the structure of one such cargo and show that it assembles in a manner that is topologically distinct from ferritin. Additionally, the authors provide evidence that metal binding promotes the assembly of the EncFer and that it does act as a ferroxidase. Altogether, there is a substantial amount of work here that will likely be viewed as a major step forward in understanding these unique bacterial organelles. I have a few suggestions and questions that are listed below:</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>108298</offset>
+      <text>1) The authors grow E. coli in minimal media with and without added iron to show that assembly is iron dependent. The output of these experiments is the ratio of decamer vs. monomer. However, we don't have information on whether the growth conditions altered either the total amount of protein produced or the total amount soluble complex/monomer. Perhaps, lower protein concentrations lead to less efficient assembly (a critical concentration is needed).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>108754</offset>
+      <text>2) There is no information regarding the reason for the use of R. rubrum encapsulins. As far as I can tell, these have not been a model for either in vivo or in vitro work. Is there even evidence that they are produced by R. rubrum? What is their size/appearance in that organism? Do they have a physiological role?</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>109070</offset>
+      <text>3) Also, how similar are the Enc and EncFer to those of M. xanthus? Are the putative iron-binding sites conserved?</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>109185</offset>
+      <text>4) I would have liked to see some mutagenesis experiments to test the models of assembly, iron binding and ferroxidase activity. These do not have to be in vivo and can be performed in vitro with the available system.</text>
+      <annotation id="2155">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T15:12:22Z</infon>
+        <location offset="109219" length="11"/>
+        <text>mutagenesis</text>
+      </annotation>
+      <annotation id="1879">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T11:32:31Z</infon>
+        <location offset="109292" length="11"/>
+        <text>ferroxidase</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>109403</offset>
+      <text>5) I would like some more phylogenetic data for the model that ferritin evolved from EncFer. Perhaps, EncFer evolved from ferritin? Do any of the existing phylogenetic analyses support one model over another.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>109612</offset>
+      <text>10.7554/eLife.18972.049</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">title</infon>
+      <offset>109636</offset>
+      <text>Author response</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>109652</offset>
+      <text>[Editors’ note: the author responses to the first round of peer review follow.]</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>109734</offset>
+      <text>In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently, and thus represent an interesting frontier in microbial cell biology. While this study significantly advances our understanding of the structural and biochemical relationship between encapsulins and EncFer, it requires significant revision prior to publication. We do, however, encourage the authors to resubmit when and if they are address the issues raised below. </text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>110334</offset>
+      <text>Reviewer #1: </text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>110348</offset>
+      <text>1) Methods: What procedures and analyses did the author use to assess whether the iron added to the various ferritin derivatives was protein coated or was simply balls of rust attached to protein fragments? If the latter, it could easily generate reactive oxygen species in air under physiological conditions. </text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>110659</offset>
+      <text>The reviewer makes an excellent point here. To ascertain whether the iron in the assays forms ‘balls of rust’ we performed transmission electron microscopy on the ferroxidase reaction mixtures after completion of the reaction to assess the formation of free, or encapsulated iron minerals. We provide an additional supplemental figure (Figure 8—figure supplement 1) and discuss the observation of iron mineral crystals and nanoparticles in the main text, subsection “Ferroxidase activity”, last paragraph. We also attempted to use a commercial luminescence-based ROS detection kit on the reactions to address the possibility that H2O2 is produced as a reaction intermediate by the EncFtn protein. We found that the results from this particular kit were inconsistent between repeats, but for the benefit of the reviewer we provide a graph of the results obtained (see Author response image 1). These results show the production of ROS by apoferritin, which is consistent with the published data on the reaction mechanism of certain ferritins; however, no significant ROS were detected for the EncFtn or encapsulin proteins.</text>
+      <annotation id="2173">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <location offset="111596" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">elife-18972-resp-fig1.jpg</infon>
+      <infon key="id">fig15</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>111792</offset>
+      <text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.037</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>111839</offset>
+      <text>We acknowledge that the reaction mechanism of the EncFtn merits further investigation in a follow up study.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>111947</offset>
+      <text>2) Results:</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>111959</offset>
+      <text>A) Critical data, such as the comparison of maximum amount of iron bound by a monomer in the dodocamer is in the Supplementary information. </text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>112100</offset>
+      <text>We acknowledge that the data for iron loading merits inclusion in the main text, we have now moved this data and other supplementary data tables to the main text.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>112263</offset>
+      <text>B) The data in Supplementary file 2, shows that the amount of iron bound by an ENCFTN decamer monomer is sub -stoichiometric, ranging from 0.18 to 0.64. In a bona fide ferritin, with ~ 2000 iron atoms/ protein cage (24 subunits), the same parameter is much, much higher. </text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>112535</offset>
+      <text>One of the central arguments of our paper is the fact that the EncFtnsH monomer must dimerize to produce a functional ferroxidase active site and that its iron binding properties are highly divergent from those of the classical ferritin nanocages. We have added additional text to the manuscript to highlight these differences (Introduction, last paragraph, and Mass spectrometry section) and discuss the functional consequences at length.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>112975</offset>
+      <text>Even an experimental situation: 24 subunit (monomer) ferritin with a biomineral prepared experimentally from apoferritin and containing, on average, only 1000 iron atoms/24 subunit cage, the equivalent parameter appears to be 1000/24 = 42. This Fe/protein ratio is 66 times more iron than in the test system described. Moreover, in nature, some ferritin protein cages contain as much as 4500 Fe atoms, several hundred times higher than the test system! Thus the significance of the experimental results in the paper are unclear. </text>
+      <annotation id="2174">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <location offset="113084" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>113505</offset>
+      <text>We have clarified this key difference in the discussion of the iron storage function of the encapsulin nanocompartment (subsection “Iron storage in encapsulin nanocompartments”, second paragraph). The key conclusion of the paper is that the iron storage and iron oxidation functions that are combined in classical ferritins are split between the encapsulin nanocompartment and the EncFtn protein.</text>
+      <annotation id="2193">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:03:16Z</infon>
+        <location offset="113608" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
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+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:03:16Z</infon>
+        <location offset="113862" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>113906</offset>
+      <text>3) Table 4: Data are shown for three proteins, Encapsulin, Enc-Ftn-10mer, and EncFTN-Enc. Missing are data for the starting material, 24 subunit ferritin or apoferritin (ferritin with the iron removed, by reduction and chelation, as a control.) </text>
+      <annotation id="2175">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <location offset="114063" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>114152</offset>
+      <text>The data for the starting material are shown in Table 5. Control data for apoferritin have been added to this table and are illustrated in Figure 8. We note that we do not reach the experimental maximum loading capacity for apoferritin; however, we also note that the EncFtn-encapsulin nanocompartment sequesters five times more iron than the ferritin under the same reaction conditions, supporting the published observations that these nanocompartments can store more iron than classical ferritin nanocages.</text>
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+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <location offset="114226" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
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+        <infon key="type">protein_state</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T16:21:26Z</infon>
+        <location offset="114376" length="11"/>
+        <text>apoferritin</text>
+      </annotation>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T18:03:16Z</infon>
+        <location offset="114438" length="15"/>
+        <text>nanocompartment</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>114661</offset>
+      <text>Reviewer #2: </text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>114675</offset>
+      <text>In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently. While the structure of the encapsulin shell has been determined, that of its cargo, the ferritin-like protein (EncFer), has remained elusive. Here, the authors provide the structure of one such cargo and show that it assembles in a manner that is topologically distinct from ferritin. Additionally, the authors provide evidence that metal binding promotes the assembly of the EncFer and that it does act as a ferroxidase. Altogether, there is a substantial amount of work here that will likely be viewed as a major step forward in understanding these unique bacterial organelles. I have a few suggestions and questions that are listed below: </text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>115551</offset>
+      <text>1) The authors grow E. coli in minimal media with and without added iron to show that assembly is iron dependent. The output of these experiments is the ratio of decamer vs. monomer. However, we don't have information on whether the growth conditions altered either the total amount of protein produced or the total amount soluble complex/monomer. Perhaps, lower protein concentrations lead to less efficient assembly (a critical concentration is needed). </text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>116008</offset>
+      <text>The reviewer makes an interesting point about growth conditions and we acknowledge that production of the protein in LB medium leads to varying protein yields and monomer/decamer proportions. We therefore adopted the use of M9 minimal medium throughout the study to give better reproducibility, which also enables better control of metal ion availability than the complex LB medium. Given the fact that the protein is produced recombinantly in E. coli it is not particularly instructive to prove the in vivoproduction of the EncFtn multimer in this host. We have added a panel to Figure 3 to show the effect of protein concentration on multimerization in vitro(Figure 3C). Our mass spectrometry results show that the protein spontaneously multimerized in the presence of iron in vitroto form decameric species and that this is metal ion concentration dependent (Figure 7).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>116881</offset>
+      <text>2) There is no information regarding the reason for the use of R. rubrum encapsulins. As far as I can tell, these have not been a model for either in vivo or in vitro work. Is there even evidence that they are produced by R. rubrum? What is their size/appearance in that organism? Do they have a physiological role? </text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>117198</offset>
+      <text>We have put a comment in the Introduction to introduce R. rubrum (last paragraph). A preliminary study in the laboratory identified encapsulins in a preparation of lipid vesicles from R. rubrum containing chromatophores. We chose to follow up on these structures in this study. We do not feel this particular information is key to the central argument of the paper.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>117564</offset>
+      <text>3) Also, how similar are the Enc and EncFer to those of M. xanthus? Are the putative iron-binding sites conserved? </text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>117680</offset>
+      <text>We have noted this in the Introduction of the manuscript.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>117738</offset>
+      <text>4) I would have liked to see some mutagenesis experiments to test the models of assembly, iron binding and ferroxidase activity. These do not have to be in vivo and can be performed in vitro with the available system. </text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>117957</offset>
+      <text>To address this question we have produced three FOC mutants of the EncFtn protein and characterized these in solution, by mass spectrometry, and crystallographically (section: Mutagenesis of the EncFtnsH Ferroxidase center). We thank the reviewer for this suggestion as it highlighted the importance of the FOC residues for assembly and activity, and our new data has provided interesting insights into the EncFtn protein.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>118380</offset>
+      <text>5) I would like some more phylogenetic data for the model that ferritin evolved from EncFer. Perhaps, EncFer evolved from ferritin? Do any of the existing phylogenetic analyses support one model over another. </text>
+    </passage>
+    <passage>
+      <infon key="section_type">REVIEW_INFO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>118590</offset>
+      <text>We now include a phylogenetic tree (Figure 13) and consider the question of ferritin evolution in the Discussion (first paragraph). None of the authors of this study are evolutionary biologists but we appreciate the difficulty inherent in tracing the history of protein folds, especially in bacterial lineages. We refer to previous studies in this section and make a suggestion that can be followed up in subsequent studies.</text>
+    </passage>
+  </document>
+</collection>
diff --git a/annotated_BioC_XML/PMC5014086_ann.xml b/annotated_BioC_XML/PMC5014086_ann.xml
new file mode 100644
index 0000000000000000000000000000000000000000..337ef2b38d9e09b7b45a7ec563a29d6ed76962b0
--- /dev/null
+++ b/annotated_BioC_XML/PMC5014086_ann.xml
@@ -0,0 +1,7047 @@
+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection>
+  <source>PMC</source>
+  <date>20201220</date>
+  <key>pmc.key</key>
+  <document>
+    <id>5014086</id>
+    <infon key="license">CC BY</infon>
+    <infon key="tt_curatable">no</infon>
+    <infon key="tt_version">2</infon>
+    <infon key="tt_round">2</infon>
+    <passage>
+      <infon key="article-id_doi">10.1016/j.str.2016.06.020</infon>
+      <infon key="article-id_pmc">5014086</infon>
+      <infon key="article-id_pmid">27524201</infon>
+      <infon key="article-id_publisher-id">S0969-2126(16)30167-8</infon>
+      <infon key="fpage">1599</infon>
+      <infon key="issue">9</infon>
+      <infon key="license">This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</infon>
+      <infon key="lpage">1605</infon>
+      <infon key="name_0">surname:Zebisch;given-names:Matthias</infon>
+      <infon key="name_1">surname:Jackson;given-names:Verity A.</infon>
+      <infon key="name_2">surname:Zhao;given-names:Yuguang</infon>
+      <infon key="name_3">surname:Jones;given-names:E. Yvonne</infon>
+      <infon key="notes">Published: August 11, 2016</infon>
+      <infon key="section_type">TITLE</infon>
+      <infon key="type">front</infon>
+      <infon key="volume">24</infon>
+      <infon key="year">2016</infon>
+      <offset>0</offset>
+      <text>Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf</text>
+      <annotation id="1">
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+        <infon key="type">evidence</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="0" length="9"/>
+        <text>Structure</text>
+      </annotation>
+      <annotation id="505">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:32Z</infon>
+        <location offset="27" length="3"/>
+        <text>Wnt</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:04:18Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="41" length="7"/>
+        <text>Kremen1</text>
+      </annotation>
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+        <infon key="score">0.9978811</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:04:27Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="97" length="4"/>
+        <text>LRP6</text>
+      </annotation>
+      <annotation id="4">
+        <infon key="score">0.99473995</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:10:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="106" length="8"/>
+        <text>Dickkopf</text>
+      </annotation>
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+    <passage>
+      <infon key="section_type">ABSTRACT</infon>
+      <infon key="type">abstract_title_1</infon>
+      <offset>115</offset>
+      <text>Summary</text>
+    </passage>
+    <passage>
+      <infon key="section_type">ABSTRACT</infon>
+      <infon key="type">abstract</infon>
+      <offset>123</offset>
+      <text>Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling. We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains. The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor. We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD. DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC. Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.</text>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="123" length="14"/>
+        <text>Kremen 1 and 2</text>
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+        <infon key="type">protein_type</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>co-receptors</text>
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+        <text>Dickkopf</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:10:08Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="189" length="3"/>
+        <text>Dkk</text>
+      </annotation>
+      <annotation id="504">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:31Z</infon>
+        <location offset="247" length="3"/>
+        <text>Wnt</text>
+      </annotation>
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+        <infon key="score">0.9986756</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:08:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="284" length="18"/>
+        <text>crystal structures</text>
+      </annotation>
+      <annotation id="10">
+        <infon key="score">0.9993445</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:05:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="310" length="10"/>
+        <text>ectodomain</text>
+      </annotation>
+      <annotation id="11">
+        <infon key="score">0.99843925</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:05:25Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="324" length="5"/>
+        <text>human</text>
+      </annotation>
+      <annotation id="12">
+        <infon key="score">0.9983398</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:04:18Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="330" length="7"/>
+        <text>Kremen1</text>
+      </annotation>
+      <annotation id="474">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:06:10Z</infon>
+        <location offset="339" length="4"/>
+        <text>KRM1</text>
+      </annotation>
+      <annotation id="476">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:06:25Z</infon>
+        <location offset="343" length="3"/>
+        <text>ECD</text>
+      </annotation>
+      <annotation id="477">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:06:40Z</infon>
+        <location offset="386" length="4"/>
+        <text>KRM1</text>
+      </annotation>
+      <annotation id="478">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:06:49Z</infon>
+        <location offset="390" length="3"/>
+        <text>ECD</text>
+      </annotation>
+      <annotation id="13">
+        <infon key="score">0.7506454</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:59:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="460" length="22"/>
+        <text>triangular arrangement</text>
+      </annotation>
+      <annotation id="14">
+        <infon key="score">0.9995679</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:07:03Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="490" length="7"/>
+        <text>Kringle</text>
+      </annotation>
+      <annotation id="15">
+        <infon key="score">0.9994825</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:07:08Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="499" length="3"/>
+        <text>WSC</text>
+      </annotation>
+      <annotation id="482">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:07:23Z</infon>
+        <location offset="508" length="3"/>
+        <text>CUB</text>
+      </annotation>
+      <annotation id="16">
+        <infon key="score">0.99839205</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:56:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="536" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="17">
+        <infon key="score">0.9995127</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:07:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="585" length="3"/>
+        <text>WSC</text>
+      </annotation>
+      <annotation id="18">
+        <infon key="score">0.84306806</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:54:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="599" length="24"/>
+        <text>hepatocyte growth factor</text>
+      </annotation>
+      <annotation id="506">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:32Z</infon>
+        <location offset="705" length="3"/>
+        <text>Wnt</text>
+      </annotation>
+      <annotation id="528">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:17:09Z</infon>
+        <location offset="709" length="11"/>
+        <text>co-receptor</text>
+      </annotation>
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+        <infon key="score">0.9976937</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:37:55Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="721" length="6"/>
+        <text>Lrp5/6</text>
+      </annotation>
+      <annotation id="20">
+        <infon key="score">0.9320832</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:10:08Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="729" length="3"/>
+        <text>Dkk</text>
+      </annotation>
+      <annotation id="21">
+        <infon key="score">0.7082609</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:12:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="738" length="3"/>
+        <text>Krm</text>
+      </annotation>
+      <annotation id="22">
+        <infon key="score">0.96125305</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:08:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="784" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="493">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:11:48Z</infon>
+        <location offset="810" length="36"/>
+        <text>β-propeller/EGF repeats (PE) 3 and 4</text>
+      </annotation>
+      <annotation id="507">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:32Z</infon>
+        <location offset="854" length="3"/>
+        <text>Wnt</text>
+      </annotation>
+      <annotation id="529">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:17:09Z</infon>
+        <location offset="858" length="11"/>
+        <text>co-receptor</text>
+      </annotation>
+      <annotation id="23">
+        <infon key="score">0.99893945</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:04:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="870" length="4"/>
+        <text>LRP6</text>
+      </annotation>
+      <annotation id="491">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:10:58Z</infon>
+        <location offset="876" length="4"/>
+        <text>LRP6</text>
+      </annotation>
+      <annotation id="492">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+      <text>Zebisch et al. describe the ectodomain structure of KREMEN 1, a receptor for Wnt antagonists of the DKK family. Apo structures and a complex with functional fragments of DKK1 and LRP6 shed light on the function of this dual-mode regulator of Wnt signaling.</text>
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+        <infon key="updated_at">2023-09-18T15:15:03Z</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="updated_at">2023-09-18T15:15:48Z</infon>
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+      <text>Introduction</text>
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+      <text>Signaling by Wnt morphogens is renowned for its fundamental roles in embryonic development, tissue homeostasis, and stem cell maintenance. Due to these functions, generation, delivery, and interpretation of Wnt signals are all heavily regulated in the animal body. Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6. Kremen proteins (Krm1 and Krm2) have been identified as additional high-affinity transmembrane receptors for Dkk. Krm and Dkk synergize in Wnt inhibition during Xenopus embryogenesis to regulate anterior-posterior patterning. Mechanistically it is thought that, in the presence of Dkk, Krm forms a ternary complex with Lrp6, which is then rapidly endocytosed. This amplifies the intrinsic Wnt antagonistic activity of Dkk by efficiently depleting the cell surface of the Wnt co-receptor. In accordance with this, Krm1−/− and Krm2−/− double knockout mice show a high bone mass phenotype typical of increased Wnt signaling, as well as growth of ectopic forelimb digits. Growth of ectopic digits is further enhanced upon additional loss of dkk expression. The Wnt antagonistic activity of Krm1 is also linked to its importance for correct thymus epithelium formation in mice. The importance of intact KRM1 for normal human development and health is highlighted by the recent finding that a homozygous mutation in the ectodomain of KRM1 leads to severe ectodermal dysplasia including oligodontia. Interestingly, the Wnt antagonistic activity of Krm is context dependent, and Krm proteins are actually dual-mode Wnt regulators. In the absence of Dkk, Krm1 and 2 change their function from inhibition to enhancement of Lrp6-mediated signaling. By direct binding to Lrp6 via the ectodomains, Krm proteins promote Lrp6 cell-surface localization and hence increase receptor availability. Further increasing the complexity of Krm functionality, it was recently found that Krm1 (but not Krm2) can also act independently of LRP5/6 and Wnt as a dependence receptor, triggering apoptosis unless bound to Dkk.</text>
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+        <infon key="identifier">PR:</infon>
+        <location offset="3743" length="6"/>
+        <text>LRP5/6</text>
+      </annotation>
+      <annotation id="95">
+        <infon key="score">0.5520213</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3754" length="3"/>
+        <text>Wnt</text>
+      </annotation>
+      <annotation id="96">
+        <infon key="score">0.99863183</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:59:36Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3812" length="8"/>
+        <text>bound to</text>
+      </annotation>
+      <annotation id="97">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:10:08Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3821" length="3"/>
+        <text>Dkk</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>3834</offset>
+      <text>Structurally, Krm1 and 2 are type I transmembrane proteins with a 40 kDa ectodomain and a flexible cytoplasmic tail consisting of 60–75 residues. The ectodomain consists of three similarly sized structural domains of around 10 kDa each: the N-terminal Kringle domain (KR) is followed by a WSC domain of unknown fold. The third structural domain is a CUB domain. An approximately 70-residue linker connects the CUB domain to the transmembrane span. An intact KR-WSC-CUB domain triplet and membrane attachment is required for Wnt antagonism. The transmembrane span and cytoplasmic tail can be replaced with a GPI linker without impact on Wnt antagonism.</text>
+      <annotation id="475">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:42:04Z</infon>
+        <location offset="3848" length="4"/>
+        <text>Krm1</text>
+      </annotation>
+      <annotation id="541">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:42:15Z</infon>
+        <location offset="3857" length="1"/>
+        <text>2</text>
+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:53:48Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3863" length="29"/>
+        <text>type I transmembrane proteins</text>
+      </annotation>
+      <annotation id="99">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:05:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="3907" length="10"/>
+        <text>ectodomain</text>
+      </annotation>
+      <annotation id="100">
+        <infon key="score">0.9392079</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:59:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>flexible</text>
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+      <annotation id="101">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:42:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="3933" length="16"/>
+        <text>cytoplasmic tail</text>
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+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:42:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3964" length="2"/>
+        <text>60</text>
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+      <annotation id="103">
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+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:42:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3967" length="2"/>
+        <text>75</text>
+      </annotation>
+      <annotation id="104">
+        <infon key="score">0.99932384</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:05:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="3984" length="10"/>
+        <text>ectodomain</text>
+      </annotation>
+      <annotation id="479">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:07:04Z</infon>
+        <location offset="4086" length="7"/>
+        <text>Kringle</text>
+      </annotation>
+      <annotation id="105">
+        <infon key="score">0.9994307</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:18:39Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4102" length="2"/>
+        <text>KR</text>
+      </annotation>
+      <annotation id="106">
+        <infon key="score">0.9988023</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:07:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4123" length="3"/>
+        <text>WSC</text>
+      </annotation>
+      <annotation id="533">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:18:34Z</infon>
+        <location offset="4184" length="3"/>
+        <text>CUB</text>
+      </annotation>
+      <annotation id="107">
+        <infon key="score">0.6716569</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T10:08:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>approximately 70-residue</text>
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+      <annotation id="108">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:44:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4224" length="6"/>
+        <text>linker</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:18:33Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4244" length="3"/>
+        <text>CUB</text>
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+      <annotation id="110">
+        <infon key="score">0.9992559</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:44:30Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4262" length="18"/>
+        <text>transmembrane span</text>
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+      <annotation id="111">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:59:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4285" length="6"/>
+        <text>intact</text>
+      </annotation>
+      <annotation id="542">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:43:37Z</infon>
+        <location offset="4292" length="10"/>
+        <text>KR-WSC-CUB</text>
+      </annotation>
+      <annotation id="517">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:32Z</infon>
+        <location offset="4358" length="3"/>
+        <text>Wnt</text>
+      </annotation>
+      <annotation id="112">
+        <infon key="score">0.999244</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:44:30Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4378" length="18"/>
+        <text>transmembrane span</text>
+      </annotation>
+      <annotation id="113">
+        <infon key="score">0.9991549</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:43:12Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4401" length="16"/>
+        <text>cytoplasmic tail</text>
+      </annotation>
+      <annotation id="543">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:44:08Z</infon>
+        <location offset="4441" length="3"/>
+        <text>GPI</text>
+      </annotation>
+      <annotation id="544">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:44:20Z</infon>
+        <location offset="4445" length="6"/>
+        <text>linker</text>
+      </annotation>
+      <annotation id="518">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:32Z</infon>
+        <location offset="4470" length="3"/>
+        <text>Wnt</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>4490</offset>
+      <text>We sought to provide structural insights into the multi-functionality of this cell-surface receptor. The structures presented here reveal the unknown fold of the WSC domain and the tight interactions of all three domains. We further succeeded in determination of a low-resolution LRP6PE3PE4-DKK1CRD2-KRM1ECD complex, defining the architecture of the Wnt inhibitory complex that leads to Lrp6 cell-surface depletion.</text>
+      <annotation id="114">
+        <infon key="score">0.9981673</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:44:35Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4595" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="115">
+        <infon key="score">0.9994917</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:07:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4652" length="3"/>
+        <text>WSC</text>
+      </annotation>
+      <annotation id="116">
+        <infon key="score">0.99919</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:45:00Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="4770" length="27"/>
+        <text>LRP6PE3PE4-DKK1CRD2-KRM1ECD</text>
+      </annotation>
+      <annotation id="117">
+        <infon key="score">0.5890333</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4840" length="3"/>
+        <text>Wnt</text>
+      </annotation>
+      <annotation id="118">
+        <infon key="score">0.6935159</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T10:08:21Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="4844" length="18"/>
+        <text>inhibitory complex</text>
+      </annotation>
+      <annotation id="119">
+        <infon key="score">0.99836403</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:04:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4877" length="4"/>
+        <text>Lrp6</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_1</infon>
+      <offset>4907</offset>
+      <text>Results</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>4915</offset>
+      <text>The recombinant production of the extracellular domain of Krm for structural studies proved challenging (see Experimental Procedures). We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown). Several crystal forms were obtained from these complexes, however, crystals always contained only KRM1 protein.</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:45:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4949" length="20"/>
+        <text>extracellular domain</text>
+      </annotation>
+      <annotation id="121">
+        <infon key="score">0.97918665</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:53:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4973" length="3"/>
+        <text>Krm</text>
+      </annotation>
+      <annotation id="545">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:45:21Z</infon>
+        <location offset="4981" length="18"/>
+        <text>structural studies</text>
+      </annotation>
+      <annotation id="546">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:46:05Z</infon>
+        <location offset="5076" length="4"/>
+        <text>KRM1</text>
+      </annotation>
+      <annotation id="547">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:46:16Z</infon>
+        <location offset="5080" length="3"/>
+        <text>ECD</text>
+      </annotation>
+      <annotation id="122">
+        <infon key="score">0.72899675</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:45:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5084" length="14"/>
+        <text>complexes with</text>
+      </annotation>
+      <annotation id="123">
+        <infon key="score">0.85313314</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:46:23Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="5099" length="6"/>
+        <text>DKK1fl</text>
+      </annotation>
+      <annotation id="548">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:46:42Z</infon>
+        <location offset="5107" length="4"/>
+        <text>DKK1</text>
+      </annotation>
+      <annotation id="549">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:47:00Z</infon>
+        <location offset="5111" length="11"/>
+        <text>Linker-CRD2</text>
+      </annotation>
+      <annotation id="550">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:47:15Z</infon>
+        <location offset="5128" length="4"/>
+        <text>DKK1</text>
+      </annotation>
+      <annotation id="551">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:47:24Z</infon>
+        <location offset="5132" length="4"/>
+        <text>CRD2</text>
+      </annotation>
+      <annotation id="124">
+        <infon key="score">0.96971107</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:47:29Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5174" length="14"/>
+        <text>gel filtration</text>
+      </annotation>
+      <annotation id="125">
+        <infon key="score">0.99780023</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:47:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5262" length="13"/>
+        <text>crystal forms</text>
+      </annotation>
+      <annotation id="126">
+        <infon key="score">0.9982065</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:47:36Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5321" length="8"/>
+        <text>crystals</text>
+      </annotation>
+      <annotation id="127">
+        <infon key="score">0.99675816</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:06:10Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="5352" length="4"/>
+        <text>KRM1</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>5366</offset>
+      <text>We solved the structure of KRM1ECD in three crystal forms at 1.9, 2.8, and 3.2 Å resolution (Table 1). The high-resolution structure is a near full-length model (Figure 1). The small, flexible, and charged 98AEHED102 loop could only be modeled in a slightly lower resolution structure and in crystal form III. The KR, WSC, and CUB are arranged in a roughly triangular fashion with tight interactions between all three domains. The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements. The structurally most similar Kringle domain is that of human plasminogen (PDB: 1PKR) with an root-mean-square deviation (RMSD) of 1.7 Å for 73 aligned Cα (Figure 1B). The KRM1 structure reveals the fold of the WSC domain for the first time. The structure is best described as a sandwich of a β1-β5-β3-β4-β2 antiparallel β sheet and a single α helix. The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198). Using the PDBeFold server, we detected a surprising yet significant homology to PAN module domains. The closest structural relative is hepatocyte growth factor (HGF, PDB: 1GP9), which superposes with an RMSD of 2.3 Å for 58 aligned Cα (Figure 1B). The CUB domain bears two glycosylation sites. Although present, the quality of the electron density around N217 did not allow modeling of the sugar moiety. In crystal form I, a calcium ion is present at the canonical position coordinated by the carboxylates of D263, D266 (bidentate), and D306, as well as the carbonyl of N309 and a water molecule. The coordination sphere deviates significantly from perfectly octahedral (not shown). This might result in the site having a low affinity and may explain why calcium is not present in the two low-resolution crystal forms. Loss of calcium has led to loop rearrangements and partial disorder in these crystal forms. The closest structural relative is the CUB_C domain of Tsg-6 (PDB: 2WNO), which superposes with KRMCUB with an RMSD of 1.6 Å for 104 Cα (Figure 1B).</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:54:11Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:54:24Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="8027" length="9"/>
+        <text>interface</text>
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:59:37Z</infon>
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+        <text>bound to</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:06:10Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8148" length="4"/>
+        <text>KRM1</text>
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+      <annotation id="519">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:32Z</infon>
+        <location offset="8184" length="3"/>
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+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:32Z</infon>
+        <location offset="8202" length="3"/>
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+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:32Z</infon>
+        <location offset="8223" length="3"/>
+        <text>Wnt</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>8251</offset>
+      <text>Low-Resolution Insight into Ternary Complex Formation</text>
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+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>8305</offset>
+      <text>Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands. One face of the rather flat DKK1CRD2 fragment binds to the third β propeller of LRP6. Mutational analyses further implied that the LRP6PE3-averted face of DKK1CRD2 bears the Krm binding site, hence suggesting how Dkk can recruit both receptors into a ternary complex.</text>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="8305" length="18"/>
+        <text>Co-crystallization</text>
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+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:55:01Z</infon>
+        <location offset="8327" length="4"/>
+        <text>LRP6</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:55:12Z</infon>
+        <location offset="8331" length="6"/>
+        <text>PE3PE4</text>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:55:27Z</infon>
+        <location offset="8343" length="4"/>
+        <text>DKK1</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:55:37Z</infon>
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+        <infon key="updated_at">2023-09-19T08:55:52Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="8395" length="4"/>
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:32Z</infon>
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+        <infon key="updated_at">2023-09-18T15:10:08Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:59:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8507" length="4"/>
+        <text>flat</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:56:40Z</infon>
+        <location offset="8512" length="4"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:56:50Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T10:00:11Z</infon>
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+        <text>binds to</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:56:21Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>third β propeller</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <text>LRP6</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>Mutational analyses</text>
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+        <infon key="type">protein</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>Krm binding site</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>Dkk</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <location offset="8718" length="9"/>
+        <text>receptors</text>
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+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>8755</offset>
+      <text>To obtain direct insight into ternary complex formation by Lrp5/6, Dkk, and Krm, we subjected an LRP6PE3PE4-DKK1fl-KRM1ECD complex to crystallization trials. Diffraction data collected from the resulting crystals were highly anisotropic with diffraction extending in the best directions to 3.5 Å and 3.7 Å but only to 6.4 Å in the third direction. Despite the lack of high-resolution diffraction, the general architecture of the ternary complex is revealed (Figure 2A). DKK1CRD2 binds to the top face of the LRP6 PE3 β propeller as described earlier for the binary complex. KRM1ECD does indeed bind on the opposite side of DKK1CRD2 with only its KR and WSC domains engaged in binding (Figure 2A). Although present in the complex subjected to crystallization, we observe no density that could correspond to CRD1 or the domain linker (L). We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure 2B). A SUMO fusion of DKK1L-CRD2 displayed a similar (slightly higher) affinity. In contrast, a SUMO fusion of DKK1CRD1-L did not display binding for concentrations tested up to 325 μM (Figure 2B).</text>
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+        <infon key="identifier">MESH:</infon>
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+        <text>Krm</text>
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:00:46Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="8852" length="25"/>
+        <text>LRP6PE3PE4-DKK1fl-KRM1ECD</text>
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+        <location offset="8889" length="22"/>
+        <text>crystallization trials</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>Diffraction data</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>crystals</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:01:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>diffraction</text>
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+        <location offset="9225" length="4"/>
+        <text>DKK1</text>
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+        <infon key="updated_at">2023-09-19T09:01:49Z</infon>
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+        <text>CRD2</text>
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T10:00:12Z</infon>
+        <location offset="9234" length="8"/>
+        <text>binds to</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:04:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9263" length="4"/>
+        <text>LRP6</text>
+      </annotation>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:18:50Z</infon>
+        <location offset="9268" length="3"/>
+        <text>PE3</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:57:24Z</infon>
+        <location offset="9272" length="11"/>
+        <text>β propeller</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:02:28Z</infon>
+        <location offset="9329" length="4"/>
+        <text>KRM1</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:02:38Z</infon>
+        <location offset="9333" length="3"/>
+        <text>ECD</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T10:00:33Z</infon>
+        <location offset="9349" length="7"/>
+        <text>bind on</text>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:02:01Z</infon>
+        <location offset="9378" length="4"/>
+        <text>DKK1</text>
+      </annotation>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:02:13Z</infon>
+        <location offset="9382" length="4"/>
+        <text>CRD2</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:18:40Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9401" length="2"/>
+        <text>KR</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:07:09Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>WSC</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T10:03:24Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>crystallization</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:57:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9528" length="7"/>
+        <text>density</text>
+      </annotation>
+      <annotation id="226">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:58:29Z</infon>
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+        <location offset="9561" length="4"/>
+        <text>CRD1</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <text>domain linker</text>
+      </annotation>
+      <annotation id="228">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:03:09Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>L</text>
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+        <infon key="type">structure_element</infon>
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+        <infon key="updated_at">2023-09-18T15:05:16Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:56:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9620" length="4"/>
+        <text>DKK1</text>
+      </annotation>
+      <annotation id="231">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:06:10Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9667" length="4"/>
+        <text>KRM1</text>
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+      <annotation id="232">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:04:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9676" length="25"/>
+        <text>surface plasmon resonance</text>
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+      <annotation id="233">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:04:40Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>SPR</text>
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+      <annotation id="234">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:04:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>affinity</text>
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+        <infon key="updated_at">2023-09-19T08:48:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>full-length</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <text>DKK1</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:03:59Z</infon>
+        <location offset="9786" length="4"/>
+        <text>KRM1</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:04:09Z</infon>
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+        <text>ECD</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:04:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9809" length="11"/>
+        <text>SUMO fusion</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:03:44Z</infon>
+        <location offset="9824" length="10"/>
+        <text>DKK1L-CRD2</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:04:31Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9873" length="8"/>
+        <text>affinity</text>
+      </annotation>
+      <annotation id="239">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:04:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9898" length="11"/>
+        <text>SUMO fusion</text>
+      </annotation>
+      <annotation id="240">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:05:18Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9913" length="10"/>
+        <text>DKK1CRD1-L</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>10014</offset>
+      <text>Overall, the DKK1-KRM1 interface is characterized by a large number of polar interactions but only few hydrophobic contacts (Figure 2C). The crystal structure gives an explanation for DKK1 loss-of-binding mutations identified previously: R191 of DKK1 forms a double salt bridge to D125 and E162 of KRM1 (Figure 2C). A charge reversal as in the mouse Dkk1 (mDkk1) R197E variant would severely disrupt the binding. Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90. Again, a charge reversal as shown before for mDkk1 K232E would be incompatible with binding. The side chain of DKK1 S192 was also predicted to be involved in Krm binding. Indeed, we found (Figure 2C) that the side chain of D201 of KRM1 forms two hydrogen bonds to the side-chain hydroxyl and the backbone amide of S192 (mouse, S198). Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively. The carbonyl of DKK1 R224 is hydrogen bonded to Y105 and W106 of KRM1. We suspect that the Dkk charge reversal mutations performed in the murine background and shown to diminish Krm binding K211E and R203E (mouse K217E and R209E) do so likely indirectly by disruption of the Dkk fold. We further validated the DKK1 binding site on KRM1 by introducing glycosylation sites at the KR (90DVS92→NVS) and WSC (189VCF191→NCS) domains pointing toward DKK (Figures 2A and 2D). Introduction of N-linked glycans in protein-protein-binding sites is an established way of disrupting protein-binding interfaces. Both ectodomain mutants were secreted comparably with the wild-type, indicating correct folding, but failed to achieve any detectable binding in SPR using full-length DKK1 as analyte. In contrast, a mutant carrying an additional N-glycan outside the interface at the CUB domain (309NQA311→NQS), was wild-type-like in DKK1 binding (Figure 2D).</text>
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+        <infon key="identifier">SO:</infon>
+        <location offset="10027" length="19"/>
+        <text>DKK1-KRM1 interface</text>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="updated_at">2023-09-19T09:05:51Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:08:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>crystal structure</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:56:12Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="10312" length="4"/>
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+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:11:06Z</infon>
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+        <infon key="updated_at">2023-09-19T09:08:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="updated_at">2023-09-19T09:56:31Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:07:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10370" length="5"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:07:25Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:56:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10461" length="4"/>
+        <text>DKK1</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="updated_at">2023-09-18T15:06:10Z</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="updated_at">2023-09-19T09:08:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">residue_name_number</infon>
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+        <infon key="updated_at">2023-09-19T09:08:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10558" length="4"/>
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+        <infon key="updated_at">2023-09-19T09:05:59Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <infon key="updated_at">2023-09-18T15:06:10Z</infon>
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+        <infon key="updated_at">2023-09-19T09:09:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="updated_at">2023-09-19T09:09:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>D90</text>
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:11:12Z</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:07:14Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10670" length="5"/>
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+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:07:29Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10676" length="5"/>
+        <text>K232E</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:56:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10736" length="4"/>
+        <text>DKK1</text>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:09:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10741" length="4"/>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:06:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10783" length="3"/>
+        <text>Krm</text>
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+      <annotation id="272">
+        <infon key="score">0.99949014</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:09:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10848" length="4"/>
+        <text>D201</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:06:10Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10856" length="4"/>
+        <text>KRM1</text>
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+        <infon key="type">bond_interaction</infon>
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+        <infon key="updated_at">2023-09-19T09:06:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10871" length="14"/>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:09:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10939" length="4"/>
+        <text>S192</text>
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+      <annotation id="276">
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+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:08:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10945" length="5"/>
+        <text>mouse</text>
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+      <annotation id="277">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:09:28Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10952" length="4"/>
+        <text>S198</text>
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+      <annotation id="278">
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+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:05:48Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10970" length="18"/>
+        <text>polar interactions</text>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:09:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11012" length="4"/>
+        <text>N140</text>
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+      <annotation id="280">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:09:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+      <annotation id="281">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:09:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11028" length="4"/>
+        <text>Y165</text>
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+      <annotation id="282">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:06:10Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11048" length="4"/>
+        <text>KRM1</text>
+      </annotation>
+      <annotation id="283">
+        <infon key="score">0.9987478</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:56:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11057" length="4"/>
+        <text>DKK1</text>
+      </annotation>
+      <annotation id="284">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:09:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11084" length="4"/>
+        <text>W206</text>
+      </annotation>
+      <annotation id="285">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:09:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11090" length="4"/>
+        <text>L190</text>
+      </annotation>
+      <annotation id="286">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:09:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11100" length="4"/>
+        <text>C189</text>
+      </annotation>
+      <annotation id="287">
+        <infon key="score">0.9989812</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:56:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11136" length="4"/>
+        <text>DKK1</text>
+      </annotation>
+      <annotation id="288">
+        <infon key="score">0.9995389</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:10:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11141" length="4"/>
+        <text>R224</text>
+      </annotation>
+      <annotation id="289">
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+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:07:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11149" length="15"/>
+        <text>hydrogen bonded</text>
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+        <location offset="12035" length="4"/>
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+      <text>Identification of a Direct LRP6-KRM1 Binding Site</text>
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+        <text>LRP6-KRM1 Binding Site</text>
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+      <text>The LRP6PE3PE4-DKK1CRD2-KRM1ECD complex structure reveals no direct interactions between KRM1 and LRP6. We constructed in silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data. An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A).</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <location offset="12224" length="4"/>
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:15:14Z</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="12306" length="10"/>
+        <text>ectodomain</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:13:50Z</infon>
+        <location offset="12318" length="12"/>
+        <text>PE1PE2PE3PE4</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:14:01Z</infon>
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+        <text>horse shoe</text>
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+        <infon key="updated_at">2023-09-19T09:13:15Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12385" length="19"/>
+        <text>electron microscopy</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>EM</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:13:23Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>small-angle X-ray scattering</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:13:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12461" length="6"/>
+        <text>PE3PE4</text>
+      </annotation>
+      <annotation id="338">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:13:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12481" length="12"/>
+        <text>superimposed</text>
+      </annotation>
+      <annotation id="339">
+        <infon key="score">0.99630725</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:18:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12498" length="3"/>
+        <text>PE4</text>
+      </annotation>
+      <annotation id="340">
+        <infon key="score">0.99660873</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:18:50Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12507" length="3"/>
+        <text>PE3</text>
+      </annotation>
+      <annotation id="341">
+        <infon key="score">0.99758327</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:08:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12518" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="589">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:14:17Z</infon>
+        <location offset="12545" length="4"/>
+        <text>LRP6</text>
+      </annotation>
+      <annotation id="590">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:14:30Z</infon>
+        <location offset="12549" length="6"/>
+        <text>PE1PE2</text>
+      </annotation>
+      <annotation id="342">
+        <infon key="score">0.9973882</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:13:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12556" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="343">
+        <infon key="score">0.9988292</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:13:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12570" length="12"/>
+        <text>superimposed</text>
+      </annotation>
+      <annotation id="344">
+        <infon key="score">0.99678135</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:25:11Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12587" length="3"/>
+        <text>PE2</text>
+      </annotation>
+      <annotation id="345">
+        <infon key="score">0.99711955</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:18:50Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12596" length="3"/>
+        <text>PE3</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>12642</offset>
+      <text>For this crude approximation of a true ternary complex, we noted very close proximity between the Ca2+-binding region of KRM1 and the top face of the PE2 β propeller of LRP6. The solvent-exposed residues R307, I308, and N309 of the central Ca2+-binding β connection loop of KRM1 would be almost ideally positioned for binding to this face, which is commonly used as a binding site on β propellers. Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure 3B). To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment. Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2 (Figure 3C). In contrast, no saturable binding was observed between KRM1 and LRP6PE3PE4. Introduction of an N-glycosylation site at N309 in KRM1ECD abolished LRP6PE1PE2 binding (Figure 3C), while binding to DKK1 was unaffected (Figure 2D). We conclude that the predicted binding site between KRM1CUB and LRP6PE2 is a strong candidate for mediating the direct Lrp6-Krm interaction, which is thought to increase Wnt responsiveness by stabilizing Lrp6 at the cell surface. Further experiments are required to pinpoint the exact binding site. Although LRP6PE1 appears somewhat out of reach in the modeled ternary complex, it cannot be excluded as the Krm binding site in the ternary complex and LRP6-Krm binary complex. The presence of DKK may govern which propeller (PE1 versus PE2) of LRP6 is available for Krm binding.</text>
+      <annotation id="346">
+        <infon key="score">0.99889165</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:14:38Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12740" length="19"/>
+        <text>Ca2+-binding region</text>
+      </annotation>
+      <annotation id="347">
+        <infon key="score">0.70204115</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:06:10Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12763" length="4"/>
+        <text>KRM1</text>
+      </annotation>
+      <annotation id="594">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:25:10Z</infon>
+        <location offset="12792" length="3"/>
+        <text>PE2</text>
+      </annotation>
+      <annotation id="595">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:25:23Z</infon>
+        <location offset="12796" length="11"/>
+        <text>β propeller</text>
+      </annotation>
+      <annotation id="348">
+        <infon key="score">0.99911326</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:04:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12811" length="4"/>
+        <text>LRP6</text>
+      </annotation>
+      <annotation id="667">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T10:04:32Z</infon>
+        <location offset="12821" length="15"/>
+        <text>solvent-exposed</text>
+      </annotation>
+      <annotation id="349">
+        <infon key="score">0.9994591</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:14:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12846" length="4"/>
+        <text>R307</text>
+      </annotation>
+      <annotation id="350">
+        <infon key="score">0.99941707</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:14:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12852" length="4"/>
+        <text>I308</text>
+      </annotation>
+      <annotation id="351">
+        <infon key="score">0.99950254</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:14:56Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12862" length="4"/>
+        <text>N309</text>
+      </annotation>
+      <annotation id="591">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:15:38Z</infon>
+        <location offset="12882" length="30"/>
+        <text>Ca2+-binding β connection loop</text>
+      </annotation>
+      <annotation id="352">
+        <infon key="score">0.8115768</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:06:10Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12916" length="4"/>
+        <text>KRM1</text>
+      </annotation>
+      <annotation id="353">
+        <infon key="score">0.998543</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:15:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13010" length="12"/>
+        <text>binding site</text>
+      </annotation>
+      <annotation id="354">
+        <infon key="score">0.99863666</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:15:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13026" length="12"/>
+        <text>β propellers</text>
+      </annotation>
+      <annotation id="355">
+        <infon key="score">0.99799347</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:15:50Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13060" length="8"/>
+        <text>arginine</text>
+      </annotation>
+      <annotation id="356">
+        <infon key="score">0.997482</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:15:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13069" length="6"/>
+        <text>lysine</text>
+      </annotation>
+      <annotation id="357">
+        <infon key="score">0.99793506</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:15:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13077" length="10"/>
+        <text>isoleucine</text>
+      </annotation>
+      <annotation id="358">
+        <infon key="score">0.99770665</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:15:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13093" length="10"/>
+        <text>asparagine</text>
+      </annotation>
+      <annotation id="359">
+        <infon key="score">0.9350921</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T10:07:14Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13124" length="13"/>
+        <text>N-X-I-(G)-R/K</text>
+      </annotation>
+      <annotation id="360">
+        <infon key="score">0.99911493</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:56:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="13160" length="4"/>
+        <text>DKK1</text>
+      </annotation>
+      <annotation id="361">
+        <infon key="score">0.999201</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:16:15Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="13169" length="4"/>
+        <text>SOST</text>
+      </annotation>
+      <annotation id="362">
+        <infon key="score">0.99911827</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:04:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="13185" length="4"/>
+        <text>LRP6</text>
+      </annotation>
+      <annotation id="363">
+        <infon key="score">0.9990943</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:58:40Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13201" length="11"/>
+        <text>propeller 1</text>
+      </annotation>
+      <annotation id="596">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:25:38Z</infon>
+        <location offset="13257" length="4"/>
+        <text>KRM1</text>
+      </annotation>
+      <annotation id="597">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:25:48Z</infon>
+        <location offset="13261" length="3"/>
+        <text>CUB</text>
+      </annotation>
+      <annotation id="665">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T10:00:12Z</infon>
+        <location offset="13265" length="8"/>
+        <text>binds to</text>
+      </annotation>
+      <annotation id="598">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:26:02Z</infon>
+        <location offset="13274" length="4"/>
+        <text>LRP6</text>
+      </annotation>
+      <annotation id="599">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:26:14Z</infon>
+        <location offset="13278" length="3"/>
+        <text>PE2</text>
+      </annotation>
+      <annotation id="364">
+        <infon key="score">0.99864656</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:04:41Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="13291" length="3"/>
+        <text>SPR</text>
+      </annotation>
+      <annotation id="365">
+        <infon key="score">0.99910635</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:12:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13323" length="9"/>
+        <text>wild-type</text>
+      </annotation>
+      <annotation id="366">
+        <infon key="score">0.9700352</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:27:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13341" length="15"/>
+        <text>GlycoCUB mutant</text>
+      </annotation>
+      <annotation id="600">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:26:33Z</infon>
+        <location offset="13360" length="4"/>
+        <text>KRM1</text>
+      </annotation>
+      <annotation id="601">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:26:50Z</infon>
+        <location offset="13364" length="3"/>
+        <text>ECD</text>
+      </annotation>
+      <annotation id="367">
+        <infon key="score">0.9965527</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:16:02Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13380" length="20"/>
+        <text>N-glycosylation site</text>
+      </annotation>
+      <annotation id="368">
+        <infon key="score">0.9995345</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:14:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13404" length="4"/>
+        <text>N309</text>
+      </annotation>
+      <annotation id="602">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:27:18Z</infon>
+        <location offset="13426" length="4"/>
+        <text>LRP6</text>
+      </annotation>
+      <annotation id="603">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:27:28Z</infon>
+        <location offset="13430" length="6"/>
+        <text>PE1PE2</text>
+      </annotation>
+      <annotation id="369">
+        <infon key="score">0.9990541</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:24:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13476" length="10"/>
+        <text>absence of</text>
+      </annotation>
+      <annotation id="370">
+        <infon key="score">0.9986665</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:10:08Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="13487" length="3"/>
+        <text>Dkk</text>
+      </annotation>
+      <annotation id="604">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:27:42Z</infon>
+        <location offset="13492" length="4"/>
+        <text>KRM1</text>
+      </annotation>
+      <annotation id="605">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:27:53Z</infon>
+        <location offset="13496" length="3"/>
+        <text>ECD</text>
+      </annotation>
+      <annotation id="371">
+        <infon key="score">0.9928445</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T10:01:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13500" length="5"/>
+        <text>bound</text>
+      </annotation>
+      <annotation id="372">
+        <infon key="score">0.9828691</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T10:01:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13533" length="2"/>
+        <text>to</text>
+      </annotation>
+      <annotation id="606">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:28:11Z</infon>
+        <location offset="13536" length="4"/>
+        <text>LRP6</text>
+      </annotation>
+      <annotation id="607">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:28:21Z</infon>
+        <location offset="13540" length="6"/>
+        <text>PE1PE2</text>
+      </annotation>
+      <annotation id="373">
+        <infon key="score">0.9926576</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:32Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:58:53Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14352" length="3"/>
+        <text>Krm</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>14374</offset>
+      <text>Apparent binding across the proposed KRM1CUB-LRP6PE2 interface is expected to be higher once Krm is also cross-linked to LRP6PE3 via DKK1CRD2 (Figure 3D). Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3. It is therefore possible that the interplay of Krm and Dkk binding can promote changes in LRP6 ectodomain conformation with functional consequences; however, such ideas await investigation.</text>
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+        <infon key="identifier">SO:</infon>
+        <location offset="14411" length="25"/>
+        <text>KRM1CUB-LRP6PE2 interface</text>
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+        <infon key="type">protein_type</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>Krm</text>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:32:33Z</infon>
+        <location offset="14495" length="4"/>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:32:43Z</infon>
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+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:32:57Z</infon>
+        <location offset="14507" length="4"/>
+        <text>DKK1</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:33:06Z</infon>
+        <location offset="14511" length="4"/>
+        <text>CRD2</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:32:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14544" length="17"/>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:13:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14566" length="28"/>
+        <text>small-angle X-ray scattering</text>
+      </annotation>
+      <annotation id="624">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:33:25Z</infon>
+        <location offset="14606" length="4"/>
+        <text>LRP6</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:33:35Z</infon>
+        <location offset="14610" length="12"/>
+        <text>PE1PE2PE3PE4</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:31:44Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14624" length="12"/>
+        <text>in isolation</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:31:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14641" length="15"/>
+        <text>in complex with</text>
+      </annotation>
+      <annotation id="396">
+        <infon key="score">0.9985501</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:33:12Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14657" length="4"/>
+        <text>Dkk1</text>
+      </annotation>
+      <annotation id="397">
+        <infon key="score">0.99890447</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:32:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14668" length="17"/>
+        <text>negative-stain EM</text>
+      </annotation>
+      <annotation id="398">
+        <infon key="score">0.9991421</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:48:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14689" length="11"/>
+        <text>full-length</text>
+      </annotation>
+      <annotation id="399">
+        <infon key="score">0.999263</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:04:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="14701" length="4"/>
+        <text>LRP6</text>
+      </annotation>
+      <annotation id="400">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:05:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="14706" length="10"/>
+        <text>ectodomain</text>
+      </annotation>
+      <annotation id="401">
+        <infon key="score">0.99760216</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T10:01:44Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14733" length="6"/>
+        <text>curved</text>
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+      <annotation id="402">
+        <infon key="score">0.9706499</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T10:01:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14741" length="13"/>
+        <text>platform-like</text>
+      </annotation>
+      <annotation id="403">
+        <infon key="score">0.99944097</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:25:11Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="14808" length="3"/>
+        <text>PE2</text>
+      </annotation>
+      <annotation id="404">
+        <infon key="score">0.9994288</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:18:50Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="14816" length="3"/>
+        <text>PE3</text>
+      </annotation>
+      <annotation id="405">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:31:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14868" length="3"/>
+        <text>Krm</text>
+      </annotation>
+      <annotation id="406">
+        <infon key="score">0.5479226</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:10:08Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14876" length="3"/>
+        <text>Dkk</text>
+      </annotation>
+      <annotation id="407">
+        <infon key="score">0.9992786</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:04:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="14911" length="4"/>
+        <text>LRP6</text>
+      </annotation>
+      <annotation id="408">
+        <infon key="score">0.9993906</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:05:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="14916" length="10"/>
+        <text>ectodomain</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>15012</offset>
+      <text>Taken together, the structural and biophysical studies we report here extend our mechanistic understanding of Wnt signal regulation. We describe the ectodomain structure of the dual Wnt regulator Krm1, providing an explanation for the detrimental effect on health and development of a homozygous KRM1 mutation. We also reveal the interaction mode of Krm-Dkk and the architecture of the ternary complex formed by Lrp5/6, Dkk, and Krm. Furthermore, the ternary crystal structure has guided in silico and biophysical analyses to suggest a direct LRP6-KRM1 interaction site. Our findings provide a solid foundation for additional studies to probe how ternary complex formation triggers internalization, whereas Krm binding in the absence of Dkk stabilizes the Wnt co-receptor at the cell surface.</text>
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+        <infon key="identifier">MESH:</infon>
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+        <text>structural and biophysical studies</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:32Z</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <text>ectodomain</text>
+      </annotation>
+      <annotation id="411">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:57:37Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>structure</text>
+      </annotation>
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:32Z</infon>
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+        <text>Wnt</text>
+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:33:57Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>Krm1</text>
+      </annotation>
+      <annotation id="413">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:06:10Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="15308" length="4"/>
+        <text>KRM1</text>
+      </annotation>
+      <annotation id="626">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:34:21Z</infon>
+        <location offset="15362" length="7"/>
+        <text>Krm-Dkk</text>
+      </annotation>
+      <annotation id="414">
+        <infon key="score">0.9881404</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:34:07Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15424" length="6"/>
+        <text>Lrp5/6</text>
+      </annotation>
+      <annotation id="415">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:10:08Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15432" length="3"/>
+        <text>Dkk</text>
+      </annotation>
+      <annotation id="416">
+        <infon key="score">0.9982216</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:34:29Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15441" length="3"/>
+        <text>Krm</text>
+      </annotation>
+      <annotation id="417">
+        <infon key="score">0.9973328</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:08:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15471" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="418">
+        <infon key="score">0.9021877</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:33:45Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15500" length="34"/>
+        <text>in silico and biophysical analyses</text>
+      </annotation>
+      <annotation id="419">
+        <infon key="score">0.97991484</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:34:38Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="15555" length="26"/>
+        <text>LRP6-KRM1 interaction site</text>
+      </annotation>
+      <annotation id="420">
+        <infon key="score">0.833017</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:34:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15719" length="3"/>
+        <text>Krm</text>
+      </annotation>
+      <annotation id="421">
+        <infon key="score">0.9991188</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:24:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15738" length="10"/>
+        <text>absence of</text>
+      </annotation>
+      <annotation id="422">
+        <infon key="score">0.9983999</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:10:08Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15749" length="3"/>
+        <text>Dkk</text>
+      </annotation>
+      <annotation id="526">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:32Z</infon>
+        <location offset="15768" length="3"/>
+        <text>Wnt</text>
+      </annotation>
+      <annotation id="532">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:17:09Z</infon>
+        <location offset="15772" length="11"/>
+        <text>co-receptor</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_1</infon>
+      <offset>15806</offset>
+      <text>Experimental Procedures</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>15830</offset>
+      <text>Large-Scale Mammalian Expression and Protein Purification</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>15888</offset>
+      <text>KrmECD fragments were cloned into pHLsec or variants thereof. Full ectodomain variants (e.g., KRM1 isoform 3, P30-T377) were well secreted into the conditioned medium (CM) of HEK293T cells, but exhibited extensive O-glycosylation (as judged from smeary bands in western blot), which would be detrimental to crystallization. Fragments truncated to the KR-WSC-CUB core gave sharp bands but were barely secreted. We therefore engineered an A23-G373 (isoform 1 numbering used throughout the article) full ectodomain construct (KRM1ECD-TEV) with a C-terminal His10 tag that contained a TEV protease cleavage site after E324. The expected sequence of the secreted protein is ETG-23APSPGLGPGPE31 … 320AVKEE324-GSENLYFQGGS-325LPQ … VPG373-THHHHHHHHHH (the isoform-2-specific PG insertion and the TEV site are underlined). This construct was well secreted and could be processed using TEV protease. However, 80%–90% of the protein eluted as aggregates from a size-exclusion column even before TEV treatment. The same applied to analog constructs for Krm1 from zebrafish, frog, and mouse. No monomeric protein at all could be obtained for several Krm2 constructs from multiple species. A KRM1ECD-TEV expressing stable GntI-deficient HEK293S cell line was generated by excision of an EcoRI-XhoI fragment, sub-cloning into pNeo-Sec-1, and selection of neomycin-resistant cells. The stable cell line showed expression levels superior to transiently transfected cells (not shown).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>17365</offset>
+      <text>Human LRP6PE1PE2, LRP6PE3PE4, and full-length DKK1 were produced in a similar way as described. Shorter constructs of DKK1 lacking the N-terminal flexible region and CRD1 were not secreted from HEK cells. However, using the approach of an N-terminal fusion to a modified SUMO protein as described earlier, we succeeded in secretory expression of a SUMO-DKK1Linker-CRD2 construct encompassing residues S141-H266. A variant of this containing a TEV cleavage site just before T181, SUMO-DKK1Linker-TEV-CRD2, was also well expressed and allowed removal of the flexible linker region.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>17945</offset>
+      <text>To obtain complexes of KRM1ECD-TEV, we (co-)transfected the stable cell line with DKK and LRP6PE3PE4 constructs described earlier. Binary and ternary KRM1ECD-DKK1fl and KRM1ECD-DKK1fl-LRP6PE3PE4 complexes were stable in gel-filtration eluting as distinct monodisperse peaks.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>18220</offset>
+      <text>Crystallization and Data Collection</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>18256</offset>
+      <text>All samples subjected to crystallization were purified from CM by affinity and size-exclusion chromatography. After treatment with TEV protease and endoglycosidase F1 overnight using mass equivalents of 1%, samples were subjected to size-exclusion chromatography in 10 mM HEPES/NaOH (pH 7.5), 150 mM NaCl. The crystals giving rise to the 1.9 Å dataset for KRM1 in crystal form I were obtained from a KRM1ECD-DKK1Linker-CRD2 complex concentrated to 12 mg/mL. Out of this complex, KRM1ECD crystallized alone in 2.0 M ammonium sulfate, 5% (v/v) iso-propanol. For cryoprotection, crystals were transferred to mother liquor mixed 1:1 with 3.4 M sodium malonate (pH 7.0). The slightly less well-ordered crystal of crystal form I and crystals of form II were obtained from a KRM1ECD-DKK1CRD2 complex using the SUMO-DKK1Linker-TEV-CRD2 construct and releasing SUMO and the DKK linker region by TEV and 3C protease treatment. Crystals of form I (2.1 Å) appeared from protein at 12 mg/mL in 1.0 M (NH4)H2PO4, 0.100 M sodium citrate (pH 5.6) and were cryoprotected by transfer to 2.9 M sodium malonate (pH 5.0). Crystals of form II grew from protein concentrated to 17 mg/mL in 1.0 M MgSO4, 0.1 M trisodium citrate (final pH 5.6). For cryoprotection, crystals were transferred to mother liquor mixed 1:3 with 3.0 M ammonium sulfate, 18% glycerol. Crystal form III appeared after 11 months in a dried-out drop of condition H5 of the Morpheus screen. The protein concentration had been 9 mg/mL. For cryoprotection, fresh liquid from Morpheus/H5 was added. The ternary complex structure was obtained from an LRP6PE3PE4-DKK1fl-KRM1ECD complex at 9 mg/mL that grew in condition E10 of the PACTpremier screen (pH approximately 6.8) over the course of 2–11 months. For cryoprotection, 10% PEG200 was added. By mistake, the crystals were incubated for 1 hr with 1 mM platinum compound in this cryosolution before cryocooling.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>20188</offset>
+      <text>Structure Determination</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>20212</offset>
+      <text>Diffraction data were collected at DIAMOND synchrotron light source at the beamlines detailed in Table 1. The structure was initially solved from crystal form III by molecular replacement (MR) with PHASER, placing models for the CUB domain (PDB: 2WNO, CUB_C domain of Tsg-6, 37% sequence identity), and the KR domain (PDB: 1PKR, Kringle 1 of plasminogen; 39% sequence identity). Traceable density for the WSC domain became immediately evident. The KRM1 structure was then built and refined by cycling between the various crystal forms.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>20748</offset>
+      <text>For the ternary complex, we obtained only a low-resolution, highly anisotropic dataset extending to Bragg spacings of 3.5 Å, 6.4 Å, and 3.7 Å along the three principle directions (&lt;I/σI&gt; = 2). All data to 3.5 Å were used during structure determination by MR. LRP6PE3PE4 (PDB: 4A0P) and KRM1ECD (both stripped of glycosylation sites) could be placed independently by PHASER, giving Z scores of &gt;10 and log likelihood gains (LLG) of &gt;200. The combined LLG was 673, increasing to 901 after rigid-body refinement. Strong electron density became apparent at glycosylation sites and close to methionines (see platinum soak above), further supporting the MR solution. Additional strong density was evident between LRP6 and KRM1, suggesting the presence of DKK1. A model of the DKK1CRD2 (PDB: 3S2K and 3S8V) could then be placed with PHASER by testing all rotation function peaks. This increased the LLG from 901 to 973 indicating a correct solution. The individually placed LRP6 and DKK models were then replaced with chains B and C from the LRP6-DKK complex in PDB: 3S2K. The structure was subjected to rigid-body refinement using single structural domains as individually positioned bodies.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>21945</offset>
+      <text>We then performed restrained refinement of the coordinates against the ellipsoidally truncated and anisotropically scaled diffraction data as obtained from the diffraction anisotropy server at UCLA. The resolution cutoffs were 3.5 Å, 6.4 Å, and 3.7 Å. Strong geometric restraints generated by PROSMART from the available high-resolution reference structures were used during refinement. No manual model building was attempted. Restrained refinement was followed by ten cycles of structure idealization. The final model had Rwork/Rfree errors of 32.5%/36.1% against the anisotropy-corrected data and 32.1%/35.5% against the unmodified but ellipsoidally truncated diffraction data.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>22631</offset>
+      <text>Surface Plasmon Resonance</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>22657</offset>
+      <text>Equilibrium experiments were performed as described before with the addition of 2 mM CaCl2 for experiments investigating the direct LRP6PE1PE2-KRM1ECD interaction.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">title_1</infon>
+      <offset>22822</offset>
+      <text>Author Contributions</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">paragraph</infon>
+      <offset>22843</offset>
+      <text>M.Z. and V.A.J. performed experiments with support from Y.Z., who generated the stable cell line. M.Z. and E.Y.J. designed the research. M.Z. wrote the paper with input from all other authors.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REF</infon>
+      <infon key="type">title</infon>
+      <offset>23036</offset>
+      <text>References</text>
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+      <text>Structural basis of Wnt signaling inhibition by Dickkopf binding to LRP5/6</text>
+    </passage>
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+      <text>Wnt antagonists bind through a short peptide to the first beta-propeller domain of LRP5/6</text>
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+      <text>Metal ion-dependent heavy chain transfer activity of Tsg-6 mediates assembly of the cumulus-oocyte matrix</text>
+    </passage>
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+    </passage>
+    <passage>
+      <infon key="section_type">KEYWORD</infon>
+      <infon key="type">title_1</infon>
+      <offset>26731</offset>
+      <text>Accession Numbers</text>
+    </passage>
+    <passage>
+      <infon key="section_type">KEYWORD</infon>
+      <infon key="type">paragraph</infon>
+      <offset>26749</offset>
+      <text>Coordinates and structure factors have been deposited in the PDB with succession numbers PDB: 5FWS, 5FWT, 5FWU, 5FWV, and 5FWW.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">title_1</infon>
+      <offset>26877</offset>
+      <text>Supplemental Information</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>26902</offset>
+      <text>Supplemental Information includes one figure and can be found with this article online at http://dx.doi.org/10.1016/j.str.2016.06.020.</text>
+    </passage>
+    <passage>
+      <infon key="file">gr1.jpg</infon>
+      <infon key="id">fig1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>27037</offset>
+      <text>Structure of Unliganded KRM1ECD</text>
+      <annotation id="423">
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+        <infon key="annotator">cleaner0</infon>
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+        <text>Unliganded</text>
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+        <location offset="27061" length="4"/>
+        <text>KRM1</text>
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+        <infon key="annotator">cleaner0</infon>
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+      <infon key="type">fig_caption</infon>
+      <offset>27069</offset>
+      <text>(A) The KRM1ECD fold (crystal form I) colored blue to red from the N to C terminus. Cysteines as ball and sticks, glycosylation sites as sticks. The bound calcium is shown as a gray sphere. The site of the F207S mutation associated with ectodermal dysplasia in humans is shown as mesh.</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:43:59Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:44:11Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:53:58Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <location offset="27387" length="4"/>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:44:47Z</infon>
+        <location offset="27391" length="3"/>
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+      <infon key="id">fig1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
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+        <infon key="type">experimental_method</infon>
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+        <infon key="updated_at">2023-09-19T08:53:58Z</infon>
+        <infon key="identifier">MESH:</infon>
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+      </annotation>
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+        <infon key="annotator">cleaner0</infon>
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+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:45:14Z</infon>
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+      </annotation>
+      <annotation id="636">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:45:30Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:45:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>Alignment scores</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">gr2.jpg</infon>
+      <infon key="id">fig2</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>27617</offset>
+      <text>Insight into Ternary Complex Formation</text>
+    </passage>
+    <passage>
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+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>27656</offset>
+      <text>(A) The structure of the ternary LRP6PE3PE4-DKK1CRD2-KRM1ECD complex. DKK1 (orange) is sandwiched between the PE3 module of LRP6 (blue) and the KR-WSC domain pair of KRM1 (green). Colored symbols indicate introduced N-glycan attachment sites (see D).</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>LRP6PE3PE4-DKK1CRD2-KRM1ECD</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:18:50Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>N-glycan attachment sites</text>
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+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
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+      <text>(B) SPR data comparing binding of full-length DKK1 and SUMO fusions of DKK1 truncations for binding to immobilized wild-type KRM1ECD.</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:47:17Z</infon>
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+      <infon key="type">fig_caption</infon>
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+      <text>(C) Close-up view of the DKK1CRD2-KRM1ECD interface. Residues involved in interface formation are shown as sticks; those mentioned in the text are labeled. Salt bridges are in pink and hydrogen bonds in black. Model bias cannot be excluded as single atoms and bonds are not resolved at 6.4–3.5 Å. See also Figure S1.</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+      <infon key="type">fig_caption</infon>
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+      <text>(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange). See also symbols in (A).</text>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:47:35Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:56:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="28395" length="4"/>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:12:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:47:52Z</infon>
+        <location offset="28431" length="4"/>
+        <text>KRM1</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:48:05Z</infon>
+        <location offset="28435" length="3"/>
+        <text>ECD</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:48:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28466" length="10"/>
+        <text>engineered</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:51:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28477" length="19"/>
+        <text>glycosylation sites</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:18:40Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28504" length="2"/>
+        <text>KR</text>
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+      <annotation id="455">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:07:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28511" length="3"/>
+        <text>WSC</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:56:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="28551" length="4"/>
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+      <annotation id="457">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:18:34Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28568" length="3"/>
+        <text>CUB</text>
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+    </passage>
+    <passage>
+      <infon key="file">gr3.jpg</infon>
+      <infon key="id">fig3</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>28614</offset>
+      <text>LRP6-KRM1 Direct Interaction and Summary</text>
+      <annotation id="643">
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+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:48:37Z</infon>
+        <location offset="28614" length="9"/>
+        <text>LRP6-KRM1</text>
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+    </passage>
+    <passage>
+      <infon key="file">gr3.jpg</infon>
+      <infon key="id">fig3</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>28655</offset>
+      <text>(A) In a construction of a ternary complex with all four β propellers of LRP6 intact, the CUB domain points via its Ca2+-binding region toward the top face of the second β propeller.</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:15:14Z</infon>
+        <location offset="28690" length="12"/>
+        <text>complex with</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:59:08Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28712" length="12"/>
+        <text>β propellers</text>
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+      <annotation id="459">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:04:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="28728" length="4"/>
+        <text>LRP6</text>
+      </annotation>
+      <annotation id="460">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T10:01:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28733" length="6"/>
+        <text>intact</text>
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+      <annotation id="534">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:18:34Z</infon>
+        <location offset="28745" length="3"/>
+        <text>CUB</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:48:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28771" length="19"/>
+        <text>Ca2+-binding region</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:59:12Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28818" length="18"/>
+        <text>second β propeller</text>
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+    </passage>
+    <passage>
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+      <infon key="id">fig3</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>28844</offset>
+      <text>(B) Close-up view of the potential interaction site. In addition, LRP6PE2 has been superimposed with DKK1 (yellow) and SOST (pink) peptide complexes of LRP6PE1.</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:48:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28879" length="16"/>
+        <text>interaction site</text>
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+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:49:12Z</infon>
+        <location offset="28910" length="4"/>
+        <text>LRP6</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:49:23Z</infon>
+        <location offset="28914" length="3"/>
+        <text>PE2</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:13:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="28927" length="12"/>
+        <text>superimposed</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:56:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="28945" length="4"/>
+        <text>DKK1</text>
+      </annotation>
+      <annotation id="466">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:16:16Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="28963" length="4"/>
+        <text>SOST</text>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:49:36Z</infon>
+        <location offset="28996" length="4"/>
+        <text>LRP6</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:49:45Z</infon>
+        <location offset="29000" length="3"/>
+        <text>PE1</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">gr3.jpg</infon>
+      <infon key="id">fig3</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>29005</offset>
+      <text>(C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309.</text>
+      <annotation id="467">
+        <infon key="score">0.7926361</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:49:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="29009" length="16"/>
+        <text>SPR measurements</text>
+      </annotation>
+      <annotation id="648">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:50:12Z</infon>
+        <location offset="29036" length="4"/>
+        <text>LRP6</text>
+      </annotation>
+      <annotation id="649">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:50:22Z</infon>
+        <location offset="29040" length="6"/>
+        <text>PE1PE2</text>
+      </annotation>
+      <annotation id="468">
+        <infon key="score">0.9989366</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:12:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29060" length="9"/>
+        <text>wild-type</text>
+      </annotation>
+      <annotation id="650">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:50:35Z</infon>
+        <location offset="29070" length="4"/>
+        <text>KRM1</text>
+      </annotation>
+      <annotation id="651">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:50:45Z</infon>
+        <location offset="29074" length="3"/>
+        <text>ECD</text>
+      </annotation>
+      <annotation id="675">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T10:06:53Z</infon>
+        <location offset="29086" length="15"/>
+        <text>GlycoCUB mutant</text>
+      </annotation>
+      <annotation id="652">
+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:51:21Z</infon>
+        <location offset="29113" length="8"/>
+        <text>N-glycan</text>
+      </annotation>
+      <annotation id="469">
+        <infon key="score">0.99950445</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:14:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29125" length="4"/>
+        <text>N309</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">gr3.jpg</infon>
+      <infon key="id">fig3</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>29131</offset>
+      <text>(D) Schematic representation of structural and biophysical findings and their implications for Wnt-dependent (left, middle) and independent (right) signaling. Conformational differences in the depictions of LRP6 are included purely for ease of representation.</text>
+      <annotation id="527">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:16:32Z</infon>
+        <location offset="29226" length="3"/>
+        <text>Wnt</text>
+      </annotation>
+      <annotation id="470">
+        <infon key="score">0.9989386</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T15:04:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29338" length="4"/>
+        <text>LRP6</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">tbl1.xml</infon>
+      <infon key="id">tbl1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_caption</infon>
+      <offset>29391</offset>
+      <text>Diffraction and Refinement Statistics</text>
+      <annotation id="471">
+        <infon key="score">0.9977757</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:57:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29391" length="37"/>
+        <text>Diffraction and Refinement Statistics</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">tbl1.xml</infon>
+      <infon key="id">tbl1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table</infon>
+      <infon key="xml">&lt;?xml version="1.0" encoding="UTF-8"?&gt;
+&lt;table xmlns:xlink="http://www.w3.org/1999/xlink" frame="hsides" rules="groups"&gt;&lt;thead&gt;&lt;tr&gt;&lt;th/&gt;&lt;th&gt;KRM1&lt;sub&gt;ECD&lt;/sub&gt;&lt;/th&gt;&lt;th&gt;KRM1&lt;sub&gt;ECD&lt;/sub&gt;&lt;/th&gt;&lt;th&gt;KRM1&lt;sub&gt;ECD&lt;/sub&gt;&lt;/th&gt;&lt;th&gt;KRM1&lt;sub&gt;ECD&lt;/sub&gt;&lt;/th&gt;&lt;th&gt;LRP6&lt;sub&gt;PE3PE4&lt;/sub&gt;-DKK&lt;sub&gt;CRD2&lt;/sub&gt;-KRM1&lt;sub&gt;ECD&lt;/sub&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td&gt;Crystal form&lt;/td&gt;&lt;td&gt;I&lt;/td&gt;&lt;td&gt;I&lt;/td&gt;&lt;td&gt;II&lt;/td&gt;&lt;td&gt;III&lt;/td&gt;&lt;td&gt;I&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;X-ray source&lt;/td&gt;&lt;td&gt;Diamond i04&lt;/td&gt;&lt;td&gt;Diamond i03&lt;/td&gt;&lt;td&gt;Diamond i03&lt;/td&gt;&lt;td&gt;Diamond i04&lt;/td&gt;&lt;td&gt;Diamond i04&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Wavelength (Å)&lt;/td&gt;&lt;td&gt;0.9793&lt;/td&gt;&lt;td&gt;0.9700&lt;/td&gt;&lt;td&gt;0.9700&lt;/td&gt;&lt;td&gt;0.9795&lt;/td&gt;&lt;td&gt;0.9795&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Space group&lt;/td&gt;&lt;td&gt;&lt;italic&gt;P&lt;/italic&gt;3&lt;sub&gt;1&lt;/sub&gt;21&lt;/td&gt;&lt;td&gt;&lt;italic&gt;P&lt;/italic&gt;3&lt;sub&gt;1&lt;/sub&gt;21&lt;/td&gt;&lt;td&gt;&lt;italic&gt;P&lt;/italic&gt;4&lt;sub&gt;3&lt;/sub&gt;&lt;/td&gt;&lt;td&gt;&lt;italic&gt;P&lt;/italic&gt;4&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;/td&gt;&lt;td&gt;&lt;italic&gt;C&lt;/italic&gt;222&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Unit cell a/α (Å/°)&lt;/td&gt;&lt;td&gt;50.9/90&lt;/td&gt;&lt;td&gt;50.5/90&lt;/td&gt;&lt;td&gt;65.8/90&lt;/td&gt;&lt;td&gt;67.8/90&lt;/td&gt;&lt;td&gt;86.9/90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;b/β (Å/°)&lt;/td&gt;&lt;td&gt;50.9/90&lt;/td&gt;&lt;td&gt;50.5/90&lt;/td&gt;&lt;td&gt;65.8/90&lt;/td&gt;&lt;td&gt;67.8/90&lt;/td&gt;&lt;td&gt;100.1/90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;c/γ (Å/°)&lt;/td&gt;&lt;td&gt;188.4/120&lt;/td&gt;&lt;td&gt;187.4/120&lt;/td&gt;&lt;td&gt;75.0/90&lt;/td&gt;&lt;td&gt;198.2/90&lt;/td&gt;&lt;td&gt;270.7/90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Wilson B factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;td&gt;31&lt;/td&gt;&lt;td&gt;41&lt;/td&gt;&lt;td&gt;76&lt;/td&gt;&lt;td&gt;77&lt;/td&gt;&lt;td&gt;NA&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Resolution range (Å)&lt;/td&gt;&lt;td&gt;47.10–1.90 (1.95–1.90)&lt;/td&gt;&lt;td&gt;62.47–2.10 (2.16–2.10)&lt;/td&gt;&lt;td&gt;75.00–2.80 (2.99–2.80)&lt;/td&gt;&lt;td&gt;67.80–3.20 (3.42–3.20)&lt;/td&gt;&lt;td&gt;67.68–3.50 (7.16–6.40, 3.92–3.50)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Unique reflections&lt;/td&gt;&lt;td&gt;23,300 (1,524)&lt;/td&gt;&lt;td&gt;17,089 (1,428)&lt;/td&gt;&lt;td&gt;7,964 (1,448)&lt;/td&gt;&lt;td&gt;8,171 (1,343)&lt;/td&gt;&lt;td&gt;8,070 (723, 645)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Average multiplicity&lt;/td&gt;&lt;td&gt;9.1 (9.2)&lt;/td&gt;&lt;td&gt;5.2 (5.3)&lt;/td&gt;&lt;td&gt;3.7 (3.7)&lt;/td&gt;&lt;td&gt;22.7 (12.6)&lt;/td&gt;&lt;td&gt;3.8 (3.5, 4.4)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Completeness (%)&lt;/td&gt;&lt;td&gt;99.8 (98.5)&lt;/td&gt;&lt;td&gt;100 (100)&lt;/td&gt;&lt;td&gt;99.8 (100)&lt;/td&gt;&lt;td&gt;98.8 (93.4)&lt;/td&gt;&lt;td&gt;51.6 (98.5, 14.1)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&amp;lt;&lt;italic&gt;I&lt;/italic&gt;/&lt;italic&gt;σI&lt;/italic&gt;&amp;gt;&lt;/td&gt;&lt;td&gt;11.4 (1.7)&lt;/td&gt;&lt;td&gt;12.0 (1.7)&lt;/td&gt;&lt;td&gt;14.9 (1.5)&lt;/td&gt;&lt;td&gt;13.1 (1.9)&lt;/td&gt;&lt;td&gt;4.6 (4.1, 2.2)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;merge&lt;/sub&gt; (%)&lt;/td&gt;&lt;td&gt;14.8 (158.3)&lt;/td&gt;&lt;td&gt;9.3 (98.0)&lt;/td&gt;&lt;td&gt;6.2 (98.9)&lt;/td&gt;&lt;td&gt;29.8 (142.2)&lt;/td&gt;&lt;td&gt;44.9 (40.5, 114.2)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;pim&lt;/sub&gt; (%)&lt;/td&gt;&lt;td&gt;15.7 (55.3)&lt;/td&gt;&lt;td&gt;10.3 (109.0)&lt;/td&gt;&lt;td&gt;3.7 (53.8)&lt;/td&gt;&lt;td&gt;6.3 (40.0)&lt;/td&gt;&lt;td&gt;24.7 (23.9, 59.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="6"&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="6"&gt;&lt;bold&gt;Refinement&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="6"&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;work&lt;/sub&gt; (%)&lt;/td&gt;&lt;td&gt;17.9&lt;/td&gt;&lt;td&gt;18.4&lt;/td&gt;&lt;td&gt;21.6&lt;/td&gt;&lt;td&gt;20.2&lt;/td&gt;&lt;td&gt;32.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt; (%)&lt;/td&gt;&lt;td&gt;22.7&lt;/td&gt;&lt;td&gt;23.2&lt;/td&gt;&lt;td&gt;30.7&lt;/td&gt;&lt;td&gt;27.1&lt;/td&gt;&lt;td&gt;35.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="6"&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="6"&gt;&lt;bold&gt;No. of Non-Hydrogen Atoms&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="6"&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Protein&lt;/td&gt;&lt;td&gt;2,260&lt;/td&gt;&lt;td&gt;2,301&lt;/td&gt;&lt;td&gt;2,102&lt;/td&gt;&lt;td&gt;2,305&lt;/td&gt;&lt;td&gt;7,730&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;N-glycans&lt;/td&gt;&lt;td&gt;42&lt;/td&gt;&lt;td&gt;42&lt;/td&gt;&lt;td&gt;28&lt;/td&gt;&lt;td&gt;28&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Water&lt;/td&gt;&lt;td&gt;79&lt;/td&gt;&lt;td&gt;54&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;td&gt;2&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Ligands&lt;/td&gt;&lt;td&gt;6&lt;/td&gt;&lt;td&gt;6&lt;/td&gt;&lt;td&gt;2&lt;/td&gt;&lt;td&gt;5&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="6"&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="6"&gt;&lt;bold&gt;Average B factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="6"&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Protein&lt;/td&gt;&lt;td&gt;63&lt;/td&gt;&lt;td&gt;65&lt;/td&gt;&lt;td&gt;108&lt;/td&gt;&lt;td&gt;84&lt;/td&gt;&lt;td&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;N-glycans&lt;/td&gt;&lt;td&gt;35&lt;/td&gt;&lt;td&gt;46&lt;/td&gt;&lt;td&gt;102&lt;/td&gt;&lt;td&gt;18&lt;/td&gt;&lt;td&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Water&lt;/td&gt;&lt;td&gt;68&lt;/td&gt;&lt;td&gt;85&lt;/td&gt;&lt;td&gt;–&lt;/td&gt;&lt;td&gt;75&lt;/td&gt;&lt;td&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Ligands&lt;/td&gt;&lt;td&gt;36&lt;/td&gt;&lt;td&gt;47&lt;/td&gt;&lt;td&gt;91&lt;/td&gt;&lt;td&gt;75&lt;/td&gt;&lt;td&gt;66&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="6"&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="6"&gt;&lt;bold&gt;RMSD from Ideality&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="6"&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Bond lengths (Å)&lt;/td&gt;&lt;td&gt;0.020&lt;/td&gt;&lt;td&gt;0.016&lt;/td&gt;&lt;td&gt;0.019&lt;/td&gt;&lt;td&gt;0.016&lt;/td&gt;&lt;td&gt;0.004&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Bond angles (°)&lt;/td&gt;&lt;td&gt;2.050&lt;/td&gt;&lt;td&gt;1.748&lt;/td&gt;&lt;td&gt;1.952&lt;/td&gt;&lt;td&gt;1.796&lt;/td&gt;&lt;td&gt;0.770&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="6"&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="6"&gt;&lt;bold&gt;Ramachandran Plot&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="6"&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Favored (%)&lt;/td&gt;&lt;td&gt;96.8&lt;/td&gt;&lt;td&gt;95.5&lt;/td&gt;&lt;td&gt;96.9&lt;/td&gt;&lt;td&gt;94.9&lt;/td&gt;&lt;td&gt;92.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Allowed (%)&lt;/td&gt;&lt;td&gt;99.7&lt;/td&gt;&lt;td&gt;100.0&lt;/td&gt;&lt;td&gt;100.0&lt;/td&gt;&lt;td&gt;99.7&lt;/td&gt;&lt;td&gt;99.8&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Number of outliers&lt;/td&gt;&lt;td&gt;1&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;td&gt;1&lt;/td&gt;&lt;td&gt;2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;PDB code&lt;/td&gt;&lt;td&gt;&lt;ext-link ext-link-type="uri" xlink:href="pdb:5FWS" id="intref0085"&gt;5FWS&lt;/ext-link&gt;&lt;/td&gt;&lt;td&gt;&lt;ext-link ext-link-type="uri" xlink:href="pdb:5FWT" id="intref0090"&gt;5FWT&lt;/ext-link&gt;&lt;/td&gt;&lt;td&gt;&lt;ext-link ext-link-type="uri" xlink:href="pdb:5FWU" id="intref0095"&gt;5FWU&lt;/ext-link&gt;&lt;/td&gt;&lt;td&gt;&lt;ext-link ext-link-type="uri" xlink:href="pdb:5FWV" id="intref0100"&gt;5FWV&lt;/ext-link&gt;&lt;/td&gt;&lt;td&gt;&lt;ext-link ext-link-type="uri" xlink:href="pdb:5FWW" id="intref0105"&gt;5FWW&lt;/ext-link&gt;&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon>
+      <offset>29429</offset>
+      <text>	KRM1ECD	KRM1ECD	KRM1ECD	KRM1ECD	LRP6PE3PE4-DKKCRD2-KRM1ECD	 	Crystal form	I	I	II	III	I	 	X-ray source	Diamond i04	Diamond i03	Diamond i03	Diamond i04	Diamond i04	 	Wavelength (Å)	0.9793	0.9700	0.9700	0.9795	0.9795	 	Space group	P3121	P3121	P43	P41212	C2221	 	Unit cell a/α (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	86.9/90	 	b/β (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	100.1/90	 	c/γ (Å/°)	188.4/120	187.4/120	75.0/90	198.2/90	270.7/90	 	Wilson B factor (Å2)	31	41	76	77	NA	 	Resolution range (Å)	47.10–1.90 (1.95–1.90)	62.47–2.10 (2.16–2.10)	75.00–2.80 (2.99–2.80)	67.80–3.20 (3.42–3.20)	67.68–3.50 (7.16–6.40, 3.92–3.50)	 	Unique reflections	23,300 (1,524)	17,089 (1,428)	7,964 (1,448)	8,171 (1,343)	8,070 (723, 645)	 	Average multiplicity	9.1 (9.2)	5.2 (5.3)	3.7 (3.7)	22.7 (12.6)	3.8 (3.5, 4.4)	 	Completeness (%)	99.8 (98.5)	100 (100)	99.8 (100)	98.8 (93.4)	51.6 (98.5, 14.1)	 	&lt;I/σI&gt;	11.4 (1.7)	12.0 (1.7)	14.9 (1.5)	13.1 (1.9)	4.6 (4.1, 2.2)	 	Rmerge (%)	14.8 (158.3)	9.3 (98.0)	6.2 (98.9)	29.8 (142.2)	44.9 (40.5, 114.2)	 	Rpim (%)	15.7 (55.3)	10.3 (109.0)	3.7 (53.8)	6.3 (40.0)	24.7 (23.9, 59.9)	 		 	Refinement	 		 	Rwork (%)	17.9	18.4	21.6	20.2	32.1	 	Rfree (%)	22.7	23.2	30.7	27.1	35.5	 		 	No. of Non-Hydrogen Atoms	 		 	Protein	2,260	2,301	2,102	2,305	7,730	 	N-glycans	42	42	28	28	0	 	Water	79	54	0	2	0	 	Ligands	6	6	2	5	0	 		 	Average B factor (Å2)	 		 	Protein	63	65	108	84	–	 	N-glycans	35	46	102	18	–	 	Water	68	85	–	75	–	 	Ligands	36	47	91	75	66	 		 	RMSD from Ideality	 		 	Bond lengths (Å)	0.020	0.016	0.019	0.016	0.004	 	Bond angles (°)	2.050	1.748	1.952	1.796	0.770	 		 	Ramachandran Plot	 		 	Favored (%)	96.8	95.5	96.9	94.9	92.3	 	Allowed (%)	99.7	100.0	100.0	99.7	99.8	 	Number of outliers	1	0	0	1	2	 	PDB code	5FWS	5FWT	5FWU	5FWV	5FWW	 	</text>
+      <annotation id="653">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:51:42Z</infon>
+        <location offset="29430" length="4"/>
+        <text>KRM1</text>
+      </annotation>
+      <annotation id="654">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:51:53Z</infon>
+        <location offset="29434" length="3"/>
+        <text>ECD</text>
+      </annotation>
+      <annotation id="655">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:52:10Z</infon>
+        <location offset="29438" length="4"/>
+        <text>KRM1</text>
+      </annotation>
+      <annotation id="656">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:52:20Z</infon>
+        <location offset="29442" length="3"/>
+        <text>ECD</text>
+      </annotation>
+      <annotation id="657">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:52:35Z</infon>
+        <location offset="29446" length="4"/>
+        <text>KRM1</text>
+      </annotation>
+      <annotation id="658">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:52:45Z</infon>
+        <location offset="29450" length="3"/>
+        <text>ECD</text>
+      </annotation>
+      <annotation id="659">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:52:57Z</infon>
+        <location offset="29454" length="4"/>
+        <text>KRM1</text>
+      </annotation>
+      <annotation id="660">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:53:06Z</infon>
+        <location offset="29458" length="3"/>
+        <text>ECD</text>
+      </annotation>
+      <annotation id="472">
+        <infon key="score">0.98330337</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T10:08:25Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="29462" length="26"/>
+        <text>LRP6PE3PE4-DKKCRD2-KRM1ECD</text>
+      </annotation>
+      <annotation id="556">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:52:06Z</infon>
+        <location offset="30724" length="5"/>
+        <text>Water</text>
+      </annotation>
+      <annotation id="557">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:52:06Z</infon>
+        <location offset="30846" length="5"/>
+        <text>Water</text>
+      </annotation>
+      <annotation id="555">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T08:51:09Z</infon>
+        <location offset="30895" length="4"/>
+        <text>RMSD</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">tbl1.xml</infon>
+      <infon key="id">tbl1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_footnote</infon>
+      <offset>31236</offset>
+      <text>Values in parentheses refer to the highest-resolution shell. An additional shell given for the ternary complex corresponds to the last shell with near-complete diffraction data. NA, not announced.</text>
+      <annotation id="473">
+        <infon key="score">0.7761474</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-19T09:57:51Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="31396" length="16"/>
+        <text>diffraction data</text>
+      </annotation>
+    </passage>
+  </document>
+</collection>
diff --git a/annotated_BioC_XML/PMC5063996_ann.xml b/annotated_BioC_XML/PMC5063996_ann.xml
new file mode 100644
index 0000000000000000000000000000000000000000..db1f4ec9a9cf991abf5fafbb42151b3e5b28105e
--- /dev/null
+++ b/annotated_BioC_XML/PMC5063996_ann.xml
@@ -0,0 +1,10670 @@
+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection>
+  <source>PMC</source>
+  <date>20230813</date>
+  <key>pmc.key</key>
+  <document>
+    <id>5063996</id>
+    <infon key="license">CC BY</infon>
+    <infon key="tt_curatable">no</infon>
+    <infon key="tt_version">2</infon>
+    <infon key="tt_round">2</infon>
+    <passage>
+      <infon key="alt-title">Mechanism of Arabinoxylanase</infon>
+      <infon key="article-id_doi">10.1074/jbc.M116.743948</infon>
+      <infon key="article-id_pmc">5063996</infon>
+      <infon key="article-id_pmid">27531750</infon>
+      <infon key="article-id_publisher-id">M116.743948</infon>
+      <infon key="fpage">22149</infon>
+      <infon key="issue">42</infon>
+      <infon key="kwd">cellulosome crystallography enzyme kinetics enzyme mechanism glycoside hydrolase</infon>
+      <infon key="license">Author's Choice—Final version free via Creative Commons CC-BY license.</infon>
+      <infon key="lpage">22159</infon>
+      <infon key="name_0">surname:Labourel;given-names:Aurore</infon>
+      <infon key="name_1">surname:Crouch;given-names:Lucy I.</infon>
+      <infon key="name_10">surname:Najmudin;given-names:Shabir</infon>
+      <infon key="name_11">surname:Baslé;given-names:Arnaud</infon>
+      <infon key="name_12">surname:Cuskin;given-names:Fiona</infon>
+      <infon key="name_2">surname:Brás;given-names:Joana L. A.</infon>
+      <infon key="name_3">surname:Jackson;given-names:Adam</infon>
+      <infon key="name_4">surname:Rogowski;given-names:Artur</infon>
+      <infon key="name_5">surname:Gray;given-names:Joseph</infon>
+      <infon key="name_6">surname:Yadav;given-names:Madhav P.</infon>
+      <infon key="name_7">surname:Henrissat;given-names:Bernard</infon>
+      <infon key="name_8">surname:Fontes;given-names:Carlos M. G. A.</infon>
+      <infon key="name_9">surname:Gilbert;given-names:Harry J.</infon>
+      <infon key="section_type">TITLE</infon>
+      <infon key="type">front</infon>
+      <infon key="volume">291</infon>
+      <infon key="year">2016</infon>
+      <offset>0</offset>
+      <text>The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans*</text>
+      <annotation id="1">
+        <infon key="score">0.99930334</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23" length="16"/>
+        <text>Arabinoxylanases</text>
+      </annotation>
+      <annotation id="925">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:47:12Z</infon>
+        <location offset="54" length="16"/>
+        <text>Highly Decorated</text>
+      </annotation>
+      <annotation id="2">
+        <infon key="score">0.9991499</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:31Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="71" length="6"/>
+        <text>Xylans</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">ABSTRACT</infon>
+      <infon key="type">abstract</infon>
+      <offset>79</offset>
+      <text>The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance. Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains. A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans. The mechanism of substrate recognition displayed by the enzyme, however, remains unclear. Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands. The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain. The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility. The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center. The −2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants. Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity. Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed. This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack.</text>
+      <annotation id="3">
+        <infon key="score">0.9984566</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="108" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="4">
+        <infon key="score">0.99672145</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="217" length="5"/>
+        <text>Xylan</text>
+      </annotation>
+      <annotation id="5">
+        <infon key="score">0.99865866</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="249" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="6">
+        <infon key="score">0.9991608</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:55Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="292" length="12"/>
+        <text>β-1,4-xylose</text>
+      </annotation>
+      <annotation id="7">
+        <infon key="score">0.99928457</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:01Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="306" length="4"/>
+        <text>Xylp</text>
+      </annotation>
+      <annotation id="8">
+        <infon key="score">0.99911076</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:08Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="348" length="15"/>
+        <text>arabinofuranose</text>
+      </annotation>
+      <annotation id="9">
+        <infon key="score">0.99916506</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="365" length="4"/>
+        <text>Araf</text>
+      </annotation>
+      <annotation id="10">
+        <infon key="score">0.9825443</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="392" length="20"/>
+        <text>penta-modular enzyme</text>
+      </annotation>
+      <annotation id="11">
+        <infon key="score">0.99902785</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:29Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="414" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="12">
+        <infon key="score">0.9981812</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="467" length="13"/>
+        <text>arabinoxylans</text>
+      </annotation>
+      <annotation id="13">
+        <infon key="score">0.9987552</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="591" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="14">
+        <infon key="score">0.9991007</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:56Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="616" length="15"/>
+        <text>arabinoxylanase</text>
+      </annotation>
+      <annotation id="15">
+        <infon key="score">0.9980863</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="647" length="15"/>
+        <text>in complex with</text>
+      </annotation>
+      <annotation id="16">
+        <infon key="score">0.50939804</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:08Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="663" length="7"/>
+        <text>ligands</text>
+      </annotation>
+      <annotation id="17">
+        <infon key="score">0.9991667</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:19Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="767" length="16"/>
+        <text>catalytic domain</text>
+      </annotation>
+      <annotation id="18">
+        <infon key="score">0.99892145</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:25Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="800" length="41"/>
+        <text>non-catalytic carbohydrate binding module</text>
+      </annotation>
+      <annotation id="19">
+        <infon key="score">0.99877536</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="871" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="20">
+        <infon key="score">0.99855906</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="929" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="21">
+        <infon key="score">0.9979739</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="953" length="15"/>
+        <text>in complex with</text>
+      </annotation>
+      <annotation id="22">
+        <infon key="score">0.998847</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="969" length="50"/>
+        <text>Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp</text>
+      </annotation>
+      <annotation id="23">
+        <infon key="score">0.9983198</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1036" length="4"/>
+        <text>Araf</text>
+      </annotation>
+      <annotation id="24">
+        <infon key="score">0.9865536</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1069" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="25">
+        <infon key="score">0.9988514</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="1083" length="11"/>
+        <text>active site</text>
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+      <annotation id="26">
+        <infon key="score">0.9991247</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:55:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="1113" length="6"/>
+        <text>pocket</text>
+      </annotation>
+      <annotation id="27">
+        <infon key="score">0.9987526</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:55:19Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="1121" length="11"/>
+        <text>−2* subsite</text>
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+      <annotation id="28">
+        <infon key="score">0.9985621</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:28:03Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="1154" length="16"/>
+        <text>catalytic center</text>
+      </annotation>
+      <annotation id="29">
+        <infon key="score">0.99883324</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:55:30Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="1176" length="11"/>
+        <text>−2* subsite</text>
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+      <annotation id="30">
+        <infon key="score">0.9991928</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:01Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1205" length="4"/>
+        <text>Xylp</text>
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+      <annotation id="31">
+        <infon key="score">0.9991721</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:38:24Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1214" length="4"/>
+        <text>Arap</text>
+      </annotation>
+      <annotation id="32">
+        <infon key="score">0.9986651</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1258" length="6"/>
+        <text>xylose</text>
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+      <annotation id="33">
+        <infon key="score">0.9984883</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:41Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1269" length="9"/>
+        <text>arabinose</text>
+      </annotation>
+      <annotation id="34">
+        <infon key="score">0.99220085</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1308" length="20"/>
+        <text>Alanine substitution</text>
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+      <annotation id="35">
+        <infon key="score">0.99950814</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1332" length="5"/>
+        <text>Glu68</text>
+      </annotation>
+      <annotation id="36">
+        <infon key="score">0.99953973</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1339" length="5"/>
+        <text>Tyr92</text>
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+      <annotation id="37">
+        <infon key="score">0.99952006</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1349" length="6"/>
+        <text>Asn139</text>
+      </annotation>
+      <annotation id="38">
+        <infon key="score">0.9983119</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1377" length="9"/>
+        <text>arabinose</text>
+      </annotation>
+      <annotation id="39">
+        <infon key="score">0.9982514</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1391" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="40">
+        <infon key="score">0.99881476</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:55:33Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="1417" length="11"/>
+        <text>−2* subsite</text>
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+      <annotation id="41">
+        <infon key="score">0.9989426</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="1474" length="11"/>
+        <text>active site</text>
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+      <annotation id="42">
+        <infon key="score">0.9970607</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1491" length="5"/>
+        <text>xylan</text>
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+      <annotation id="43">
+        <infon key="score">0.99551225</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:48:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1575" length="15"/>
+        <text>solvent-exposed</text>
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+      <annotation id="44">
+        <infon key="score">0.9989027</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:30Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="1610" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="45">
+        <infon key="score">0.9959501</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1644" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="46">
+        <infon key="score">0.9521629</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1719" length="13"/>
+        <text>endo-xylanase</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">title_1</infon>
+      <offset>1757</offset>
+      <text>Introduction</text>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>1770</offset>
+      <text>The plant cell wall is an important biological substrate. This complex composite structure is depolymerized by microorganisms that occupy important highly competitive ecological niches, whereas the process makes an important contribution to the carbon cycle. Lignocellulosic degradation is also of continued interest to environmentally sensitive industries such as the biofuels and biorefinery sectors, where the use of sustainable or renewable substrates is of increasing importance. Given that the plant cell wall is the most abundant source of renewable organic carbon on the planet, this macromolecular substrate has substantial industrial potential.</text>
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+        <infon key="score">0.9988167</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1774" length="5"/>
+        <text>plant</text>
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+      <annotation id="48">
+        <infon key="score">0.9986657</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:43:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1881" length="14"/>
+        <text>microorganisms</text>
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+      <annotation id="49">
+        <infon key="score">0.9988594</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2270" length="5"/>
+        <text>plant</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>2425</offset>
+      <text>An example of the chemical complexity of the plant cell wall is provided by xylan, which is the major hemicellulosic component. This polysaccharide comprises a backbone of β-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with α-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated. In addition, the Araf side chain decorations can also be esterified to ferulic acid that, in some species, provide a chemical link between hemicellulose and lignin. The precise structure of xylans varies between plant species, in particular in different tissues and during cellular differentiation. In specialized plant tissues, such as the outer layer of cereal grains, xylans are extremely complex, and side chains may comprise a range of other sugars including l- and d-galactose and β- and α-Xylp units. Indeed, in these cereal brans, xylans have very few backbone Xylp units that are undecorated, and the side chains can contain up to six sugars.</text>
+      <annotation id="50">
+        <infon key="score">0.9986474</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2470" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="51">
+        <infon key="score">0.97633845</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2501" length="5"/>
+        <text>xylan</text>
+      </annotation>
+      <annotation id="52">
+        <infon key="score">0.90193385</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:28:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2558" length="14"/>
+        <text>polysaccharide</text>
+      </annotation>
+      <annotation id="53">
+        <infon key="score">0.99882466</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:28:55Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2597" length="14"/>
+        <text>β-1,4-d-xylose</text>
+      </annotation>
+      <annotation id="1005">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:41:15Z</infon>
+        <location offset="2630" length="8"/>
+        <text>pyranose</text>
+      </annotation>
+      <annotation id="54">
+        <infon key="score">0.99762434</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:02Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2654" length="4"/>
+        <text>Xylp</text>
+      </annotation>
+      <annotation id="55">
+        <infon key="score">0.99699223</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:29:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2690" length="28"/>
+        <text>4-O-methyl-d-glucuronic acid</text>
+      </annotation>
+      <annotation id="56">
+        <infon key="score">0.99859554</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:29:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2720" length="4"/>
+        <text>GlcA</text>
+      </annotation>
+      <annotation id="57">
+        <infon key="score">0.9988452</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:30:05Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2751" length="19"/>
+        <text>α-l-arabinofuranose</text>
+      </annotation>
+      <annotation id="58">
+        <infon key="score">0.99826837</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2772" length="4"/>
+        <text>Araf</text>
+      </annotation>
+      <annotation id="59">
+        <infon key="score">0.92519265</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:28:43Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2800" length="14"/>
+        <text>polysaccharide</text>
+      </annotation>
+      <annotation id="60">
+        <infon key="score">0.99387217</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2868" length="4"/>
+        <text>Araf</text>
+      </annotation>
+      <annotation id="61">
+        <infon key="score">0.99838805</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:38:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2922" length="12"/>
+        <text>ferulic acid</text>
+      </annotation>
+      <annotation id="62">
+        <infon key="score">0.72233874</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:29:25Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="2990" length="13"/>
+        <text>hemicellulose</text>
+      </annotation>
+      <annotation id="899">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:29:37Z</infon>
+        <location offset="3008" length="6"/>
+        <text>lignin</text>
+      </annotation>
+      <annotation id="63">
+        <infon key="score">0.48770082</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3041" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="64">
+        <infon key="score">0.99851996</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3063" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="65">
+        <infon key="score">0.99830014</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3165" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="66">
+        <infon key="score">0.9969138</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:43:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3207" length="6"/>
+        <text>cereal</text>
+      </annotation>
+      <annotation id="67">
+        <infon key="score">0.70049024</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3222" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="68">
+        <infon key="score">0.99178773</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:38:37Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3298" length="6"/>
+        <text>sugars</text>
+      </annotation>
+      <annotation id="69">
+        <infon key="score">0.998679</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:29:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3315" length="18"/>
+        <text>l- and d-galactose</text>
+      </annotation>
+      <annotation id="70">
+        <infon key="score">0.9987858</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:29:11Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3338" length="13"/>
+        <text>β- and α-Xylp</text>
+      </annotation>
+      <annotation id="71">
+        <infon key="score">0.9947548</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:43:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3376" length="6"/>
+        <text>cereal</text>
+      </annotation>
+      <annotation id="72">
+        <infon key="score">0.57896805</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3390" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="73">
+        <infon key="score">0.99786454</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:02Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3420" length="4"/>
+        <text>Xylp</text>
+      </annotation>
+      <annotation id="74">
+        <infon key="score">0.93305</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:38:41Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3495" length="6"/>
+        <text>sugars</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>3517</offset>
+      <text>Reflecting the chemical and physical complexity of the plant cell wall, microorganisms that utilize these composite structures express a large number of polysaccharide-degrading enzymes, primarily glycoside hydrolases, but also polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. These carbohydrate active enzymes are grouped into sequence-based families in the CAZy database. With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8. The extensive decoration of the xylan backbone generally restricts the capacity of these enzymes to attack the polysaccharide prior to removal of the side chains by a range of α-glucuronidases, α-arabinofuranosidases, and esterases. Two xylanases, however, utilize the side chains as essential specificity determinants and thus target decorated forms of the hemicellulose. The GH30 glucuronoxylanases require the Xylp bound at the −2 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites −1 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively). The GH5 arabinoxylanase (CtXyl5A) derived from Clostridium thermocellum displays an absolute requirement for xylans that contain Araf side chains. In this enzyme, the key specificity determinant is the Araf appended to O3 of the Xylp bound in the active site (−1 subsite). The reaction products generated from arabinoxylans, however, suggest that Araf can be accommodated at subsites distal to the active site.</text>
+      <annotation id="75">
+        <infon key="score">0.99864966</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3572" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="76">
+        <infon key="score">0.99864155</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:43:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3589" length="14"/>
+        <text>microorganisms</text>
+      </annotation>
+      <annotation id="77">
+        <infon key="score">0.65709233</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:30:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3670" length="32"/>
+        <text>polysaccharide-degrading enzymes</text>
+      </annotation>
+      <annotation id="78">
+        <infon key="score">0.9984101</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:30:27Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3714" length="20"/>
+        <text>glycoside hydrolases</text>
+      </annotation>
+      <annotation id="79">
+        <infon key="score">0.9986268</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:30:38Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3745" length="21"/>
+        <text>polysaccharide lyases</text>
+      </annotation>
+      <annotation id="80">
+        <infon key="score">0.99855816</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:30:43Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3768" length="22"/>
+        <text>carbohydrate esterases</text>
+      </annotation>
+      <annotation id="81">
+        <infon key="score">0.99858</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:30:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3796" length="35"/>
+        <text>lytic polysaccharide monooxygenases</text>
+      </annotation>
+      <annotation id="82">
+        <infon key="score">0.99558616</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:00Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3839" length="27"/>
+        <text>carbohydrate active enzymes</text>
+      </annotation>
+      <annotation id="83">
+        <infon key="score">0.99593437</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3946" length="5"/>
+        <text>xylan</text>
+      </annotation>
+      <annotation id="84">
+        <infon key="score">0.99739957</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3988" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="85">
+        <infon key="score">0.99709374</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:07Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4012" length="21"/>
+        <text>endo-acting xylanases</text>
+      </annotation>
+      <annotation id="86">
+        <infon key="score">0.9923669</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:19Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4072" length="19"/>
+        <text>glycoside hydrolase</text>
+      </annotation>
+      <annotation id="87">
+        <infon key="score">0.99262285</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:27Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4093" length="2"/>
+        <text>GH</text>
+      </annotation>
+      <annotation id="88">
+        <infon key="score">0.9738185</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:44Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4096" length="1"/>
+        <text>5</text>
+      </annotation>
+      <annotation id="89">
+        <infon key="score">0.9936046</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4107" length="4"/>
+        <text>GH10</text>
+      </annotation>
+      <annotation id="90">
+        <infon key="score">0.99484766</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4116" length="4"/>
+        <text>GH11</text>
+      </annotation>
+      <annotation id="91">
+        <infon key="score">0.9940958</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4156" length="3"/>
+        <text>GH8</text>
+      </annotation>
+      <annotation id="92">
+        <infon key="score">0.9956683</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4193" length="5"/>
+        <text>xylan</text>
+      </annotation>
+      <annotation id="93">
+        <infon key="score">0.99571615</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:28:43Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4272" length="14"/>
+        <text>polysaccharide</text>
+      </annotation>
+      <annotation id="94">
+        <infon key="score">0.99883527</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:32:36Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4337" length="16"/>
+        <text>α-glucuronidases</text>
+      </annotation>
+      <annotation id="95">
+        <infon key="score">0.9988317</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:32:43Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4355" length="22"/>
+        <text>α-arabinofuranosidases</text>
+      </annotation>
+      <annotation id="96">
+        <infon key="score">0.9990785</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:32:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4383" length="9"/>
+        <text>esterases</text>
+      </annotation>
+      <annotation id="97">
+        <infon key="score">0.99923325</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:33:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4398" length="9"/>
+        <text>xylanases</text>
+      </annotation>
+      <annotation id="98">
+        <infon key="score">0.9843167</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:29:26Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4519" length="13"/>
+        <text>hemicellulose</text>
+      </annotation>
+      <annotation id="99">
+        <infon key="score">0.99851674</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:33:15Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4538" length="4"/>
+        <text>GH30</text>
+      </annotation>
+      <annotation id="100">
+        <infon key="score">0.99904984</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:33:19Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4543" length="18"/>
+        <text>glucuronoxylanases</text>
+      </annotation>
+      <annotation id="101">
+        <infon key="score">0.99920505</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:02Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4574" length="4"/>
+        <text>Xylp</text>
+      </annotation>
+      <annotation id="102">
+        <infon key="score">0.9013893</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:33:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4579" length="8"/>
+        <text>bound at</text>
+      </annotation>
+      <annotation id="103">
+        <infon key="score">0.99698335</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:55:43Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4592" length="2"/>
+        <text>−2</text>
+      </annotation>
+      <annotation id="104">
+        <infon key="score">0.94409853</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:29:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4608" length="4"/>
+        <text>GlcA</text>
+      </annotation>
+      <annotation id="105">
+        <infon key="score">0.9941329</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:30:28Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4655" length="20"/>
+        <text>glycoside hydrolases</text>
+      </annotation>
+      <annotation id="1015">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:56:02Z</infon>
+        <location offset="4687" length="18"/>
+        <text>subsites −1 and +1</text>
+      </annotation>
+      <annotation id="106">
+        <infon key="score">0.9901659</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:56:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4711" length="8"/>
+        <text>subsites</text>
+      </annotation>
+      <annotation id="107">
+        <infon key="score">0.9971566</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:40:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4865" length="3"/>
+        <text>GH5</text>
+      </annotation>
+      <annotation id="108">
+        <infon key="score">0.9978956</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4869" length="15"/>
+        <text>arabinoxylanase</text>
+      </annotation>
+      <annotation id="109">
+        <infon key="score">0.99816823</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:30Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4886" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="110">
+        <infon key="score">0.9986105</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:33:35Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4908" length="24"/>
+        <text>Clostridium thermocellum</text>
+      </annotation>
+      <annotation id="111">
+        <infon key="score">0.99725866</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4970" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="112">
+        <infon key="score">0.99785656</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4990" length="4"/>
+        <text>Araf</text>
+      </annotation>
+      <annotation id="113">
+        <infon key="score">0.99804723</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5063" length="4"/>
+        <text>Araf</text>
+      </annotation>
+      <annotation id="114">
+        <infon key="score">0.999025</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:02Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5090" length="4"/>
+        <text>Xylp</text>
+      </annotation>
+      <annotation id="115">
+        <infon key="score">0.9891261</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:33:45Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5095" length="8"/>
+        <text>bound in</text>
+      </annotation>
+      <annotation id="116">
+        <infon key="score">0.99886334</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5108" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="117">
+        <infon key="score">0.9914019</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:56:14Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5121" length="10"/>
+        <text>−1 subsite</text>
+      </annotation>
+      <annotation id="118">
+        <infon key="score">0.99909043</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5171" length="13"/>
+        <text>arabinoxylans</text>
+      </annotation>
+      <annotation id="119">
+        <infon key="score">0.99802303</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="5208" length="4"/>
+        <text>Araf</text>
+      </annotation>
+      <annotation id="120">
+        <infon key="score">0.99847084</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:56:18Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5236" length="8"/>
+        <text>subsites</text>
+      </annotation>
+      <annotation id="121">
+        <infon key="score">0.99856865</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5259" length="11"/>
+        <text>active site</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>5280</offset>
+      <text>CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1. Previous studies of Fn3 domains have indicated that they might function as ligand-binding modules, as a compact form of peptide linkers or spacers between other domains, as cellulose-disrupting modules, or as proteins that help large enzyme complexes remain soluble. The dockerin domain recruits the enzyme into the cellulosome, a multienzyme plant cell wall degrading complex presented on the surface of C. thermocellum. CtCBM6 stabilizes CtGH5, and CtCBM62 binds to d-galactopyranose and l-arabinopyranose. The function of the CtCBM13 and Fn3 modules remains unclear. Similarly, the mechanism of substrate recognition and its impact on specificity are key unresolved issues. This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity. The data show that the plasticity in substrate recognition enables the enzyme to hydrolyze highly complex xylans that are not accessible to classical GH10 and GH11 endo-xylanases.</text>
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+        <infon key="score">0.99918216</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:30Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="5280" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="123">
+        <infon key="score">0.67755586</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:40:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5344" length="3"/>
+        <text>GH5</text>
+      </annotation>
+      <annotation id="124">
+        <infon key="score">0.9980315</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:35:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5348" length="16"/>
+        <text>catalytic module</text>
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+      <annotation id="125">
+        <infon key="score">0.860335</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5366" length="5"/>
+        <text>CtGH5</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="5380" length="42"/>
+        <text>non-catalytic carbohydrate binding modules</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:11Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5424" length="4"/>
+        <text>CBMs</text>
+      </annotation>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>6</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:40Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5455" length="6"/>
+        <text>CtCBM6</text>
+      </annotation>
+      <annotation id="130">
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="5464" length="2"/>
+        <text>13</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="5468" length="7"/>
+        <text>CtCBM13</text>
+      </annotation>
+      <annotation id="132">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:18Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5482" length="2"/>
+        <text>62</text>
+      </annotation>
+      <annotation id="133">
+        <infon key="score">0.8562604</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5486" length="7"/>
+        <text>CtCBM62</text>
+      </annotation>
+      <annotation id="134">
+        <infon key="score">0.9989775</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:53Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5496" length="18"/>
+        <text>fibronectin type 3</text>
+      </annotation>
+      <annotation id="135">
+        <infon key="score">0.9988217</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:03Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5516" length="3"/>
+        <text>Fn3</text>
+      </annotation>
+      <annotation id="136">
+        <infon key="score">0.99874604</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5546" length="8"/>
+        <text>dockerin</text>
+      </annotation>
+      <annotation id="137">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5590" length="3"/>
+        <text>Fn3</text>
+      </annotation>
+      <annotation id="138">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:51:22Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5645" length="22"/>
+        <text>ligand-binding modules</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5743" length="28"/>
+        <text>cellulose-disrupting modules</text>
+      </annotation>
+      <annotation id="140">
+        <infon key="score">0.9987042</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5841" length="8"/>
+        <text>dockerin</text>
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+      <annotation id="141">
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+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:32Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="5886" length="11"/>
+        <text>cellulosome</text>
+      </annotation>
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+        <infon key="score">0.98729646</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5913" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="143">
+        <infon key="score">0.99846244</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5975" length="15"/>
+        <text>C. thermocellum</text>
+      </annotation>
+      <annotation id="144">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:40Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="5992" length="6"/>
+        <text>CtCBM6</text>
+      </annotation>
+      <annotation id="145">
+        <infon key="score">0.6251633</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6010" length="5"/>
+        <text>CtGH5</text>
+      </annotation>
+      <annotation id="146">
+        <infon key="score">0.59856856</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:36Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6021" length="7"/>
+        <text>CtCBM62</text>
+      </annotation>
+      <annotation id="147">
+        <infon key="score">0.99911016</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:43Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="6038" length="17"/>
+        <text>d-galactopyranose</text>
+      </annotation>
+      <annotation id="148">
+        <infon key="score">0.99909335</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:47Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="6060" length="17"/>
+        <text>l-arabinopyranose</text>
+      </annotation>
+      <annotation id="149">
+        <infon key="score">0.98460996</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6099" length="7"/>
+        <text>CtCBM13</text>
+      </annotation>
+      <annotation id="905">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:04Z</infon>
+        <location offset="6111" length="3"/>
+        <text>Fn3</text>
+      </annotation>
+      <annotation id="150">
+        <infon key="score">0.9985752</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6272" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="151">
+        <infon key="score">0.99908304</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:39:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6293" length="6"/>
+        <text>mature</text>
+      </annotation>
+      <annotation id="152">
+        <infon key="score">0.9991793</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:30Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="6300" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="153">
+        <infon key="score">0.99882346</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:39:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6308" length="7"/>
+        <text>lacking</text>
+      </annotation>
+      <annotation id="907">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:10Z</infon>
+        <location offset="6331" length="8"/>
+        <text>dockerin</text>
+      </annotation>
+      <annotation id="154">
+        <infon key="score">0.7470989</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:39:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6348" length="11"/>
+        <text>CtXyl5A-Doc</text>
+      </annotation>
+      <annotation id="893">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:04Z</infon>
+        <location offset="6377" length="15"/>
+        <text>in complex with</text>
+      </annotation>
+      <annotation id="910">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:40:19Z</infon>
+        <location offset="6393" length="7"/>
+        <text>ligands</text>
+      </annotation>
+      <annotation id="155">
+        <infon key="score">0.9989073</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="6559" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="156">
+        <infon key="score">0.99873036</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6603" length="4"/>
+        <text>GH10</text>
+      </annotation>
+      <annotation id="157">
+        <infon key="score">0.99821556</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6612" length="4"/>
+        <text>GH11</text>
+      </annotation>
+      <annotation id="158">
+        <infon key="score">0.9986395</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:35:35Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6617" length="14"/>
+        <text>endo-xylanases</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">zbc0441653440001.jpg</infon>
+      <infon key="id">F1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>6633</offset>
+      <text>Molecular architecture of GH5_34 enzymes. Modules prefaced by GH, CBM, or CE are modules in the indicated glycoside hydrolase, carbohydrate binding module, or carbohydrate esterase families, respectively. Laminin_3_G domain belongs to the concanavalin A lectin superfamily, and FN3 denotes a fibronectin type 3 domain. Segments labeled D are dockerin domains.</text>
+      <annotation id="958">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:36:54Z</infon>
+        <location offset="6659" length="6"/>
+        <text>GH5_34</text>
+      </annotation>
+      <annotation id="159">
+        <infon key="score">0.7534902</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:41:23Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6695" length="2"/>
+        <text>GH</text>
+      </annotation>
+      <annotation id="160">
+        <infon key="score">0.9863507</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:51:27Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6699" length="3"/>
+        <text>CBM</text>
+      </annotation>
+      <annotation id="161">
+        <infon key="score">0.99045384</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:51:31Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6707" length="2"/>
+        <text>CE</text>
+      </annotation>
+      <annotation id="162">
+        <infon key="score">0.99766254</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6739" length="19"/>
+        <text>glycoside hydrolase</text>
+      </annotation>
+      <annotation id="163">
+        <infon key="score">0.7450091</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:42:37Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6760" length="27"/>
+        <text>carbohydrate binding module</text>
+      </annotation>
+      <annotation id="164">
+        <infon key="score">0.9983574</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:42:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6792" length="21"/>
+        <text>carbohydrate esterase</text>
+      </annotation>
+      <annotation id="911">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:41:42Z</infon>
+        <location offset="6838" length="11"/>
+        <text>Laminin_3_G</text>
+      </annotation>
+      <annotation id="165">
+        <infon key="score">0.99794406</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:41:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6872" length="33"/>
+        <text>concanavalin A lectin superfamily</text>
+      </annotation>
+      <annotation id="166">
+        <infon key="score">0.998723</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6911" length="3"/>
+        <text>FN3</text>
+      </annotation>
+      <annotation id="167">
+        <infon key="score">0.9979355</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:51:37Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="6925" length="25"/>
+        <text>fibronectin type 3 domain</text>
+      </annotation>
+      <annotation id="908">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:10Z</infon>
+        <location offset="6975" length="8"/>
+        <text>dockerin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_1</infon>
+      <offset>6993</offset>
+      <text>Results</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_4</infon>
+      <offset>7001</offset>
+      <text>Substrate Specificity of CtXyl5A</text>
+      <annotation id="168">
+        <infon key="score">0.99469876</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:30Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7026" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>7034</offset>
+      <text>Previous studies showed that CtXyl5A is an arabinoxylan-specific xylanase that generates xylooligosaccharides with an arabinose linked O3 to the reducing end xylose. The enzyme is active against both wheat and rye arabinoxylans (abbreviated as WAX and RAX, respectively). It was proposed that arabinose decorations make productive interactions with a pocket (−2*) that is abutted onto the active site or −1 subsite. Arabinose side chains of the other backbone xylose units in the oligosaccharides generated by CtXyl5A were essentially random. These data suggest that O3, and possibly O2, on the xylose residues at subsites distal to the active site and −2* pocket are solvent-exposed, implying that the enzyme can access highly decorated xylans. To test this hypothesis, the activity of CtXyl5A against xylans from cereal brans was assessed. CtXyl5a was incubated with a range of xylans for 16 h at 60 °C, and the limit products were visualized by TLC. These xylans are highly decorated not only with Araf and GlcA units but also with l-Gal, d-Gal, and d-Xyl. Indeed, very few xylose units in the backbone of bran xylans lack side chains. The data presented in Table 1 showed that CtXyl5A was active against corn bran xylan (CX). In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues). The limit products generated by CtXyl5A from CX consisted of an extensive range of oligosaccharides. These data support the view that in subsites out with the active site the O2 and O3 groups of the bound xylose units are solvent-exposed and will thus tolerate decoration.</text>
+      <annotation id="169">
+        <infon key="score">0.999027</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7063" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="170">
+        <infon key="score">0.9967934</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:34:39Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7077" length="30"/>
+        <text>arabinoxylan-specific xylanase</text>
+      </annotation>
+      <annotation id="171">
+        <infon key="score">0.9990753</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7123" length="20"/>
+        <text>xylooligosaccharides</text>
+      </annotation>
+      <annotation id="172">
+        <infon key="score">0.99847776</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7152" length="9"/>
+        <text>arabinose</text>
+      </annotation>
+      <annotation id="173">
+        <infon key="score">0.99852186</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7192" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="174">
+        <infon key="score">0.8807422</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:44:28Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7234" length="5"/>
+        <text>wheat</text>
+      </annotation>
+      <annotation id="175">
+        <infon key="score">0.99000096</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:44:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7244" length="3"/>
+        <text>rye</text>
+      </annotation>
+      <annotation id="176">
+        <infon key="score">0.99898416</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7248" length="13"/>
+        <text>arabinoxylans</text>
+      </annotation>
+      <annotation id="177">
+        <infon key="score">0.99872786</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:23Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7278" length="3"/>
+        <text>WAX</text>
+      </annotation>
+      <annotation id="178">
+        <infon key="score">0.9978104</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:27Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7286" length="3"/>
+        <text>RAX</text>
+      </annotation>
+      <annotation id="179">
+        <infon key="score">0.9987662</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7327" length="9"/>
+        <text>arabinose</text>
+      </annotation>
+      <annotation id="180">
+        <infon key="score">0.99883765</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:56:24Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7385" length="6"/>
+        <text>pocket</text>
+      </annotation>
+      <annotation id="1016">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:56:40Z</infon>
+        <location offset="7393" length="3"/>
+        <text>−2*</text>
+      </annotation>
+      <annotation id="181">
+        <infon key="score">0.9989691</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7423" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="182">
+        <infon key="score">0.99868107</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:56:44Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7438" length="10"/>
+        <text>−1 subsite</text>
+      </annotation>
+      <annotation id="183">
+        <infon key="score">0.99863005</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7450" length="9"/>
+        <text>Arabinose</text>
+      </annotation>
+      <annotation id="184">
+        <infon key="score">0.99874973</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7494" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="185">
+        <infon key="score">0.9984124</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:47Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7514" length="16"/>
+        <text>oligosaccharides</text>
+      </annotation>
+      <annotation id="186">
+        <infon key="score">0.99849904</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7544" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="187">
+        <infon key="score">0.9846201</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7629" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="188">
+        <infon key="score">0.99893147</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:56:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7648" length="8"/>
+        <text>subsites</text>
+      </annotation>
+      <annotation id="189">
+        <infon key="score">0.9989191</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7671" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="190">
+        <infon key="score">0.99832433</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:56:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7687" length="10"/>
+        <text>−2* pocket</text>
+      </annotation>
+      <annotation id="191">
+        <infon key="score">0.99759704</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:48:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7702" length="15"/>
+        <text>solvent-exposed</text>
+      </annotation>
+      <annotation id="192">
+        <infon key="score">0.9976566</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7772" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="193">
+        <infon key="score">0.99891055</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7821" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="194">
+        <infon key="score">0.9827283</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7837" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="195">
+        <infon key="score">0.9425561</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:43:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7849" length="6"/>
+        <text>cereal</text>
+      </annotation>
+      <annotation id="196">
+        <infon key="score">0.9973623</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="7876" length="7"/>
+        <text>CtXyl5a</text>
+      </annotation>
+      <annotation id="197">
+        <infon key="score">0.9663724</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:05:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7888" length="9"/>
+        <text>incubated</text>
+      </annotation>
+      <annotation id="198">
+        <infon key="score">0.9815502</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7914" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="199">
+        <infon key="score">0.9964102</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:47:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7982" length="3"/>
+        <text>TLC</text>
+      </annotation>
+      <annotation id="200">
+        <infon key="score">0.9857853</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7993" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="201">
+        <infon key="score">0.99912196</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8035" length="4"/>
+        <text>Araf</text>
+      </annotation>
+      <annotation id="202">
+        <infon key="score">0.9990427</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:29:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8044" length="4"/>
+        <text>GlcA</text>
+      </annotation>
+      <annotation id="203">
+        <infon key="score">0.99907273</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:20Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8069" length="5"/>
+        <text>l-Gal</text>
+      </annotation>
+      <annotation id="204">
+        <infon key="score">0.9989554</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:27Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8076" length="5"/>
+        <text>d-Gal</text>
+      </annotation>
+      <annotation id="205">
+        <infon key="score">0.999036</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8087" length="5"/>
+        <text>d-Xyl</text>
+      </annotation>
+      <annotation id="206">
+        <infon key="score">0.9979019</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8111" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="207">
+        <infon key="score">0.9713136</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8148" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="208">
+        <infon key="score">0.9989698</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8215" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="912">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:08Z</infon>
+        <location offset="8242" length="4"/>
+        <text>corn</text>
+      </annotation>
+      <annotation id="209">
+        <infon key="score">0.9789818</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8252" length="5"/>
+        <text>xylan</text>
+      </annotation>
+      <annotation id="210">
+        <infon key="score">0.9837578</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8259" length="2"/>
+        <text>CX</text>
+      </annotation>
+      <annotation id="211">
+        <infon key="score">0.99865055</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:35:36Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8284" length="14"/>
+        <text>endo-xylanases</text>
+      </annotation>
+      <annotation id="212">
+        <infon key="score">0.99723804</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8304" length="4"/>
+        <text>GH10</text>
+      </annotation>
+      <annotation id="213">
+        <infon key="score">0.9968777</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8313" length="4"/>
+        <text>GH11</text>
+      </annotation>
+      <annotation id="214">
+        <infon key="score">0.7958819</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8340" length="2"/>
+        <text>CX</text>
+      </annotation>
+      <annotation id="919">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:47:40Z</infon>
+        <location offset="8359" length="7"/>
+        <text>lack of</text>
+      </annotation>
+      <annotation id="215">
+        <infon key="score">0.99779606</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8379" length="6"/>
+        <text>xylose</text>
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+      <annotation id="216">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="8413" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="992">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:02:58Z</infon>
+        <location offset="8451" length="7"/>
+        <text>bind to</text>
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+      <annotation id="217">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8475" length="6"/>
+        <text>xylose</text>
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+      <annotation id="218">
+        <infon key="score">0.99850285</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
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+        <location offset="8525" length="7"/>
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+      <annotation id="219">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8538" length="2"/>
+        <text>CX</text>
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+      <annotation id="220">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T11:03:41Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="8576" length="16"/>
+        <text>oligosaccharides</text>
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+      <annotation id="221">
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:56:58Z</infon>
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+        <location offset="8630" length="8"/>
+        <text>subsites</text>
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+      <annotation id="222">
+        <infon key="score">0.9986953</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
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+      <annotation id="224">
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:48:31Z</infon>
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+        <location offset="8715" length="15"/>
+        <text>solvent-exposed</text>
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+    <passage>
+      <infon key="file">T1.xml</infon>
+      <infon key="id">T1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_caption</infon>
+      <offset>8773</offset>
+      <text>Kinetics of GH5_34 arabinoxylanases</text>
+      <annotation id="993">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:03:30Z</infon>
+        <location offset="8773" length="8"/>
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+      <annotation id="959">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:36:54Z</infon>
+        <location offset="8785" length="6"/>
+        <text>GH5_34</text>
+      </annotation>
+      <annotation id="225">
+        <infon key="score">0.99908423</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:24Z</infon>
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+    <passage>
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+      <infon key="id">T1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_caption</infon>
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+      <text>ND, not determined; NA, no activity.</text>
+    </passage>
+    <passage>
+      <infon key="file">T1.xml</infon>
+      <infon key="id">T1</infon>
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+      <infon key="xml">&lt;?xml version="1.0" encoding="UTF-8"?&gt;
+&lt;table frame="hsides" rules="groups"&gt;&lt;thead valign="bottom"&gt;&lt;tr&gt;&lt;th align="center" rowspan="2" colspan="1"&gt;Enzyme&lt;/th&gt;&lt;th align="center" rowspan="2" colspan="1"&gt;Variant&lt;/th&gt;&lt;th align="center" rowspan="1" colspan="3"&gt;&lt;italic&gt;k&lt;/italic&gt;&lt;sub&gt;cat&lt;/sub&gt;/&lt;italic&gt;K&lt;sub&gt;m&lt;/sub&gt;&lt;/italic&gt;&lt;hr/&gt;&lt;/th&gt;&lt;/tr&gt;&lt;tr&gt;&lt;th align="center" rowspan="1" colspan="1"&gt;WAX&lt;/th&gt;&lt;th align="center" rowspan="1" colspan="1"&gt;RAX&lt;/th&gt;&lt;th align="center" rowspan="1" colspan="1"&gt;CX&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign="top"&gt;&lt;tr&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td align="center" rowspan="1" colspan="3"&gt;&lt;italic&gt;min&lt;/italic&gt;&lt;sup&gt;−&lt;italic&gt;1&lt;/italic&gt;&lt;/sup&gt;
+&lt;italic&gt;mg&lt;/italic&gt;&lt;sup&gt;−&lt;italic&gt;1&lt;/italic&gt;&lt;/sup&gt;
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+</infon>
+      <offset>8846</offset>
+      <text>Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	</text>
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+        <infon key="updated_at">2023-09-18T13:50:05Z</infon>
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+        <text>kcat</text>
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+        <infon key="updated_at">2023-09-18T10:45:23Z</infon>
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+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9136" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="1040">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:29:09Z</infon>
+        <location offset="9144" length="10"/>
+        <text>CtGH5-CBM6</text>
+      </annotation>
+      <annotation id="237">
+        <infon key="score">0.55628043</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9156" length="5"/>
+        <text>N135A</text>
+      </annotation>
+      <annotation id="884">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <location offset="9174" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="1041">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:29:26Z</infon>
+        <location offset="9182" length="10"/>
+        <text>CtGH5-CBM6</text>
+      </annotation>
+      <annotation id="238">
+        <infon key="score">0.86166924</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:54:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9194" length="5"/>
+        <text>N139A</text>
+      </annotation>
+      <annotation id="936">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:50:32Z</infon>
+        <location offset="9211" length="5"/>
+        <text>AcGH5</text>
+      </annotation>
+      <annotation id="239">
+        <infon key="score">0.9990313</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:27Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9217" length="9"/>
+        <text>Wild type</text>
+      </annotation>
+      <annotation id="240">
+        <infon key="score">0.95552933</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:47:40Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9243" length="5"/>
+        <text>GpGH5</text>
+      </annotation>
+      <annotation id="241">
+        <infon key="score">0.99901223</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:27Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9249" length="9"/>
+        <text>Wild type</text>
+      </annotation>
+      <annotation id="931">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:41Z</infon>
+        <location offset="9277" length="5"/>
+        <text>VbGH5</text>
+      </annotation>
+      <annotation id="242">
+        <infon key="score">0.9988765</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9283" length="9"/>
+        <text>Wild type</text>
+      </annotation>
+      <annotation id="930">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:40Z</infon>
+        <location offset="9304" length="5"/>
+        <text>VbGH5</text>
+      </annotation>
+      <annotation id="243">
+        <infon key="score">0.9978617</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9310" length="4"/>
+        <text>D45A</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>9333</offset>
+      <text>To explore whether substrate bound only at −2* and −1 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study. HPAEC analysis of the smallest oligosaccharide fraction (pool 4) contained two species with retention times of 14.0 min (oligosaccharide 1) and 20.8 min (oligosaccharide 2) (Fig. 2). Positive mode electrospray mass spectrometry showed that pool 4 contained exclusively molecular ions with a m/z = 305 [M + Na]+, which corresponds to a pentose-pentose disaccharide (molecular mass = 282 Da) as a sodium ion adduct, whereas a dimer of the disaccharide with a sodium adduct (m/z = 587 [2M+Na]+) was also evident. The monosaccharide composition of pool 4 determined by TFA hydrolysis contained xylose and arabinose in a 3:1 ratio. This suggests that the two oligosaccharides consist of two disaccharides: one consisting of two xylose residues and the other consisting of an arabinose linked to a xylose. Treatment of pool 4 with the nonspecific arabinofuranosidase, CjAbf51A, resulted in the loss of oligosaccharide 2 and the production of both xylose and arabinose, indicative of a disaccharide of xylose and arabinose. Incubation of pool 4 with a β-1,3-xylosidase (XynB) converted oligosaccharide 1 into xylose, demonstrating that this molecule is the disaccharide β-1,3-xylobiose. This view is supported by the inability of a β-1,4-specific xylosidase to hydrolyze oligosaccharide 1 or oligosaccharide 2 (data not shown). The crucial importance of occupancy of the −2* pocket for catalytic competence is illustrated by the inability of the enzyme to hydrolyze linear β-1,4-xylooligosaccharides. The generation of Araf-Xylp and Xyl-β-1,3-Xyl as reaction products demonstrates that occupancy of the −2 subsite is not essential for catalytic activity, which is in contrast to all endo-acting xylanases where this subsite plays a critical role in enzyme activity. Indeed, the data demonstrate that −2* plays a more important role in productive substrate binding than the −2 subsite. Unfortunately, the inability to generate highly purified (Xyl-β-1,4)n-[β-1,3-Xyl/Ara]-Xyl oligosaccharides from arabinoxylans prevented the precise binding energies at the negative subsites to be determined.</text>
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+        <infon key="score">0.76418567</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:35:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="9362" length="13"/>
+        <text>bound only at</text>
+      </annotation>
+      <annotation id="245">
+        <infon key="score">0.99534464</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:57:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9376" length="3"/>
+        <text>−2*</text>
+      </annotation>
+      <annotation id="246">
+        <infon key="score">0.9800586</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:57:06Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9384" length="2"/>
+        <text>−1</text>
+      </annotation>
+      <annotation id="247">
+        <infon key="score">0.9986288</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:57:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="9394" length="17"/>
+        <text>negative subsites</text>
+      </annotation>
+      <annotation id="248">
+        <infon key="score">0.9944119</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="9430" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="921">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:39Z</infon>
+        <location offset="9461" length="2"/>
+        <text>CX</text>
+      </annotation>
+      <annotation id="249">
+        <infon key="score">0.93394166</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9480" length="15"/>
+        <text>arabinoxylanase</text>
+      </annotation>
+      <annotation id="250">
+        <infon key="score">0.99740463</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:59:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9514" length="29"/>
+        <text>size exclusion chromatography</text>
+      </annotation>
+      <annotation id="251">
+        <infon key="score">0.99753416</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T11:03:40Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9582" length="16"/>
+        <text>oligosaccharides</text>
+      </annotation>
+      <annotation id="252">
+        <infon key="score">0.9985832</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:58:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9655" length="5"/>
+        <text>HPAEC</text>
+      </annotation>
+      <annotation id="253">
+        <infon key="score">0.998097</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:38:46Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="9686" length="15"/>
+        <text>oligosaccharide</text>
+      </annotation>
+      <annotation id="998">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:39:31Z</infon>
+        <location offset="9776" length="15"/>
+        <text>oligosaccharide</text>
+      </annotation>
+      <annotation id="999">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:39:32Z</infon>
+        <location offset="9809" length="15"/>
+        <text>oligosaccharide</text>
+      </annotation>
+      <annotation id="254">
+        <infon key="score">0.9654604</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:58:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="9838" length="44"/>
+        <text>Positive mode electrospray mass spectrometry</text>
+      </annotation>
+      <annotation id="949">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:16:00Z</infon>
+        <location offset="9990" length="7"/>
+        <text>pentose</text>
+      </annotation>
+      <annotation id="950">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:16:00Z</infon>
+        <location offset="9998" length="7"/>
+        <text>pentose</text>
+      </annotation>
+      <annotation id="255">
+        <infon key="score">0.7560745</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T11:03:20Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10006" length="12"/>
+        <text>disaccharide</text>
+      </annotation>
+      <annotation id="256">
+        <infon key="score">0.8210523</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T11:03:20Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10092" length="12"/>
+        <text>disaccharide</text>
+      </annotation>
+      <annotation id="257">
+        <infon key="score">0.9970212</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:59:12Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10220" length="14"/>
+        <text>TFA hydrolysis</text>
+      </annotation>
+      <annotation id="258">
+        <infon key="score">0.9957487</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10245" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="259">
+        <infon key="score">0.9935015</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10256" length="9"/>
+        <text>arabinose</text>
+      </annotation>
+      <annotation id="260">
+        <infon key="score">0.9950472</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T11:03:41Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10309" length="16"/>
+        <text>oligosaccharides</text>
+      </annotation>
+      <annotation id="261">
+        <infon key="score">0.9897843</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T11:04:03Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10341" length="13"/>
+        <text>disaccharides</text>
+      </annotation>
+      <annotation id="262">
+        <infon key="score">0.9872656</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10378" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="263">
+        <infon key="score">0.97808754</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10425" length="9"/>
+        <text>arabinose</text>
+      </annotation>
+      <annotation id="264">
+        <infon key="score">0.9635059</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10447" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="947">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:15:03Z</infon>
+        <location offset="10484" length="31"/>
+        <text>nonspecific arabinofuranosidase</text>
+      </annotation>
+      <annotation id="265">
+        <infon key="score">0.9518543</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:54:51Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10517" length="8"/>
+        <text>CjAbf51A</text>
+      </annotation>
+      <annotation id="997">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:39:31Z</infon>
+        <location offset="10551" length="15"/>
+        <text>oligosaccharide</text>
+      </annotation>
+      <annotation id="266">
+        <infon key="score">0.9971559</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10596" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="267">
+        <infon key="score">0.9963881</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10607" length="9"/>
+        <text>arabinose</text>
+      </annotation>
+      <annotation id="944">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T11:03:20Z</infon>
+        <location offset="10634" length="12"/>
+        <text>disaccharide</text>
+      </annotation>
+      <annotation id="268">
+        <infon key="score">0.9972711</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10650" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="269">
+        <infon key="score">0.9962746</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10661" length="9"/>
+        <text>arabinose</text>
+      </annotation>
+      <annotation id="270">
+        <infon key="score">0.9963164</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:55:06Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10700" length="16"/>
+        <text>β-1,3-xylosidase</text>
+      </annotation>
+      <annotation id="271">
+        <infon key="score">0.5966584</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:55:11Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10718" length="4"/>
+        <text>XynB</text>
+      </annotation>
+      <annotation id="1000">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:39:32Z</infon>
+        <location offset="10734" length="15"/>
+        <text>oligosaccharide</text>
+      </annotation>
+      <annotation id="272">
+        <infon key="score">0.9976882</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10757" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="945">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T11:03:20Z</infon>
+        <location offset="10805" length="12"/>
+        <text>disaccharide</text>
+      </annotation>
+      <annotation id="273">
+        <infon key="score">0.9992453</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <text>β-1,3-xylobiose</text>
+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:59:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10880" length="25"/>
+        <text>β-1,4-specific xylosidase</text>
+      </annotation>
+      <annotation id="1001">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:39:32Z</infon>
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:39:32Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:55:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <text>β-1,4-xylooligosaccharides</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:55:27Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:07Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <infon key="score">0.99076325</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+      <annotation id="951">
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+      <annotation id="888">
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:42Z</infon>
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+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>11762</offset>
+      <text>Identification of the disaccharide reaction products generated from CX. The smallest reaction products were purified by size exclusion chromatography and analyzed by HPAEC (A) and positive mode ESI-MS (B), respectively. The samples were treated with a nonspecific arabinofuranosidase (CjAbf51A) and a GH3 xylosidase (XynB) that targeted β-1,3-xylosidic bonds. X, xylose; A, arabinose. The m/z = 305 species denotes a pentose disaccharide as a sodium adduct [M + Na]+, whereas the m/z = 587 signal corresponds to an ESI-MS dimer of the pentose disaccharide also as a sodium adduct [2M + Na]+.</text>
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+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11784" length="12"/>
+        <text>disaccharide</text>
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+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11830" length="2"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:59:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11882" length="29"/>
+        <text>size exclusion chromatography</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:58:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11928" length="5"/>
+        <text>HPAEC</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="11956" length="6"/>
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+      <annotation id="946">
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:15:02Z</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="12047" length="8"/>
+        <text>CjAbf51A</text>
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+      <annotation id="948">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:15:31Z</infon>
+        <location offset="12063" length="14"/>
+        <text>GH3 xylosidase</text>
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+      <annotation id="291">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:55:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12079" length="4"/>
+        <text>XynB</text>
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+      <annotation id="292">
+        <infon key="score">0.99745184</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12125" length="6"/>
+        <text>xylose</text>
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+      <annotation id="293">
+        <infon key="score">0.99641883</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12136" length="9"/>
+        <text>arabinose</text>
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+      <annotation id="294">
+        <infon key="score">0.9981957</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:16:00Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12179" length="7"/>
+        <text>pentose</text>
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+        <infon key="score">0.76520646</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T11:03:20Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12187" length="12"/>
+        <text>disaccharide</text>
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+      <annotation id="296">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:14:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12277" length="6"/>
+        <text>ESI-MS</text>
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+      <annotation id="297">
+        <infon key="score">0.9983165</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:15:59Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12297" length="7"/>
+        <text>pentose</text>
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+      <annotation id="298">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T11:03:20Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12305" length="12"/>
+        <text>disaccharide</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_4</infon>
+      <offset>12357</offset>
+      <text>Crystal Structure of the Catalytic Module of CtXyl5A in Complex with Ligands</text>
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+        <infon key="score">0.9987371</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12357" length="17"/>
+        <text>Crystal Structure</text>
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+      <annotation id="300">
+        <infon key="score">0.998405</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:35:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12382" length="16"/>
+        <text>Catalytic Module</text>
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+      <annotation id="301">
+        <infon key="score">0.95691925</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12402" length="7"/>
+        <text>CtXyl5A</text>
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+      <annotation id="302">
+        <infon key="score">0.99861926</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12410" length="15"/>
+        <text>in Complex with</text>
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+      <annotation id="303">
+        <infon key="score">0.46196067</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:40:17Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12426" length="7"/>
+        <text>Ligands</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>12434</offset>
+      <text>To understand the structural basis for the biochemical properties of CtXyl5A, the crystal structure of the enzyme with ligands that occupy the substrate binding cleft and the critical −2* subsite were sought. The data presented in Fig. 3A show the structure of the CtXyl5A derivative CtGH5-CtCBM6 in complex with arabinose bound in the −2* pocket. Interestingly, the bound arabinose was in the pyranose conformation rather than in its furanose form found in arabinoxylans. O1 was facing toward the active site −1 subsite, indicative of the bound arabinose being in the right orientation to be linked to the xylan backbone via an α-1,3 linkage. As discussed on below, the axial O4 of the Arap did not interact with the −2* subsite, suggesting that the pocket might be capable of binding a xylose molecule. Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the −2* subsite in its pyranose conformation (Fig. 3B). These crystal structures support the biochemical data presented above showing that the enzyme generated β-1,3-xylobiose from CX, which would require the disaccharide to bind at the −1 and −2* subsites. A third product complex was generated by co-crystallizing the nucleophile inactive mutant CtGH5E279S-CtCBM6 with a WAX-derived oligosaccharide (Fig. 3C). The data revealed a pentasaccharide bound to the enzyme, comprising β-1,4-xylotetraose with an Araf linked α-1,3 to the reducing end xylose. The xylotetraose was positioned in subsites −1 to −4 and the Araf in the −2* pocket. Analysis of the three structures showed that O1, O2, O3, and the endocyclic oxygen occupied identical positions in the Arap, Araf, and Xylp ligands bound in the −2* subsite and thus made identical interactions with the pocket. O1 makes a polar contact with Nδ2 of Asn139, O2 is within hydrogen bonding distance with Oδ1 of Asn139 and the backbone N of Asn135, and O3 interacts with the N of Gly136 and Oϵ2 of Glu68. Although O4 of Arap does not make a direct interaction with the enzyme, O4 and O5 of Xylp and Araf, respectively, form hydrogen bonds with Oϵ1 of Glu68. Finally Tyr92 makes apolar parallel interactions with the pyranose or furanose rings of the three sugars.</text>
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+        <infon key="identifier">PR:</infon>
+        <location offset="12503" length="7"/>
+        <text>CtXyl5A</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <text>substrate binding cleft</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:50:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12682" length="9"/>
+        <text>structure</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12699" length="7"/>
+        <text>CtXyl5A</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:29:50Z</infon>
+        <location offset="12718" length="12"/>
+        <text>CtGH5-CtCBM6</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12747" length="9"/>
+        <text>arabinose</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:33:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>bound in</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:57:34Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12770" length="10"/>
+        <text>−2* pocket</text>
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+      <annotation id="314">
+        <infon key="score">0.9988469</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:17:09Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12801" length="5"/>
+        <text>bound</text>
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+        <infon key="type">chemical</infon>
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+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12807" length="9"/>
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+      <annotation id="1004">
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:41:14Z</infon>
+        <location offset="12828" length="8"/>
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+      <annotation id="1008">
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:41:26Z</infon>
+        <location offset="12869" length="8"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12892" length="13"/>
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+      <annotation id="317">
+        <infon key="score">0.99867976</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12932" length="11"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:57:38Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12944" length="10"/>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12974" length="5"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12980" length="9"/>
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+        <infon key="updated_at">2023-09-18T10:24:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="updated_at">2023-09-18T13:40:21Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13222" length="6"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:21:15Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="13247" length="7"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:50:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13259" length="8"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13273" length="6"/>
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:40:56Z</infon>
+        <location offset="13296" length="7"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:40:26Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13304" length="5"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:33:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13315" length="8"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="13328" length="11"/>
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:41:15Z</infon>
+        <location offset="13347" length="8"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:50:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13386" length="18"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:41:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13484" length="15"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13505" length="2"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T11:03:20Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13533" length="12"/>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:58:09Z</infon>
+        <location offset="13561" length="19"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:05:47Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="13623" length="16"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:36:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13644" length="20"/>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:36:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13665" length="6"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:09:45Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="13672" length="10"/>
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+      <annotation id="903">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:40Z</infon>
+        <location offset="13683" length="6"/>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:23Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13697" length="3"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:39:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13709" length="15"/>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:41:37Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13756" length="15"/>
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+      <annotation id="344">
+        <infon key="score">0.9987526</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:19:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13772" length="8"/>
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+        <infon key="score">0.99912447</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:41:41Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13804" length="18"/>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13831" length="4"/>
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+      <annotation id="347">
+        <infon key="score">0.9987872</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13869" length="6"/>
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+      <annotation id="348">
+        <infon key="score">0.99923956</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:41:45Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13881" length="12"/>
+        <text>xylotetraose</text>
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+      <annotation id="349">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:58:13Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13912" length="17"/>
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+      <annotation id="350">
+        <infon key="score">0.9991596</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13938" length="4"/>
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+      <annotation id="351">
+        <infon key="score">0.9983297</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:58:16Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="13950" length="10"/>
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+      <annotation id="352">
+        <infon key="score">0.9958412</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:50:27Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13984" length="10"/>
+        <text>structures</text>
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+        <infon key="score">0.99901795</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:40:22Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14081" length="4"/>
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+        <infon key="score">0.9989477</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14087" length="4"/>
+        <text>Araf</text>
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+      <annotation id="355">
+        <infon key="score">0.9988612</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:02Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14097" length="4"/>
+        <text>Xylp</text>
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+      <annotation id="356">
+        <infon key="score">0.9988979</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:33:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14110" length="8"/>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:58:18Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="14123" length="11"/>
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+      <annotation id="358">
+        <infon key="score">0.9976653</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:58:22Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="14181" length="6"/>
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+      <annotation id="359">
+        <infon key="score">0.9838765</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:20:15Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14200" length="13"/>
+        <text>polar contact</text>
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+      <annotation id="360">
+        <infon key="score">0.9994537</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14226" length="6"/>
+        <text>Asn139</text>
+      </annotation>
+      <annotation id="361">
+        <infon key="score">0.99667907</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:20:11Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14247" length="16"/>
+        <text>hydrogen bonding</text>
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+      <annotation id="362">
+        <infon key="score">0.999443</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14285" length="6"/>
+        <text>Asn139</text>
+      </annotation>
+      <annotation id="363">
+        <infon key="score">0.99943477</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:23:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14314" length="6"/>
+        <text>Asn135</text>
+      </annotation>
+      <annotation id="364">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:06:25Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14353" length="6"/>
+        <text>Gly136</text>
+      </annotation>
+      <annotation id="365">
+        <infon key="score">0.99930656</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14371" length="5"/>
+        <text>Glu68</text>
+      </annotation>
+      <annotation id="366">
+        <infon key="score">0.99909437</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:40:22Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14393" length="4"/>
+        <text>Arap</text>
+      </annotation>
+      <annotation id="367">
+        <infon key="score">0.9990483</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:02Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14463" length="4"/>
+        <text>Xylp</text>
+      </annotation>
+      <annotation id="368">
+        <infon key="score">0.9990252</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14472" length="4"/>
+        <text>Araf</text>
+      </annotation>
+      <annotation id="369">
+        <infon key="score">0.9971278</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:19:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14497" length="14"/>
+        <text>hydrogen bonds</text>
+      </annotation>
+      <annotation id="370">
+        <infon key="score">0.99928504</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14524" length="5"/>
+        <text>Glu68</text>
+      </annotation>
+      <annotation id="371">
+        <infon key="score">0.9993992</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14539" length="5"/>
+        <text>Tyr92</text>
+      </annotation>
+      <annotation id="372">
+        <infon key="score">0.97813207</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:20:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14558" length="21"/>
+        <text>parallel interactions</text>
+      </annotation>
+      <annotation id="1007">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:41:15Z</infon>
+        <location offset="14589" length="8"/>
+        <text>pyranose</text>
+      </annotation>
+      <annotation id="1009">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:41:27Z</infon>
+        <location offset="14601" length="8"/>
+        <text>furanose</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">zbc0441653440003.jpg</infon>
+      <infon key="id">F3</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>14671</offset>
+      <text>Representation of the residues involved in the ligands recognition at the −2* subsite. The protein backbone is represented as a cartoon in gray. Interacting residues are represented as stick in blue, and the catalytic residues and the mutated glutamate (into a serine) are in magenta. A, CtGH5-CBM6 in complex with an arabinopyranose. B, CtGH5-CBM6 in complex with a xylopyranose. C, CtGH5E279S-CBM6 in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose). The xylan backbone is shown transparently for more clarity. Densities shown in blue are RefMac maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ. The figure and all other structural figures were made with PyMOL unless otherwise stated.</text>
+      <annotation id="373">
+        <infon key="score">0.99794537</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:58:28Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="14745" length="11"/>
+        <text>−2* subsite</text>
+      </annotation>
+      <annotation id="374">
+        <infon key="score">0.99496317</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:58:36Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="14879" length="18"/>
+        <text>catalytic residues</text>
+      </annotation>
+      <annotation id="1018">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:58:46Z</infon>
+        <location offset="14906" length="7"/>
+        <text>mutated</text>
+      </annotation>
+      <annotation id="375">
+        <infon key="score">0.9964224</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:21:34Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="14914" length="9"/>
+        <text>glutamate</text>
+      </annotation>
+      <annotation id="376">
+        <infon key="score">0.9966878</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:21:37Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="14932" length="6"/>
+        <text>serine</text>
+      </annotation>
+      <annotation id="1043">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:30:17Z</infon>
+        <location offset="14959" length="10"/>
+        <text>CtGH5-CBM6</text>
+      </annotation>
+      <annotation id="377">
+        <infon key="score">0.96348566</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14970" length="15"/>
+        <text>in complex with</text>
+      </annotation>
+      <annotation id="378">
+        <infon key="score">0.9985078</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:41:51Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="14989" length="15"/>
+        <text>arabinopyranose</text>
+      </annotation>
+      <annotation id="1044">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:30:42Z</infon>
+        <location offset="15009" length="10"/>
+        <text>CtGH5-CBM6</text>
+      </annotation>
+      <annotation id="379">
+        <infon key="score">0.9178238</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15020" length="15"/>
+        <text>in complex with</text>
+      </annotation>
+      <annotation id="380">
+        <infon key="score">0.9992086</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:41:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15038" length="12"/>
+        <text>xylopyranose</text>
+      </annotation>
+      <annotation id="381">
+        <infon key="score">0.99710983</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:09:51Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15055" length="10"/>
+        <text>CtGH5E279S</text>
+      </annotation>
+      <annotation id="939">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:51:34Z</infon>
+        <location offset="15066" length="4"/>
+        <text>CBM6</text>
+      </annotation>
+      <annotation id="382">
+        <infon key="score">0.9848954</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15071" length="15"/>
+        <text>in complex with</text>
+      </annotation>
+      <annotation id="383">
+        <infon key="score">0.99923825</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:42:01Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15089" length="15"/>
+        <text>pentasaccharide</text>
+      </annotation>
+      <annotation id="384">
+        <infon key="score">0.99916303</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:42:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15106" length="17"/>
+        <text>β1,4-xylotetraose</text>
+      </annotation>
+      <annotation id="385">
+        <infon key="score">0.999108</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:25:05Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15132" length="6"/>
+        <text>l-Araf</text>
+      </annotation>
+      <annotation id="386">
+        <infon key="score">0.9986161</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15171" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="387">
+        <infon key="score">0.9975291</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15184" length="5"/>
+        <text>xylan</text>
+      </annotation>
+      <annotation id="388">
+        <infon key="score">0.9933159</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:22:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15240" length="9"/>
+        <text>Densities</text>
+      </annotation>
+      <annotation id="952">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:22:32Z</infon>
+        <location offset="15275" length="48"/>
+        <text>maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>15430</offset>
+      <text>The importance of the interactions between the ligands and the side chains of the residues in the −2* pocket were evaluated by alanine substitution of these amino acids. The mutants E68A, Y92A, and N139A were all inactive (Table 1), demonstrating the importance of the interactions of these residues with the substrate and reinforcing the critical role the −2* subsite plays in the activity of the enzyme. N135A retained wild type activity because the O2 of the sugars interacts with the backbone N of Asn135 and not with the side chain. Because the hydroxyls of Xylp or Araf in the −2* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration. This may explain why the kcat/Km for CtXyl5A against WAX was 2-fold higher than against CX (Table 1). WAX is likely to have a higher concentration of single Araf decorations compared with CX and thus contain more substrate available to the arabinoxylanase.</text>
+      <annotation id="389">
+        <infon key="score">0.99893385</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:58:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="15528" length="10"/>
+        <text>−2* pocket</text>
+      </annotation>
+      <annotation id="390">
+        <infon key="score">0.9966867</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:23:04Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15557" length="20"/>
+        <text>alanine substitution</text>
+      </annotation>
+      <annotation id="391">
+        <infon key="score">0.9985077</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:22:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15604" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="392">
+        <infon key="score">0.9990114</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:08Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15612" length="4"/>
+        <text>E68A</text>
+      </annotation>
+      <annotation id="393">
+        <infon key="score">0.9989949</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15618" length="4"/>
+        <text>Y92A</text>
+      </annotation>
+      <annotation id="394">
+        <infon key="score">0.99898463</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:54:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15628" length="5"/>
+        <text>N139A</text>
+      </annotation>
+      <annotation id="395">
+        <infon key="score">0.99903464</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:23:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15643" length="8"/>
+        <text>inactive</text>
+      </annotation>
+      <annotation id="396">
+        <infon key="score">0.99899477</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:58:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="15787" length="11"/>
+        <text>−2* subsite</text>
+      </annotation>
+      <annotation id="397">
+        <infon key="score">0.9990736</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:17Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15836" length="5"/>
+        <text>N135A</text>
+      </annotation>
+      <annotation id="398">
+        <infon key="score">0.99906486</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:27Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15851" length="9"/>
+        <text>wild type</text>
+      </annotation>
+      <annotation id="399">
+        <infon key="score">0.9993063</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:23:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15932" length="6"/>
+        <text>Asn135</text>
+      </annotation>
+      <annotation id="400">
+        <infon key="score">0.9992543</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:02Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15993" length="4"/>
+        <text>Xylp</text>
+      </annotation>
+      <annotation id="401">
+        <infon key="score">0.99923706</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16001" length="4"/>
+        <text>Araf</text>
+      </annotation>
+      <annotation id="402">
+        <infon key="score">0.9989507</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:58:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="16013" length="10"/>
+        <text>−2* pocket</text>
+      </annotation>
+      <annotation id="926">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:48:32Z</infon>
+        <location offset="16032" length="15"/>
+        <text>solvent-exposed</text>
+      </annotation>
+      <annotation id="403">
+        <infon key="score">0.99911714</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="16053" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="404">
+        <infon key="score">0.9991146</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16072" length="15"/>
+        <text>arabinoxylanase</text>
+      </annotation>
+      <annotation id="405">
+        <infon key="score">0.99835086</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16105" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="406">
+        <infon key="score">0.9980102</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16143" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="407">
+        <infon key="score">0.9981377</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16153" length="9"/>
+        <text>arabinose</text>
+      </annotation>
+      <annotation id="1012">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:50:52Z</infon>
+        <location offset="16203" length="4"/>
+        <text>kcat</text>
+      </annotation>
+      <annotation id="1013">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:51:00Z</infon>
+        <location offset="16208" length="2"/>
+        <text>Km</text>
+      </annotation>
+      <annotation id="408">
+        <infon key="score">0.9909752</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16215" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="409">
+        <infon key="score">0.9985014</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:23Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16231" length="3"/>
+        <text>WAX</text>
+      </annotation>
+      <annotation id="410">
+        <infon key="score">0.7723853</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16266" length="2"/>
+        <text>CX</text>
+      </annotation>
+      <annotation id="411">
+        <infon key="score">0.99858814</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:23Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16280" length="3"/>
+        <text>WAX</text>
+      </annotation>
+      <annotation id="412">
+        <infon key="score">0.99835324</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16335" length="4"/>
+        <text>Araf</text>
+      </annotation>
+      <annotation id="413">
+        <infon key="score">0.926579</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16366" length="2"/>
+        <text>CX</text>
+      </annotation>
+      <annotation id="414">
+        <infon key="score">0.99903345</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16418" length="15"/>
+        <text>arabinoxylanase</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>16441</offset>
+      <text>In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92. O3 (O1 of the Araf at the −2* subsite) makes a polar contact with Nδ2 of Asn139; the endocyclic oxygen hydrogens bonds with the OH of Tyr255. The Xylp in the active site makes strong parallel apolar interactions with Phe310. Substrate recognition in the active site is conserved between CtXyl5A and the closest GH5 structural homolog, the endoglucanase BaCel5A (PDB code 1qi2) as noted previously.</text>
+      <annotation id="415">
+        <infon key="score">0.99896586</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="16448" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="416">
+        <infon key="score">0.99930596</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16463" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="417">
+        <infon key="score">0.9991194</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:23:44Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16475" length="8"/>
+        <text>α-d-Xylp</text>
+      </annotation>
+      <annotation id="418">
+        <infon key="score">0.99690115</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:19:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16594" length="14"/>
+        <text>hydrogen bonds</text>
+      </annotation>
+      <annotation id="419">
+        <infon key="score">0.9994248</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:23:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16625" length="6"/>
+        <text>His253</text>
+      </annotation>
+      <annotation id="420">
+        <infon key="score">0.9993506</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:24:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16643" length="6"/>
+        <text>Glu171</text>
+      </annotation>
+      <annotation id="421">
+        <infon key="score">0.9857984</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:20:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16698" length="13"/>
+        <text>polar contact</text>
+      </annotation>
+      <annotation id="422">
+        <infon key="score">0.99934465</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:23:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16727" length="6"/>
+        <text>Tyr255</text>
+      </annotation>
+      <annotation id="423">
+        <infon key="score">0.9993443</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:23:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16744" length="6"/>
+        <text>Ser279</text>
+      </annotation>
+      <annotation id="424">
+        <infon key="score">0.99672544</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:19:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16795" length="14"/>
+        <text>hydrogen bonds</text>
+      </annotation>
+      <annotation id="425">
+        <infon key="score">0.9993864</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:24:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16822" length="6"/>
+        <text>Asn170</text>
+      </annotation>
+      <annotation id="426">
+        <infon key="score">0.9994099</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16839" length="5"/>
+        <text>Tyr92</text>
+      </annotation>
+      <annotation id="427">
+        <infon key="score">0.999243</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16860" length="4"/>
+        <text>Araf</text>
+      </annotation>
+      <annotation id="428">
+        <infon key="score">0.9981232</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:59:03Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="16872" length="11"/>
+        <text>−2* subsite</text>
+      </annotation>
+      <annotation id="429">
+        <infon key="score">0.9942347</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:20:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16893" length="13"/>
+        <text>polar contact</text>
+      </annotation>
+      <annotation id="430">
+        <infon key="score">0.9994549</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16919" length="6"/>
+        <text>Asn139</text>
+      </annotation>
+      <annotation id="431">
+        <infon key="score">0.9965925</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:24:38Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16949" length="15"/>
+        <text>hydrogens bonds</text>
+      </annotation>
+      <annotation id="432">
+        <infon key="score">0.9993518</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:23:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16980" length="6"/>
+        <text>Tyr255</text>
+      </annotation>
+      <annotation id="433">
+        <infon key="score">0.9992919</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:02Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16992" length="4"/>
+        <text>Xylp</text>
+      </annotation>
+      <annotation id="434">
+        <infon key="score">0.99828696</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17004" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="435">
+        <infon key="score">0.8674073</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:24:31Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17029" length="28"/>
+        <text>parallel apolar interactions</text>
+      </annotation>
+      <annotation id="436">
+        <infon key="score">0.9994276</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:24:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17063" length="6"/>
+        <text>Phe310</text>
+      </annotation>
+      <annotation id="437">
+        <infon key="score">0.9986855</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17100" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="438">
+        <infon key="score">0.9959131</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:36:24Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17115" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="439">
+        <infon key="score">0.99930716</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17133" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="440">
+        <infon key="score">0.99881834</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:40:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17157" length="3"/>
+        <text>GH5</text>
+      </annotation>
+      <annotation id="441">
+        <infon key="score">0.9991805</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:34:45Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17185" length="13"/>
+        <text>endoglucanase</text>
+      </annotation>
+      <annotation id="442">
+        <infon key="score">0.9994155</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:45:00Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17199" length="7"/>
+        <text>BaCel5A</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">zbc0441653440004.jpg</infon>
+      <infon key="id">F4</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>17270</offset>
+      <text>Comparison of the ligand recognition at the distal negative subsites between CtGH5E279S-CBM6, the cellulase BaCel5A, and the xylanase GH10.
+A–C show CtGH5E279S-CBM6 is in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose). A, Poseview representation highlighting the hydrogen bonding and the hydrophobic interactions that occur in the negative subsites. C, density of the ligand shown in blue is RefMac maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ. D and E display BaCel5A in complex with deoxy-2-fluoro-β-d-cellotrioside (PDB code 1qi2), and F and G show CmXyn10B in complex with a xylotriose (PDB code 1uqy). The ligand are represented as sticks. B, D, and F are surface representations (CtGH5E279S-CBM6 in gray, BaCel5A in cyan, and the xylanase GH10 in light brown). C, E, and G show the protein backbone as a cartoon representation with the interacting residues represented as sticks. The black dashes represent the hydrogen bonds.</text>
+      <annotation id="443">
+        <infon key="score">0.99856913</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:59:11Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17321" length="17"/>
+        <text>negative subsites</text>
+      </annotation>
+      <annotation id="444">
+        <infon key="score">0.9817981</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:10:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17347" length="10"/>
+        <text>CtGH5E279S</text>
+      </annotation>
+      <annotation id="445">
+        <infon key="score">0.8062502</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:51:34Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17358" length="4"/>
+        <text>CBM6</text>
+      </annotation>
+      <annotation id="446">
+        <infon key="score">0.9989459</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:26:17Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17368" length="9"/>
+        <text>cellulase</text>
+      </annotation>
+      <annotation id="447">
+        <infon key="score">0.999137</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:45:06Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17378" length="7"/>
+        <text>BaCel5A</text>
+      </annotation>
+      <annotation id="448">
+        <infon key="score">0.99901605</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:34:52Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17395" length="8"/>
+        <text>xylanase</text>
+      </annotation>
+      <annotation id="449">
+        <infon key="score">0.99872714</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17404" length="4"/>
+        <text>GH10</text>
+      </annotation>
+      <annotation id="450">
+        <infon key="score">0.97036284</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:10:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17419" length="10"/>
+        <text>CtGH5E279S</text>
+      </annotation>
+      <annotation id="451">
+        <infon key="score">0.79618585</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:51:34Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17430" length="4"/>
+        <text>CBM6</text>
+      </annotation>
+      <annotation id="452">
+        <infon key="score">0.9977508</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17438" length="15"/>
+        <text>in complex with</text>
+      </annotation>
+      <annotation id="453">
+        <infon key="score">0.9992555</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:42:12Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="17456" length="15"/>
+        <text>pentasaccharide</text>
+      </annotation>
+      <annotation id="454">
+        <infon key="score">0.99919766</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:42:14Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="17473" length="17"/>
+        <text>β1,4-xylotetraose</text>
+      </annotation>
+      <annotation id="455">
+        <infon key="score">0.99904007</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:25:05Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="17499" length="6"/>
+        <text>l-Araf</text>
+      </annotation>
+      <annotation id="456">
+        <infon key="score">0.9987269</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="17538" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="457">
+        <infon key="score">0.9961971</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:20:12Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17591" length="16"/>
+        <text>hydrogen bonding</text>
+      </annotation>
+      <annotation id="458">
+        <infon key="score">0.99668086</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:25:53Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17616" length="24"/>
+        <text>hydrophobic interactions</text>
+      </annotation>
+      <annotation id="459">
+        <infon key="score">0.9985068</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:59:14Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="17659" length="17"/>
+        <text>negative subsites</text>
+      </annotation>
+      <annotation id="460">
+        <infon key="score">0.9700168</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:25:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17681" length="7"/>
+        <text>density</text>
+      </annotation>
+      <annotation id="953">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:25:42Z</infon>
+        <location offset="17727" length="48"/>
+        <text>maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ</text>
+      </annotation>
+      <annotation id="461">
+        <infon key="score">0.999212</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:45:09Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17793" length="7"/>
+        <text>BaCel5A</text>
+      </annotation>
+      <annotation id="462">
+        <infon key="score">0.99365395</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17801" length="15"/>
+        <text>in complex with</text>
+      </annotation>
+      <annotation id="463">
+        <infon key="score">0.9992079</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:42:17Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="17817" length="32"/>
+        <text>deoxy-2-fluoro-β-d-cellotrioside</text>
+      </annotation>
+      <annotation id="464">
+        <infon key="score">0.9983261</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:45:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17884" length="8"/>
+        <text>CmXyn10B</text>
+      </annotation>
+      <annotation id="894">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:04Z</infon>
+        <location offset="17893" length="15"/>
+        <text>in complex with</text>
+      </annotation>
+      <annotation id="465">
+        <infon key="score">0.9992083</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:42:26Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="17911" length="10"/>
+        <text>xylotriose</text>
+      </annotation>
+      <annotation id="466">
+        <infon key="score">0.9557879</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:10:11Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18018" length="10"/>
+        <text>CtGH5E279S</text>
+      </annotation>
+      <annotation id="467">
+        <infon key="score">0.9193694</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:51:34Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18029" length="4"/>
+        <text>CBM6</text>
+      </annotation>
+      <annotation id="468">
+        <infon key="score">0.99914455</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:45:17Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18043" length="7"/>
+        <text>BaCel5A</text>
+      </annotation>
+      <annotation id="469">
+        <infon key="score">0.9989845</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:34:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18068" length="8"/>
+        <text>xylanase</text>
+      </annotation>
+      <annotation id="470">
+        <infon key="score">0.9984413</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18077" length="4"/>
+        <text>GH10</text>
+      </annotation>
+      <annotation id="471">
+        <infon key="score">0.9951364</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:19:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18249" length="14"/>
+        <text>hydrogen bonds</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>18283</offset>
+      <text>The capacity of CtXyl5A to act on the highly decorated xylan CX indicates that O3 and possibly O2 of the backbone Xylp units are solvent-exposed. This is consistent with the interaction of the xylotetraose backbone with the enzyme distal to the active site. A surface representation of the enzyme (Fig. 4B) shows that O3 and O2 of xylose units at subsites −2 to −4 are solvent-exposed and are thus available for decoration. Indeed, these pyranose sugars make very weak apolar interactions with the arabinoxylanase. At −2, Xylp makes planar apolar interactions with the Araf bound to the −2* subsite (Fig. 4C). Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69. Comparison of the distal negative subsites of CtXyl5A with BaCel5A and a typical GH10 xylanase (CmXyn10B, PDB code 1uqy) highlights the paucity of interactions between the arabinoxylanase and its substrate out with the active site (Fig. 4). Thus, the cellulase contains three negative subsites and the sugars bound in the −2 and −3 subsites make a total of 9 polar interactions with the enzyme (Fig. 4, D and E). The GH10 xylanase also contains a −2 subsite that, similar to the cellulase, makes numerous interactions with the substrate (Fig. 4, F and G).</text>
+      <annotation id="472">
+        <infon key="score">0.99838185</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18299" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="473">
+        <infon key="score">0.99818134</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18338" length="5"/>
+        <text>xylan</text>
+      </annotation>
+      <annotation id="474">
+        <infon key="score">0.7490116</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18344" length="2"/>
+        <text>CX</text>
+      </annotation>
+      <annotation id="475">
+        <infon key="score">0.9990195</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:02Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18397" length="4"/>
+        <text>Xylp</text>
+      </annotation>
+      <annotation id="476">
+        <infon key="score">0.9982736</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:48:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18412" length="15"/>
+        <text>solvent-exposed</text>
+      </annotation>
+      <annotation id="477">
+        <infon key="score">0.99916756</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:42:30Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18476" length="12"/>
+        <text>xylotetraose</text>
+      </annotation>
+      <annotation id="478">
+        <infon key="score">0.9988993</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18528" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="479">
+        <infon key="score">0.998987</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18614" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="1019">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:59:33Z</infon>
+        <location offset="18630" length="17"/>
+        <text>subsites −2 to −4</text>
+      </annotation>
+      <annotation id="480">
+        <infon key="score">0.99836254</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:48:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18652" length="15"/>
+        <text>solvent-exposed</text>
+      </annotation>
+      <annotation id="481">
+        <infon key="score">0.9971443</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:41:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18721" length="8"/>
+        <text>pyranose</text>
+      </annotation>
+      <annotation id="482">
+        <infon key="score">0.846215</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:42:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18730" length="6"/>
+        <text>sugars</text>
+      </annotation>
+      <annotation id="1031">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:07:27Z</infon>
+        <location offset="18752" length="19"/>
+        <text>apolar interactions</text>
+      </annotation>
+      <annotation id="483">
+        <infon key="score">0.9933171</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18781" length="15"/>
+        <text>arabinoxylanase</text>
+      </annotation>
+      <annotation id="484">
+        <infon key="score">0.98697215</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:59:38Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18801" length="2"/>
+        <text>−2</text>
+      </annotation>
+      <annotation id="485">
+        <infon key="score">0.9984598</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:02Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18805" length="4"/>
+        <text>Xylp</text>
+      </annotation>
+      <annotation id="1030">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:07:11Z</infon>
+        <location offset="18816" length="26"/>
+        <text>planar apolar interactions</text>
+      </annotation>
+      <annotation id="486">
+        <infon key="score">0.99914765</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18852" length="4"/>
+        <text>Araf</text>
+      </annotation>
+      <annotation id="487">
+        <infon key="score">0.9965229</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:19:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18857" length="8"/>
+        <text>bound to</text>
+      </annotation>
+      <annotation id="488">
+        <infon key="score">0.99492747</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:59:41Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18870" length="11"/>
+        <text>−2* subsite</text>
+      </annotation>
+      <annotation id="489">
+        <infon key="score">0.9988463</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:02Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18893" length="4"/>
+        <text>Xylp</text>
+      </annotation>
+      <annotation id="1020">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:00:00Z</infon>
+        <location offset="18901" length="18"/>
+        <text>subsites −2 and −3</text>
+      </annotation>
+      <annotation id="490">
+        <infon key="score">0.9847268</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:26:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18945" length="19"/>
+        <text>hydrophobic contact</text>
+      </annotation>
+      <annotation id="491">
+        <infon key="score">0.9995246</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:06:30Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18970" length="6"/>
+        <text>Val318</text>
+      </annotation>
+      <annotation id="492">
+        <infon key="score">0.8751849</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:00:03Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18982" length="2"/>
+        <text>−3</text>
+      </annotation>
+      <annotation id="493">
+        <infon key="score">0.9987112</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:02Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18985" length="4"/>
+        <text>Xylp</text>
+      </annotation>
+      <annotation id="1021">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:00:26Z</infon>
+        <location offset="18996" length="26"/>
+        <text>planar apolar interactions</text>
+      </annotation>
+      <annotation id="494">
+        <infon key="score">0.99950683</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:06:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19028" length="6"/>
+        <text>Ala137</text>
+      </annotation>
+      <annotation id="495">
+        <infon key="score">0.99882287</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="19048" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="496">
+        <infon key="score">0.65771174</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:00:33Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19058" length="2"/>
+        <text>−4</text>
+      </annotation>
+      <annotation id="1029">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:06:50Z</infon>
+        <location offset="19067" length="24"/>
+        <text>parallel apolar contacts</text>
+      </annotation>
+      <annotation id="497">
+        <infon key="score">0.99949133</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:06:36Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19097" length="5"/>
+        <text>Trp69</text>
+      </annotation>
+      <annotation id="498">
+        <infon key="score">0.9983335</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:00:44Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19129" length="17"/>
+        <text>negative subsites</text>
+      </annotation>
+      <annotation id="499">
+        <infon key="score">0.9988159</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="19150" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="500">
+        <infon key="score">0.9990707</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:45:23Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="19163" length="7"/>
+        <text>BaCel5A</text>
+      </annotation>
+      <annotation id="501">
+        <infon key="score">0.9989895</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19185" length="4"/>
+        <text>GH10</text>
+      </annotation>
+      <annotation id="502">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:35:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19190" length="8"/>
+        <text>xylanase</text>
+      </annotation>
+      <annotation id="503">
+        <infon key="score">0.9923934</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="19200" length="8"/>
+        <text>CmXyn10B</text>
+      </annotation>
+      <annotation id="504">
+        <infon key="score">0.99495846</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19276" length="15"/>
+        <text>arabinoxylanase</text>
+      </annotation>
+      <annotation id="505">
+        <infon key="score">0.9988996</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19323" length="11"/>
+        <text>active site</text>
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+      <annotation id="506">
+        <infon key="score">0.99886346</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:26:17Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19355" length="9"/>
+        <text>cellulase</text>
+      </annotation>
+      <annotation id="507">
+        <infon key="score">0.9983138</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:00:38Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19380" length="17"/>
+        <text>negative subsites</text>
+      </annotation>
+      <annotation id="508">
+        <infon key="score">0.98889786</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:42:36Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="19406" length="6"/>
+        <text>sugars</text>
+      </annotation>
+      <annotation id="509">
+        <infon key="score">0.98305774</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:33:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="score">0.9832241</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:00:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19426" length="18"/>
+        <text>−2 and −3 subsites</text>
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+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:26:11Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19463" length="18"/>
+        <text>polar interactions</text>
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+      <annotation id="512">
+        <infon key="score">0.99883157</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19521" length="4"/>
+        <text>GH10</text>
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+      <annotation id="513">
+        <infon key="score">0.99209</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:35:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19526" length="8"/>
+        <text>xylanase</text>
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+      <annotation id="514">
+        <infon key="score">0.99729645</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:00:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19551" length="10"/>
+        <text>−2 subsite</text>
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+      <annotation id="515">
+        <infon key="score">0.99879384</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:26:17Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19583" length="9"/>
+        <text>cellulase</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_4</infon>
+      <offset>19682</offset>
+      <text>The Influence of the Modular Architecture of CtXyl5A on Catalytic Activity</text>
+      <annotation id="516">
+        <infon key="score">0.98137945</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="19727" length="7"/>
+        <text>CtXyl5A</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>19757</offset>
+      <text>CtXyl5A, in addition to its catalytic module, contains three CBMs (CtCBM6, CtCBM13, and CtCBM62) and a fibronectin domain (CtFn3). A previous study showed that although the CBM6 bound in an exo-mode to xylo- and cellulooligosaccharides, the primary role of this module was to stabilize the structure of the GH5 catalytic module. To explore the contribution of the other non-catalytic modules to CtXyl5A function, the activity of a series of truncated derivatives of the arabinoxylanase were assessed. The data in Table 1 show that removal of CtCBM62 caused a modest increase in activity against both WAX and CX, whereas deletion of the Fn3 domain had no further impact on catalytic performance. Truncation of CtCBM13, however, caused a 4–5-fold reduction in activity against both substrates. Members of CBM13 have been shown to bind to xylans, mannose, and galactose residues in complex glycans, hinting that the function of CtCBM13 is to increase the proximity of substrate to the catalytic module of CtXyl5A. Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown). It would appear, therefore, that CtCBM13 makes a structural contribution to the function of CtXyl5A.</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="19757" length="7"/>
+        <text>CtXyl5A</text>
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+      <annotation id="518">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:35:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19785" length="16"/>
+        <text>catalytic module</text>
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+      <annotation id="519">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:12Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19818" length="4"/>
+        <text>CBMs</text>
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+      <annotation id="520">
+        <infon key="score">0.8389996</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:40Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19824" length="6"/>
+        <text>CtCBM6</text>
+      </annotation>
+      <annotation id="521">
+        <infon key="score">0.9042827</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19832" length="7"/>
+        <text>CtCBM13</text>
+      </annotation>
+      <annotation id="522">
+        <infon key="score">0.9175341</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:36Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19845" length="7"/>
+        <text>CtCBM62</text>
+      </annotation>
+      <annotation id="523">
+        <infon key="score">0.9989512</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:51:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19860" length="18"/>
+        <text>fibronectin domain</text>
+      </annotation>
+      <annotation id="524">
+        <infon key="score">0.99350643</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:45:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19880" length="5"/>
+        <text>CtFn3</text>
+      </annotation>
+      <annotation id="525">
+        <infon key="score">0.9994</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:51:34Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19930" length="4"/>
+        <text>CBM6</text>
+      </annotation>
+      <annotation id="526">
+        <infon key="score">0.93146616</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:33:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19935" length="8"/>
+        <text>bound in</text>
+      </annotation>
+      <annotation id="527">
+        <infon key="score">0.9521939</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:36:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19947" length="8"/>
+        <text>exo-mode</text>
+      </annotation>
+      <annotation id="528">
+        <infon key="score">0.986774</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:42:44Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="19959" length="33"/>
+        <text>xylo- and cellulooligosaccharides</text>
+      </annotation>
+      <annotation id="529">
+        <infon key="score">0.99714065</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:40:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20064" length="3"/>
+        <text>GH5</text>
+      </annotation>
+      <annotation id="530">
+        <infon key="score">0.99528027</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:35:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="20068" length="16"/>
+        <text>catalytic module</text>
+      </annotation>
+      <annotation id="531">
+        <infon key="score">0.99527496</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:51:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="20127" length="21"/>
+        <text>non-catalytic modules</text>
+      </annotation>
+      <annotation id="532">
+        <infon key="score">0.99911505</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="20152" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="1032">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:08:05Z</infon>
+        <location offset="20198" length="9"/>
+        <text>truncated</text>
+      </annotation>
+      <annotation id="533">
+        <infon key="score">0.99889565</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20227" length="15"/>
+        <text>arabinoxylanase</text>
+      </annotation>
+      <annotation id="534">
+        <infon key="score">0.96990776</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:26:35Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20288" length="10"/>
+        <text>removal of</text>
+      </annotation>
+      <annotation id="535">
+        <infon key="score">0.7631579</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:36Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="20299" length="7"/>
+        <text>CtCBM62</text>
+      </annotation>
+      <annotation id="536">
+        <infon key="score">0.99899715</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:23Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20357" length="3"/>
+        <text>WAX</text>
+      </annotation>
+      <annotation id="537">
+        <infon key="score">0.99888617</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20365" length="2"/>
+        <text>CX</text>
+      </annotation>
+      <annotation id="538">
+        <infon key="score">0.8609744</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:26:40Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20377" length="11"/>
+        <text>deletion of</text>
+      </annotation>
+      <annotation id="539">
+        <infon key="score">0.9990049</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="20393" length="3"/>
+        <text>Fn3</text>
+      </annotation>
+      <annotation id="540">
+        <infon key="score">0.9974022</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:26:38Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20452" length="10"/>
+        <text>Truncation</text>
+      </annotation>
+      <annotation id="541">
+        <infon key="score">0.99084926</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="20466" length="7"/>
+        <text>CtCBM13</text>
+      </annotation>
+      <annotation id="542">
+        <infon key="score">0.979066</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:52:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="20560" length="5"/>
+        <text>CBM13</text>
+      </annotation>
+      <annotation id="543">
+        <infon key="score">0.9990688</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20593" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="544">
+        <infon key="score">0.99915814</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:42:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20601" length="7"/>
+        <text>mannose</text>
+      </annotation>
+      <annotation id="545">
+        <infon key="score">0.9989704</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:42:52Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20614" length="9"/>
+        <text>galactose</text>
+      </annotation>
+      <annotation id="546">
+        <infon key="score">0.777462</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:42:54Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20636" length="15"/>
+        <text>complex glycans</text>
+      </annotation>
+      <annotation id="547">
+        <infon key="score">0.9620865</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="20682" length="7"/>
+        <text>CtCBM13</text>
+      </annotation>
+      <annotation id="548">
+        <infon key="score">0.99735713</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:35:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="20739" length="16"/>
+        <text>catalytic module</text>
+      </annotation>
+      <annotation id="549">
+        <infon key="score">0.9991768</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="20759" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="550">
+        <infon key="score">0.9986268</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:26:43Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20768" length="15"/>
+        <text>Binding studies</text>
+      </annotation>
+      <annotation id="551">
+        <infon key="score">0.9760868</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="20806" length="7"/>
+        <text>CtCBM13</text>
+      </annotation>
+      <annotation id="552">
+        <infon key="score">0.99847585</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:42:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20860" length="7"/>
+        <text>glycans</text>
+      </annotation>
+      <annotation id="553">
+        <infon key="score">0.99904245</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:24Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20878" length="3"/>
+        <text>WAX</text>
+      </annotation>
+      <annotation id="554">
+        <infon key="score">0.99910945</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20883" length="2"/>
+        <text>CX</text>
+      </annotation>
+      <annotation id="555">
+        <infon key="score">0.999185</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20887" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="556">
+        <infon key="score">0.9991738</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:43:00Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20895" length="7"/>
+        <text>mannose</text>
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+      <annotation id="557">
+        <infon key="score">0.99903405</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:43:03Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20904" length="9"/>
+        <text>galactose</text>
+      </annotation>
+      <annotation id="558">
+        <infon key="score">0.99697894</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:26:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20919" length="15"/>
+        <text>birchwood xylan</text>
+      </annotation>
+      <annotation id="559">
+        <infon key="score">0.9985886</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:43:07Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20936" length="2"/>
+        <text>BX</text>
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+      <annotation id="560">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="20991" length="7"/>
+        <text>CtCBM13</text>
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+      <annotation id="561">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="21050" length="7"/>
+        <text>CtXyl5A</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_4</infon>
+      <offset>21061</offset>
+      <text>Crystal Structure of CtXyl5A-D</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>Crystal Structure</text>
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+      <annotation id="563">
+        <infon key="score">0.9985922</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:27:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21082" length="9"/>
+        <text>CtXyl5A-D</text>
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+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>21092</offset>
+      <text>To explore further the role of the non-catalytic modules in CtXyl5A the crystal structure of CtXyl5A extending from CtGH5 to CtCBM62 was sought. To obtain a construct that could potentially be crystallized, the protein was generated without the C-terminal dockerin domain because it is known to be unstable and prone to cleavage. Using this construct (CtXyl5A-D) the crystal structure of the arabinoxylanase was determined by molecular replacement to a resolution of 2.64 Å with Rwork and Rfree at 23.7% and 27.8%, respectively. The structure comprises a continuous polypeptide extending from Ala36 to Trp742 displaying four modules GH5-CBM6-CBM13-Fn3. Although there was some electron density for CtCBM62, it was not sufficient to confidently build the module (Fig. 5). Further investigation of the crystal packing revealed a large solvent channel adjacent to the area the CBM62 occupies. We postulate that the reason for the poor electron density is due to the CtCBM62 being mobile compared with the rest of the protein. The structures of CtGH5 and CtCBM6 have been described previously.</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="21185" length="7"/>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:10Z</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="21444" length="9"/>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>crystal structure</text>
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+      <annotation id="574">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21484" length="15"/>
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+      <annotation id="575">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:06:00Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21518" length="21"/>
+        <text>molecular replacement</text>
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+      <annotation id="576">
+        <infon key="score">0.9985116</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:28:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21571" length="5"/>
+        <text>Rwork</text>
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+      <annotation id="577">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:28:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21581" length="5"/>
+        <text>Rfree</text>
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+      <annotation id="578">
+        <infon key="score">0.99822944</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:27:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21625" length="9"/>
+        <text>structure</text>
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+      <annotation id="579">
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+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:28:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21685" length="15"/>
+        <text>Ala36 to Trp742</text>
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+      <annotation id="1045">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:31:31Z</infon>
+        <location offset="21725" length="18"/>
+        <text>GH5-CBM6-CBM13-Fn3</text>
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+      <annotation id="580">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:27:52Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21769" length="16"/>
+        <text>electron density</text>
+      </annotation>
+      <annotation id="581">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:36Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="21790" length="7"/>
+        <text>CtCBM62</text>
+      </annotation>
+      <annotation id="1022">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:01:19Z</infon>
+        <location offset="21892" length="15"/>
+        <text>crystal packing</text>
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+      <annotation id="582">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:01:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="21925" length="15"/>
+        <text>solvent channel</text>
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+      <annotation id="583">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:52:00Z</infon>
+        <infon key="identifier">SO:</infon>
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+      <annotation id="584">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:28:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>electron density</text>
+      </annotation>
+      <annotation id="585">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:36Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22055" length="7"/>
+        <text>CtCBM62</text>
+      </annotation>
+      <annotation id="586">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:36:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22069" length="6"/>
+        <text>mobile</text>
+      </annotation>
+      <annotation id="587">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:27:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:02Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22133" length="5"/>
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+      <annotation id="589">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:41Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>CtCBM6</text>
+      </annotation>
+    </passage>
+    <passage>
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+      <infon key="id">F5</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>22184</offset>
+      <text>Surface representation of the tetra-modular arabinoxylanase and zoom view on the CtGH5 loop. The blue module is the CtGH5 catalytic domain, the green module corresponds to the CtCBM6, the yellow module is the CtCBM13, and the salmon module is the fibronectin domain. Surfaces are semitransparent with the protein backbone represented as a cartoon. The CtGH5 loop is stabilized between the CtCBM6 and the CtCBM13 modules. The black dashes represent the hydrogen bonds. The protein backbone is represented as cartoon, and interacting residues are shown as sticks.</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:58Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+      <annotation id="596">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:05Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:52:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22431" length="18"/>
+        <text>fibronectin domain</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:02Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22536" length="5"/>
+        <text>CtGH5</text>
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+      <annotation id="599">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:52:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22542" length="4"/>
+        <text>loop</text>
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+      <annotation id="600">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:41Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22573" length="6"/>
+        <text>CtCBM6</text>
+      </annotation>
+      <annotation id="601">
+        <infon key="score">0.9829951</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22588" length="7"/>
+        <text>CtCBM13</text>
+      </annotation>
+      <annotation id="602">
+        <infon key="score">0.99495596</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:19:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22636" length="14"/>
+        <text>hydrogen bonds</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>22746</offset>
+      <text>CtCBM13 extends from Gly567 to Pro648. Typical of CBM13 proteins CtCBM13 displays a β-trefoil fold comprising the canonical pseudo 3-fold symmetry with a 3-fold repeating unit of 40–50 amino acid residues characteristic of the Ricin superfamily. Each repeat contains two pairs of antiparallel β-strands. A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).</text>
+      <annotation id="603">
+        <infon key="score">0.9912047</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22746" length="7"/>
+        <text>CtCBM13</text>
+      </annotation>
+      <annotation id="604">
+        <infon key="score">0.79160744</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:29:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22767" length="16"/>
+        <text>Gly567 to Pro648</text>
+      </annotation>
+      <annotation id="940">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:29:04Z</infon>
+        <location offset="22796" length="5"/>
+        <text>CBM13</text>
+      </annotation>
+      <annotation id="605">
+        <infon key="score">0.97495085</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22811" length="7"/>
+        <text>CtCBM13</text>
+      </annotation>
+      <annotation id="606">
+        <infon key="score">0.99881685</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:32:18Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22830" length="14"/>
+        <text>β-trefoil fold</text>
+      </annotation>
+      <annotation id="607">
+        <infon key="score">0.99027336</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:52:13Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22900" length="21"/>
+        <text>3-fold repeating unit</text>
+      </annotation>
+      <annotation id="608">
+        <infon key="score">0.78913796</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:29:24Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22925" length="16"/>
+        <text>40–50 amino acid</text>
+      </annotation>
+      <annotation id="609">
+        <infon key="score">0.998422</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:35:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22973" length="17"/>
+        <text>Ricin superfamily</text>
+      </annotation>
+      <annotation id="610">
+        <infon key="score">0.8408495</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:52:19Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22997" length="6"/>
+        <text>repeat</text>
+      </annotation>
+      <annotation id="611">
+        <infon key="score">0.9983705</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:32:15Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23026" length="22"/>
+        <text>antiparallel β-strands</text>
+      </annotation>
+      <annotation id="612">
+        <infon key="score">0.9987341</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:06:09Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23052" length="11"/>
+        <text>Dali search</text>
+      </annotation>
+      <annotation id="941">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:29:39Z</infon>
+        <location offset="23102" length="5"/>
+        <text>CBM13</text>
+      </annotation>
+      <annotation id="613">
+        <infon key="score">0.998098</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:31:37Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23123" length="26"/>
+        <text>root mean square deviation</text>
+      </annotation>
+      <annotation id="614">
+        <infon key="score">0.99859744</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23255" length="15"/>
+        <text>C. thermocellum</text>
+      </annotation>
+      <annotation id="955">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:30:06Z</infon>
+        <location offset="23271" length="21"/>
+        <text>exo-β-1,3-galactanase</text>
+      </annotation>
+      <annotation id="615">
+        <infon key="score">0.9984226</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:30:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23310" length="24"/>
+        <text>Streptomyces avermitilis</text>
+      </annotation>
+      <annotation id="956">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:30:39Z</infon>
+        <location offset="23335" length="23"/>
+        <text>β-l-arabinopyranosidase</text>
+      </annotation>
+      <annotation id="616">
+        <infon key="score">0.9983331</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:30:17Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23376" length="21"/>
+        <text>Streptomyces lividans</text>
+      </annotation>
+      <annotation id="617">
+        <infon key="score">0.8736156</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:47:34Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="23398" length="12"/>
+        <text>xylanase 10A</text>
+      </annotation>
+      <annotation id="1010">
+        <infon key="type">species</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:49:01Z</infon>
+        <location offset="23433" length="33"/>
+        <text>Streptomyces olivaceoviridis E-86</text>
+      </annotation>
+      <annotation id="618">
+        <infon key="score">0.7191514</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:49:07Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="23467" length="12"/>
+        <text>xylanase 10A</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>23509</offset>
+      <text>The Fn3 module displays a typical β-sandwich fold with the two sheets comprising, primarily, three antiparallel strands in the order β1-β2-β5 in β-sheet 1 and β4-β3-β6 in β-sheet 2. Although β-sheet 2 presents a cleft-like topology, typical of endo-binding CBMs, the surface lacks aromatic residues that play a key role in ligand recognition, and in the context of the full-length enzyme, the cleft abuts into CtCBM13 and thus would not be able to accommodate an extended polysaccharide chain (see below).</text>
+      <annotation id="906">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:04Z</infon>
+        <location offset="23513" length="3"/>
+        <text>Fn3</text>
+      </annotation>
+      <annotation id="619">
+        <infon key="score">0.9970993</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:32:51Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23543" length="15"/>
+        <text>β-sandwich fold</text>
+      </annotation>
+      <annotation id="1014">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:52:42Z</infon>
+        <location offset="23572" length="6"/>
+        <text>sheets</text>
+      </annotation>
+      <annotation id="620">
+        <infon key="score">0.9945593</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:52:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23608" length="20"/>
+        <text>antiparallel strands</text>
+      </annotation>
+      <annotation id="621">
+        <infon key="score">0.99866545</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:32:37Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23642" length="8"/>
+        <text>β1-β2-β5</text>
+      </annotation>
+      <annotation id="622">
+        <infon key="score">0.9991293</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:32:40Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23654" length="9"/>
+        <text>β-sheet 1</text>
+      </annotation>
+      <annotation id="623">
+        <infon key="score">0.9986183</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:32:42Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23668" length="8"/>
+        <text>β4-β3-β6</text>
+      </annotation>
+      <annotation id="624">
+        <infon key="score">0.99922734</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:32:44Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23680" length="9"/>
+        <text>β-sheet 2</text>
+      </annotation>
+      <annotation id="625">
+        <infon key="score">0.999304</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:32:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23700" length="9"/>
+        <text>β-sheet 2</text>
+      </annotation>
+      <annotation id="626">
+        <infon key="score">0.6592578</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:01:28Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23721" length="5"/>
+        <text>cleft</text>
+      </annotation>
+      <annotation id="627">
+        <infon key="score">0.732665</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:31:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23753" length="17"/>
+        <text>endo-binding CBMs</text>
+      </annotation>
+      <annotation id="628">
+        <infon key="score">0.9990554</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:07:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23878" length="11"/>
+        <text>full-length</text>
+      </annotation>
+      <annotation id="629">
+        <infon key="score">0.66877615</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:49:11Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="23890" length="6"/>
+        <text>enzyme</text>
+      </annotation>
+      <annotation id="630">
+        <infon key="score">0.9982305</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:01:38Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23902" length="5"/>
+        <text>cleft</text>
+      </annotation>
+      <annotation id="631">
+        <infon key="score">0.998874</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="23919" length="7"/>
+        <text>CtCBM13</text>
+      </annotation>
+      <annotation id="632">
+        <infon key="score">0.9986582</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:28:43Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="23981" length="14"/>
+        <text>polysaccharide</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>24045</offset>
+      <text>In the structure of CtXyl5A-D, the four modules form a three-leaf clover-like structure (Fig. 5). Between the interfaces of CtGH5-CBM6-CBM13 there are a number of interactions that maintain the modules in a fixed position relative to each other. The interaction of CtGH5 and CtCBM6, which buries a substantial apolar solvent-exposed surface of the two modules, has been described previously. The polar interactions between these two modules comprise 14 hydrogen bonds and 5 salt bridges. The apolar and polar interactions between these two modules likely explaining why they do not fold independently compared with other glycoside hydrolases that contain CBMs. CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of ∼450, 350, and 500 Å2, respectively, to form a compact heterotetramer. With respect to the CtCBM6-CBM13 interface, the linker (SPISTGTIP) between the two modules, extending from Ser514 to Pro522, adopts a fixed conformation. Such sequences are normally extremely flexible; however, the two Ile residues make extensive apolar contacts within the linker and with the two CBMs, leading to conformational stabilization. The interactions between CtGH5 and the two CBMs, which are mediated by the tip of the loop between β-7 and α-7 (loop 7) of CtGH5, not only stabilize the trimodular clover-like structure but also make a contribution to catalytic function. Central to the interactions between the three modules is Trp285, which is intercalated between the two CBMs. The Nϵ of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5). Indeed, loop 7 is completely disordered in the truncated derivative of CtXyl5A comprising CtGH5 and CtCBM6, demonstrating that the interactions with CtCBM13 stabilize the conformation of this loop. Although the tip of loop 7 does not directly contribute to the topology of the active site, it is only ∼12 Å from the catalytic nucleophile Glu279. Thus, any perturbation of the loop (through the removal of CtCBM13) is likely to influence the electrostatic and apolar environment of the catalytic apparatus, which could explain the reduction in activity associated with the deletion of CtCBM13.</text>
+      <annotation id="633">
+        <infon key="score">0.99554837</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:51:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24052" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="634">
+        <infon key="score">0.99779385</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:27:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="24065" length="9"/>
+        <text>CtXyl5A-D</text>
+      </annotation>
+      <annotation id="635">
+        <infon key="score">0.7459186</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:52:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24085" length="7"/>
+        <text>modules</text>
+      </annotation>
+      <annotation id="636">
+        <infon key="score">0.9990779</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:01:43Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24155" length="10"/>
+        <text>interfaces</text>
+      </annotation>
+      <annotation id="1046">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:32:00Z</infon>
+        <location offset="24169" length="16"/>
+        <text>CtGH5-CBM6-CBM13</text>
+      </annotation>
+      <annotation id="637">
+        <infon key="score">0.9977719</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:02Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24310" length="5"/>
+        <text>CtGH5</text>
+      </annotation>
+      <annotation id="638">
+        <infon key="score">0.99530184</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:41Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24320" length="6"/>
+        <text>CtCBM6</text>
+      </annotation>
+      <annotation id="1023">
+        <infon key="type">site</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:02:09Z</infon>
+        <location offset="24355" length="30"/>
+        <text>apolar solvent-exposed surface</text>
+      </annotation>
+      <annotation id="639">
+        <infon key="score">0.9960426</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:26:11Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:05:50Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:53:02Z</infon>
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+        <location offset="25560" length="7"/>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:08:51Z</infon>
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+        <location offset="25661" length="14"/>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25706" length="6"/>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25717" length="6"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="25727" length="7"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:02:58Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:41Z</infon>
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+        <location offset="25791" length="6"/>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25815" length="6"/>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25827" length="6"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:37:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25892" length="9"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="25935" length="5"/>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="26037" length="4"/>
+        <text>loop</text>
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+      <annotation id="689">
+        <infon key="score">0.9990918</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:34:18Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26063" length="6"/>
+        <text>loop 7</text>
+      </annotation>
+      <annotation id="690">
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26122" length="11"/>
+        <text>active site</text>
+      </annotation>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:34:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26183" length="6"/>
+        <text>Glu279</text>
+      </annotation>
+      <annotation id="692">
+        <infon key="score">0.9958968</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:34:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26221" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="693">
+        <infon key="score">0.5024981</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:34:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26239" length="7"/>
+        <text>removal</text>
+      </annotation>
+      <annotation id="694">
+        <infon key="score">0.99695325</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26250" length="7"/>
+        <text>CtCBM13</text>
+      </annotation>
+      <annotation id="1027">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:04:06Z</infon>
+        <location offset="26417" length="8"/>
+        <text>deletion</text>
+      </annotation>
+      <annotation id="904">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:05Z</infon>
+        <location offset="26429" length="7"/>
+        <text>CtCBM13</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>26451</offset>
+      <text>Similar to the interactions between CtCBM6 and CtCBM13, there are extensive hydrophobic interactions between CtCBM13 and CtFn3, resulting in very little flexibility between these modules. As stated above, the absence of CtCBM62 in the structure suggests that the module can adopt multiple positions with respect to the rest of the protein. The CtCBM62, by binding to its ligands (d-Galp and l-Arap) in plant cell walls, may be able to recruit the enzyme onto its target substrate. Xylans are not generally thought to contain such sugars. d-Galp, however, has been detected in xylans in the outer layer of cereal grains and in eucalyptus trees, which are substrates used by CtXyl5A. Thus, CtCBM62 may direct the enzyme to particularly complex xylans containing d-Galp at the non-reducing termini of the side chains, consistent with the open substrate binding cleft of the arabinoxylanase that is optimized to bind highly decorated forms of the hemicellulose. In general CBMs have little influence on enzyme activity against soluble substrates but have a significant impact on glycans within plant cell walls. Thus, the role of CBM62 will likely only be evident against insoluble composite substrates.</text>
+      <annotation id="695">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:41Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26487" length="6"/>
+        <text>CtCBM6</text>
+      </annotation>
+      <annotation id="696">
+        <infon key="score">0.983107</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26498" length="7"/>
+        <text>CtCBM13</text>
+      </annotation>
+      <annotation id="697">
+        <infon key="score">0.99704546</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:25:53Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26527" length="24"/>
+        <text>hydrophobic interactions</text>
+      </annotation>
+      <annotation id="698">
+        <infon key="score">0.994769</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26560" length="7"/>
+        <text>CtCBM13</text>
+      </annotation>
+      <annotation id="699">
+        <infon key="score">0.99780685</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:35:31Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26572" length="5"/>
+        <text>CtFn3</text>
+      </annotation>
+      <annotation id="700">
+        <infon key="score">0.7309727</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:53:10Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26630" length="7"/>
+        <text>modules</text>
+      </annotation>
+      <annotation id="701">
+        <infon key="score">0.9990598</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:07:30Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26660" length="10"/>
+        <text>absence of</text>
+      </annotation>
+      <annotation id="702">
+        <infon key="score">0.9950388</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:36Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26671" length="7"/>
+        <text>CtCBM62</text>
+      </annotation>
+      <annotation id="703">
+        <infon key="score">0.997883</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:35:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26686" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="704">
+        <infon key="score">0.9134587</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:53:13Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26714" length="6"/>
+        <text>module</text>
+      </annotation>
+      <annotation id="705">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:36Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26795" length="7"/>
+        <text>CtCBM62</text>
+      </annotation>
+      <annotation id="1028">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:05:12Z</infon>
+        <location offset="26807" length="10"/>
+        <text>binding to</text>
+      </annotation>
+      <annotation id="706">
+        <infon key="score">0.99920434</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:35:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="26831" length="6"/>
+        <text>d-Galp</text>
+      </annotation>
+      <annotation id="707">
+        <infon key="score">0.99922657</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:43:19Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="26842" length="6"/>
+        <text>l-Arap</text>
+      </annotation>
+      <annotation id="708">
+        <infon key="score">0.998511</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26853" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="709">
+        <infon key="score">0.99731666</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="26932" length="6"/>
+        <text>Xylans</text>
+      </annotation>
+      <annotation id="710">
+        <infon key="score">0.9827069</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:43:11Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="26981" length="6"/>
+        <text>sugars</text>
+      </annotation>
+      <annotation id="711">
+        <infon key="score">0.9991762</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:35:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="26989" length="6"/>
+        <text>d-Galp</text>
+      </annotation>
+      <annotation id="712">
+        <infon key="score">0.99790454</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="27027" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="713">
+        <infon key="score">0.99599224</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:43:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27056" length="6"/>
+        <text>cereal</text>
+      </annotation>
+      <annotation id="714">
+        <infon key="score">0.81789005</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:35:51Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27077" length="16"/>
+        <text>eucalyptus trees</text>
+      </annotation>
+      <annotation id="715">
+        <infon key="score">0.99418384</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="27124" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="716">
+        <infon key="score">0.99656916</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:37Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27139" length="7"/>
+        <text>CtCBM62</text>
+      </annotation>
+      <annotation id="717">
+        <infon key="score">0.99837226</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="27193" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="718">
+        <infon key="score">0.9991806</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:35:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="27211" length="6"/>
+        <text>d-Galp</text>
+      </annotation>
+      <annotation id="719">
+        <infon key="score">0.9913663</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:06:30Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27286" length="4"/>
+        <text>open</text>
+      </annotation>
+      <annotation id="720">
+        <infon key="score">0.9990905</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:36:11Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27291" length="23"/>
+        <text>substrate binding cleft</text>
+      </annotation>
+      <annotation id="721">
+        <infon key="score">0.9991543</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27322" length="15"/>
+        <text>arabinoxylanase</text>
+      </annotation>
+      <annotation id="898">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:29:26Z</infon>
+        <location offset="27394" length="13"/>
+        <text>hemicellulose</text>
+      </annotation>
+      <annotation id="722">
+        <infon key="score">0.99875224</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:13Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27420" length="4"/>
+        <text>CBMs</text>
+      </annotation>
+      <annotation id="723">
+        <infon key="score">0.998137</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:43:24Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="27526" length="7"/>
+        <text>glycans</text>
+      </annotation>
+      <annotation id="724">
+        <infon key="score">0.99851733</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27541" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="725">
+        <infon key="score">0.8947423</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:53:17Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27577" length="5"/>
+        <text>CBM62</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_4</infon>
+      <offset>27651</offset>
+      <text>Exploring GH5 Subfamily 34</text>
+      <annotation id="726">
+        <infon key="score">0.9395437</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:36:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27661" length="16"/>
+        <text>GH5 Subfamily 34</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>27678</offset>
+      <text>CtXyl5A is a member of a seven-protein subfamily of GH5, GH5_34. Four of these proteins are distinct, whereas the other three members are essentially identical (derived from different strains of C. thermocellum). To investigate further the substrate specificity within this subfamily, recombinant forms of three members of GH5_34 that were distinct from CtXyl5A were generated. AcGH5 has a similar molecular architecture to CtXyl5A with the exception of an additional carbohydrate esterase family 6 module at the C terminus (Fig. 1). The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function. The Verrucomicobiae enzyme also has an N-terminal GH43 subfamily 10 (GH43_10) catalytic module. The fungal GH5_34, GpGH5, unlike the two bacterial homologs, comprises a single GH5 catalytic module lacking all of the other accessory modules (Fig. 1). GpGh5 is particularly interesting as Gonapodya prolifera is the only fungus of the several hundred fungal genomes that encodes a GH5_34 enzyme. In fact there are four potential GH5_34 sequences in the G. prolifera genome, all of which show high sequence homology to Clostridium GH5_34 sequences. G. prolifera and Clostridium occupy similar environments, suggesting that the GpGH5_34 gene was acquired from a Clostridium species, which was followed by duplication of the gene in the fungal genome. The sequence identity of the GH5_34 catalytic modules with CtXyl5A ranged from 55 to 80% (supplemental Fig. S1). All the GH5_34 enzymes were active on the arabinoxylans RAX, WAX, and CX but displayed no activity on BX (Table 1 and Fig. 6) and are thus defined as arabinoxylanases. The limit products generated by CtXyl5A, AcGH5, and GpGH5 comprised a range of oligosaccharides with some high molecular weight material. The oligosaccharides with low degrees of polymerization were absent in the VbGH5 reaction products. However, the enzyme generated a large amount of arabinose, which was not produced by the other arabinoxylanases. Given that GH43_10 is predominantly an arabinofuranosidase subfamily of GH43, the arabinose generated by VbGH5 is likely mediated by the N-terminal catalytic module (see below). Kinetic analysis showed that AcGH5 displayed similar activity to CtXyl5A against both WAX and RAX and was 2-fold less active against CX. When initially measuring the activity of wild type VbGH5 against the different substrates, no clear data could be obtained, regardless of the concentration of enzyme used the reaction appeared to cease after a few minutes. We hypothesized that the N-terminal GH43_10 rapidly removed single arabinose decorations from the arabinoxylans depleting the substrate available to the arabinoxylanase, explaining why this activity was short lived. To test this hypothesis, the conserved catalytic base (Asp45) of the GH43_10 module of VbGH5 was substituted with alanine, which is predicted to inactivate this catalytic module. The D45A mutant did not produce arabinose consistent with the arabinofuranosidase activity displayed by the GH43_10 module in the wild type enzyme (Fig. 6). The kinetics of the GH5_34 arabinoxylanase catalytic module was now measurable, and activities were determined to be between ∼6- and 10-fold lower than that of CtXyl5A. Interestingly, the fungal arabinoxylanase displays the highest activities against WAX and RAX, ∼4- and 6-fold higher, respectively, than CtXyl5A; however, there is very little difference in the activity between the eukaryotic and prokaryotic enzymes against CX. Attempts to express individual modules of a variety of truncations of AcGH5 and VbGH5 were unsuccessful. This may indicate that the individual modules can only fold correctly when incorporated into the full-length enzyme, demonstrating the importance of intermodule interactions to maintain the structural integrity of these enzymes.</text>
+      <annotation id="727">
+        <infon key="score">0.99605703</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="27678" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="728">
+        <infon key="score">0.99675363</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:40:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27730" length="3"/>
+        <text>GH5</text>
+      </annotation>
+      <annotation id="957">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:36:53Z</infon>
+        <location offset="27735" length="6"/>
+        <text>GH5_34</text>
+      </annotation>
+      <annotation id="729">
+        <infon key="score">0.9986108</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <text>arabinose</text>
+      </annotation>
+      <annotation id="773">
+        <infon key="score">0.9989502</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="29910" length="16"/>
+        <text>arabinoxylanases</text>
+      </annotation>
+      <annotation id="977">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:42:40Z</infon>
+        <location offset="29939" length="7"/>
+        <text>GH43_10</text>
+      </annotation>
+      <annotation id="942">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:54:58Z</infon>
+        <location offset="29967" length="19"/>
+        <text>arabinofuranosidase</text>
+      </annotation>
+      <annotation id="981">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:42:23Z</infon>
+        <location offset="30000" length="4"/>
+        <text>GH43</text>
+      </annotation>
+      <annotation id="895">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <location offset="30010" length="9"/>
+        <text>arabinose</text>
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+      <annotation id="932">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:42Z</infon>
+        <location offset="30033" length="5"/>
+        <text>VbGH5</text>
+      </annotation>
+      <annotation id="900">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:35:55Z</infon>
+        <location offset="30076" length="16"/>
+        <text>catalytic module</text>
+      </annotation>
+      <annotation id="937">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:50:32Z</infon>
+        <location offset="30135" length="5"/>
+        <text>AcGH5</text>
+      </annotation>
+      <annotation id="885">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <location offset="30171" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="914">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:24Z</infon>
+        <location offset="30192" length="3"/>
+        <text>WAX</text>
+      </annotation>
+      <annotation id="917">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:28Z</infon>
+        <location offset="30200" length="3"/>
+        <text>RAX</text>
+      </annotation>
+      <annotation id="922">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:39Z</infon>
+        <location offset="30239" length="2"/>
+        <text>CX</text>
+      </annotation>
+      <annotation id="928">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:27Z</infon>
+        <location offset="30284" length="9"/>
+        <text>wild type</text>
+      </annotation>
+      <annotation id="933">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:42Z</infon>
+        <location offset="30294" length="5"/>
+        <text>VbGH5</text>
+      </annotation>
+      <annotation id="978">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:42:40Z</infon>
+        <location offset="30502" length="7"/>
+        <text>GH43_10</text>
+      </annotation>
+      <annotation id="896">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <location offset="30533" length="9"/>
+        <text>arabinose</text>
+      </annotation>
+      <annotation id="889">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:42Z</infon>
+        <location offset="30564" length="13"/>
+        <text>arabinoxylans</text>
+      </annotation>
+      <annotation id="890">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:58Z</infon>
+        <location offset="30619" length="15"/>
+        <text>arabinoxylanase</text>
+      </annotation>
+      <annotation id="984">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:44:44Z</infon>
+        <location offset="30711" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="982">
+        <infon key="type">residue_name_number</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:44:04Z</infon>
+        <location offset="30737" length="5"/>
+        <text>Asp45</text>
+      </annotation>
+      <annotation id="979">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:45:12Z</infon>
+        <location offset="30751" length="7"/>
+        <text>GH43_10</text>
+      </annotation>
+      <annotation id="934">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:42Z</infon>
+        <location offset="30769" length="5"/>
+        <text>VbGH5</text>
+      </annotation>
+      <annotation id="985">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:44:57Z</infon>
+        <location offset="30779" length="16"/>
+        <text>substituted with</text>
+      </annotation>
+      <annotation id="983">
+        <infon key="type">residue_name</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:44:24Z</infon>
+        <location offset="30796" length="7"/>
+        <text>alanine</text>
+      </annotation>
+      <annotation id="901">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:35:55Z</infon>
+        <location offset="30843" length="16"/>
+        <text>catalytic module</text>
+      </annotation>
+      <annotation id="927">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:23Z</infon>
+        <location offset="30865" length="4"/>
+        <text>D45A</text>
+      </annotation>
+      <annotation id="986">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:45:25Z</infon>
+        <location offset="30870" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="897">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <location offset="30893" length="9"/>
+        <text>arabinose</text>
+      </annotation>
+      <annotation id="943">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:54:58Z</infon>
+        <location offset="30923" length="19"/>
+        <text>arabinofuranosidase</text>
+      </annotation>
+      <annotation id="980">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:43:05Z</infon>
+        <location offset="30969" length="7"/>
+        <text>GH43_10</text>
+      </annotation>
+      <annotation id="929">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:27Z</infon>
+        <location offset="30991" length="9"/>
+        <text>wild type</text>
+      </annotation>
+      <annotation id="987">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:45:51Z</infon>
+        <location offset="31022" length="8"/>
+        <text>kinetics</text>
+      </annotation>
+      <annotation id="968">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:36:54Z</infon>
+        <location offset="31038" length="6"/>
+        <text>GH5_34</text>
+      </annotation>
+      <annotation id="891">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:58Z</infon>
+        <location offset="31045" length="15"/>
+        <text>arabinoxylanase</text>
+      </annotation>
+      <annotation id="902">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:35:55Z</infon>
+        <location offset="31061" length="16"/>
+        <text>catalytic module</text>
+      </annotation>
+      <annotation id="886">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:31Z</infon>
+        <location offset="31178" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="974">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:38:21Z</infon>
+        <location offset="31206" length="6"/>
+        <text>fungal</text>
+      </annotation>
+      <annotation id="892">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:58Z</infon>
+        <location offset="31213" length="15"/>
+        <text>arabinoxylanase</text>
+      </annotation>
+      <annotation id="915">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:24Z</infon>
+        <location offset="31269" length="3"/>
+        <text>WAX</text>
+      </annotation>
+      <annotation id="918">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:28Z</infon>
+        <location offset="31277" length="3"/>
+        <text>RAX</text>
+      </annotation>
+      <annotation id="887">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:32Z</infon>
+        <location offset="31324" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="988">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:46:09Z</infon>
+        <location offset="31402" length="10"/>
+        <text>eukaryotic</text>
+      </annotation>
+      <annotation id="989">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:46:19Z</infon>
+        <location offset="31417" length="11"/>
+        <text>prokaryotic</text>
+      </annotation>
+      <annotation id="923">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:39Z</infon>
+        <location offset="31445" length="2"/>
+        <text>CX</text>
+      </annotation>
+      <annotation id="938">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:50:32Z</infon>
+        <location offset="31519" length="5"/>
+        <text>AcGH5</text>
+      </annotation>
+      <annotation id="935">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:42Z</infon>
+        <location offset="31529" length="5"/>
+        <text>VbGH5</text>
+      </annotation>
+      <annotation id="995">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:07:19Z</infon>
+        <location offset="31651" length="11"/>
+        <text>full-length</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">zbc0441653440006.jpg</infon>
+      <infon key="id">F6</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>31787</offset>
+      <text>Products profile generated of GH5_34 enzymes. The enzymes at 1 μm were incubated with the four different xylans at 1% in 50 mm sodium phosphate buffer for 16 h at 37 °C (GpGH5, VbGH5, and AcGH5) or 60 °C. The limit products were separated by TLC. The xylooligosaccharide standards (X) are indicated by their degrees of polymerization.</text>
+      <annotation id="969">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:36:54Z</infon>
+        <location offset="31817" length="6"/>
+        <text>GH5_34</text>
+      </annotation>
+      <annotation id="774">
+        <infon key="score">0.93129015</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:46:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="31858" length="9"/>
+        <text>incubated</text>
+      </annotation>
+      <annotation id="775">
+        <infon key="score">0.99875844</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="31892" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="776">
+        <infon key="score">0.9989768</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:47:40Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="31957" length="5"/>
+        <text>GpGH5</text>
+      </annotation>
+      <annotation id="777">
+        <infon key="score">0.9987948</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:42Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="31964" length="5"/>
+        <text>VbGH5</text>
+      </annotation>
+      <annotation id="778">
+        <infon key="score">0.9988304</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:50:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="31975" length="5"/>
+        <text>AcGH5</text>
+      </annotation>
+      <annotation id="779">
+        <infon key="score">0.9960063</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:47:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="32029" length="3"/>
+        <text>TLC</text>
+      </annotation>
+      <annotation id="780">
+        <infon key="score">0.99918824</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:43:43Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="32038" length="19"/>
+        <text>xylooligosaccharide</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">title_1</infon>
+      <offset>32125</offset>
+      <text>Discussion</text>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>32136</offset>
+      <text>A characteristic feature of enzymes that attack the plant cell wall is their complex molecular architecture. The CBMs in these enzymes generally play a role in substrate targeting and are appended to the catalytic modules through flexible linker sequences. CtXyl5A provides a rare visualization of the structure of multiple modules within a single enzyme. The central feature of these data is the structural role played by two of the CBMs, CtCBM6 and CtCBM13, in maintaining the active conformation of the catalytic module, CtGH5. The crystallographic data described here are supported by biochemical data showing either that these two modules do not bind to glycans (CtCBM13) or that the recognition of the non-reducing end of xylan or cellulose chains (CtCBM6) is unlikely to be biologically significant. It should be emphasized, however, that glycan binding and substrate targeting may only be evident in the full-length enzyme acting on highly complex structures such as the plant cell wall, as observed recently by a CBM46 module in the Bacillus xyloglucanase/mixed linked glucanase BhCel5B.</text>
+      <annotation id="781">
+        <infon key="score">0.99862134</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="32188" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="782">
+        <infon key="score">0.9987355</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:13Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32249" length="4"/>
+        <text>CBMs</text>
+      </annotation>
+      <annotation id="783">
+        <infon key="score">0.99867666</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:53:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32340" length="17"/>
+        <text>catalytic modules</text>
+      </annotation>
+      <annotation id="784">
+        <infon key="score">0.865277</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:53:30Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32366" length="25"/>
+        <text>flexible linker sequences</text>
+      </annotation>
+      <annotation id="785">
+        <infon key="score">0.5641079</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="32393" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="786">
+        <infon key="score">0.99627954</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:51:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="32438" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="787">
+        <infon key="score">0.99776864</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:13Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32570" length="4"/>
+        <text>CBMs</text>
+      </annotation>
+      <annotation id="788">
+        <infon key="score">0.86189497</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:41Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32576" length="6"/>
+        <text>CtCBM6</text>
+      </annotation>
+      <annotation id="789">
+        <infon key="score">0.95701545</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32587" length="7"/>
+        <text>CtCBM13</text>
+      </annotation>
+      <annotation id="790">
+        <infon key="score">0.99906987</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:38:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="32615" length="6"/>
+        <text>active</text>
+      </annotation>
+      <annotation id="791">
+        <infon key="score">0.9981731</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:35:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32642" length="16"/>
+        <text>catalytic module</text>
+      </annotation>
+      <annotation id="792">
+        <infon key="score">0.9962853</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:02Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32660" length="5"/>
+        <text>CtGH5</text>
+      </annotation>
+      <annotation id="793">
+        <infon key="score">0.99705863</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:51:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="32671" length="21"/>
+        <text>crystallographic data</text>
+      </annotation>
+      <annotation id="794">
+        <infon key="score">0.998901</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:43:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="32795" length="7"/>
+        <text>glycans</text>
+      </annotation>
+      <annotation id="795">
+        <infon key="score">0.9889486</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:37:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32804" length="7"/>
+        <text>CtCBM13</text>
+      </annotation>
+      <annotation id="796">
+        <infon key="score">0.99855596</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="32864" length="5"/>
+        <text>xylan</text>
+      </annotation>
+      <annotation id="797">
+        <infon key="score">0.99709713</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:43:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="32873" length="9"/>
+        <text>cellulose</text>
+      </annotation>
+      <annotation id="798">
+        <infon key="score">0.9898572</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:41Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32891" length="6"/>
+        <text>CtCBM6</text>
+      </annotation>
+      <annotation id="799">
+        <infon key="score">0.9853876</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:43:56Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="32982" length="6"/>
+        <text>glycan</text>
+      </annotation>
+      <annotation id="800">
+        <infon key="score">0.99907523</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:07:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="33048" length="11"/>
+        <text>full-length</text>
+      </annotation>
+      <annotation id="801">
+        <infon key="score">0.9986217</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="33115" length="5"/>
+        <text>plant</text>
+      </annotation>
+      <annotation id="802">
+        <infon key="score">0.9963482</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:53:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="33158" length="5"/>
+        <text>CBM46</text>
+      </annotation>
+      <annotation id="803">
+        <infon key="score">0.9411532</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:47:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="33178" length="8"/>
+        <text>Bacillus</text>
+      </annotation>
+      <annotation id="804">
+        <infon key="score">0.99831474</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:35:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="33187" length="13"/>
+        <text>xyloglucanase</text>
+      </annotation>
+      <annotation id="805">
+        <infon key="score">0.9972825</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:35:29Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="33201" length="22"/>
+        <text>mixed linked glucanase</text>
+      </annotation>
+      <annotation id="806">
+        <infon key="score">0.9992636</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:47:30Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="33224" length="7"/>
+        <text>BhCel5B</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>33233</offset>
+      <text>CtXyl5A is a member of GH5 that contains 6644 members. These proteins have been subdivided into 51 subfamilies based on sequence similarity. CtXyl5A is a member of subfamily GH5_34. Here we have explored the substrate specificity of the other members of this subfamily. Despite differences in sequence identity all of the homologs were shown to be arabinoxylanases. Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the −2* subsite, which are 1,3-linked to the xylose positioned in the active site. The presence of a CBM62 in CtXyl5A and AcGH5 suggests that these enzymes target highly complex xylans that contain d-galactose in their side chains. The absence of a “non-structural” CBM in GpGH5 may indicate that this arabinoxylanase is designed to target simpler arabinoxylans present in the endosperm of cereals. Although the characterization of all members of GH5_34 suggests that this subfamily is monospecific, differences in specificity are observed in other subfamilies of GHs including GH43 and GH5. Thus, as new members of GH5_34 are identified from genomic sequence data and subsequently characterized, the specificity of this family may require reinterpretation.</text>
+      <annotation id="807">
+        <infon key="score">0.9979913</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="33233" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="808">
+        <infon key="score">0.94887006</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:40:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="33256" length="3"/>
+        <text>GH5</text>
+      </annotation>
+      <annotation id="809">
+        <infon key="score">0.9977837</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="33374" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="970">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:36:54Z</infon>
+        <location offset="33407" length="6"/>
+        <text>GH5_34</text>
+      </annotation>
+      <annotation id="810">
+        <infon key="score">0.99929583</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="33581" length="16"/>
+        <text>arabinoxylanases</text>
+      </annotation>
+      <annotation id="971">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:36:54Z</infon>
+        <location offset="33667" length="6"/>
+        <text>GH5_34</text>
+      </annotation>
+      <annotation id="811">
+        <infon key="score">0.99110305</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:04:24Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="33688" length="24"/>
+        <text>specificity determinants</text>
+      </annotation>
+      <annotation id="812">
+        <infon key="score">0.99954575</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="33713" length="5"/>
+        <text>Glu68</text>
+      </annotation>
+      <annotation id="813">
+        <infon key="score">0.9995704</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="33720" length="5"/>
+        <text>Tyr92</text>
+      </annotation>
+      <annotation id="814">
+        <infon key="score">0.99954885</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="33731" length="6"/>
+        <text>Asn139</text>
+      </annotation>
+      <annotation id="815">
+        <infon key="score">0.9985304</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="33781" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="816">
+        <infon key="score">0.99864835</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="33791" length="9"/>
+        <text>arabinose</text>
+      </annotation>
+      <annotation id="817">
+        <infon key="score">0.9986283</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:04:29Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="33808" length="11"/>
+        <text>−2* subsite</text>
+      </annotation>
+      <annotation id="818">
+        <infon key="score">0.9983754</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="33849" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="819">
+        <infon key="score">0.99908876</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:27:57Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="33874" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="820">
+        <infon key="score">0.99881303</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:53:39Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="33905" length="5"/>
+        <text>CBM62</text>
+      </annotation>
+      <annotation id="821">
+        <infon key="score">0.9981012</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="33914" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+      <annotation id="822">
+        <infon key="score">0.9863192</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:50:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="33926" length="5"/>
+        <text>AcGH5</text>
+      </annotation>
+      <annotation id="823">
+        <infon key="score">0.99910295</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="33982" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="824">
+        <infon key="score">0.9990118</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:44:04Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="34002" length="11"/>
+        <text>d-galactose</text>
+      </annotation>
+      <annotation id="825">
+        <infon key="score">0.70076585</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:07:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="34040" length="10"/>
+        <text>absence of</text>
+      </annotation>
+      <annotation id="826">
+        <infon key="score">0.9968041</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:53:44Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="34070" length="3"/>
+        <text>CBM</text>
+      </annotation>
+      <annotation id="827">
+        <infon key="score">0.9990013</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:47:40Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="34077" length="5"/>
+        <text>GpGH5</text>
+      </annotation>
+      <annotation id="828">
+        <infon key="score">0.99921906</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="34106" length="15"/>
+        <text>arabinoxylanase</text>
+      </annotation>
+      <annotation id="829">
+        <infon key="score">0.9991093</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="34152" length="13"/>
+        <text>arabinoxylans</text>
+      </annotation>
+      <annotation id="830">
+        <infon key="score">0.9977469</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:44:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="34194" length="7"/>
+        <text>cereals</text>
+      </annotation>
+      <annotation id="972">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:36:54Z</infon>
+        <location offset="34251" length="6"/>
+        <text>GH5_34</text>
+      </annotation>
+      <annotation id="831">
+        <infon key="score">0.9990747</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:35:33Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="34368" length="3"/>
+        <text>GHs</text>
+      </annotation>
+      <annotation id="832">
+        <infon key="score">0.97536546</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:49:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="34382" length="4"/>
+        <text>GH43</text>
+      </annotation>
+      <annotation id="833">
+        <infon key="score">0.993919</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:40:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="34391" length="3"/>
+        <text>GH5</text>
+      </annotation>
+      <annotation id="973">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:36:54Z</infon>
+        <location offset="34420" length="6"/>
+        <text>GH5_34</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>34568</offset>
+      <text>An intriguing feature of VbGH5 is that the limited products generated by this enzymes are much larger than those produced by the other arabinoxylanases. This suggests that although arabinose decorations contribute to enzyme specificity (VbGH5 is not active on xylans lacking arabinose side chains), the enzyme requires other specificity determinants that occur less frequently in arabinoxylans. This has some resonance with a recently described GH98 xylanase that also exploits specificity determinants that occur infrequently and are only evident in highly complex xylans (e.g. CX).</text>
+      <annotation id="834">
+        <infon key="score">0.9990096</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:42Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="34593" length="5"/>
+        <text>VbGH5</text>
+      </annotation>
+      <annotation id="835">
+        <infon key="score">0.9992551</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="34703" length="16"/>
+        <text>arabinoxylanases</text>
+      </annotation>
+      <annotation id="836">
+        <infon key="score">0.9983157</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="34749" length="9"/>
+        <text>arabinose</text>
+      </annotation>
+      <annotation id="837">
+        <infon key="score">0.999131</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:49:42Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="34805" length="5"/>
+        <text>VbGH5</text>
+      </annotation>
+      <annotation id="838">
+        <infon key="score">0.9985896</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="34828" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="839">
+        <infon key="score">0.9986557</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="34843" length="9"/>
+        <text>arabinose</text>
+      </annotation>
+      <annotation id="840">
+        <infon key="score">0.9989998</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="34948" length="13"/>
+        <text>arabinoxylans</text>
+      </annotation>
+      <annotation id="841">
+        <infon key="score">0.99906427</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:35:38Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="35013" length="4"/>
+        <text>GH98</text>
+      </annotation>
+      <annotation id="842">
+        <infon key="score">0.9987771</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:35:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="35018" length="8"/>
+        <text>xylanase</text>
+      </annotation>
+      <annotation id="843">
+        <infon key="score">0.9987264</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:33Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="35134" length="6"/>
+        <text>xylans</text>
+      </annotation>
+      <annotation id="924">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:39Z</infon>
+        <location offset="35147" length="2"/>
+        <text>CX</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>35152</offset>
+      <text>To conclude, this study provides the molecular basis for the specificity displayed by arabinoxylanases. Substrate specificity is dominated by the pocket that binds single arabinose or xylose side chains. The open xylan binding cleft explains why the enzyme is able to attack highly decorated forms of the hemicellulose. It is also evident that appending additional catalytic modules and CBMs onto the core components of these enzymes generates bespoke arabinoxylanases with activities optimized for specific functions. The specificities of the arabinoxylanases described here are distinct from the classical endo-xylanases and thus have the potential to contribute to the toolbox of biocatalysts required by industries that exploit the plant cell wall as a sustainable substrate.</text>
+      <annotation id="844">
+        <infon key="score">0.9992999</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="35238" length="16"/>
+        <text>arabinoxylanases</text>
+      </annotation>
+      <annotation id="845">
+        <infon key="score">0.99896705</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:05:33Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="35298" length="6"/>
+        <text>pocket</text>
+      </annotation>
+      <annotation id="846">
+        <infon key="score">0.99837506</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:42Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="35323" length="9"/>
+        <text>arabinose</text>
+      </annotation>
+      <annotation id="847">
+        <infon key="score">0.99813867</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="35336" length="6"/>
+        <text>xylose</text>
+      </annotation>
+      <annotation id="848">
+        <infon key="score">0.9551583</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:38:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="35360" length="4"/>
+        <text>open</text>
+      </annotation>
+      <annotation id="849">
+        <infon key="score">0.99909574</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:49:02Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="35365" length="19"/>
+        <text>xylan binding cleft</text>
+      </annotation>
+      <annotation id="850">
+        <infon key="score">0.9953093</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:29:26Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="35457" length="13"/>
+        <text>hemicellulose</text>
+      </annotation>
+      <annotation id="851">
+        <infon key="score">0.9972641</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:53:50Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="35517" length="17"/>
+        <text>catalytic modules</text>
+      </annotation>
+      <annotation id="852">
+        <infon key="score">0.99897206</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:36:13Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="35539" length="4"/>
+        <text>CBMs</text>
+      </annotation>
+      <annotation id="853">
+        <infon key="score">0.9993069</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="35604" length="16"/>
+        <text>arabinoxylanases</text>
+      </annotation>
+      <annotation id="854">
+        <infon key="score">0.9992654</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="35696" length="16"/>
+        <text>arabinoxylanases</text>
+      </annotation>
+      <annotation id="855">
+        <infon key="score">0.9987754</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:35:36Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="35760" length="14"/>
+        <text>endo-xylanases</text>
+      </annotation>
+      <annotation id="856">
+        <infon key="score">0.9984994</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="35888" length="5"/>
+        <text>plant</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_1</infon>
+      <offset>35932</offset>
+      <text>Experimental Procedures</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_4</infon>
+      <offset>35956</offset>
+      <text>Cloning, Expression, and Purification of Components of CtXyl5A</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>36019</offset>
+      <text>All recombinant forms of CtXyl5A used in this study were expressed in the cytoplasm of Escherichia coli because they lacked a signal peptide. DNA encoding CtGH5-CtCBM6 and CtXyl5A-D (CtXyl5A lacking the C-terminal dockerin domain (CtGH5-CtCBM6-CtCBM13-Fn3-CtCBM62)) were described previously. DNA encoding CtGH5-CtCBM6-CtCBM13-Fn3 and CtGH5-CtCBM6-CtCBM13 and mature Acetivibrio cellulolyticus GH5 (AcGH5) were amplified by PCR using plasmid encoding the full-length C. thermocellum arabinoxylanase or A. cellulolyticus genomic DNA as the respective templates. DNA encoding the G. prolifera GH5 (GpGH5) and V. bacterium GH5 (VbGH5) were initially generated by GeneArt® gene synthesis (Thermo Fisher Scientific). DNA encoding VbGH5 lacking the C-terminal cell surface anchoring residues was also amplified by PCR using the synthesized nucleic acid as the template. All the primers used in the PCRs required restriction sites and plasmids used are listed inj supplemental Table S1. All constructs were cloned such that the encoded proteins contain a C-terminal His6 tag. Site-directed mutagenesis was carried out using the PCR-based QuikChange method (Stratagene) deploying the primers listed in supplemental Table S1.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>37237</offset>
+      <text>To express the recombinant proteins, E. coli strain BL21(DE3), harboring appropriate recombinant plasmids, was cultured to mid-exponential phase in Luria broth at 37 °C. Isopropyl β-d-galactopyranoside at 1 mm was then added to induce recombinant gene expression, and the culture incubated for a further 18 h at 16 °C. The recombinant proteins were purified to &gt;90% electrophoretic purity by immobilized metal ion affinity chromatography using TalonTM (Clontech), cobalt-based matrix, and elution with 100 mm imidazole, as described previously. When preparing the selenomethionine derivative of CtXyl5A-D for crystallography, the proteins were expressed in E. coli B834 (DE3), a methionine auxotroph, cultured in medium comprising 1 liter of SelenoMet Medium BaseTM, 50 ml of SelenoMetTM nutrient mix (Molecular Dimensions), and 4 ml of a 10 mg/ml solution of l-selenomethionine. Recombinant gene expression and protein purification were as described above except that all purification buffers were supplemented with 10 mm β-mercaptoethanol.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_4</infon>
+      <offset>38285</offset>
+      <text>Enzyme Assays</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>38299</offset>
+      <text>CtXyl5A-D and its derivatives were assayed for enzyme activity using the method of Miller to detect the release of reducing sugar. The standard assay was carried out in 50 mm sodium phosphate buffer, pH 7.0, containing 0.1 mg/ml BSA and at substrate concentrations ranging from 1 to 6 mg/ml. The pH and temperature optima were previously determined to be 7 and 60 °C, respectively, for the CtXyl5A-D and its derivatives. The optimum temperature for the other enzymes was found to be 37 °C, and pH optima of 5, 7, and 4 were determined for AcGH5, GpGH5 and VbGH5, respectively. All enzymes were assayed for activity at their individual temperature and pH optimum. A FLUOstar Omega microplate reader (BMG Labtech) was used to measure activity in 96-well plates. Overnight assays to assess end point products were carried out with 6 mg/ml substrate and 1 μm enzyme concentrations. The identification of potential reaction products was also assessed by HPAEC or TLC using methodology described previously.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_4</infon>
+      <offset>39304</offset>
+      <text>Oligosaccharide Analysis</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>39329</offset>
+      <text>Approximately 5 g of CX or WAX were digested to completion (no further increase in reducing sugar and change in the HPAEC product profile) with 3 μm of CtXyl5A-D at 60 °C for 48 h. The oligosaccharide products were purified by size exclusion chromatography using a Bio-Gel P2 column as described previously. The structures of the oligosaccharides were analyzed by positive ion-mode infusion/offline electrospray ionization (ESI)-MS following either dilution with 30% acetonitrile or via desalting as described previously </text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_4</infon>
+      <offset>39853</offset>
+      <text>Crystallography</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>39869</offset>
+      <text>Purified SeMet CtXyl5A-D was concentrated and stored in 5 mm DTT, 2 mm CaCl2. Crystals of seleno-l-methionine-containing protein were obtained by hanging drop vapor diffusion in 40% (v/v) 2-methyl-2,4-pentandiol. The data were collected on Beamlines ID14-1 and ID14-4 at the European Synchrotron Radiation Facility (Grenoble, France) to a resolution of 2.64 Å. The data were processed using the programs iMOSFLM and SCALA from the CCP4 suite (Collaborative Computational Project, Number 4, 1994). The crystal belongs to the orthorhombic space group (P21212). The structure was solved by molecular replacement using independently solved structures of some of the modules of the CtXyl5A: CtGH5-CBM6 (PDB code 2y8k), Fn3 (PDB code 3mpc), and CtCBM62 (PDB codes 2y8m, 2yfz, and 2y9s) using PHASER. The CtCBM13 domain was built de novo. BUCCANEER and PHENIX were initially used for auto building. The structure was completed by iterative cycles of manual rebuilding in COOT in tandem with refinement with RefMac5. The final values for Rwork and Rfree) were 23.73 and 27.80%) using TLS and restraining refinement to amino acid residues 36–373 representing the CtGH5 module, 374–516 for the CtCBM6, 517–652 for CtCBM13, and 653–742 for CtFn3. Stereochemistry was assessed with COOT and PDBSUM (with 677 residues (96%) in preferred, 22 in allowed regions (3%), and 6 outliers (1%) in the Ramachandran plot).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>41279</offset>
+      <text>To obtain structures of CtGH5-CBM6 in complex with ligand the protein was crystallized using the sitting drop vapor phase diffusion method with an equal volume (100 nl) of protein and reservoir solution (unless otherwise stated), using the robotic nanodrop dispensing systems (mosquitoR LCP; TTPLabTech). Crystals of the protein (10 mg/ml) co-crystallized with arabinose (300 mm) were obtained in 1 m ammonium sulfate, 0.1 m Bis-Tris, pH 5.5, and 1% PEG 3350. Crystals with xylose (300 mm) grew in 100 mm sodium/potassium phosphate, 100 mm MES, pH 6.5, and 2 m sodium chloride. To obtain crystals of the arabinoxylanase in complex with an oligosaccharide, the nucleophile mutant E279S was used and mixed with a range of arabinoxylooligosaccharides that was generated by digestion of WAX with CtGH5-CBM6 (see above) and thereafter by 100 nm of the Cellvibrio japonicus GH43 exo-1,4-β-xylosidase. Only the inclusion of the largest purified oligosaccharide generated crystals of the arabinoxylanase. Crystals of CtGH5E279S-CBM6 were obtained by mixing an equal volume (100 nl) of the protein (11 mg/ml)/oligosaccharide (10 mm) solution and mother liquor solution consisting of 100 mm Tris-Bicine, pH 8.5, 12.5% (w/v) polyethylene glycol with an average molecular mass of 1,000 Da, 12.5% (w/v) polyethylene glycol with an average molecular mass of 3,350 Da and 12.5% (R,S)-2-methyl-2,4-pentanediol (racemic). Crystallographic data were collected on Beamlines IO2, IO4-1, and I24 at the DIAMOND Light Source (Harwell, UK). The data were processed using XDS The crystal structures were solved by molecular replacement using MolRep with CtGH5-CtCBM6 (PDB code 5AK1) as the search model. The refinement was done in RefMac5, and COOT was used for model (re)building. The final model were validated using Molprobity. The data collection and refinement statistics are listed in Table 2.</text>
+    </passage>
+    <passage>
+      <infon key="file">T2.xml</infon>
+      <infon key="id">T2</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_caption</infon>
+      <offset>43158</offset>
+      <text>Data collection and refinement statistics</text>
+      <annotation id="990">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:51:14Z</infon>
+        <location offset="43158" length="41"/>
+        <text>Data collection and refinement statistics</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">T2.xml</infon>
+      <infon key="id">T2</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_caption</infon>
+      <offset>43200</offset>
+      <text>The values in parentheses are for highest resolution shell.</text>
+    </passage>
+    <passage>
+      <infon key="file">T2.xml</infon>
+      <infon key="id">T2</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table</infon>
+      <infon key="xml">&lt;?xml version="1.0" encoding="UTF-8"?&gt;
+&lt;table xmlns:xlink="http://www.w3.org/1999/xlink" frame="hsides" rules="groups"&gt;&lt;thead valign="bottom"&gt;&lt;tr&gt;&lt;th rowspan="1" colspan="1"/&gt;&lt;th align="center" rowspan="1" colspan="1"&gt;CtXyl5A&lt;sub&gt;-D&lt;/sub&gt;&lt;/th&gt;&lt;th align="center" rowspan="1" colspan="1"&gt;GH5-CBM6-&lt;italic&gt;Arap&lt;/italic&gt;&lt;/th&gt;&lt;th align="center" rowspan="1" colspan="1"&gt;GH5-CBM6-&lt;italic&gt;Xylp&lt;/italic&gt;&lt;/th&gt;&lt;th align="center" rowspan="1" colspan="1"&gt;GH5-CBM6- (&lt;italic&gt;Araf&lt;/italic&gt;-Xyl&lt;italic&gt;p&lt;/italic&gt;&lt;sub&gt;4&lt;/sub&gt;)&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign="top"&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;&lt;bold&gt;Data collection&lt;/bold&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    Source&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;ESRF-ID14-1&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;Diamond I04–1&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;Diamond I24&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;Diamond I02&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    Wavelength (Å)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;0.9334&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;0.9173&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;0.9772&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;0.9791&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    Space group&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    Cell dimensions&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;        &lt;italic&gt;a&lt;/italic&gt;, &lt;italic&gt;b&lt;/italic&gt;, &lt;italic&gt;c&lt;/italic&gt; (Å)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;147.4, 191.7, 50.7&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;67.1, 72.4, 109.1&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;67.9, 72.5, 109.5&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;76.3, 123.2, 125.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;        α, β, γ (°)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;90, 90, 90&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;90, 90, 90&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;90, 90, 90&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;90, 90, 90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    No. of measured reflections&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;244,475 (29,324)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;224,842 (11,281)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;152,004 (4,996)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;463,237 (23,068)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    No. of independent reflections&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;42246 (5,920)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;63,523 (3,175)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;42,716 (2,334)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;140,288 (6,879)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    Resolution (Å)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;50.70–2.64 (2.78–2.64)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;44.85–1.65 (1.68–1.65)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;45.16–1.90 (1.94–1.90)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;48.43–1.65 (1.68–1.65)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;merge&lt;/sub&gt; (%)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;16.5 (69.5)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;6.7 (65.1)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;2.8 (8.4)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;5.7 (74.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    CC&lt;sub&gt;1/2&lt;/sub&gt;&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;0.985 (0.478)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;0.998 (0.594)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;0.999 (0.982)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;0.998 (0.484)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    &lt;italic&gt;I&lt;/italic&gt;/σ&lt;italic&gt;I&lt;/italic&gt;&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;8.0 (2.0)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;13 (1.6)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;26.6 (8.0)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;11.2 (1.6)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    Completeness (%)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;98.5 (96.4)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;98.5 (99.4)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;98.6 (85.0)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;98.8 (99.4)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    Redundancy&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;5.8 (5.0)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;3.5 (3.6)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;3.6 (2.1)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;3.3 (3.4)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="5" rowspan="1"&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;&lt;bold&gt;Refinement&lt;/bold&gt;&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;work&lt;/sub&gt;/&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt;&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;23.7/27.8&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;12.2/17.0&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;12.9/16.1&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;14.5/19.9&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    No. atoms&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;        Protein&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;5446&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;3790&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;3729&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;7333&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;        Ligand&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;19&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;20&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;20&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;92&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;        Water&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;227&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;579&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;601&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;923&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    B-factors&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;        Protein&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;41.6&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;17.8&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;15.8&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;21.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;        Ligand&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;65.0&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;19.4&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;24.2&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;39.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;        Water&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;35.4&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;38.5&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;32.2&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;37.6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    R.m.s deviations&lt;/td&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;td rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;        Bond lengths (Å)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;0.008&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;0.015&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;0.012&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;0.012&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;        Bond angles (°)&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;1.233&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;1.502&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;1.624&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;1.554&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;    Protein Data Bank code&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;&lt;ext-link ext-link-type="pdb" xlink:href="5G56"&gt;5G56&lt;/ext-link&gt;&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;&lt;ext-link ext-link-type="pdb" xlink:href="5LA0"&gt;5LA0&lt;/ext-link&gt;&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;&lt;ext-link ext-link-type="pdb" xlink:href="5LA1"&gt;5LA1&lt;/ext-link&gt;&lt;/td&gt;&lt;td align="left" rowspan="1" colspan="1"&gt;&lt;ext-link ext-link-type="pdb" xlink:href="2LA2"&gt;2LA2&lt;/ext-link&gt;&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon>
+      <offset>43260</offset>
+      <text>	CtXyl5A-D	GH5-CBM6-Arap	GH5-CBM6-Xylp	GH5-CBM6- (Araf-Xylp4)	 	Data collection					 	    Source	ESRF-ID14-1	Diamond I04–1	Diamond I24	Diamond I02	 	    Wavelength (Å)	0.9334	0.9173	0.9772	0.9791	 	    Space group	P21212	P212121	P212121	P212121	 	    Cell dimensions					 	        a, b, c (Å)	147.4, 191.7, 50.7	67.1, 72.4, 109.1	67.9, 72.5, 109.5	76.3, 123.2, 125.4	 	        α, β, γ (°)	90, 90, 90	90, 90, 90	90, 90, 90	90, 90, 90	 	    No. of measured reflections	244,475 (29,324)	224,842 (11,281)	152,004 (4,996)	463,237 (23,068)	 	    No. of independent reflections	42246 (5,920)	63,523 (3,175)	42,716 (2,334)	140,288 (6,879)	 	    Resolution (Å)	50.70–2.64 (2.78–2.64)	44.85–1.65 (1.68–1.65)	45.16–1.90 (1.94–1.90)	48.43–1.65 (1.68–1.65)	 	    Rmerge (%)	16.5 (69.5)	6.7 (65.1)	2.8 (8.4)	5.7 (74.9)	 	    CC1/2	0.985 (0.478)	0.998 (0.594)	0.999 (0.982)	0.998 (0.484)	 	    I/σI	8.0 (2.0)	13 (1.6)	26.6 (8.0)	11.2 (1.6)	 	    Completeness (%)	98.5 (96.4)	98.5 (99.4)	98.6 (85.0)	98.8 (99.4)	 	    Redundancy	5.8 (5.0)	3.5 (3.6)	3.6 (2.1)	3.3 (3.4)	 		 	Refinement					 	    Rwork/Rfree	23.7/27.8	12.2/17.0	12.9/16.1	14.5/19.9	 	    No. atoms					 	        Protein	5446	3790	3729	7333	 	        Ligand	19	20	20	92	 	        Water	227	579	601	923	 	    B-factors					 	        Protein	41.6	17.8	15.8	21.0	 	        Ligand	65.0	19.4	24.2	39.5	 	        Water	35.4	38.5	32.2	37.6	 	    R.m.s deviations					 	        Bond lengths (Å)	0.008	0.015	0.012	0.012	 	        Bond angles (°)	1.233	1.502	1.624	1.554	 	    Protein Data Bank code	5G56	5LA0	5LA1	2LA2	 	</text>
+      <annotation id="954">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:27:43Z</infon>
+        <location offset="43261" length="9"/>
+        <text>CtXyl5A-D</text>
+      </annotation>
+      <annotation id="857">
+        <infon key="score">0.9927343</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:52:03Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="43271" length="13"/>
+        <text>GH5-CBM6-Arap</text>
+      </annotation>
+      <annotation id="858">
+        <infon key="score">0.99599856</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:52:14Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="43285" length="13"/>
+        <text>GH5-CBM6-Xylp</text>
+      </annotation>
+      <annotation id="991">
+        <infon key="type">complex_assembly</infon>
+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:52:48Z</infon>
+        <location offset="43299" length="22"/>
+        <text>GH5-CBM6- (Araf-Xylp4)</text>
+      </annotation>
+      <annotation id="859">
+        <infon key="score">0.91044104</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:52:52Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44339" length="5"/>
+        <text>Rwork</text>
+      </annotation>
+      <annotation id="860">
+        <infon key="score">0.8382509</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T12:52:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="44345" length="5"/>
+        <text>Rfree</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">title_1</infon>
+      <offset>45145</offset>
+      <text>Author Contributions</text>
+    </passage>
+    <passage>
+      <infon key="section_type">AUTH_CONT</infon>
+      <infon key="type">paragraph</infon>
+      <offset>45166</offset>
+      <text>A. L. obtained crystals of the GH5-CBM6 complex. L. I. C. analyzed the biochemistry of GH5 subfamilies. J. L. A. B. obtained crystals of CtXyl5A-D. A. J. analyzed the biochemistry of GH5-CBM6 and obtained crystals of GH5-CBM6. A. R. analyzed the biochemistry of GH5-CBM6 products. J. G. performed mass spectrometry. M. P. Y. provided the substrate. B. H. performed analysis of GH5 sequences. C. M. G. A. F. designed the experiments. H. J. G. designed the experiments, analyzed data, and contributed to writing the paper. S. N. solved the structure of CtXyl5A-D and contributed to writing the paper. A. B. used crystallography to solve GH5-CBM6 structures. F. C. analyzed the biochemistry of GH5-CBM6 mutants, obtained crystals of GH5-CBM6, and contributed to writing the paper.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">title_1</infon>
+      <offset>45944</offset>
+      <text>Supplementary Material</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>45967</offset>
+      <text>This work was supported in part by European Research Council Grant 322820 (to H. J. G. and B. H.), Biotechnology and Biological Research Council Grants BB/K020358/1 and BB/K001949/1 (to H. J. G.), Wellcome Trust Grant RES/0120/7613 (to H. J. G.), Agence Nationale de la Recherche Grant ANR 12-BIME-0006-01 (to B. H.), and Fundação para a Ciência e Tecnologia Grants PTDC/BIAPRO/103980/2008 and PTDC/BIAMIC/5947/2014 (to C. M. G. A. F.). The authors declare that they have no conflicts of interest with the contents of this article.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46502</offset>
+      <text>This article contains supplemental Table S1 and Fig. S1.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46559</offset>
+      <text>GH</text>
+      <annotation id="861">
+        <infon key="score">0.9930426</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="46559" length="2"/>
+        <text>GH</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46562</offset>
+      <text>glycoside hydrolase</text>
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+        <infon key="score">0.9983388</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:31:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="46562" length="19"/>
+        <text>glycoside hydrolase</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46582</offset>
+      <text>CtXyl5A</text>
+      <annotation id="863">
+        <infon key="score">0.9968766</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:32Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="46582" length="7"/>
+        <text>CtXyl5A</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46590</offset>
+      <text>C. thermocellum arabinoxylanase</text>
+      <annotation id="864">
+        <infon key="score">0.9984484</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:38:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="46590" length="15"/>
+        <text>C. thermocellum</text>
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+        <infon key="score">0.998863</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:25:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="46606" length="15"/>
+        <text>arabinoxylanase</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46622</offset>
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+        <infon key="score">0.99871993</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:53:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="46622" length="3"/>
+        <text>CBM</text>
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+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46626</offset>
+      <text>non-catalytic carbohydrate binding module</text>
+      <annotation id="867">
+        <infon key="score">0.82269233</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:26:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="46626" length="41"/>
+        <text>non-catalytic carbohydrate binding module</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46668</offset>
+      <text>Fn</text>
+      <annotation id="868">
+        <infon key="score">0.40625653</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:54:48Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="46668" length="2"/>
+        <text>Fn</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46671</offset>
+      <text>fibronectin</text>
+      <annotation id="869">
+        <infon key="score">0.6230605</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:35:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="46671" length="11"/>
+        <text>fibronectin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46683</offset>
+      <text>WAX</text>
+      <annotation id="870">
+        <infon key="score">0.9978181</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:24Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="46683" length="3"/>
+        <text>WAX</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46687</offset>
+      <text>wheat arabinoxylan</text>
+      <annotation id="871">
+        <infon key="score">0.91318005</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:44:28Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="46687" length="5"/>
+        <text>wheat</text>
+      </annotation>
+      <annotation id="872">
+        <infon key="score">0.9990382</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:44:12Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="46693" length="12"/>
+        <text>arabinoxylan</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46706</offset>
+      <text>RAX</text>
+      <annotation id="873">
+        <infon key="score">0.87364924</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:28Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="46706" length="3"/>
+        <text>RAX</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46710</offset>
+      <text>rye arabinoxylan</text>
+      <annotation id="874">
+        <infon key="score">0.8627195</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:44:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="46710" length="3"/>
+        <text>rye</text>
+      </annotation>
+      <annotation id="875">
+        <infon key="score">0.99903655</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:44:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="46714" length="12"/>
+        <text>arabinoxylan</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46727</offset>
+      <text>CX</text>
+      <annotation id="876">
+        <infon key="score">0.9888843</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:46:39Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="46727" length="2"/>
+        <text>CX</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46730</offset>
+      <text>corn bran xylan</text>
+      <annotation id="877">
+        <infon key="score">0.6373213</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:45:09Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="46730" length="4"/>
+        <text>corn</text>
+      </annotation>
+      <annotation id="878">
+        <infon key="score">0.8507954</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="46740" length="5"/>
+        <text>xylan</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46746</offset>
+      <text>HPAEC</text>
+      <annotation id="879">
+        <infon key="score">0.8287563</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:58:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="46746" length="5"/>
+        <text>HPAEC</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46752</offset>
+      <text>high performance anion exchange chromatography</text>
+      <annotation id="880">
+        <infon key="score">0.99853724</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:06:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="46752" length="46"/>
+        <text>high performance anion exchange chromatography</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46799</offset>
+      <text>PDB</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46803</offset>
+      <text>Protein Data Bank</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46821</offset>
+      <text>BX</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46824</offset>
+      <text>birchwood xylan</text>
+      <annotation id="881">
+        <infon key="score">0.96442974</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T13:44:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="46824" length="9"/>
+        <text>birchwood</text>
+      </annotation>
+      <annotation id="882">
+        <infon key="score">0.99584645</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:24:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="46834" length="5"/>
+        <text>xylan</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46840</offset>
+      <text>ESI</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46844</offset>
+      <text>electrospray ionization.</text>
+      <annotation id="883">
+        <infon key="score">0.99849916</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T14:06:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="46844" length="23"/>
+        <text>electrospray ionization</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>46869</offset>
+      <text>The abbreviations used are: </text>
+    </passage>
+    <passage>
+      <infon key="section_type">REF</infon>
+      <infon key="type">title</infon>
+      <offset>46898</offset>
+      <text>References</text>
+    </passage>
+    <passage>
+      <infon key="fpage">444</infon>
+      <infon key="lpage">455</infon>
+      <infon key="name_0">surname:Gilbert;given-names:H. J.</infon>
+      <infon key="pub-id_pmid">20406913</infon>
+      <infon key="section_type">REF</infon>
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+      <infon key="type">ref</infon>
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+      <infon key="year">2010</infon>
+      <offset>46909</offset>
+      <text>The biochemistry and structural biology of plant cell wall deconstruction</text>
+    </passage>
+    <passage>
+      <infon key="fpage">316</infon>
+      <infon key="lpage">317</infon>
+      <infon key="name_0">surname:Himmel;given-names:M. E.</infon>
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+    <passage>
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+      <infon key="section_type">REF</infon>
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+    <passage>
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+    <passage>
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+    <passage>
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diff --git a/annotated_BioC_XML/PMC5173035_ann.xml b/annotated_BioC_XML/PMC5173035_ann.xml
new file mode 100644
index 0000000000000000000000000000000000000000..f372c9415c196019239c69c6c3e0dc8e56e6aaf4
--- /dev/null
+++ b/annotated_BioC_XML/PMC5173035_ann.xml
@@ -0,0 +1,8885 @@
+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection>
+  <source>PMC</source>
+  <date>20230815</date>
+  <key>pmc.key</key>
+  <document>
+    <id>5173035</id>
+    <infon key="license">CC BY</infon>
+    <infon key="tt_curatable">no</infon>
+    <infon key="tt_version">2</infon>
+    <infon key="tt_round">2</infon>
+    <passage>
+      <infon key="article-id_doi">10.18632/oncotarget.9692</infon>
+      <infon key="article-id_pmc">5173035</infon>
+      <infon key="article-id_pmid">27259995</infon>
+      <infon key="article-id_publisher-id">9692</infon>
+      <infon key="fpage">40965</infon>
+      <infon key="issue">27</infon>
+      <infon key="kwd">DNA N6-adenine methyltransferase M1.HpyAVI substrate recognition AdoMet-binding Helicobacter pylori Immunology and Microbiology Section Immune response Immunity</infon>
+      <infon key="license">This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</infon>
+      <infon key="lpage">40977</infon>
+      <infon key="name_0">surname:Ma;given-names:Bo</infon>
+      <infon key="name_1">surname:Ma;given-names:Ji</infon>
+      <infon key="name_2">surname:Liu;given-names:Dong</infon>
+      <infon key="name_3">surname:Guo;given-names:Ling</infon>
+      <infon key="name_4">surname:Chen;given-names:Huiling</infon>
+      <infon key="name_5">surname:Ding;given-names:Jingjin</infon>
+      <infon key="name_6">surname:Liu;given-names:Wei</infon>
+      <infon key="name_7">surname:Zhang;given-names:Hongquan</infon>
+      <infon key="section_type">TITLE</infon>
+      <infon key="type">front</infon>
+      <infon key="volume">7</infon>
+      <infon key="year">2016</infon>
+      <offset>0</offset>
+      <text>Biochemical and structural characterization of a DNA N6-adenine methyltransferase from Helicobacter pylori </text>
+      <annotation id="1">
+        <infon key="score">0.9988349</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:48:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="0" length="43"/>
+        <text>Biochemical and structural characterization</text>
+      </annotation>
+      <annotation id="2">
+        <infon key="score">0.9984956</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:48:27Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="49" length="32"/>
+        <text>DNA N6-adenine methyltransferase</text>
+      </annotation>
+      <annotation id="3">
+        <infon key="score">0.99830234</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:48:33Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="87" length="19"/>
+        <text>Helicobacter pylori</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">ABSTRACT</infon>
+      <infon key="type">abstract</infon>
+      <offset>108</offset>
+      <text>DNA N6-methyladenine modification plays an important role in regulating a variety of biological functions in bacteria. However, the mechanism of sequence-specific recognition in N6-methyladenine modification remains elusive. M1.HpyAVI, a DNA N6-adenine methyltransferase from Helicobacter pylori, shows more promiscuous substrate specificity than other enzymes. Here, we present the crystal structures of cofactor-free and AdoMet-bound structures of this enzyme, which were determined at resolutions of 3.0 Å and 3.1 Å, respectively. The core structure of M1.HpyAVI resembles the canonical AdoMet-dependent MTase fold, while the putative DNA binding regions considerably differ from those of the other MTases, which may account for the substrate promiscuity of this enzyme. Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity. Taken together, our data revealed the structural basis underlying DNA N6-adenine methyltransferase substrate promiscuity.</text>
+      <annotation id="683">
+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:12:13Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+      <annotation id="697">
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+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>structures</text>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
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+      <text>INTRODUCTION</text>
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+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
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+      <text>DNA methylation is a common form of modification on nucleic acids occurring in both prokaryotes and eukaryotes. Such a modification creates a signature motif recognized by DNA-interacting proteins and functions as a mechanism to regulate gene expression. DNA methylation is mediated by DNA methyltransferases (MTases), which catalyze the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to a given position of a particular DNA base within a specific DNA sequence.</text>
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:14:12Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:29Z</infon>
+        <location offset="1537" length="15"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>MTases</text>
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:14:24Z</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="updated_at">2023-09-18T08:33:18Z</infon>
+        <location offset="1749" length="3"/>
+        <text>DNA</text>
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+      <infon key="section_type">INTRO</infon>
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+      <offset>1763</offset>
+      <text>Three classes of DNA MTases have been identified to transfer a methyl group to different positions of DNA bases. C5-cytosine MTases, for example, methylate C5 of cytosine (m5C). In eukaryotes, m5C plays an important role in gene expression, chromatin organization, genome maintenance and parental imprinting, and is involved in a variety of human diseases including cancer. By contrast, the functions of the prokaryotic DNA cytosine MTase remain unknown. N4-cytosine MTases, which are frequently present in thermophilic or mesophilic bacteria, transfer a methyl group to the exocyclic amino group of cytosine (4mC). N4 methylation seems to be primarily a component of bacterial immune system against invasion by foreign DNA, such as conjugative plasmids and bacteriophages. The third group, N6-adenine MTases methylate the exocyclic amino groups of adenine (6mA), which exists in prokaryotes as a signal for genome defense, DNA replication and repair, regulation of gene expression, control of transposition and host-pathogen interactions. Recent studies utilizing new sequencing approaches have showed the existence of 6mA in several eukaryotic species. DNA 6mA modification is associated with important biological processes including nucleosome distribution close to the transcription start sites in Chlamydomonas, carrying heritable epigenetic information in C.elegans or controlling development of Drosophila.</text>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="1780" length="10"/>
+        <text>DNA MTases</text>
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="updated_at">2023-09-15T15:14:33Z</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="type">residue_name</infon>
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+        <text>cytosine</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="1935" length="3"/>
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+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1944" length="10"/>
+        <text>eukaryotes</text>
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+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>human</text>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:52:43Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2183" length="18"/>
+        <text>DNA cytosine MTase</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:52:53Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2218" length="18"/>
+        <text>N4-cytosine MTases</text>
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+        <infon key="type">taxonomy_domain</infon>
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+        <infon key="updated_at">2023-09-15T14:53:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>thermophilic</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:53:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2286" length="10"/>
+        <text>mesophilic</text>
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+        <infon key="type">taxonomy_domain</infon>
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+        <infon key="updated_at">2023-09-15T14:48:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2297" length="8"/>
+        <text>bacteria</text>
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:56:49Z</infon>
+        <location offset="2318" length="6"/>
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+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:52Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:53:58Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>4mC</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:27:38Z</infon>
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+        <text>N4 methylation</text>
+      </annotation>
+      <annotation id="43">
+        <infon key="score">0.99867094</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:53:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2431" length="9"/>
+        <text>bacterial</text>
+      </annotation>
+      <annotation id="687">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:14:44Z</infon>
+        <location offset="2483" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="677">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:56:28Z</infon>
+        <location offset="2521" length="14"/>
+        <text>bacteriophages</text>
+      </annotation>
+      <annotation id="44">
+        <infon key="score">0.99827415</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:53:24Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2554" length="17"/>
+        <text>N6-adenine MTases</text>
+      </annotation>
+      <annotation id="45">
+        <infon key="score">0.9711873</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:54:08Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2612" length="7"/>
+        <text>adenine</text>
+      </annotation>
+      <annotation id="46">
+        <infon key="score">0.98714834</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:54:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2621" length="3"/>
+        <text>6mA</text>
+      </annotation>
+      <annotation id="47">
+        <infon key="score">0.9985636</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2643" length="11"/>
+        <text>prokaryotes</text>
+      </annotation>
+      <annotation id="688">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:14:54Z</infon>
+        <location offset="2687" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="48">
+        <infon key="score">0.96919215</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:54:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2883" length="3"/>
+        <text>6mA</text>
+      </annotation>
+      <annotation id="49">
+        <infon key="score">0.99854434</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:27:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2898" length="10"/>
+        <text>eukaryotic</text>
+      </annotation>
+      <annotation id="820">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:33:46Z</infon>
+        <location offset="2918" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="50">
+        <infon key="score">0.9743464</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:54:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2922" length="3"/>
+        <text>6mA</text>
+      </annotation>
+      <annotation id="51">
+        <infon key="score">0.84729904</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:54:37Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3065" length="13"/>
+        <text>Chlamydomonas</text>
+      </annotation>
+      <annotation id="52">
+        <infon key="score">0.99722385</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:54:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3125" length="9"/>
+        <text>C.elegans</text>
+      </annotation>
+      <annotation id="53">
+        <infon key="score">0.9626723</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:54:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3165" length="10"/>
+        <text>Drosophila</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>3177</offset>
+      <text>All the three types of methylation exist in prokaryotes, and most DNA MTases are components of the restriction-modification (R-M) systems. The R-M systems are composed of two enzymes displaying opposing activities. “R” stands for a restriction endonuclease cleaving specific DNA sequences, while “M” symbolizes a modification methyltransferase rendering these sequences resistant to cleavage. The cooperation of these two enzymes provides a defensive mechanism to protect bacteria from infection by bacteriophages. The R-M systems are classified into three types based on specific structural features, position of DNA cleavage and cofactor requirements. In types I and III, the DNA adenine or cytosine methyltransferase is part of a multi-subunit enzyme that catalyzes both restriction and modification. By contrast, two separate enzymes exist in type II systems, where a restriction endonuclease and a DNA adenine or cytosine methyltransferase recognize the same targets.</text>
+      <annotation id="815">
+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:31:49Z</infon>
+        <location offset="3200" length="11"/>
+        <text>methylation</text>
+      </annotation>
+      <annotation id="54">
+        <infon key="score">0.99844944</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3221" length="11"/>
+        <text>prokaryotes</text>
+      </annotation>
+      <annotation id="55">
+        <infon key="score">0.99861634</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3243" length="10"/>
+        <text>DNA MTases</text>
+      </annotation>
+      <annotation id="56">
+        <infon key="score">0.9980486</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:56:19Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3409" length="24"/>
+        <text>restriction endonuclease</text>
+      </annotation>
+      <annotation id="689">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:15:09Z</infon>
+        <location offset="3452" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="57">
+        <infon key="score">0.99867034</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:56:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3490" length="30"/>
+        <text>modification methyltransferase</text>
+      </annotation>
+      <annotation id="58">
+        <infon key="score">0.99824834</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:48:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3649" length="8"/>
+        <text>bacteria</text>
+      </annotation>
+      <annotation id="59">
+        <infon key="score">0.99335164</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:56:28Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3676" length="14"/>
+        <text>bacteriophages</text>
+      </annotation>
+      <annotation id="690">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:15:18Z</infon>
+        <location offset="3791" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="60">
+        <infon key="score">0.99397075</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:56:38Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3855" length="41"/>
+        <text>DNA adenine or cytosine methyltransferase</text>
+      </annotation>
+      <annotation id="61">
+        <infon key="score">0.99784696</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:56:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4049" length="24"/>
+        <text>restriction endonuclease</text>
+      </annotation>
+      <annotation id="62">
+        <infon key="score">0.9003789</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:56:45Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4080" length="41"/>
+        <text>DNA adenine or cytosine methyltransferase</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>4158</offset>
+      <text>To date, a number of bacterial DNA MTases have been structurally characterized, covering enzymes from all the three classes. All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base. Conserved amino acid motifs have been identified from reported structures, including ten motifs (I-X) in cytosine MTases and nine motifs (I-VIII and X) in adenine MTases, all of which are arranged in an almost constant order. According to the linear arrangement of three conserved domains, exocyclic amino MTases are subdivided into six groups (namely α, β, γ, ζ, δ and ε). N6-adenine and N4-cytosine MTases, in particular, are closely related by sharing common structural features. Despite the considerable similarity among bacterial MTases, some differences were observed among the enzymes from various species. For example, the structural regions of MTases beyond the catalytic domain are rather variable, such as the C-terminal domain of M.TaqI, the extended arm of M.MboIIA and M.RsrI, the helix bundle of EcoDam, and so on.</text>
+      <annotation id="63">
+        <infon key="score">0.9985929</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:53:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4179" length="9"/>
+        <text>bacterial</text>
+      </annotation>
+      <annotation id="64">
+        <infon key="score">0.9986365</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4189" length="10"/>
+        <text>DNA MTases</text>
+      </annotation>
+      <annotation id="65">
+        <infon key="score">0.9897859</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:02:45Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4210" length="26"/>
+        <text>structurally characterized</text>
+      </annotation>
+      <annotation id="66">
+        <infon key="score">0.9991573</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4293" length="6"/>
+        <text>MTases</text>
+      </annotation>
+      <annotation id="67">
+        <infon key="score">0.9947482</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4403" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="828">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:36:18Z</infon>
+        <location offset="4420" length="16"/>
+        <text>catalytic domain</text>
+      </annotation>
+      <annotation id="68">
+        <infon key="score">0.9982283</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:35:08Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4451" length="11"/>
+        <text>active site</text>
+      </annotation>
+      <annotation id="852">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:56:49Z</infon>
+        <location offset="4467" length="6"/>
+        <text>methyl</text>
+      </annotation>
+      <annotation id="69">
+        <infon key="score">0.9962698</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4498" length="6"/>
+        <text>AdoMet</text>
+      </annotation>
+      <annotation id="821">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:34:46Z</infon>
+        <location offset="4528" length="31"/>
+        <text>target (DNA)-recognition domain</text>
+      </annotation>
+      <annotation id="733">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:23:59Z</infon>
+        <location offset="4561" length="3"/>
+        <text>TRD</text>
+      </annotation>
+      <annotation id="691">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:15:33Z</infon>
+        <location offset="4626" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="692">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:15:43Z</infon>
+        <location offset="4670" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="70">
+        <infon key="score">0.9656195</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4699" length="9"/>
+        <text>Conserved</text>
+      </annotation>
+      <annotation id="71">
+        <infon key="score">0.99657625</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:58:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4762" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="824">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:35:22Z</infon>
+        <location offset="4796" length="3"/>
+        <text>I-X</text>
+      </annotation>
+      <annotation id="72">
+        <infon key="score">0.99850804</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:58:34Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4804" length="15"/>
+        <text>cytosine MTases</text>
+      </annotation>
+      <annotation id="822">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:35:02Z</infon>
+        <location offset="4837" length="6"/>
+        <text>I-VIII</text>
+      </annotation>
+      <annotation id="823">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:35:13Z</infon>
+        <location offset="4848" length="1"/>
+        <text>X</text>
+      </annotation>
+      <annotation id="73">
+        <infon key="score">0.9986151</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:58:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4854" length="14"/>
+        <text>adenine MTases</text>
+      </annotation>
+      <annotation id="74">
+        <infon key="score">0.8387143</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4970" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="75">
+        <infon key="score">0.9985342</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:58:44Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="4989" length="22"/>
+        <text>exocyclic amino MTases</text>
+      </annotation>
+      <annotation id="76">
+        <infon key="score">0.99915075</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:00:07Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5051" length="1"/>
+        <text>α</text>
+      </annotation>
+      <annotation id="77">
+        <infon key="score">0.9991696</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:00:15Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5054" length="1"/>
+        <text>β</text>
+      </annotation>
+      <annotation id="78">
+        <infon key="score">0.9992083</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:00:25Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5057" length="1"/>
+        <text>γ</text>
+      </annotation>
+      <annotation id="79">
+        <infon key="score">0.9991721</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:00:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5060" length="1"/>
+        <text>ζ</text>
+      </annotation>
+      <annotation id="80">
+        <infon key="score">0.99910283</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:00:40Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5063" length="1"/>
+        <text>δ</text>
+      </annotation>
+      <annotation id="81">
+        <infon key="score">0.9991536</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:00:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5069" length="1"/>
+        <text>ε</text>
+      </annotation>
+      <annotation id="678">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:59:23Z</infon>
+        <location offset="5073" length="33"/>
+        <text>N6-adenine and N4-cytosine MTases</text>
+      </annotation>
+      <annotation id="82">
+        <infon key="score">0.99840933</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:53:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5224" length="9"/>
+        <text>bacterial</text>
+      </annotation>
+      <annotation id="83">
+        <infon key="score">0.9991761</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5234" length="6"/>
+        <text>MTases</text>
+      </annotation>
+      <annotation id="84">
+        <infon key="score">0.999046</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5352" length="6"/>
+        <text>MTases</text>
+      </annotation>
+      <annotation id="827">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:36:17Z</infon>
+        <location offset="5370" length="16"/>
+        <text>catalytic domain</text>
+      </annotation>
+      <annotation id="829">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:36:35Z</infon>
+        <location offset="5420" length="17"/>
+        <text>C-terminal domain</text>
+      </annotation>
+      <annotation id="711">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:44:49Z</infon>
+        <location offset="5441" length="6"/>
+        <text>M.TaqI</text>
+      </annotation>
+      <annotation id="713">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:05Z</infon>
+        <location offset="5469" length="8"/>
+        <text>M.MboIIA</text>
+      </annotation>
+      <annotation id="716">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:22Z</infon>
+        <location offset="5482" length="6"/>
+        <text>M.RsrI</text>
+      </annotation>
+      <annotation id="831">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:36:51Z</infon>
+        <location offset="5494" length="12"/>
+        <text>helix bundle</text>
+      </annotation>
+      <annotation id="85">
+        <infon key="score">0.99908864</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:02:49Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="5510" length="6"/>
+        <text>EcoDam</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>5553</offset>
+      <text>DNA methylation is thought to influence bacterial virulence. DNA adenine methyltransferase has been shown to play a crucial role in colonization of deep tissue sites in Salmonella typhimurium and Aeromonas hydrophila. Importantly, DNA adenine methylation is a global regulator of genes expressed during infection and inhibitors of DNA adenine methylation are likely to have a broad antimicrobial action. Dam was considered a promising target for antimicrobial drug development.</text>
+      <annotation id="671">
+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:29Z</infon>
+        <location offset="5553" length="15"/>
+        <text>DNA methylation</text>
+      </annotation>
+      <annotation id="86">
+        <infon key="score">0.9987413</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:53:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="5593" length="9"/>
+        <text>bacterial</text>
+      </annotation>
+      <annotation id="87">
+        <infon key="score">0.99834085</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:37:53Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5614" length="29"/>
+        <text>DNA adenine methyltransferase</text>
+      </annotation>
+      <annotation id="88">
+        <infon key="score">0.9982779</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:03:43Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5722" length="22"/>
+        <text>Salmonella typhimurium</text>
+      </annotation>
+      <annotation id="89">
+        <infon key="score">0.9983058</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:03:47Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5749" length="20"/>
+        <text>Aeromonas hydrophila</text>
+      </annotation>
+      <annotation id="680">
+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:07:59Z</infon>
+        <location offset="5784" length="23"/>
+        <text>DNA adenine methylation</text>
+      </annotation>
+      <annotation id="693">
+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:16:42Z</infon>
+        <location offset="5884" length="23"/>
+        <text>DNA adenine methylation</text>
+      </annotation>
+      <annotation id="90">
+        <infon key="score">0.9145081</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:16:58Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="5957" length="3"/>
+        <text>Dam</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>6031</offset>
+      <text>Helicobacter pylori is a Gram-negative bacterium that persistently colonizes in human stomach worldwide. It is a major pathogen of gastritis and peptic ulcer diseases as well as a cancer-causing factor for gastric cancer. H. pylori is involved in 90% of all gastric malignancies, infecting nearly 50% of the world's population and is the most crucial etiologic agent for gastric adenocarcinoma. H. pylori strains possess a few R-M systems like other bacteria to function as defensive systems. H. pylori 26695, for example, has 23 R-M systems. Methyltransferases were suggested to be involved in H. pylori pathogenicity. M1.HpyAVI is a DNA adenine MTase that belongs to the type II R-M system. This system contains two DNA MTases named M1.HpyAVI and M2.HpyAVI, and a putative restriction enzyme. M1.HpyAVI encoded by ORF hp0050 is an N6-adenine methyltransferase belonging to the β-class MTase. It has been reported recently that this enzyme recognizes the sequence of 5′-GAGG-3′, 5′-GGAG-3′ or 5′-GAAG-3′ and methylates adenines in these sequences. Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5′-GAAG-3′ seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5′-GAGG-3′. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.Here, we report the crystal structure of M1.HpyAVI from H. pylori 26695, which is the first determined N6-adenine MTase structure in H. pylori. The structure reveals a similar architecture as the canonical fold of homologous proteins, but displays several differences in the loop regions and TRD. Based on structural and biochemical analyses, we then identified two conserved amino acids, D29 at the catalytic site and E216 close to the C-terminus, as crucial residues for cofactor binding and methyltransferase activity of M1.HpyAVI. In addition, a non-conserved amino acid, P41, seems to play a key role in substrate recognition.</text>
+      <annotation id="91">
+        <infon key="score">0.9981073</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:48:33Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6031" length="19"/>
+        <text>Helicobacter pylori</text>
+      </annotation>
+      <annotation id="92">
+        <infon key="score">0.9708807</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:03:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6056" length="23"/>
+        <text>Gram-negative bacterium</text>
+      </annotation>
+      <annotation id="93">
+        <infon key="score">0.9986656</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:54:51Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6111" length="5"/>
+        <text>human</text>
+      </annotation>
+      <annotation id="94">
+        <infon key="score">0.9977002</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:04:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6253" length="9"/>
+        <text>H. pylori</text>
+      </annotation>
+      <annotation id="95">
+        <infon key="score">0.9980526</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:04:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6426" length="9"/>
+        <text>H. pylori</text>
+      </annotation>
+      <annotation id="96">
+        <infon key="score">0.9977798</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:48:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="6481" length="8"/>
+        <text>bacteria</text>
+      </annotation>
+      <annotation id="97">
+        <infon key="score">0.9855281</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:05:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6524" length="15"/>
+        <text>H. pylori 26695</text>
+      </annotation>
+      <annotation id="98">
+        <infon key="score">0.99871564</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:24:53Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6574" length="18"/>
+        <text>Methyltransferases</text>
+      </annotation>
+      <annotation id="99">
+        <infon key="score">0.99808294</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:04:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6626" length="9"/>
+        <text>H. pylori</text>
+      </annotation>
+      <annotation id="721">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:46:13Z</infon>
+        <location offset="6651" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="100">
+        <infon key="score">0.9983508</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:24:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6666" length="17"/>
+        <text>DNA adenine MTase</text>
+      </annotation>
+      <annotation id="101">
+        <infon key="score">0.9968161</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6749" length="10"/>
+        <text>DNA MTases</text>
+      </annotation>
+      <annotation id="699">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:44:17Z</infon>
+        <location offset="6766" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="720">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:45Z</infon>
+        <location offset="6780" length="9"/>
+        <text>M2.HpyAVI</text>
+      </annotation>
+      <annotation id="102">
+        <infon key="score">0.8533181</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:25:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6806" length="18"/>
+        <text>restriction enzyme</text>
+      </annotation>
+      <annotation id="700">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:44:17Z</infon>
+        <location offset="6826" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="103">
+        <infon key="score">0.995255</infon>
+        <infon key="type">gene</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:05:36Z</infon>
+        <infon key="identifier">GENE:</infon>
+        <location offset="6851" length="6"/>
+        <text>hp0050</text>
+      </annotation>
+      <annotation id="104">
+        <infon key="score">0.9986347</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:31:18Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6864" length="28"/>
+        <text>N6-adenine methyltransferase</text>
+      </annotation>
+      <annotation id="105">
+        <infon key="score">0.998701</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:05:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="6910" length="13"/>
+        <text>β-class MTase</text>
+      </annotation>
+      <annotation id="106">
+        <infon key="score">0.97984064</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:07:36Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="6999" length="11"/>
+        <text>5′-GAGG-3′,</text>
+      </annotation>
+      <annotation id="107">
+        <infon key="score">0.9948631</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:07:34Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7011" length="10"/>
+        <text>5′-GGAG-3′</text>
+      </annotation>
+      <annotation id="108">
+        <infon key="score">0.997119</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:07:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7025" length="10"/>
+        <text>5′-GAAG-3′</text>
+      </annotation>
+      <annotation id="109">
+        <infon key="score">0.65228903</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:07:27Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7051" length="8"/>
+        <text>adenines</text>
+      </annotation>
+      <annotation id="816">
+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:31:49Z</infon>
+        <location offset="7091" length="11"/>
+        <text>methylation</text>
+      </annotation>
+      <annotation id="110">
+        <infon key="score">0.64126253</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:07:28Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="7119" length="8"/>
+        <text>adenines</text>
+      </annotation>
+      <annotation id="111">
+        <infon key="score">0.9987367</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:05:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7182" length="17"/>
+        <text>N6-adenine MTases</text>
+      </annotation>
+      <annotation id="817">
+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:31:49Z</infon>
+        <location offset="7205" length="11"/>
+        <text>methylation</text>
+      </annotation>
+      <annotation id="112">
+        <infon key="score">0.99674714</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:07:10Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7229" length="10"/>
+        <text>5′-GAAG-3′</text>
+      </annotation>
+      <annotation id="701">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:44:17Z</infon>
+        <location offset="7272" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="113">
+        <infon key="score">0.9982695</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:25:33Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7332" length="8"/>
+        <text>H.pylori</text>
+      </annotation>
+      <annotation id="114">
+        <infon key="score">0.9978259</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:07:08Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="7373" length="10"/>
+        <text>5′-GAGG-3′</text>
+      </annotation>
+      <annotation id="115">
+        <infon key="score">0.8282734</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:07:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7529" length="9"/>
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+        <infon key="score">0.9972285</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:28:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7574" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="702">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:44:17Z</infon>
+        <location offset="7595" length="9"/>
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+      </annotation>
+      <annotation id="117">
+        <infon key="score">0.9966917</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:05:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7610" length="15"/>
+        <text>H. pylori 26695</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:25:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7657" length="16"/>
+        <text>N6-adenine MTase</text>
+      </annotation>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:07:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7674" length="9"/>
+        <text>structure</text>
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:04:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7687" length="9"/>
+        <text>H. pylori</text>
+      </annotation>
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+        <infon key="score">0.99784255</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:07:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7702" length="9"/>
+        <text>structure</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:01Z</infon>
+        <location offset="7829" length="4"/>
+        <text>loop</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:23:59Z</infon>
+        <location offset="7846" length="3"/>
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+        <infon key="score">0.9985078</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:06:45Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="7860" length="35"/>
+        <text>structural and biochemical analyses</text>
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+      <annotation id="123">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7920" length="9"/>
+        <text>conserved</text>
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+      <annotation id="124">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7943" length="3"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="7954" length="14"/>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="7973" length="4"/>
+        <text>E216</text>
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+      <annotation id="814">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:31:07Z</infon>
+        <location offset="8048" length="17"/>
+        <text>methyltransferase</text>
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+        <infon key="type">protein</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="updated_at">2023-09-18T09:25:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8104" length="13"/>
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+        <location offset="8130" length="3"/>
+        <text>P41</text>
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+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_1</infon>
+      <offset>8207</offset>
+      <text>RESULTS</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>8215</offset>
+      <text>Overall structure</text>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>structure</text>
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+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>8233</offset>
+      <text>Recombinant full-length M1.HpyAVI was produced as a soluble protein in Escherichia coli, but was quite unstable and tended to aggregate in low salt environment. The protein, however, remained fully soluble in a buffer containing higher concentration of sodium chloride (&gt;300 mM), which prompted that M1.HpyAVI is likely a halophilic protein.</text>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8245" length="11"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:44:17Z</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="8304" length="16"/>
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+        <infon key="identifier">CHEBI:</infon>
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+      <annotation id="705">
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+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:44:17Z</infon>
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+        <infon key="updated_at">2023-09-15T15:08:57Z</infon>
+        <location offset="8555" length="10"/>
+        <text>halophilic</text>
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+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>8575</offset>
+      <text>The cofactor-free and AdoMet-bound proteins were crystallized at different conditions. Both structures were determined by means of molecular replacement, and refined to 3.0 Å and 3.1 Å, respectively. Statistics of X-ray data collection and structure refinement were summarized in Table 1.</text>
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+        <infon key="updated_at">2023-09-15T14:49:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8597" length="12"/>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="8624" length="12"/>
+        <text>crystallized</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:28:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8667" length="10"/>
+        <text>structures</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:09:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8706" length="21"/>
+        <text>molecular replacement</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:09:15Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8789" length="21"/>
+        <text>X-ray data collection</text>
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+      <annotation id="139">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:09:17Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="8815" length="20"/>
+        <text>structure refinement</text>
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+    </passage>
+    <passage>
+      <infon key="file">T1.xml</infon>
+      <infon key="id">T1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_title_caption</infon>
+      <offset>8866</offset>
+      <text>Data collection and structure refinement statistics of M1.HpyAVI</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:28:27Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="8886" length="31"/>
+        <text>structure refinement statistics</text>
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+      <annotation id="141">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="8921" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">T1.xml</infon>
+      <infon key="id">T1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table</infon>
+      <infon key="xml">&lt;?xml version="1.0" encoding="UTF-8"?&gt;
+&lt;table frame="box" rules="all"&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align="left" valign="middle" rowspan="1" colspan="1"/&gt;&lt;th align="left" valign="middle" rowspan="1" colspan="1"&gt;M1.HpyAVI&lt;/th&gt;&lt;th align="left" valign="middle" rowspan="1" colspan="1"&gt;M1.HpyAVI-AdoMet complex&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;&lt;bold&gt;Data collection&lt;/bold&gt;&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"/&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;Wavelength (Å)&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;1.0000&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;0.97772&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;Space group&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;&lt;italic&gt;P&lt;/italic&gt;4&lt;sub&gt;3&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;&lt;italic&gt;P&lt;/italic&gt;6&lt;sub&gt;5&lt;/sub&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;Unit-cell parameters (Å, ˚)&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;&lt;italic&gt;a&lt;/italic&gt; = &lt;italic&gt;b&lt;/italic&gt; = 69.73, &lt;italic&gt;c&lt;/italic&gt; = 532.75&lt;break/&gt;&lt;italic&gt;α = β = γ&lt;/italic&gt; = 90&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;&lt;italic&gt;a = b&lt;/italic&gt; = 135.60, &lt;italic&gt;c&lt;/italic&gt; = 265.15&lt;break/&gt;&lt;italic&gt;α = β&lt;/italic&gt; = 90, &lt;italic&gt;γ&lt;/italic&gt; = 120&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;Resolution range (Å) &lt;xref ref-type="table-fn" rid="tfn_001"&gt;&lt;sup&gt;a&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;49.09-3.00 (3.09-3.00)&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;48.91-3.10 (3.18-3.10)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;Unique reflections &lt;xref ref-type="table-fn" rid="tfn_001"&gt;&lt;sup&gt;a&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;27243&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;49833&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;Multiplicity &lt;xref ref-type="table-fn" rid="tfn_001"&gt;&lt;sup&gt;a&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;3.7 (3.8)&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;5.6 (4.0)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;Completeness (%)&lt;xref ref-type="table-fn" rid="tfn_001"&gt;&lt;sup&gt;a&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;98.7 (98.9)&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;99.7 (97.8)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;Mean &lt;italic&gt;I/δ&lt;/italic&gt; (&lt;italic&gt;I&lt;/italic&gt;) &lt;xref ref-type="table-fn" rid="tfn_001"&gt;&lt;sup&gt;a&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;12.1 (3.4)&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;14.0 (1.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;Solvent content (%)&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;58.67&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;61.96&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;merge&lt;/sub&gt;
+&lt;xref ref-type="table-fn" rid="tfn_001"&gt;&lt;sup&gt;a&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;0.073 (0.378)&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;0.106 (0.769)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;&lt;bold&gt;Structure refinement&lt;/bold&gt;&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"/&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;work&lt;/sub&gt;&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;0.251&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;0.221&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt;&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;0.308&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;0.276&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;R.m.s.d., bond lengths (Å)&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;0.007&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;0.007&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;R.m.s.d., bond angles (˚)&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;1.408&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;1.651&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;&lt;bold&gt;Ramachandran plot&lt;/bold&gt;&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"/&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;Favoured region (%)&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;89.44&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;91.44&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;Allowed region (%)&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;9.58&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;7.11&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;Outliers (%)&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;0.99&lt;/td&gt;&lt;td align="left" valign="middle" rowspan="1" colspan="1"&gt;1.45&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon>
+      <offset>8931</offset>
+      <text>	M1.HpyAVI	M1.HpyAVI-AdoMet complex	 	Data collection			 	Wavelength (Å)	1.0000	0.97772	 	Space group	P43212	P65	 	Unit-cell parameters (Å, ˚)	a = b = 69.73, c = 532.75α = β = γ = 90	a = b = 135.60, c = 265.15α = β = 90, γ = 120	 	Resolution range (Å) a	49.09-3.00 (3.09-3.00)	48.91-3.10 (3.18-3.10)	 	Unique reflections a	27243	49833	 	Multiplicity a	3.7 (3.8)	5.6 (4.0)	 	Completeness (%)a	98.7 (98.9)	99.7 (97.8)	 	Mean I/δ (I) a	12.1 (3.4)	14.0 (1.9)	 	Solvent content (%)	58.67	61.96	 	Rmergea	0.073 (0.378)	0.106 (0.769)	 	Structure refinement			 	Rwork	0.251	0.221	 	Rfree	0.308	0.276	 	R.m.s.d., bond lengths (Å)	0.007	0.007	 	R.m.s.d., bond angles (˚)	1.408	1.651	 	Ramachandran plot			 	Favoured region (%)	89.44	91.44	 	Allowed region (%)	9.58	7.11	 	Outliers (%)	0.99	1.45	 	</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:10:21Z</infon>
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+        <text>M1.HpyAVI-AdoMet</text>
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+        <infon key="type">evidence</infon>
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+    </passage>
+    <passage>
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+      <infon key="id">T1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_footnote</infon>
+      <offset>9745</offset>
+      <text>Values in parentheses are statistics of the highest resolution shell.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>9815</offset>
+      <text>Four and eight protein monomers resided in the asymmetric units of the two crystal structures. Some amino acids, particularly those within two loops (residues 32-61 and 152-172) in both structures, were poorly defined in electron density and had to be omitted from the refined models. Details of invisible amino acids are given in Table S1.</text>
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+        <text>monomers</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:10:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10001" length="10"/>
+        <text>structures</text>
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+      <annotation id="149">
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:10:37Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>electron density</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>10156</offset>
+      <text>The two structures are very similar to each other (Figure 1) and could be well overlaid with an RMSD of 0.76 Å on 191 Cα atoms. The overall architecture of M1.HpyAVI revealed in these structures resembles the AdoMet-dependent MTase fold in which a twisted seven-stranded β-sheet flanked by six α-helices forms the structural core. Like the reported structures of the larger domain of MTases, three helices (αA, αB and αZ) are located at one face of the central β-sheet, while the other three αD, αE and αC sit at the other side. All these conserved structural motifs form a typical α/β Rossmann fold. The catalytic motif DPPY lies in a loop connecting αD and β4, and the cofactor AdoMet binds in a neighboring cavity. The loop (residues 136-166) located between β7 and αZ corresponds to a highly diverse region in other MTases that is involved in target DNA recognition. The hairpin loop (residues 101-133) bridging β6 and β7, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII. The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA. This loop, however, was well ordered in an M.TaqI-DNA complex structure and was shown to play a crucial role in DNA methylation by contacting the flipping adenine and recognizing specific DNA sequence.</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:25:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10695" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="783">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:27:27Z</infon>
+        <location offset="10738" length="17"/>
+        <text>α/β Rossmann fold</text>
+      </annotation>
+      <annotation id="809">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:29:40Z</infon>
+        <location offset="10761" length="15"/>
+        <text>catalytic motif</text>
+      </annotation>
+      <annotation id="780">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:27:09Z</infon>
+        <location offset="10777" length="4"/>
+        <text>DPPY</text>
+      </annotation>
+      <annotation id="788">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:01Z</infon>
+        <location offset="10792" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="754">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:24:28Z</infon>
+        <location offset="10808" length="2"/>
+        <text>αD</text>
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+      <annotation id="750">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:24:17Z</infon>
+        <location offset="10815" length="2"/>
+        <text>β4</text>
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+      <annotation id="157">
+        <infon key="score">0.99921036</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10836" length="6"/>
+        <text>AdoMet</text>
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+      <annotation id="158">
+        <infon key="score">0.96160835</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:35:16Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10866" length="6"/>
+        <text>cavity</text>
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+      <annotation id="789">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:01Z</infon>
+        <location offset="10878" length="4"/>
+        <text>loop</text>
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+      <annotation id="159">
+        <infon key="score">0.99757195</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:26:15Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10893" length="7"/>
+        <text>136-166</text>
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+      <annotation id="801">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:43Z</infon>
+        <location offset="10918" length="2"/>
+        <text>β7</text>
+      </annotation>
+      <annotation id="771">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:25:55Z</infon>
+        <location offset="10925" length="2"/>
+        <text>αZ</text>
+      </annotation>
+      <annotation id="160">
+        <infon key="score">0.9987655</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:30:35Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10945" length="14"/>
+        <text>highly diverse</text>
+      </annotation>
+      <annotation id="161">
+        <infon key="score">0.9991721</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10976" length="6"/>
+        <text>MTases</text>
+      </annotation>
+      <annotation id="695">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:28:47Z</infon>
+        <location offset="11010" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="806">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:29:16Z</infon>
+        <location offset="11031" length="12"/>
+        <text>hairpin loop</text>
+      </annotation>
+      <annotation id="162">
+        <infon key="score">0.9974844</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:26:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11054" length="7"/>
+        <text>101-133</text>
+      </annotation>
+      <annotation id="804">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:56Z</infon>
+        <location offset="11072" length="2"/>
+        <text>β6</text>
+      </annotation>
+      <annotation id="802">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:43Z</infon>
+        <location offset="11079" length="2"/>
+        <text>β7</text>
+      </annotation>
+      <annotation id="163">
+        <infon key="score">0.9946524</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:36:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11109" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="784">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:27:47Z</infon>
+        <location offset="11120" length="12"/>
+        <text>minor groove</text>
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+      <annotation id="714">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:05Z</infon>
+        <location offset="11187" length="8"/>
+        <text>M.MboIIA</text>
+      </annotation>
+      <annotation id="717">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:22Z</infon>
+        <location offset="11197" length="6"/>
+        <text>M.RsrI</text>
+      </annotation>
+      <annotation id="722">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:47:39Z</infon>
+        <location offset="11208" length="7"/>
+        <text>M.pvuII</text>
+      </annotation>
+      <annotation id="164">
+        <infon key="score">0.7994238</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:30:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11221" length="7"/>
+        <text>missing</text>
+      </annotation>
+      <annotation id="790">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:01Z</infon>
+        <location offset="11229" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="165">
+        <infon key="score">0.99738026</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:26:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11244" length="5"/>
+        <text>33-58</text>
+      </annotation>
+      <annotation id="166">
+        <infon key="score">0.9981224</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:28:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11258" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="707">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:44:17Z</infon>
+        <location offset="11271" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="800">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:26Z</infon>
+        <location offset="11296" length="6"/>
+        <text>loop I</text>
+      </annotation>
+      <annotation id="712">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:44:50Z</infon>
+        <location offset="11306" length="6"/>
+        <text>M.TaqI</text>
+      </annotation>
+      <annotation id="167">
+        <infon key="score">0.99823296</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:29:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11344" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="694">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:26:42Z</infon>
+        <location offset="11354" length="11"/>
+        <text>without DNA</text>
+      </annotation>
+      <annotation id="785">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:01Z</infon>
+        <location offset="11372" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="880">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:29:06Z</infon>
+        <location offset="11391" length="12"/>
+        <text>well ordered</text>
+      </annotation>
+      <annotation id="723">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:48:16Z</infon>
+        <location offset="11410" length="28"/>
+        <text>M.TaqI-DNA complex structure</text>
+      </annotation>
+      <annotation id="672">
+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:29Z</infon>
+        <location offset="11479" length="15"/>
+        <text>DNA methylation</text>
+      </annotation>
+      <annotation id="168">
+        <infon key="score">0.65298414</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:02:22Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11522" length="7"/>
+        <text>adenine</text>
+      </annotation>
+      <annotation id="696">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:28:58Z</infon>
+        <location offset="11555" length="3"/>
+        <text>DNA</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">oncotarget-07-40965-g001.jpg</infon>
+      <infon key="id">F1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>11634</offset>
+      <text>Overall structure of M1.HpyAVI</text>
+      <annotation id="169">
+        <infon key="score">0.9969964</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:29:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11642" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="708">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:44:17Z</infon>
+        <location offset="11655" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">oncotarget-07-40965-g001.jpg</infon>
+      <infon key="id">F1</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>11665</offset>
+      <text>A. Free form B. AdoMet-bound form. Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY. Rainbow coloring from blue through green to red indicates the N- to C-terminal position of the residues in the model. The α-helices and β-strands are labelled and numbered according to the commonly numbering rule for the known MTases. The AdoMet molecule is shown in green.</text>
+      <annotation id="170">
+        <infon key="score">0.99928844</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:30:17Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11668" length="4"/>
+        <text>Free</text>
+      </annotation>
+      <annotation id="171">
+        <infon key="score">0.9989874</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11681" length="12"/>
+        <text>AdoMet-bound</text>
+      </annotation>
+      <annotation id="709">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:44:17Z</infon>
+        <location offset="11718" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="172">
+        <infon key="score">0.99276835</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:44Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11742" length="22"/>
+        <text>AdoMet-dependent MTase</text>
+      </annotation>
+      <annotation id="759">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:25:13Z</infon>
+        <location offset="11795" length="7"/>
+        <text>β-sheet</text>
+      </annotation>
+      <annotation id="762">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:25:33Z</infon>
+        <location offset="11818" length="9"/>
+        <text>α-helices</text>
+      </annotation>
+      <annotation id="757">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:24:59Z</infon>
+        <location offset="11829" length="2"/>
+        <text>αA</text>
+      </annotation>
+      <annotation id="767">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:25:43Z</infon>
+        <location offset="11833" length="2"/>
+        <text>αB</text>
+      </annotation>
+      <annotation id="769">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:25:54Z</infon>
+        <location offset="11837" length="2"/>
+        <text>αZ</text>
+      </annotation>
+      <annotation id="755">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:24:28Z</infon>
+        <location offset="11856" length="2"/>
+        <text>αD</text>
+      </annotation>
+      <annotation id="772">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:26:06Z</infon>
+        <location offset="11860" length="2"/>
+        <text>αE</text>
+      </annotation>
+      <annotation id="774">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:26:16Z</infon>
+        <location offset="11864" length="2"/>
+        <text>αC</text>
+      </annotation>
+      <annotation id="173">
+        <infon key="score">0.9992446</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11899" length="6"/>
+        <text>AdoMet</text>
+      </annotation>
+      <annotation id="174">
+        <infon key="score">0.99239594</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:31:27Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11909" length="8"/>
+        <text>bound in</text>
+      </annotation>
+      <annotation id="175">
+        <infon key="score">0.9498381</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:31:30Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11920" length="6"/>
+        <text>cavity</text>
+      </annotation>
+      <annotation id="176">
+        <infon key="score">0.9990746</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11936" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="779">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:27:08Z</infon>
+        <location offset="11968" length="4"/>
+        <text>DPPY</text>
+      </annotation>
+      <annotation id="764">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:25:33Z</infon>
+        <location offset="12096" length="9"/>
+        <text>α-helices</text>
+      </annotation>
+      <annotation id="777">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:26:57Z</infon>
+        <location offset="12110" length="9"/>
+        <text>β-strands</text>
+      </annotation>
+      <annotation id="177">
+        <infon key="score">0.99922407</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>MTases</text>
+      </annotation>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12213" length="6"/>
+        <text>AdoMet</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>12286</offset>
+      <text>Dimeric state of M1.HpyAVI in crystal and solution</text>
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+      <text>Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state. Increasing number of dimeric DNA MTases, however, has been identified from later studies. For instance, M.DpnII, M.RsrI, M.KpnI, and M.MboIIA have been found as dimers in solution. In addition, several MTases including M.MboIIA, M.RsrI and TTH0409 form tightly associated dimers in crystal structures. Nonetheless, some DNA MTases such as M.CcrMI and the Bacillus amyloliquefaciens MTase dissociate from dimer into monomer upon DNA-binding.</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">oligomeric_state</infon>
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+        <infon key="updated_at">2023-09-15T15:50:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+      <annotation id="189">
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+        <infon key="type">protein_type</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="12651" length="10"/>
+        <text>DNA MTases</text>
+      </annotation>
+      <annotation id="190">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:49:40Z</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12735" length="6"/>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:49:49Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12743" length="6"/>
+        <text>M.KpnI</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:05Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12755" length="8"/>
+        <text>M.MboIIA</text>
+      </annotation>
+      <annotation id="194">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:49:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12783" length="6"/>
+        <text>dimers</text>
+      </annotation>
+      <annotation id="195">
+        <infon key="score">0.9991013</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12824" length="6"/>
+        <text>MTases</text>
+      </annotation>
+      <annotation id="196">
+        <infon key="score">0.98035747</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:05Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12841" length="8"/>
+        <text>M.MboIIA</text>
+      </annotation>
+      <annotation id="197">
+        <infon key="score">0.88078374</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12851" length="6"/>
+        <text>M.RsrI</text>
+      </annotation>
+      <annotation id="198">
+        <infon key="score">0.9984231</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:27:51Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12862" length="7"/>
+        <text>TTH0409</text>
+      </annotation>
+      <annotation id="199">
+        <infon key="score">0.998779</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:49:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12894" length="6"/>
+        <text>dimers</text>
+      </annotation>
+      <annotation id="200">
+        <infon key="score">0.9989133</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:50:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12904" length="18"/>
+        <text>crystal structures</text>
+      </annotation>
+      <annotation id="201">
+        <infon key="score">0.9965406</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12942" length="10"/>
+        <text>DNA MTases</text>
+      </annotation>
+      <annotation id="202">
+        <infon key="score">0.9439273</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:50:11Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12961" length="7"/>
+        <text>M.CcrMI</text>
+      </annotation>
+      <annotation id="203">
+        <infon key="score">0.99796015</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:50:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12977" length="26"/>
+        <text>Bacillus amyloliquefaciens</text>
+      </annotation>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:25:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="13004" length="5"/>
+        <text>MTase</text>
+      </annotation>
+      <annotation id="205">
+        <infon key="score">0.9988293</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:49:53Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13026" length="5"/>
+        <text>dimer</text>
+      </annotation>
+      <annotation id="206">
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:49:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13037" length="7"/>
+        <text>monomer</text>
+      </annotation>
+      <annotation id="207">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:36:57Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="13050" length="3"/>
+        <text>DNA</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>13063</offset>
+      <text>According to the arrangement of the three conserved domains, M1.HpyAVI belongs to the β-subgroup, in which a conserved motif NXXTX9−11AXRXFSXXHX4WX6−9 YXFXLX3RX9−26NPX1−6NVWX29−34A has been identified at the dimerization interface in crystal structures. Most of conserved amino acids within that motif are present in the sequence of M1.HpyAVI (Figure 2A), implying dimerization of this protein. In agreement, a dimer of M1.HpyAVI was observed in our crystal structures with the two monomers related by a two-fold axis (Figure 2B and 2C). An area of ~1900 Å2 was buried at the dimeric interface, taking up ca 17% of the total area. The dimeric architecture was greatly stabilized by hydrogen bonds and salt bridges formed among residues R86, D93 and E96. In addition, comparison of the dimer structure of M1.HpyAVI with some other β-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3).</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:14Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:50:56Z</infon>
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+        <text>β-subgroup</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13172" length="9"/>
+        <text>conserved</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:26:35Z</infon>
+        <location offset="13188" length="55"/>
+        <text>NXXTX9−11AXRXFSXXHX4WX6−9 YXFXLX3RX9−26NPX1−6NVWX29−34A</text>
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+        <infon key="identifier">SO:</infon>
+        <location offset="13271" length="22"/>
+        <text>dimerization interface</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:29:28Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>crystal structures</text>
+      </annotation>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13325" length="9"/>
+        <text>conserved</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="13396" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:42:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>dimerization</text>
+      </annotation>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:49:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="13474" length="5"/>
+        <text>dimer</text>
+      </annotation>
+      <annotation id="640">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="13483" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
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+        <infon key="identifier">PR:</infon>
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+        <text>M1.HpyAVI</text>
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+        <infon key="type">oligomeric_state</infon>
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+        <infon key="updated_at">2023-09-15T15:49:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
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+      <text>M1.HpyAVI exists as dimer in crystal and solution</text>
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+        <infon key="updated_at">2023-09-15T15:49:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:31:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14254" length="14"/>
+        <text>cofactor-bound</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="14269" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:10:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>monomers</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:42:13Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
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+        <location offset="14410" length="9"/>
+        <text>M1.HpyAVI</text>
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+        <infon key="type">oligomeric_state</infon>
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+        <infon key="updated_at">2023-09-15T15:49:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14431" length="5"/>
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+        <infon key="updated_at">2023-09-18T08:42:17Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="14450" length="4"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:43:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>Elution profiles</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="14613" length="9"/>
+        <text>M1.HpyAVI</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>14635</offset>
+      <text>To probe the oligomeric form of M1.HpyAVI in solution, different concentrations of purified enzyme was loaded onto a Superdex 75 10/300 column. The protein was eluted at ~10 ml regardless of the protein concentrations, corresponding to a dimeric molecular mass of 54 kDa (Figure 2D).</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="14667" length="9"/>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:50:01Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="14873" length="7"/>
+        <text>dimeric</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:42:55Z</infon>
+        <location offset="14881" length="14"/>
+        <text>molecular mass</text>
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+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
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+      <text>Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other β-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution. This variance might be caused by an addition of 100 mM arginine before cell lysis to keep protein solubility and also by later replacement of arginine with 30% glycerol by dialysis. These treatments probably changed protein conformation somehow and also the oligomeric state.</text>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="updated_at">2023-09-18T09:26:04Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15013" length="14"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:43:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="15089" length="24"/>
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+        <infon key="updated_at">2023-09-18T08:43:18Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:03:15Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <text>gel-filtration chromatography</text>
+      </annotation>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
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+      </annotation>
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+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:43:31Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="15205" length="9"/>
+        <text>monomeric</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:37:03Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15289" length="8"/>
+        <text>arginine</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:44:00Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <text>arginine</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
+        <location offset="15394" length="8"/>
+        <text>glycerol</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>15513</offset>
+      <text>Structure comparisons</text>
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+        <infon key="score">0.9956933</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <location offset="15513" length="21"/>
+        <text>Structure comparisons</text>
+      </annotation>
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+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>15535</offset>
+      <text>As a β-class N6 adenine MTase, the M1.HpyAVI structure displayed a good similarity with M.MboIIA (PDB ID 1G60) and M.RsrI (PDB ID 1NW7), which are falling into the same subgroup. Superimposition of M1.HpyAVI onto them gave RMSDs of 1.63 Å and 1.9 Å on 168 and 190 Cα atoms, respectively. The most striking structural difference was found to locate on the TRD region (residues 133-163 in M1.HpyAVI) (Figure 3A–3C), where the secondary structures vary among these structures. By comparison with the other two enzymes that possess protruding arms containing several α-helices and/or β-strands, the TRD of M1.HpyAVI is much shorter in length (Figure S1), wrapping more closely around the structural core and lacking apparent secondary structures. Given the proposed role of the TRD for DNA interaction at the major groove, some differences of DNA recognition mode can be expected. Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.</text>
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+        <infon key="updated_at">2023-09-18T08:25:33Z</infon>
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+        <infon key="updated_at">2023-09-18T09:35:00Z</infon>
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+        <text>β4</text>
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+        <infon key="type">evidence</infon>
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+        <infon key="updated_at">2023-09-18T09:29:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">evidence</infon>
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+        <infon key="updated_at">2023-09-18T09:29:51Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16573" length="10"/>
+        <text>structures</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="type">protein</infon>
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+        <infon key="identifier">PR:</infon>
+        <location offset="16656" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
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+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="16763" length="9"/>
+        <text>H. pylori</text>
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+      <annotation id="290">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:31:09Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16785" length="8"/>
+        <text>flexible</text>
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+    <passage>
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+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>16816</offset>
+      <text>Structural comparisons between M1.HpyAVI and other DNA MTases</text>
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+        <infon key="type">experimental_method</infon>
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+        <text>Structural comparisons</text>
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+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="16847" length="9"/>
+        <text>M1.HpyAVI</text>
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+      <annotation id="292">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16867" length="10"/>
+        <text>DNA MTases</text>
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+    <passage>
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+      <infon key="id">F3</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>16878</offset>
+      <text>A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.</text>
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+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="16881" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
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+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:05Z</infon>
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+        <text>M.MboIIA</text>
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+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:22Z</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:21:05Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <text>TTHA0409</text>
+      </annotation>
+      <annotation id="294">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:21:39Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16932" length="4"/>
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+      <annotation id="679">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:01:22Z</infon>
+        <location offset="16941" length="6"/>
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+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="16949" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="881">
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+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:32:38Z</infon>
+        <location offset="16976" length="13"/>
+        <text>long disorder</text>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:23:59Z</infon>
+        <location offset="16990" length="3"/>
+        <text>TRD</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:31:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17020" length="14"/>
+        <text>structure-rich</text>
+      </annotation>
+      <annotation id="738">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:23:59Z</infon>
+        <location offset="17035" length="3"/>
+        <text>TRD</text>
+      </annotation>
+      <annotation id="296">
+        <infon key="score">0.9921058</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:05Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17042" length="8"/>
+        <text>M.MboIIA</text>
+      </annotation>
+      <annotation id="719">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:22Z</infon>
+        <location offset="17052" length="6"/>
+        <text>M.RsrI</text>
+      </annotation>
+      <annotation id="297">
+        <infon key="score">0.99877506</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:21:05Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17063" length="8"/>
+        <text>TTHA0409</text>
+      </annotation>
+      <annotation id="834">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:46:25Z</infon>
+        <location offset="17086" length="18"/>
+        <text>DNA-binding domain</text>
+      </annotation>
+      <annotation id="298">
+        <infon key="score">0.99922466</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:21:39Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17108" length="4"/>
+        <text>DpnM</text>
+      </annotation>
+      <annotation id="299">
+        <infon key="score">0.9894016</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:01:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17117" length="6"/>
+        <text>M.TaqI</text>
+      </annotation>
+      <annotation id="739">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:23:59Z</infon>
+        <location offset="17156" length="3"/>
+        <text>TRD</text>
+      </annotation>
+      <annotation id="646">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="17163" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="300">
+        <infon key="score">0.97637516</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:05Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17174" length="8"/>
+        <text>M.MboIIA</text>
+      </annotation>
+      <annotation id="301">
+        <infon key="score">0.84092015</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17184" length="6"/>
+        <text>M.RsrI</text>
+      </annotation>
+      <annotation id="302">
+        <infon key="score">0.9985129</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:21:05Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17195" length="8"/>
+        <text>TTHA0409</text>
+      </annotation>
+      <annotation id="751">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:24:17Z</infon>
+        <location offset="17234" length="2"/>
+        <text>β4</text>
+      </annotation>
+      <annotation id="756">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:24:28Z</infon>
+        <location offset="17241" length="2"/>
+        <text>αD</text>
+      </annotation>
+      <annotation id="303">
+        <infon key="score">0.99199146</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:05Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17247" length="8"/>
+        <text>M.MboIIA</text>
+      </annotation>
+      <annotation id="304">
+        <infon key="score">0.98690724</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17260" length="6"/>
+        <text>M.RsrI</text>
+      </annotation>
+      <annotation id="835">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:46:25Z</infon>
+        <location offset="17280" length="18"/>
+        <text>DNA-binding domain</text>
+      </annotation>
+      <annotation id="305">
+        <infon key="score">0.9993131</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:21:39Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17302" length="4"/>
+        <text>DpnM</text>
+      </annotation>
+      <annotation id="830">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:36:35Z</infon>
+        <location offset="17326" length="17"/>
+        <text>C-terminal domain</text>
+      </annotation>
+      <annotation id="306">
+        <infon key="score">0.99063236</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:01:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17347" length="6"/>
+        <text>M.TaqI</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>17375</offset>
+      <text>N6-adenine and N4-cytosine MTases, in particular, are closely related by sharing common structural features. Structural comparison between M1.HpyAVI and a putative β-class N4 cytosine MTase named TTHA0409 (PDB ID 2ZIF) showed a good similarity as well, giving an RMSD of 1.73 Å on 164 Cα atoms (Figure 3D). Exactly like the above comparison, the most significant difference exists in the TRD, where the structures vary in terms of length and presence of α-helices (Figure S1).</text>
+      <annotation id="730">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:23:01Z</infon>
+        <location offset="17375" length="33"/>
+        <text>N6-adenine and N4-cytosine MTases</text>
+      </annotation>
+      <annotation id="307">
+        <infon key="score">0.9988351</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:03:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17484" length="21"/>
+        <text>Structural comparison</text>
+      </annotation>
+      <annotation id="308">
+        <infon key="score">0.9828641</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17514" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="309">
+        <infon key="score">0.99869984</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:20:39Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17539" length="25"/>
+        <text>β-class N4 cytosine MTase</text>
+      </annotation>
+      <annotation id="310">
+        <infon key="score">0.9993332</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:21:04Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17571" length="8"/>
+        <text>TTHA0409</text>
+      </annotation>
+      <annotation id="311">
+        <infon key="score">0.998705</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:20:45Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17638" length="4"/>
+        <text>RMSD</text>
+      </annotation>
+      <annotation id="740">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:23:59Z</infon>
+        <location offset="17763" length="3"/>
+        <text>TRD</text>
+      </annotation>
+      <annotation id="312">
+        <infon key="score">0.9986004</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:20:49Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17778" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="766">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:25:33Z</infon>
+        <location offset="17829" length="9"/>
+        <text>α-helices</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>17863</offset>
+      <text>M1.HpyAVI displayed a considerable structural dissimilarity in comparison with N6-adenine MTases from other subgroups including the α-class DpnM (PDB ID 2DPM) and the γ-class M.TaqI (PDB ID 2ADM). Both comparisons gave RMSDs above 3.0 Å (Figure 3E and 3F). These two enzymes lack a counterpart loop present in the TRD of M1.HpyAVI, but instead rely on an extra domain for DNA binding and sequence recognition.</text>
+      <annotation id="313">
+        <infon key="score">0.71178216</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17863" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="314">
+        <infon key="score">0.9987031</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:05:55Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17942" length="17"/>
+        <text>N6-adenine MTases</text>
+      </annotation>
+      <annotation id="727">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:21:34Z</infon>
+        <location offset="17995" length="7"/>
+        <text>α-class</text>
+      </annotation>
+      <annotation id="315">
+        <infon key="score">0.99924123</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:21:39Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18003" length="4"/>
+        <text>DpnM</text>
+      </annotation>
+      <annotation id="728">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:21:58Z</infon>
+        <location offset="18030" length="7"/>
+        <text>γ-class</text>
+      </annotation>
+      <annotation id="316">
+        <infon key="score">0.992579</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:01:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18038" length="6"/>
+        <text>M.TaqI</text>
+      </annotation>
+      <annotation id="317">
+        <infon key="score">0.9981804</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:45:56Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18082" length="5"/>
+        <text>RMSDs</text>
+      </annotation>
+      <annotation id="318">
+        <infon key="score">0.9785749</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:32:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18138" length="4"/>
+        <text>lack</text>
+      </annotation>
+      <annotation id="729">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:22:41Z</infon>
+        <location offset="18145" length="16"/>
+        <text>counterpart loop</text>
+      </annotation>
+      <annotation id="741">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:23:59Z</infon>
+        <location offset="18177" length="3"/>
+        <text>TRD</text>
+      </annotation>
+      <annotation id="319">
+        <infon key="score">0.9628337</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18184" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="320">
+        <infon key="score">0.60606277</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:37:19Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="18235" length="3"/>
+        <text>DNA</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>18281</offset>
+      <text>Collectively, M1.HpyAVI possesses a long disordered TRD, which is in sharp contrast to the secondary structure-rich TRD in other β-class N6 adenine or N4 cytosine MTases or the extra DNA binding domain present in DNA MTases from other subgroups. This striking difference may be a significant determinant of the wider substrate spectrum of this H. pylori enzyme.</text>
+      <annotation id="321">
+        <infon key="score">0.9905677</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18295" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="322">
+        <infon key="score">0.8527761</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:32:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18317" length="15"/>
+        <text>long disordered</text>
+      </annotation>
+      <annotation id="742">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:23:59Z</infon>
+        <location offset="18333" length="3"/>
+        <text>TRD</text>
+      </annotation>
+      <annotation id="323">
+        <infon key="score">0.84751034</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:32:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18372" length="24"/>
+        <text>secondary structure-rich</text>
+      </annotation>
+      <annotation id="743">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:23:59Z</infon>
+        <location offset="18397" length="3"/>
+        <text>TRD</text>
+      </annotation>
+      <annotation id="731">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:23:19Z</infon>
+        <location offset="18410" length="40"/>
+        <text>β-class N6 adenine or N4 cytosine MTases</text>
+      </annotation>
+      <annotation id="324">
+        <infon key="score">0.99855304</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18494" length="10"/>
+        <text>DNA MTases</text>
+      </annotation>
+      <annotation id="325">
+        <infon key="score">0.99791336</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:04:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18625" length="9"/>
+        <text>H. pylori</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>18646</offset>
+      <text>AdoMet-binding pocket</text>
+      <annotation id="326">
+        <infon key="score">0.99897456</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:47:32Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18646" length="21"/>
+        <text>AdoMet-binding pocket</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>18668</offset>
+      <text>The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases. A hydrogen bond between D29 in the catalytic motif DPPY and the amino group of bound AdoMet is preserved as other MTase structures. Residues D8 and A9 from hydrogen-bonds with N6 and N1 of the purine ring, respectively, and E216 also locates at hydrogen bonding distance with O2′ and O3′ of the ribose. In addition, H168, T200 and S198 contact the terminal carboxyl of AdoMet. Superposition of M1.HpyAVI with the five structures shown in Figure 3 reveals that the orientation of cofactor is rather conserved except for M.TaqI (Figure 4B). The different conformation of the bound cofactor observed in M.TaqI might be attributable to the absence of corresponding residues of the conserved AdoMet-binding motif FXGXG in that structure.</text>
+      <annotation id="327">
+        <infon key="score">0.9991107</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:35:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18672" length="23"/>
+        <text>cofactor binding pocket</text>
+      </annotation>
+      <annotation id="328">
+        <infon key="score">0.9654598</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18699" length="9"/>
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+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:06:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18735" length="3"/>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18747" length="7"/>
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+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:23Z</infon>
+        <infon key="identifier">MESH:</infon>
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+        <infon key="updated_at">2023-09-18T08:48:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18833" length="13"/>
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+        <infon key="type">residue_name_number</infon>
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+        <infon key="updated_at">2023-09-15T14:50:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:29:41Z</infon>
+        <location offset="18866" length="15"/>
+        <text>catalytic motif</text>
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+      <annotation id="781">
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:27:09Z</infon>
+        <location offset="18882" length="4"/>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:33:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18910" length="5"/>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:26:15Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18945" length="5"/>
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+        <infon key="score">0.9978751</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:26:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18951" length="10"/>
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+        <infon key="score">0.9995264</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:48:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18972" length="2"/>
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+        <infon key="score">0.99946505</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:48:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18979" length="2"/>
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+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:47:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18987" length="14"/>
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+        <infon key="score">0.99749845</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:37:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="19024" length="6"/>
+        <text>purine</text>
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+        <infon key="type">residue_name_number</infon>
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+        <infon key="updated_at">2023-09-15T14:50:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19055" length="4"/>
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+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:47:51Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19076" length="16"/>
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+      <annotation id="348">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:37:35Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="19126" length="6"/>
+        <text>ribose</text>
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+        <infon key="score">0.9994992</infon>
+        <infon key="type">residue_name_number</infon>
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+        <infon key="updated_at">2023-09-18T08:48:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19147" length="4"/>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:48:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19153" length="4"/>
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+      <annotation id="351">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:48:51Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19162" length="4"/>
+        <text>S198</text>
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+      <annotation id="352">
+        <infon key="score">0.99901843</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="19200" length="6"/>
+        <text>AdoMet</text>
+      </annotation>
+      <annotation id="353">
+        <infon key="score">0.99861515</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:50:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19208" length="13"/>
+        <text>Superposition</text>
+      </annotation>
+      <annotation id="354">
+        <infon key="score">0.95827633</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="19225" length="9"/>
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+        <infon key="score">0.9965333</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:26:22Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19249" length="10"/>
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+      <annotation id="356">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:26:30Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19322" length="16"/>
+        <text>rather conserved</text>
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+      <annotation id="357">
+        <infon key="score">0.9970794</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:01:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="19350" length="6"/>
+        <text>M.TaqI</text>
+      </annotation>
+      <annotation id="878">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:26:54Z</infon>
+        <location offset="19404" length="5"/>
+        <text>bound</text>
+      </annotation>
+      <annotation id="358">
+        <infon key="score">0.9969217</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:01:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="19431" length="6"/>
+        <text>M.TaqI</text>
+      </annotation>
+      <annotation id="359">
+        <infon key="score">0.9959515</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:26:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19467" length="10"/>
+        <text>absence of</text>
+      </annotation>
+      <annotation id="360">
+        <infon key="score">0.99926144</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19508" length="9"/>
+        <text>conserved</text>
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+      <annotation id="675">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <location offset="19518" length="6"/>
+        <text>AdoMet</text>
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+      <annotation id="836">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:48:19Z</infon>
+        <location offset="19539" length="5"/>
+        <text>FXGXG</text>
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+      <annotation id="361">
+        <infon key="score">0.9978817</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:29:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19553" length="9"/>
+        <text>structure</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">oncotarget-07-40965-g004.jpg</infon>
+      <infon key="id">F4</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>19568</offset>
+      <text>Structural and biochemical analyses define two conserved residues D29 and E216 to be the key sites for AdoMet binding</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:03:29Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19568" length="35"/>
+        <text>Structural and biochemical analyses</text>
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+      <annotation id="363">
+        <infon key="score">0.8775193</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19615" length="9"/>
+        <text>conserved</text>
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+      <annotation id="364">
+        <infon key="score">0.99950695</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19634" length="3"/>
+        <text>D29</text>
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+      <annotation id="365">
+        <infon key="score">0.9995067</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="19642" length="4"/>
+        <text>E216</text>
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+      <annotation id="366">
+        <infon key="score">0.9987552</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="19671" length="6"/>
+        <text>AdoMet</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">oncotarget-07-40965-g004.jpg</infon>
+      <infon key="id">F4</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>19686</offset>
+      <text>A. The cofactor-binding cavity of M1.HpyAVI. Residues (yellow) that form direct hydrogen bonds with AdoMet (green) are indicated, distance of the hydrogen bond is marked. B. Superposition of AdoMet in the structures of M1.HpyAVI (green), DpnM (yellow) and M.TaqI (orange). The AdoMet terminal carboxyl of M.TaqI reveals different orientations. C. Cofactor binding affinity of wt-/mutants M1.HpyAVI proteins analyzed by microscale thermophoresis (MST). The binding affinity was determined between fluorescently labelled M1.HpyAVI protein and unlabeled AdoMet. The bound fraction is shown on the y-axis against the protein concentration. AdoMet (15 nM to 1 mM) was titrated into a fixed concentration of M1.HpyAVI wt/mutant proteins (800 nM). The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated. Measurements were made with 40% LED and 40% laser power at 25°C. D. DNA methyltransferase activity of wide type protein and the mutants is quantified using radioactive assay. [3H]-methyl transferred to duplex DNA containing 5′-GAGG-3′ was quantified by Beckman LS6500 for 10 min, experiments were repeated for three times and data were corrected by subtraction of the background. E. Superposition of M1.HpyAVI (green) with M.MboIIA (cyan) and M.RsrI (magenta). Residues D29 and E216 are conserved through all the DNA MTases mentioned in Figure 3 (not shown in Figure 4).</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:35:31Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="19693" length="23"/>
+        <text>cofactor-binding cavity</text>
+      </annotation>
+      <annotation id="647">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="19720" length="9"/>
+        <text>M1.HpyAVI</text>
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+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:41:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19766" length="14"/>
+        <text>hydrogen bonds</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="19786" length="6"/>
+        <text>AdoMet</text>
+      </annotation>
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+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:48:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19832" length="13"/>
+        <text>hydrogen bond</text>
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+        <infon key="score">0.9977174</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:50:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="19860" length="13"/>
+        <text>Superposition</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <text>titrated</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
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+        <infon key="type">protein_state</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:52:11Z</infon>
+        <location offset="20573" length="13"/>
+        <text>M1.HpyAVI-D8A</text>
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+      <annotation id="842">
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:52:32Z</infon>
+        <location offset="20601" length="14"/>
+        <text>M1.HpyAVI-D29A</text>
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+      <annotation id="843">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:52:53Z</infon>
+        <location offset="20624" length="15"/>
+        <text>M1.HpyAVI-H168A</text>
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+      <annotation id="839">
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:51:07Z</infon>
+        <location offset="20655" length="15"/>
+        <text>M1.HpyAVI-S198A</text>
+      </annotation>
+      <annotation id="840">
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:51:26Z</infon>
+        <location offset="20686" length="15"/>
+        <text>M1.HpyAVI-T200A</text>
+      </annotation>
+      <annotation id="844">
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+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:53:13Z</infon>
+        <location offset="20717" length="15"/>
+        <text>M1.HpyAVI-E216A</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:53:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20853" length="21"/>
+        <text>DNA methyltransferase</text>
+      </annotation>
+      <annotation id="395">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:33:25Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20887" length="9"/>
+        <text>wide type</text>
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+      <annotation id="396">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:49:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="20913" length="7"/>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:53:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="20941" length="17"/>
+        <text>radioactive assay</text>
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+      <annotation id="845">
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:54:19Z</infon>
+        <location offset="20960" length="11"/>
+        <text>[3H]-methyl</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
+        <location offset="20994" length="3"/>
+        <text>DNA</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
+        <location offset="21009" length="10"/>
+        <text>5′-GAGG-3′</text>
+      </annotation>
+      <annotation id="400">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:50:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21168" length="13"/>
+        <text>Superposition</text>
+      </annotation>
+      <annotation id="653">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="21185" length="9"/>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <text>M.MboIIA</text>
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+      <annotation id="402">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:45:23Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <text>M.RsrI</text>
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+      <annotation id="403">
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+        <infon key="type">residue_name_number</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>E216</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21272" length="9"/>
+        <text>conserved</text>
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+      <annotation id="406">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21298" length="10"/>
+        <text>DNA MTases</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>21373</offset>
+      <text>To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay. As shown in Figure 4C, by contrast to the wild type enzyme, most mutants displayed variable reduction of KD value, among them the D29A and E216A mutants displayed no protein-AdoMet affinity at all.</text>
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+        <infon key="updated_at">2023-09-18T08:55:21Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21445" length="14"/>
+        <text>single mutants</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:03:36Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21463" length="9"/>
+        <text>replacing</text>
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+      <annotation id="409">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:48:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21473" length="2"/>
+        <text>D8</text>
+      </annotation>
+      <annotation id="410">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21477" length="3"/>
+        <text>D29</text>
+      </annotation>
+      <annotation id="411">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:48:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21482" length="4"/>
+        <text>H168</text>
+      </annotation>
+      <annotation id="412">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:48:51Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21488" length="4"/>
+        <text>S198</text>
+      </annotation>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:48:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21494" length="4"/>
+        <text>T200</text>
+      </annotation>
+      <annotation id="414">
+        <infon key="score">0.99935776</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21500" length="4"/>
+        <text>E216</text>
+      </annotation>
+      <annotation id="415">
+        <infon key="score">0.9922931</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:59:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="21510" length="7"/>
+        <text>alanine</text>
+      </annotation>
+      <annotation id="416">
+        <infon key="score">0.9984741</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:30:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21541" length="23"/>
+        <text>ligand binding affinity</text>
+      </annotation>
+      <annotation id="417">
+        <infon key="score">0.9987993</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:50:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21571" length="25"/>
+        <text>microscale thermophoresis</text>
+      </annotation>
+      <annotation id="418">
+        <infon key="score">0.9986884</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:50:09Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21598" length="3"/>
+        <text>MST</text>
+      </annotation>
+      <annotation id="419">
+        <infon key="score">0.99891734</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:55:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21652" length="9"/>
+        <text>wild type</text>
+      </annotation>
+      <annotation id="420">
+        <infon key="score">0.8193448</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:49:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21675" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="421">
+        <infon key="score">0.9980161</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:54:45Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21715" length="2"/>
+        <text>KD</text>
+      </annotation>
+      <annotation id="422">
+        <infon key="score">0.9979571</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:55:40Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21740" length="4"/>
+        <text>D29A</text>
+      </annotation>
+      <annotation id="423">
+        <infon key="score">0.99816173</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:55:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21749" length="5"/>
+        <text>E216A</text>
+      </annotation>
+      <annotation id="424">
+        <infon key="score">0.95574015</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:49:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21755" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="425">
+        <infon key="score">0.8913477</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:55:36Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21776" length="23"/>
+        <text>protein-AdoMet affinity</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>21808</offset>
+      <text>The results suggested that the hydrogen bonds formed by D29 and E216 with AdoMet were most crucial interactions for cofactor binding. Mutation of the two residues may directly prevent the methyl transfer reaction of M1.HpyAVI. The importance of D29 is preserved because it belongs to the catalytic active site DPPY, but the residue E216 has not been fully investigated even being a conserved amino acid throughout MTases (Figure 4E). E216 is the last residue of β2, which contacts the two hydroxyls of the ribose of AdoMet. Replacement of this residue by alanine completely abolishes the key hydrogen bonds for AdoMet-binding, and very likely blocks the methyl transfer reaction. To confirm this notion, [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the mutants. As shown in Figure 4D, the result of radiological assay agreed well with the MST measurement. The D29A and E216A mutants showed little or no methyl transfer activity, while other mutants exhibited reduced methyltransferase activity.</text>
+      <annotation id="426">
+        <infon key="score">0.99543846</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:41:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21839" length="14"/>
+        <text>hydrogen bonds</text>
+      </annotation>
+      <annotation id="427">
+        <infon key="score">0.99947375</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21864" length="3"/>
+        <text>D29</text>
+      </annotation>
+      <annotation id="428">
+        <infon key="score">0.9994604</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="21872" length="4"/>
+        <text>E216</text>
+      </annotation>
+      <annotation id="429">
+        <infon key="score">0.9984509</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="21882" length="6"/>
+        <text>AdoMet</text>
+      </annotation>
+      <annotation id="430">
+        <infon key="score">0.99554175</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:55:56Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="21942" length="8"/>
+        <text>Mutation</text>
+      </annotation>
+      <annotation id="853">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:56:49Z</infon>
+        <location offset="21996" length="6"/>
+        <text>methyl</text>
+      </annotation>
+      <annotation id="654">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="22024" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="431">
+        <infon key="score">0.99947006</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22053" length="3"/>
+        <text>D29</text>
+      </annotation>
+      <annotation id="432">
+        <infon key="score">0.93916696</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:56:00Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22096" length="21"/>
+        <text>catalytic active site</text>
+      </annotation>
+      <annotation id="433">
+        <infon key="score">0.99586785</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:27:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22118" length="4"/>
+        <text>DPPY</text>
+      </annotation>
+      <annotation id="434">
+        <infon key="score">0.9994399</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22140" length="4"/>
+        <text>E216</text>
+      </annotation>
+      <annotation id="435">
+        <infon key="score">0.99761105</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22190" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="825">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:35:41Z</infon>
+        <location offset="22200" length="10"/>
+        <text>amino acid</text>
+      </annotation>
+      <annotation id="436">
+        <infon key="score">0.998909</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22222" length="6"/>
+        <text>MTases</text>
+      </annotation>
+      <annotation id="437">
+        <infon key="score">0.9994332</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22242" length="4"/>
+        <text>E216</text>
+      </annotation>
+      <annotation id="846">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:56:11Z</infon>
+        <location offset="22270" length="2"/>
+        <text>β2</text>
+      </annotation>
+      <annotation id="438">
+        <infon key="score">0.51602983</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:37:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="22314" length="6"/>
+        <text>ribose</text>
+      </annotation>
+      <annotation id="439">
+        <infon key="score">0.9988117</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="22324" length="6"/>
+        <text>AdoMet</text>
+      </annotation>
+      <annotation id="440">
+        <infon key="score">0.99830014</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:03:41Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22332" length="11"/>
+        <text>Replacement</text>
+      </annotation>
+      <annotation id="441">
+        <infon key="score">0.9955804</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:59:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22363" length="7"/>
+        <text>alanine</text>
+      </annotation>
+      <annotation id="442">
+        <infon key="score">0.9938001</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:41:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22400" length="14"/>
+        <text>hydrogen bonds</text>
+      </annotation>
+      <annotation id="443">
+        <infon key="score">0.9766973</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="22419" length="6"/>
+        <text>AdoMet</text>
+      </annotation>
+      <annotation id="847">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:56:49Z</infon>
+        <location offset="22462" length="6"/>
+        <text>methyl</text>
+      </annotation>
+      <annotation id="885">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:04:00Z</infon>
+        <location offset="22512" length="29"/>
+        <text>[3H]AdoMet radiological assay</text>
+      </annotation>
+      <annotation id="854">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:56:49Z</infon>
+        <location offset="22570" length="6"/>
+        <text>methyl</text>
+      </annotation>
+      <annotation id="444">
+        <infon key="score">0.87604487</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:49:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22602" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="445">
+        <infon key="score">0.9986948</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:04:05Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22648" length="18"/>
+        <text>radiological assay</text>
+      </annotation>
+      <annotation id="446">
+        <infon key="score">0.9986708</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:50:09Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22688" length="3"/>
+        <text>MST</text>
+      </annotation>
+      <annotation id="447">
+        <infon key="score">0.998423</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:57:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22709" length="4"/>
+        <text>D29A</text>
+      </annotation>
+      <annotation id="448">
+        <infon key="score">0.99846244</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:57:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22718" length="5"/>
+        <text>E216A</text>
+      </annotation>
+      <annotation id="449">
+        <infon key="score">0.98877406</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:49:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22724" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="855">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:56:49Z</infon>
+        <location offset="22752" length="6"/>
+        <text>methyl</text>
+      </annotation>
+      <annotation id="837">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:49:46Z</infon>
+        <location offset="22790" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="450">
+        <infon key="score">0.6297416</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:31:07Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22816" length="17"/>
+        <text>methyltransferase</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>22847</offset>
+      <text>As mentioned previously, FXGXG is a conserved AdoMet-binding motif of DNA MTases. We also made mutants of “FMGSG” to alanine for every amino acid, and found that the F195A mutant was insoluble probably due to decreasing the local hydrophobicity upon this mutation. We subsequently investigated the ligand binding affinity and methyl transfer reaction of the other mutants using MST and a radiological assay. We found that G197 played a crucial role in AdoMet-binding, while mutagenesis of M196 and G199 did not influence cofactor binding and catalytic activity (Figure S2A and B). G197 is a conserved residue throughout the DNA MTases, and replacing by alanine at this site likely change the local conformation of cofactor-binding pocket. Mutagenesis on this glycine residue in M.EcoKI or M.EcoP15I also abolished the AdoMet-binding activity. Although mutational study could not tell the role of F195 in ligand binding due to the insolubility of the F195A mutant, structural analysis suggested the importance of this residue in AdoMet-binding. The phenyl ring of F195 forms a perpendicular π-stacking interaction with the purine ring of AdoMet, which stabilizes the orientation of AdoMet bound in the pocket of M1.HpyAVI (Figure S2C). In a separate scenario, mutagenesis of this residue in M.EcoRV has been proven to play an important role in AdoMet binding.</text>
+      <annotation id="863">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:59:23Z</infon>
+        <location offset="22872" length="5"/>
+        <text>FXGXG</text>
+      </annotation>
+      <annotation id="451">
+        <infon key="score">0.99913895</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22883" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="676">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <location offset="22893" length="6"/>
+        <text>AdoMet</text>
+      </annotation>
+      <annotation id="452">
+        <infon key="score">0.99815035</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="22917" length="10"/>
+        <text>DNA MTases</text>
+      </annotation>
+      <annotation id="453">
+        <infon key="score">0.7042762</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:49:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="22942" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="454">
+        <infon key="score">0.73182875</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:07:45Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="22954" length="5"/>
+        <text>FMGSG</text>
+      </annotation>
+      <annotation id="455">
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+        <infon key="identifier">SO:</infon>
+        <location offset="22964" length="7"/>
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+      <annotation id="826">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:35:41Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:07:56Z</infon>
+        <infon key="identifier">MESH:</infon>
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+      <annotation id="457">
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">residue_name_number</infon>
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+        <infon key="type">residue_name</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="23606" length="7"/>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+      <annotation id="477">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:04:55Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23699" length="16"/>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:58:33Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23743" length="4"/>
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+      <annotation id="479">
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:08:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23797" length="5"/>
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+      <annotation id="480">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:49:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23803" length="6"/>
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+      <annotation id="481">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:05:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23811" length="19"/>
+        <text>structural analysis</text>
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+      <annotation id="482">
+        <infon key="score">0.995095</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="23875" length="6"/>
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+      <annotation id="483">
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:58:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="23910" length="4"/>
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+      <annotation id="484">
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+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:06:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="23937" length="22"/>
+        <text>π-stacking interaction</text>
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+      <annotation id="485">
+        <infon key="score">0.99898654</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="23984" length="6"/>
+        <text>AdoMet</text>
+      </annotation>
+      <annotation id="486">
+        <infon key="score">0.9984652</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="24028" length="6"/>
+        <text>AdoMet</text>
+      </annotation>
+      <annotation id="487">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:58:44Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24035" length="8"/>
+        <text>bound in</text>
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+      <annotation id="488">
+        <infon key="score">0.99786323</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:58:41Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24048" length="6"/>
+        <text>pocket</text>
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+      <annotation id="655">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="24058" length="9"/>
+        <text>M1.HpyAVI</text>
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+      <annotation id="489">
+        <infon key="score">0.9978897</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:58:52Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="24106" length="11"/>
+        <text>mutagenesis</text>
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+      <annotation id="490">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:58:16Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24137" length="7"/>
+        <text>M.EcoRV</text>
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+      <annotation id="491">
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="24190" length="6"/>
+        <text>AdoMet</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>24211</offset>
+      <text>Potential DNA-binding sites</text>
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+        <infon key="score">0.99896115</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="24221" length="17"/>
+        <text>DNA-binding sites</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>24239</offset>
+      <text>The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58). The hairpin loop between β6 and β7 strands that carries a conserved HRRY sequence signature in the middle is proposed to insert into the minor groove of the bound DNA. As aforementioned, the TRD of M1.HpyAVI shows striking difference from the other DNA MTases, and the relaxed specificity of substrate recognition may be at least partially attributable to the disordered TRD.</text>
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+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:35:39Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24252" length="18"/>
+        <text>DNA binding region</text>
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+      <annotation id="494">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24274" length="9"/>
+        <text>M1.HpyAVI</text>
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+      <annotation id="807">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:29:18Z</infon>
+        <location offset="24297" length="12"/>
+        <text>hairpin loop</text>
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+      <annotation id="495">
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+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:06:30Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24319" length="7"/>
+        <text>101-133</text>
+      </annotation>
+      <annotation id="744">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:23:59Z</infon>
+        <location offset="24333" length="3"/>
+        <text>TRD</text>
+      </annotation>
+      <annotation id="496">
+        <infon key="score">0.997704</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:06:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24347" length="7"/>
+        <text>136-166</text>
+      </annotation>
+      <annotation id="497">
+        <infon key="score">0.99897075</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24363" length="15"/>
+        <text>highly flexible</text>
+      </annotation>
+      <annotation id="792">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:01Z</infon>
+        <location offset="24379" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="498">
+        <infon key="score">0.9976094</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:06:36Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24394" length="5"/>
+        <text>33-58</text>
+      </annotation>
+      <annotation id="808">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:29:18Z</infon>
+        <location offset="24406" length="12"/>
+        <text>hairpin loop</text>
+      </annotation>
+      <annotation id="805">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:57Z</infon>
+        <location offset="24427" length="2"/>
+        <text>β6</text>
+      </annotation>
+      <annotation id="803">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:43Z</infon>
+        <location offset="24434" length="2"/>
+        <text>β7</text>
+      </annotation>
+      <annotation id="499">
+        <infon key="score">0.9982666</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:13Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24460" length="9"/>
+        <text>conserved</text>
+      </annotation>
+      <annotation id="864">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:59:48Z</infon>
+        <location offset="24470" length="4"/>
+        <text>HRRY</text>
+      </annotation>
+      <annotation id="500">
+        <infon key="score">0.8395535</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:27:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="24539" length="12"/>
+        <text>minor groove</text>
+      </annotation>
+      <annotation id="501">
+        <infon key="score">0.9990257</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:33:30Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24559" length="5"/>
+        <text>bound</text>
+      </annotation>
+      <annotation id="502">
+        <infon key="score">0.9979765</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:37:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="24565" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="745">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:23:59Z</infon>
+        <location offset="24593" length="3"/>
+        <text>TRD</text>
+      </annotation>
+      <annotation id="503">
+        <infon key="score">0.94398093</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24600" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="504">
+        <infon key="score">0.99851084</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="24651" length="10"/>
+        <text>DNA MTases</text>
+      </annotation>
+      <annotation id="505">
+        <infon key="score">0.99915683</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24762" length="10"/>
+        <text>disordered</text>
+      </annotation>
+      <annotation id="746">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:23:59Z</infon>
+        <location offset="24773" length="3"/>
+        <text>TRD</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>24784</offset>
+      <text>In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either. This loop, however, was largely stabilized upon DNA binding, as observed in the protein-DNA complex structures of M.TaqI (PDB ID 2IBS), M.HhaI (PDB ID 1MHT) and M.HaeIII (PDB ID 1DCT). The well-ordered loop in those structures directly contacts the flipping adenine and forms hydrogen bond with neighboring bases. These observations implied that the corresponding loop in other MTases, e.g. M1.HpyAVI, is likely responsible for reducing sequence recognition specificity and thus plays crucial roles in catalysis.</text>
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+        <infon key="score">0.99875844</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24801" length="15"/>
+        <text>highly flexible</text>
+      </annotation>
+      <annotation id="793">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:01Z</infon>
+        <location offset="24817" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="782">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:27:09Z</infon>
+        <location offset="24848" length="4"/>
+        <text>DPPY</text>
+      </annotation>
+      <annotation id="507">
+        <infon key="score">0.66211873</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24862" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="508">
+        <infon key="score">0.9986974</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:01:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24894" length="16"/>
+        <text>electron density</text>
+      </annotation>
+      <annotation id="813">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:30:32Z</infon>
+        <location offset="24943" length="5"/>
+        <text>loops</text>
+      </annotation>
+      <annotation id="509">
+        <infon key="score">0.99905926</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24956" length="12"/>
+        <text>AdoMet-bound</text>
+      </annotation>
+      <annotation id="510">
+        <infon key="score">0.9975859</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:01:28Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="24969" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="511">
+        <infon key="score">0.9956278</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:47:39Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24983" length="7"/>
+        <text>M.PvuII</text>
+      </annotation>
+      <annotation id="512">
+        <infon key="score">0.9992005</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:21:39Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="24992" length="4"/>
+        <text>DpnM</text>
+      </annotation>
+      <annotation id="513">
+        <infon key="score">0.99534625</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:01:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25000" length="6"/>
+        <text>M.TaqI</text>
+      </annotation>
+      <annotation id="794">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:01Z</infon>
+        <location offset="25040" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="514">
+        <infon key="score">0.9867502</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:37:56Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="25083" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="882">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:33:56Z</infon>
+        <location offset="25115" length="30"/>
+        <text>protein-DNA complex structures</text>
+      </annotation>
+      <annotation id="515">
+        <infon key="score">0.99605507</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:01:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25149" length="6"/>
+        <text>M.TaqI</text>
+      </annotation>
+      <annotation id="516">
+        <infon key="score">0.99500823</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:42Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25171" length="6"/>
+        <text>M.HhaI</text>
+      </annotation>
+      <annotation id="517">
+        <infon key="score">0.996617</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:50Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25196" length="8"/>
+        <text>M.HaeIII</text>
+      </annotation>
+      <annotation id="866">
+        <infon key="type">protein_state</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:01:07Z</infon>
+        <location offset="25224" length="12"/>
+        <text>well-ordered</text>
+      </annotation>
+      <annotation id="795">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:01Z</infon>
+        <location offset="25237" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="518">
+        <infon key="score">0.99599034</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:01:37Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="25251" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="519">
+        <infon key="score">0.7158421</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:01:23Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25293" length="7"/>
+        <text>adenine</text>
+      </annotation>
+      <annotation id="520">
+        <infon key="score">0.9972713</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:48:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="25311" length="13"/>
+        <text>hydrogen bond</text>
+      </annotation>
+      <annotation id="796">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:01Z</infon>
+        <location offset="25399" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="521">
+        <infon key="score">0.9991862</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="25413" length="6"/>
+        <text>MTases</text>
+      </annotation>
+      <annotation id="656">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="25426" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>25548</offset>
+      <text>Key residue for wider spectrum of substrate recognition</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>25604</offset>
+      <text>Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment. Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A). Then, a [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the wide type protein and the mutants. As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.</text>
+      <annotation id="522">
+        <infon key="score">0.6295532</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25637" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="523">
+        <infon key="score">0.9984703</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:27:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="25679" length="16"/>
+        <text>N6 adenine MTase</text>
+      </annotation>
+      <annotation id="524">
+        <infon key="score">0.989359</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:38:21Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25719" length="7"/>
+        <text>adenine</text>
+      </annotation>
+      <annotation id="525">
+        <infon key="score">0.9972758</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:02:21Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="25730" length="10"/>
+        <text>5′-GAGG-3′</text>
+      </annotation>
+      <annotation id="526">
+        <infon key="score">0.9922677</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:02:24Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="25741" length="10"/>
+        <text>5′-GGAG-3′</text>
+      </annotation>
+      <annotation id="527">
+        <infon key="score">0.9940481</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:07:28Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25764" length="8"/>
+        <text>adenines</text>
+      </annotation>
+      <annotation id="528">
+        <infon key="score">0.99715126</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:38:05Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="25776" length="10"/>
+        <text>5′-GAAG-3′</text>
+      </annotation>
+      <annotation id="529">
+        <infon key="score">0.99270916</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:38:31Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25862" length="7"/>
+        <text>adenine</text>
+      </annotation>
+      <annotation id="530">
+        <infon key="score">0.9974624</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:02:26Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="25873" length="10"/>
+        <text>5′-GAGG-3′</text>
+      </annotation>
+      <annotation id="657">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="25899" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="867">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:01:59Z</infon>
+        <location offset="25943" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="658">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="25988" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="531">
+        <infon key="score">0.99622726</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:04:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26025" length="9"/>
+        <text>H. pylori</text>
+      </annotation>
+      <annotation id="532">
+        <infon key="score">0.9988635</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:03:12Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26047" length="27"/>
+        <text>multiple sequence alignment</text>
+      </annotation>
+      <annotation id="533">
+        <infon key="score">0.9983783</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:05:06Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26085" length="19"/>
+        <text>sequence comparison</text>
+      </annotation>
+      <annotation id="534">
+        <infon key="score">0.9984338</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:05:10Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26109" length="19"/>
+        <text>structural analysis</text>
+      </annotation>
+      <annotation id="535">
+        <infon key="score">0.9994925</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26154" length="3"/>
+        <text>P41</text>
+      </annotation>
+      <annotation id="536">
+        <infon key="score">0.99946815</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:03:55Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26159" length="4"/>
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+      </annotation>
+      <annotation id="537">
+        <infon key="score">0.9995011</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:03:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26165" length="4"/>
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+      </annotation>
+      <annotation id="538">
+        <infon key="score">0.99950707</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:04:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26174" length="4"/>
+        <text>T166</text>
+      </annotation>
+      <annotation id="539">
+        <infon key="score">0.9943739</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:05:15Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26197" length="8"/>
+        <text>replaced</text>
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+      <annotation id="540">
+        <infon key="score">0.99705803</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:02:27Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26209" length="6"/>
+        <text>serine</text>
+      </annotation>
+      <annotation id="541">
+        <infon key="score">0.997026</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:02:33Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26217" length="9"/>
+        <text>threonine</text>
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+      <annotation id="542">
+        <infon key="score">0.9970084</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:02:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26228" length="9"/>
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+        <infon key="score">0.9967733</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:02:37Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26242" length="6"/>
+        <text>valine</text>
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+      <annotation id="869">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:03:01Z</infon>
+        <location offset="26284" length="29"/>
+        <text>[3H]AdoMet radiological assay</text>
+      </annotation>
+      <annotation id="857">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:56:49Z</infon>
+        <location offset="26342" length="6"/>
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+      <annotation id="544">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:34:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26374" length="9"/>
+        <text>wide type</text>
+      </annotation>
+      <annotation id="545">
+        <infon key="score">0.98206836</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:49:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26400" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="868">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:02:08Z</infon>
+        <location offset="26450" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="546">
+        <infon key="score">0.99718624</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:02:15Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="26463" length="10"/>
+        <text>5′-GAGG-3′</text>
+      </annotation>
+      <annotation id="547">
+        <infon key="score">0.9764722</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:02:17Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="26477" length="11"/>
+        <text>5′-GAAG-3′,</text>
+      </annotation>
+      <annotation id="548">
+        <infon key="score">0.9746132</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:49:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26497" length="7"/>
+        <text>mutants</text>
+      </annotation>
+      <annotation id="858">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:56:49Z</infon>
+        <location offset="26538" length="6"/>
+        <text>methyl</text>
+      </annotation>
+      <annotation id="549">
+        <infon key="score">0.9992687</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:49:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26579" length="2"/>
+        <text>wt</text>
+      </annotation>
+      <annotation id="659">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="26582" length="9"/>
+        <text>M1.HpyAVI</text>
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+      <annotation id="550">
+        <infon key="score">0.9540184</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:02:19Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="26631" length="11"/>
+        <text>5′-GGAG-3′,</text>
+      </annotation>
+      <annotation id="859">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:56:49Z</infon>
+        <location offset="26647" length="6"/>
+        <text>methyl</text>
+      </annotation>
+      <annotation id="551">
+        <infon key="score">0.99837804</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:03:42Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26679" length="4"/>
+        <text>P41S</text>
+      </annotation>
+      <annotation id="552">
+        <infon key="score">0.99891484</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:49:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26684" length="6"/>
+        <text>mutant</text>
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+      <annotation id="553">
+        <infon key="score">0.9990494</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:55:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="26733" length="9"/>
+        <text>wild type</text>
+      </annotation>
+      <annotation id="660">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="26743" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">oncotarget-07-40965-g005.jpg</infon>
+      <infon key="id">F5</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>26782</offset>
+      <text>Sequence alignment, structural analysis and radioactive methyl transfer activity define the key residue for wider substrate specificity of M1.HpyAVI</text>
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+        <infon key="score">0.99885774</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:45:06Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26782" length="18"/>
+        <text>Sequence alignment</text>
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+      <annotation id="555">
+        <infon key="score">0.99821293</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:04:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26802" length="19"/>
+        <text>structural analysis</text>
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+      <annotation id="556">
+        <infon key="score">0.97248703</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:04:22Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26826" length="36"/>
+        <text>radioactive methyl transfer activity</text>
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+      <annotation id="661">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="26921" length="9"/>
+        <text>M1.HpyAVI</text>
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+    </passage>
+    <passage>
+      <infon key="file">oncotarget-07-40965-g005.jpg</infon>
+      <infon key="id">F5</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
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+      <text>A. Sequence alignment of M1.HpyAVI from 50 H. pylori strains including 26695 revealed several variant residues. Residues P41, N111, K165 and T166 of M1.HpyAVI from strain 26695 were chosen based on structural analysis and sequence alignment (shown in red arrow). Amino-acid conservation is depicted using WebLogo (Crooks et al, 2004). B., C., D. Methyl transfer reactions were performed using wt-M1.HpyAVI, M1.HpyAVI-P41S, M1.HpyAVI-N111T, and M1.HpyAVI-K165R T166V, respectively. Radioactivity incorporated into the duplex DNA containing 5′-GAGG-3′, 5′-GAAG-3′ or 5′-GGAG-3′ was quantified by Beckman LS6500 for 10 min. The experiments were repeated for three times and data were corrected by subtraction of the background.</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:45:06Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26934" length="18"/>
+        <text>Sequence alignment</text>
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+      <annotation id="662">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="26956" length="9"/>
+        <text>M1.HpyAVI</text>
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+      <annotation id="558">
+        <infon key="score">0.997293</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:04:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26974" length="9"/>
+        <text>H. pylori</text>
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+      <annotation id="559">
+        <infon key="score">0.99947685</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27052" length="3"/>
+        <text>P41</text>
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+      <annotation id="560">
+        <infon key="score">0.99948114</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:03:56Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27057" length="4"/>
+        <text>N111</text>
+      </annotation>
+      <annotation id="561">
+        <infon key="score">0.99947375</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:03:59Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27063" length="4"/>
+        <text>K165</text>
+      </annotation>
+      <annotation id="562">
+        <infon key="score">0.9994906</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:04:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27072" length="4"/>
+        <text>T166</text>
+      </annotation>
+      <annotation id="663">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="27080" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="563">
+        <infon key="score">0.39663774</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:04:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27102" length="5"/>
+        <text>26695</text>
+      </annotation>
+      <annotation id="564">
+        <infon key="score">0.99588466</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:04:56Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27129" length="19"/>
+        <text>structural analysis</text>
+      </annotation>
+      <annotation id="565">
+        <infon key="score">0.9985169</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:45:06Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27153" length="18"/>
+        <text>sequence alignment</text>
+      </annotation>
+      <annotation id="566">
+        <infon key="score">0.8075491</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:07:03Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27236" length="7"/>
+        <text>WebLogo</text>
+      </annotation>
+      <annotation id="860">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:56:49Z</infon>
+        <location offset="27277" length="6"/>
+        <text>Methyl</text>
+      </annotation>
+      <annotation id="567">
+        <infon key="score">0.998982</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:49:41Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27324" length="2"/>
+        <text>wt</text>
+      </annotation>
+      <annotation id="664">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="27327" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="870">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:05:35Z</infon>
+        <location offset="27338" length="14"/>
+        <text>M1.HpyAVI-P41S</text>
+      </annotation>
+      <annotation id="871">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:05:53Z</infon>
+        <location offset="27354" length="15"/>
+        <text>M1.HpyAVI-N111T</text>
+      </annotation>
+      <annotation id="873">
+        <infon key="type">mutant</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:06:47Z</infon>
+        <location offset="27375" length="21"/>
+        <text>M1.HpyAVI-K165R T166V</text>
+      </annotation>
+      <annotation id="568">
+        <infon key="score">0.9894895</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:38:38Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="27455" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="872">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:06:14Z</infon>
+        <location offset="27470" length="10"/>
+        <text>5′-GAGG-3′</text>
+      </annotation>
+      <annotation id="569">
+        <infon key="score">0.7406915</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:06:17Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="27482" length="10"/>
+        <text>5′-GAAG-3′</text>
+      </annotation>
+      <annotation id="570">
+        <infon key="score">0.8041685</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:06:19Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="27496" length="10"/>
+        <text>5′-GGAG-3′</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>27668</offset>
+      <text>Our experimental data identified P41 as a key residue determining the recognition of GGAG of M1.HpyAVI. This amino acid locates in the highly flexible loop between residues 33 and 58, which is involved in DNA binding and substrate recognition as shown above. Replacement by serine at this position definitely changes the local conformation and hydrophobicity, and probably some structural properties of the whole loop, which may in turn result in reduced specificity for sequence recognition of the enzyme from strain 26695.</text>
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+        <infon key="score">0.9995059</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27701" length="3"/>
+        <text>P41</text>
+      </annotation>
+      <annotation id="572">
+        <infon key="score">0.9565232</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:07:25Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27753" length="4"/>
+        <text>GGAG</text>
+      </annotation>
+      <annotation id="665">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="27761" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="573">
+        <infon key="score">0.998413</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27803" length="15"/>
+        <text>highly flexible</text>
+      </annotation>
+      <annotation id="797">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:01Z</infon>
+        <location offset="27819" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="574">
+        <infon key="score">0.95510674</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:06:42Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="27841" length="9"/>
+        <text>33 and 58</text>
+      </annotation>
+      <annotation id="874">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:07:49Z</infon>
+        <location offset="27873" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="575">
+        <infon key="score">0.99823356</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:08:14Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="27927" length="11"/>
+        <text>Replacement</text>
+      </annotation>
+      <annotation id="576">
+        <infon key="score">0.99676514</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:08:08Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="27942" length="6"/>
+        <text>serine</text>
+      </annotation>
+      <annotation id="798">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:01Z</infon>
+        <location offset="28081" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="577">
+        <infon key="score">0.5137013</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:08:04Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="28186" length="5"/>
+        <text>26695</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">title_1</infon>
+      <offset>28193</offset>
+      <text>DISCUSSION</text>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>28204</offset>
+      <text>Although the DNA-bound structure of previous investigation on a γ-class N6-adenine MTase revealed that the target adenine was rotated out of DNA helix, details of the methyl transfer process were still unclear. Additionally, recent studies reported the importance of N6-methyladenine in some eukaryotic species, but until now there has not been any N6-adenine MTases being identified in eukaryotes. Biochemical and structural characterization of M1.HpyAVI provides a new model for uncovering the methyl transfer mechanism and for investigating the N6-methyladenine in eukaryotes.</text>
+      <annotation id="578">
+        <infon key="score">0.99879885</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:08:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28217" length="9"/>
+        <text>DNA-bound</text>
+      </annotation>
+      <annotation id="579">
+        <infon key="score">0.9980544</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:30:25Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28227" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="580">
+        <infon key="score">0.9987933</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:08:39Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="28268" length="24"/>
+        <text>γ-class N6-adenine MTase</text>
+      </annotation>
+      <annotation id="581">
+        <infon key="score">0.69096255</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:38:49Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28318" length="7"/>
+        <text>adenine</text>
+      </annotation>
+      <annotation id="582">
+        <infon key="score">0.9610856</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:38:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="28345" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="861">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:56:49Z</infon>
+        <location offset="28371" length="6"/>
+        <text>methyl</text>
+      </annotation>
+      <annotation id="583">
+        <infon key="score">0.9975112</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:55:25Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="28471" length="16"/>
+        <text>N6-methyladenine</text>
+      </annotation>
+      <annotation id="584">
+        <infon key="score">0.99863404</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:27:44Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28496" length="10"/>
+        <text>eukaryotic</text>
+      </annotation>
+      <annotation id="585">
+        <infon key="score">0.9988544</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:05:55Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="28553" length="17"/>
+        <text>N6-adenine MTases</text>
+      </annotation>
+      <annotation id="586">
+        <infon key="score">0.99871874</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28591" length="10"/>
+        <text>eukaryotes</text>
+      </annotation>
+      <annotation id="587">
+        <infon key="score">0.99865556</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:08:43Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="28603" length="43"/>
+        <text>Biochemical and structural characterization</text>
+      </annotation>
+      <annotation id="588">
+        <infon key="score">0.9822526</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="28650" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="862">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:56:49Z</infon>
+        <location offset="28700" length="6"/>
+        <text>methyl</text>
+      </annotation>
+      <annotation id="589">
+        <infon key="score">0.99716395</infon>
+        <infon key="type">ptm</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:55:25Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="28752" length="16"/>
+        <text>N6-methyladenine</text>
+      </annotation>
+      <annotation id="590">
+        <infon key="score">0.9985537</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28772" length="10"/>
+        <text>eukaryotes</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>28788</offset>
+      <text>Oligomeric state of DNA MTases was long accepted as monomer, but our study indicated here that M1.HpyAVI exists as a dimer both in crystal and solution. Interestingly, some other β-class DNA exocyclic MTases showed similar oligomeric state in crystal and in solution, indicating that dimer may be the functional state shared by a subgroup of DNA MTases.</text>
+      <annotation id="591">
+        <infon key="score">0.998248</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="28808" length="10"/>
+        <text>DNA MTases</text>
+      </annotation>
+      <annotation id="592">
+        <infon key="score">0.9987644</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:49:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28840" length="7"/>
+        <text>monomer</text>
+      </annotation>
+      <annotation id="666">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="28883" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="593">
+        <infon key="score">0.998727</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:49:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28905" length="5"/>
+        <text>dimer</text>
+      </annotation>
+      <annotation id="594">
+        <infon key="score">0.9870143</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:09:24Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="28919" length="7"/>
+        <text>crystal</text>
+      </annotation>
+      <annotation id="595">
+        <infon key="score">0.99645835</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:09:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="28967" length="28"/>
+        <text>β-class DNA exocyclic MTases</text>
+      </annotation>
+      <annotation id="596">
+        <infon key="score">0.9813897</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:09:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29031" length="7"/>
+        <text>crystal</text>
+      </annotation>
+      <annotation id="597">
+        <infon key="score">0.9987569</infon>
+        <infon key="type">oligomeric_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:49:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29072" length="5"/>
+        <text>dimer</text>
+      </annotation>
+      <annotation id="598">
+        <infon key="score">0.9984802</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:51:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="29130" length="10"/>
+        <text>DNA MTases</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>29145</offset>
+      <text>The highly flexible region (residues 33-58) and TRD (residues 133-163) of M1.HpyAVI are supposed to interact with DNA at minor and major grooves, respectively. These two structural characteristics may account for the substrate promiscuity of this enzyme. And residue P41 might be a key residue partially determining the substrate spectrum of M1.HpyAVI.</text>
+      <annotation id="599">
+        <infon key="score">0.9987935</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:19Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29149" length="15"/>
+        <text>highly flexible</text>
+      </annotation>
+      <annotation id="600">
+        <infon key="score">0.99759173</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:09:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29182" length="5"/>
+        <text>33-58</text>
+      </annotation>
+      <annotation id="747">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:23:59Z</infon>
+        <location offset="29193" length="3"/>
+        <text>TRD</text>
+      </annotation>
+      <annotation id="601">
+        <infon key="score">0.9977372</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:09:37Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29207" length="7"/>
+        <text>133-163</text>
+      </annotation>
+      <annotation id="602">
+        <infon key="score">0.76505375</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29219" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="603">
+        <infon key="score">0.9985341</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:38:58Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="29259" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="875">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:09:53Z</infon>
+        <location offset="29266" length="23"/>
+        <text>minor and major grooves</text>
+      </annotation>
+      <annotation id="604">
+        <infon key="score">0.9994406</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:16Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29412" length="3"/>
+        <text>P41</text>
+      </annotation>
+      <annotation id="667">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="29487" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>29498</offset>
+      <text>The missing loop between residues 33 and 58 may need DNA binding so as to form a stable conformation, which is similar to the condition of M.TaqI. Crystallization of M1.HpyAVI-DNA complex warrants future investigations, with the purpose of revealing the mechanism behind the wider substrate specificity of this enzyme.</text>
+      <annotation id="605">
+        <infon key="score">0.66722006</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:34:09Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29502" length="7"/>
+        <text>missing</text>
+      </annotation>
+      <annotation id="799">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:28:02Z</infon>
+        <location offset="29510" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="606">
+        <infon key="score">0.71347064</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:06:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29532" length="9"/>
+        <text>33 and 58</text>
+      </annotation>
+      <annotation id="607">
+        <infon key="score">0.9982004</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:39:02Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="29551" length="3"/>
+        <text>DNA</text>
+      </annotation>
+      <annotation id="608">
+        <infon key="score">0.9979321</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:34:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29579" length="6"/>
+        <text>stable</text>
+      </annotation>
+      <annotation id="609">
+        <infon key="score">0.99098516</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:01:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29637" length="6"/>
+        <text>M.TaqI</text>
+      </annotation>
+      <annotation id="610">
+        <infon key="score">0.9987587</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:10:23Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="29645" length="15"/>
+        <text>Crystallization</text>
+      </annotation>
+      <annotation id="611">
+        <infon key="score">0.96516407</infon>
+        <infon key="type">complex_assembly</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:10:16Z</infon>
+        <infon key="identifier">GO:</infon>
+        <location offset="29664" length="13"/>
+        <text>M1.HpyAVI-DNA</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>29817</offset>
+      <text>DNA methylation plays an important role in bacterial pathogenicity. DNA adenine methylation was known to regulate the expression of some virulence genes in bacteria including H.pylori. Inhibitors of DNA adenine methylation may have a broad antimicrobial action by targeting DNA adenine methyltransferase. As an important biological modification, DNA methylation directly influences bacterial survival. Knockout of M1.HpyAVI largely prevents the growth of H. pylori. Importantly, H. pylori is involved in 90% of all gastric malignancies. Appropriate antibiotic regimens could successfully cure gastric diseases caused by H.pylori infection. However, eradication of H. pylori infection remains a big challenge for the significantly increasing prevalence of its resistance to antibiotics. The development of new drugs targeting adenine MTases such as M1.HpyAVI offers a new opportunity for inhibition of H. pylori infection. Residues that play crucial roles for catalytic activity like D29 or E216 may influence the H.pylori survival. Small molecules targeting these highly conserved residues are likely to emerge less drug resistance.</text>
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+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:29Z</infon>
+        <location offset="29817" length="15"/>
+        <text>DNA methylation</text>
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+        <infon key="score">0.9985331</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:53:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>bacterial</text>
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+      <annotation id="877">
+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:11:03Z</infon>
+        <location offset="29885" length="23"/>
+        <text>DNA adenine methylation</text>
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+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:48:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29973" length="8"/>
+        <text>bacteria</text>
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+      <annotation id="614">
+        <infon key="score">0.9971612</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:25:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="29992" length="8"/>
+        <text>H.pylori</text>
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+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:11:02Z</infon>
+        <location offset="30016" length="23"/>
+        <text>DNA adenine methylation</text>
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:37:54Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30091" length="29"/>
+        <text>DNA adenine methyltransferase</text>
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+      <annotation id="674">
+        <infon key="type">ptm</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:29Z</infon>
+        <location offset="30163" length="15"/>
+        <text>DNA methylation</text>
+      </annotation>
+      <annotation id="616">
+        <infon key="score">0.99820757</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:53:03Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="30199" length="9"/>
+        <text>bacterial</text>
+      </annotation>
+      <annotation id="617">
+        <infon key="score">0.7532001</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T10:05:20Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30219" length="11"/>
+        <text>Knockout of</text>
+      </annotation>
+      <annotation id="668">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="30231" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="618">
+        <infon key="score">0.9971716</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:04:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30272" length="9"/>
+        <text>H. pylori</text>
+      </annotation>
+      <annotation id="619">
+        <infon key="score">0.99724466</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:04:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30296" length="9"/>
+        <text>H. pylori</text>
+      </annotation>
+      <annotation id="620">
+        <infon key="score">0.9974534</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:25:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30437" length="8"/>
+        <text>H.pylori</text>
+      </annotation>
+      <annotation id="621">
+        <infon key="score">0.9974273</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:04:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30481" length="9"/>
+        <text>H. pylori</text>
+      </annotation>
+      <annotation id="622">
+        <infon key="score">0.99812746</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:58:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30642" length="14"/>
+        <text>adenine MTases</text>
+      </annotation>
+      <annotation id="669">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <location offset="30665" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="623">
+        <infon key="score">0.9971631</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T15:04:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30718" length="9"/>
+        <text>H. pylori</text>
+      </annotation>
+      <annotation id="624">
+        <infon key="score">0.9993161</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="30800" length="3"/>
+        <text>D29</text>
+      </annotation>
+      <annotation id="625">
+        <infon key="score">0.99932635</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="30807" length="4"/>
+        <text>E216</text>
+      </annotation>
+      <annotation id="626">
+        <infon key="score">0.9974186</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:25:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30830" length="8"/>
+        <text>H.pylori</text>
+      </annotation>
+      <annotation id="627">
+        <infon key="score">0.9989734</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:11:57Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="30881" length="16"/>
+        <text>highly conserved</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>30950</offset>
+      <text>In summary, the structure of M1.HpyAVI is featured with a disordered TRD and a key residue P41that located in the putative DNA binding region that may associate with the wider substrate specificity. Residues D29 and E216 were identified to play a crucial role in cofactor binding. As the first crystal structure of N6-adenine MTase in H.pylori, this model may shed light on design of new antibiotics to interfere the growth and pathogenesis of H.pylori in human.</text>
+      <annotation id="628">
+        <infon key="score">0.9974125</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:12:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="30966" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="629">
+        <infon key="score">0.8668125</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:49:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30979" length="9"/>
+        <text>M1.HpyAVI</text>
+      </annotation>
+      <annotation id="630">
+        <infon key="score">0.9987343</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:00:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="31008" length="10"/>
+        <text>disordered</text>
+      </annotation>
+      <annotation id="748">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T08:23:59Z</infon>
+        <location offset="31019" length="3"/>
+        <text>TRD</text>
+      </annotation>
+      <annotation id="884">
+        <infon key="type">residue_name_number</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:36:37Z</infon>
+        <location offset="31041" length="3"/>
+        <text>P41</text>
+      </annotation>
+      <annotation id="631">
+        <infon key="score">0.9987941</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:35:43Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="31073" length="18"/>
+        <text>DNA binding region</text>
+      </annotation>
+      <annotation id="632">
+        <infon key="score">0.9995447</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="31158" length="3"/>
+        <text>D29</text>
+      </annotation>
+      <annotation id="633">
+        <infon key="score">0.9995259</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:50:11Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="31166" length="4"/>
+        <text>E216</text>
+      </annotation>
+      <annotation id="634">
+        <infon key="score">0.9988655</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:12:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="31244" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="635">
+        <infon key="score">0.9980763</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:27:09Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="31265" length="16"/>
+        <text>N6-adenine MTase</text>
+      </annotation>
+      <annotation id="636">
+        <infon key="score">0.9978216</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:25:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="31285" length="8"/>
+        <text>H.pylori</text>
+      </annotation>
+      <annotation id="637">
+        <infon key="score">0.99823475</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-18T09:25:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="31394" length="8"/>
+        <text>H.pylori</text>
+      </annotation>
+      <annotation id="638">
+        <infon key="score">0.99853706</infon>
+        <infon key="type">species</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:54:51Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="31406" length="5"/>
+        <text>human</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_1</infon>
+      <offset>31413</offset>
+      <text>MATERIALS AND METHODS</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>31435</offset>
+      <text>Protein expression and purification</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>31471</offset>
+      <text>The ORF encoding M1.HpyAVI was inserted into the expression plasmid pET22b (Novagen, Massachusetts, USA) to produce a recombinant protein containing a C-terminal His-tag. In order to produce soluble protein, a chaperone plasmid PG-KJE8 (TaKaRa, Dalian, China) was co-expressed with M1.HpyAVI. The recombinant protein was purified with a three-step chromatography protocol using a Ni-NTA affinity column, a HiLoad 16/60 Superdex 200 column and a mono-S HR 5/5 column (1ml) (GE Healthcare, Uppsala, Sweden). Mutants of M1.HpyAVI were generated using the Muta-direct Site-directed Mutagenesis kit (SBS Genetech, Beijing, China) and produced using the same protocol with wide type protein.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>32157</offset>
+      <text>Crystallization and data collection</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>32193</offset>
+      <text>Crystallization trials were carried out for both the AdoMet-free and AdoMet-bound proteins using the hanging drop vapor diffusion technique. Crystals used for diffraction data collection of the apoprotein were grown under the condition of 1.0 M Bis-Tris, pH 9.0, 1.4 M ammonium tartrate, and the optimal crystallization condition for AdoMet-bound protein was 1.0 M Bis-Tris, pH 6.0, 14% PEG2000, 0.2 M lithium sulfate. X-ray diffraction data were collected at 100 K on beamline BL17U1 at the Shanghai Synchrotron Radiation Facility (SSRF) using an ADSC Quantum 315r CCD detector. All data were indexed, integrated and scaled using the XDS program.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>32841</offset>
+      <text>Structure determination and refinement</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>32880</offset>
+      <text>The structure of ligand-free M1.HpyAVI was determined by means of molecular replacement using the M.MboIIA (PDB ID 1G60) as a search model. Automated structure determination using Phaser gave a solution showing four subunits sitting in the asymmetric unit. The model was refined using the COOT graphics package manually and phenix.refine. The AdoMet-bound structure was determined by means of molecular replacement using the refined model of the apoprotein, and refined in the same way. Statistics from the data collection and structure refinement are summarized in Table 1. All figures representing the M1.HpyAVI structures were generated using the molecular visualization program PyMol.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>33569</offset>
+      <text>Detection of protein dimerization</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>33603</offset>
+      <text>The interface information of M1.HpyAVI free form and AdoMet-bound form structures were analyzed using the PDBePISA (Proteins, Interface, Structures and Assemblies) web server.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>33779</offset>
+      <text>The protein molecular weight was determined by gel filtration using a FPLC system coupled to a Superdex 75 HR 10 / 30 column. The sizing standard was calibrated using the gel filtration calibration kit LMW (GE Healthcare, Uppsala, Sweden).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>34019</offset>
+      <text>Binding affinity quantification via microscale thermophoresis (MST)</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>34087</offset>
+      <text>Microscale thermophoresis was performed using the NT115 nanotemper technologies. M1.HpyAVI-wt and M1.HpyAVI-mutant proteins were fluorescently labeled using the protein label kit according to manufacturer's protocol. Affinity measurements were performed by using MST buffer (0.05% Tween-20 added as final concentration). A solution of unlabeled AdoMet was serially diluted from 1 mM to 15 nM. Equal volume of 0.8 μM labeled protein was mixed with the AdoMet and loaded into the silica capillaries. This binding curve can directly be fitted with the nonlinear solution of the law of mass action, with the dissociation constant (KD) as a result. Measurement was performed at 25°C using 40% LED power and 40%IR-laser power. The dissociation constant was calculated using the Nano-temper Analysis software.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>34892</offset>
+      <text>Radioactive methyltransferase analysis</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>34931</offset>
+      <text>Several different DNA duplexes containing single site of 5′- GAGG-3′, 5′- GAAG-3′ or 5′-GGAG-3′ were used as substrate for methyl transfer reaction (Table S2). 0.1 μM of enzyme and 2 μM of S-[methyl-3H] adenosly methionine (China Isotope and Radiation Corporation, Beijing, China) were incubated at 37°C for 5 min, and then 5 μM of DNA substrate was added to initiate the reaction. Aliquots (20 μl) were taken out at 4-min time intervals and quenched with 2 N HCl. Subsequently, DNA of the mixture was purified using a DNA purification column (TIANGEN, Beijing, China) and the scintillation counting of tritiated DNA was quantified by Beckman LS6500 for 10 min. The background radioactivity was determined by omitting the enzyme from the reaction solution. All the reactions were performed in triplicate.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">title_1</infon>
+      <offset>35753</offset>
+      <text>SUPPLEMENTARY FIGURES AND TABLES</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>35786</offset>
+      <text>CONFLICTS OF INTEREST</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>35808</offset>
+      <text>The authors declare that they have no conflicts of interest.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>35869</offset>
+      <text>ACCESSION CODES</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">footnote</infon>
+      <offset>35885</offset>
+      <text>Atomic coordinates and structure factors for apo-M1.HpyAVI and cofactor-bound M1.HpyAVI have been deposited in the PDB, with accession codes 5HEK and 5HFJ respectively.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">REF</infon>
+      <infon key="type">title</infon>
+      <offset>36054</offset>
+      <text>REFERENCES</text>
+    </passage>
+    <passage>
+      <infon key="fpage">274</infon>
+      <infon key="lpage">293</infon>
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+      <infon key="pub-id_pmid">11933228</infon>
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diff --git a/annotated_BioC_XML/PMC5603727_ann.xml b/annotated_BioC_XML/PMC5603727_ann.xml
new file mode 100644
index 0000000000000000000000000000000000000000..6c050e3d5610fa913b3273ec40ddb2763f5bba21
--- /dev/null
+++ b/annotated_BioC_XML/PMC5603727_ann.xml
@@ -0,0 +1,13110 @@
+<?xml version="1.0" encoding="UTF-8"?>
+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection>
+  <source>PMC</source>
+  <date>20201222</date>
+  <key>pmc.key</key>
+  <document>
+    <id>5603727</id>
+    <infon key="license">CC BY</infon>
+    <infon key="tt_curatable">no</infon>
+    <infon key="tt_version">2</infon>
+    <infon key="tt_round">2</infon>
+    <passage>
+      <infon key="article-id_doi">10.1038/ncomms11032</infon>
+      <infon key="article-id_pii">ncomms11032</infon>
+      <infon key="article-id_pmc">5603727</infon>
+      <infon key="article-id_pmid">27010430</infon>
+      <infon key="elocation-id">11032</infon>
+      <infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon>
+      <infon key="name_0">surname:Janowski;given-names:Robert</infon>
+      <infon key="name_1">surname:Heinz;given-names:Gitta A.</infon>
+      <infon key="name_10">surname:Niessing;given-names:Dierk</infon>
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+      <infon key="name_12">surname:Sattler;given-names:Michael</infon>
+      <infon key="name_2">surname:Schlundt;given-names:Andreas</infon>
+      <infon key="name_3">surname:Wommelsdorf;given-names:Nina</infon>
+      <infon key="name_4">surname:Brenner;given-names:Sven</infon>
+      <infon key="name_5">surname:Gruber;given-names:Andreas R.</infon>
+      <infon key="name_6">surname:Blank;given-names:Michael</infon>
+      <infon key="name_7">surname:Buch;given-names:Thorsten</infon>
+      <infon key="name_8">surname:Buhmann;given-names:Raymund</infon>
+      <infon key="name_9">surname:Zavolan;given-names:Mihaela</infon>
+      <infon key="section_type">TITLE</infon>
+      <infon key="type">front</infon>
+      <infon key="volume">7</infon>
+      <infon key="year">2016</infon>
+      <offset>0</offset>
+      <text>Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40</text>
+      <annotation id="1">
+        <infon key="score">0.99516344</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:54Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="0" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="2">
+        <infon key="score">0.99766684</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:49:06Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="34" length="8"/>
+        <text>hexaloop</text>
+      </annotation>
+      <annotation id="3">
+        <infon key="score">0.99906087</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="60" length="6"/>
+        <text>3′-UTR</text>
+      </annotation>
+      <annotation id="4">
+        <infon key="score">0.9972155</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="70" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">ABSTRACT</infon>
+      <infon key="type">abstract</infon>
+      <offset>77</offset>
+      <text>The RNA-binding protein Roquin is required to prevent autoimmunity. Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40). A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin. Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE). Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin. Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin.</text>
+      <annotation id="5">
+        <infon key="score">0.9985682</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:18:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="81" length="19"/>
+        <text>RNA-binding protein</text>
+      </annotation>
+      <annotation id="6">
+        <infon key="score">0.9993561</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="101" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="7">
+        <infon key="score">0.999308</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="145" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="1156">
+        <infon key="type">protein_type</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:13:29Z</infon>
+        <location offset="244" length="23"/>
+        <text>costimulatory receptors</text>
+      </annotation>
+      <annotation id="1060">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:03:31Z</infon>
+        <location offset="276" length="44"/>
+        <text>tumor necrosis factor receptor superfamily 4</text>
+      </annotation>
+      <annotation id="8">
+        <infon key="score">0.99900836</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:48Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="322" length="6"/>
+        <text>Tnfrs4</text>
+      </annotation>
+      <annotation id="9">
+        <infon key="score">0.99922776</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:57Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="332" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="10">
+        <infon key="score">0.997199</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:41Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="341" length="26"/>
+        <text>constitutive decay element</text>
+      </annotation>
+      <annotation id="11">
+        <infon key="score">0.9984106</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:47Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="369" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="12">
+        <infon key="score">0.99896395</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:18Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="396" length="15"/>
+        <text>triloop hairpin</text>
+      </annotation>
+      <annotation id="13">
+        <infon key="score">0.9992754</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="453" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="14">
+        <infon key="score">0.9988718</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:10:38Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="473" length="12"/>
+        <text>SELEX assays</text>
+      </annotation>
+      <annotation id="15">
+        <infon key="score">0.9985797</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="506" length="21"/>
+        <text>U-rich hexaloop motif</text>
+      </annotation>
+      <annotation id="16">
+        <infon key="score">0.98554564</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:13Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="545" length="25"/>
+        <text>alternative decay element</text>
+      </annotation>
+      <annotation id="17">
+        <infon key="score">0.99670464</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:19Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="572" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="18">
+        <infon key="score">0.9987191</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:58:23Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="578" length="18"/>
+        <text>Crystal structures</text>
+      </annotation>
+      <annotation id="19">
+        <infon key="score">0.9983607</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:10:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="601" length="3"/>
+        <text>NMR</text>
+      </annotation>
+      <annotation id="20">
+        <infon key="score">0.99633616</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:34Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="624" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+      <annotation id="21">
+        <infon key="score">0.9992803</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="633" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+      <annotation id="22">
+        <infon key="score">0.9987386</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:49:11Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="655" length="9"/>
+        <text>hexaloops</text>
+      </annotation>
+      <annotation id="23">
+        <infon key="score">0.9965312</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:10:45Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="672" length="5"/>
+        <text>SELEX</text>
+      </annotation>
+      <annotation id="24">
+        <infon key="score">0.9472056</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:19Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="686" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="25">
+        <infon key="score">0.402406</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:19Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="700" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="26">
+        <infon key="score">0.9993161</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:57Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="732" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="27">
+        <infon key="score">0.9978993</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="737" length="6"/>
+        <text>3′-UTR</text>
+      </annotation>
+      <annotation id="1100">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:19Z</infon>
+        <location offset="784" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="1064">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:48Z</infon>
+        <location offset="797" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="28">
+        <infon key="score">0.99933356</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:57Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="844" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="29">
+        <infon key="score">0.9993765</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="852" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="30">
+        <infon key="score">0.9838305</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:49:29Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="905" length="21"/>
+        <text>hexaloop cis elements</text>
+      </annotation>
+      <annotation id="31">
+        <infon key="score">0.9847647</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="976" length="14"/>
+        <text>messenger RNAs</text>
+      </annotation>
+      <annotation id="32">
+        <infon key="score">0.99931824</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="994" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">ABSTRACT</infon>
+      <infon key="type">abstract</infon>
+      <offset>1004</offset>
+      <text> Roquin is an RNA-binding protein that prevents autoimmunity by limiting expression of receptors such as Ox40. Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques.</text>
+      <annotation id="33">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="1005" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="34">
+        <infon key="score">0.9987923</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:18:06Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1018" length="19"/>
+        <text>RNA-binding protein</text>
+      </annotation>
+      <annotation id="35">
+        <infon key="score">0.9930662</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:57Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="1109" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="36">
+        <infon key="score">0.99891186</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:52Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="1145" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="37">
+        <infon key="score">0.99862254</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:58:26Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1149" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="1098">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:14Z</infon>
+        <location offset="1184" length="25"/>
+        <text>alternative decay element</text>
+      </annotation>
+      <annotation id="38">
+        <infon key="score">0.9992798</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="1254" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="39">
+        <infon key="score">0.9988141</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:28:13Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="1267" length="37"/>
+        <text>structural and biochemical techniques</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>1306</offset>
+      <text>Posttranscriptional gene regulation is involved in a wide range of cellular functions and its critical importance has been described for many developmental and differentiation processes. Consistently, mutations of factors involved in posttranscriptional gene regulation pathways were found associated with a number of genetically inherited diseases. The Roquin protein is essential in T cells for the prevention of autoimmune disease. This is evident from the so-called sanroque mutation in Roquin-1, a single amino acid exchange from Met199 to Arg that causes the development of systemic lupus erythematosus-like symptoms in homozygous mice. The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells. Consistently, CD4+ and CD8+ T cells with the combined deletion of Roquin-encoding genes are spontaneously activated and CD4+ T-helper cells preferentially differentiate into the Th1, Tfh or Th17 subsets. Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs).</text>
+      <annotation id="40">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="1660" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="41">
+        <infon key="score">0.998584</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:35Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="1797" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+      <annotation id="42">
+        <infon key="score">0.9994506</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:11:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1841" length="6"/>
+        <text>Met199</text>
+      </annotation>
+      <annotation id="43">
+        <infon key="score">0.9090607</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:11:44Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="1851" length="3"/>
+        <text>Arg</text>
+      </annotation>
+      <annotation id="44">
+        <infon key="score">0.9979857</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:12:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="1943" length="4"/>
+        <text>mice</text>
+      </annotation>
+      <annotation id="45">
+        <infon key="score">0.9948087</infon>
+        <infon key="type">gene</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:12:18Z</infon>
+        <infon key="identifier">GENE:</infon>
+        <location offset="1953" length="5"/>
+        <text>Rc3h1</text>
+      </annotation>
+      <annotation id="46">
+        <infon key="score">0.9945793</infon>
+        <infon key="type">gene</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:12:23Z</infon>
+        <infon key="identifier">GENE:</infon>
+        <location offset="1963" length="5"/>
+        <text>Rc3h2</text>
+      </annotation>
+      <annotation id="47">
+        <infon key="score">0.99835825</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:35Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="1989" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+      <annotation id="48">
+        <infon key="score">0.99819595</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:19:11Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2002" length="8"/>
+        <text>Roquin-2</text>
+      </annotation>
+      <annotation id="49">
+        <infon key="score">0.99857664</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:12:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2023" length="11"/>
+        <text>vertebrates</text>
+      </annotation>
+      <annotation id="50">
+        <infon key="score">0.99825436</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:12:08Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2107" length="4"/>
+        <text>mice</text>
+      </annotation>
+      <annotation id="51">
+        <infon key="score">0.9137891</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:16:53Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2220" length="11"/>
+        <text>deletion of</text>
+      </annotation>
+      <annotation id="52">
+        <infon key="score">0.99057406</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2232" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="53">
+        <infon key="score">0.9987157</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:35Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2370" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+      <annotation id="54">
+        <infon key="score">0.9979331</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:12:51Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2451" length="24"/>
+        <text>co-stimulatory receptors</text>
+      </annotation>
+      <annotation id="55">
+        <infon key="score">0.99867827</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:50:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2484" length="4"/>
+        <text>Icos</text>
+      </annotation>
+      <annotation id="56">
+        <infon key="score">0.99837804</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:57Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2490" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="57">
+        <infon key="score">0.9986387</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:53:21Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2499" length="6"/>
+        <text>CTLA-4</text>
+      </annotation>
+      <annotation id="58">
+        <infon key="score">0.9791268</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:13:45Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2511" length="9"/>
+        <text>cytokines</text>
+      </annotation>
+      <annotation id="59">
+        <infon key="score">0.91296864</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:14:28Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2529" length="18"/>
+        <text>interleukin (IL)-6</text>
+      </annotation>
+      <annotation id="1157">
+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:15:54Z</infon>
+        <location offset="2552" length="22"/>
+        <text>tumour necrosis factor</text>
+      </annotation>
+      <annotation id="60">
+        <infon key="score">0.9902424</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:16:00Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="2582" length="21"/>
+        <text>transcription factors</text>
+      </annotation>
+      <annotation id="61">
+        <infon key="score">0.9894326</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:16:06Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2612" length="4"/>
+        <text>IRF4</text>
+      </annotation>
+      <annotation id="62">
+        <infon key="score">0.65273774</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:16:10Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2618" length="5"/>
+        <text>IκBNS</text>
+      </annotation>
+      <annotation id="63">
+        <infon key="score">0.963164</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:16:15Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2628" length="4"/>
+        <text>IκBζ</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>2652</offset>
+      <text>We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA. The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin. At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain. The structural basis for CDE recognition by the Roquin-2 ROQ domain has also been recently reported.</text>
+      <annotation id="64">
+        <infon key="score">0.9882385</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:16:32Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2678" length="30"/>
+        <text>structural and functional data</text>
+      </annotation>
+      <annotation id="65">
+        <infon key="score">0.99861914</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:35Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2716" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+      <annotation id="66">
+        <infon key="score">0.9994229</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2725" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+      <annotation id="67">
+        <infon key="score">0.9990214</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:16:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="2736" length="8"/>
+        <text>bound to</text>
+      </annotation>
+      <annotation id="68">
+        <infon key="score">0.9817135</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:42Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2757" length="26"/>
+        <text>constitutive decay element</text>
+      </annotation>
+      <annotation id="69">
+        <infon key="score">0.94088954</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2785" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="70">
+        <infon key="score">0.9986866</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:09Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2793" length="15"/>
+        <text>short stem loop</text>
+      </annotation>
+      <annotation id="71">
+        <infon key="score">0.99715364</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:13Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2810" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="1237">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:52Z</infon>
+        <location offset="2844" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="72">
+        <infon key="score">0.99731666</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:28Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2863" length="23"/>
+        <text>3′-untranslated regions</text>
+      </annotation>
+      <annotation id="73">
+        <infon key="score">0.99472845</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:34Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2888" length="7"/>
+        <text>3′-UTRs</text>
+      </annotation>
+      <annotation id="74">
+        <infon key="score">0.9919091</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:01Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="2921" length="3"/>
+        <text>Tnf</text>
+      </annotation>
+      <annotation id="75">
+        <infon key="score">0.9994338</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2936" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+      <annotation id="76">
+        <infon key="score">0.9923204</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:43Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="2957" length="26"/>
+        <text>extended winged helix fold</text>
+      </annotation>
+      <annotation id="77">
+        <infon key="score">0.9763267</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:52Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3041" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="78">
+        <infon key="score">0.9922246</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:03Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3103" length="3"/>
+        <text>Tnf</text>
+      </annotation>
+      <annotation id="79">
+        <infon key="score">0.8482103</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="3107" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="80">
+        <infon key="score">0.9985152</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:52Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3111" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="81">
+        <infon key="score">0.99448186</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:20:06Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3120" length="15"/>
+        <text>structural data</text>
+      </annotation>
+      <annotation id="82">
+        <infon key="score">0.983335</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:23:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3140" length="19"/>
+        <text>mutational analysis</text>
+      </annotation>
+      <annotation id="83">
+        <infon key="score">0.9987087</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:18:31Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="3236" length="4"/>
+        <text>loop</text>
+      </annotation>
+      <annotation id="84">
+        <infon key="score">0.9761445</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:18:33Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="3245" length="4"/>
+        <text>stem</text>
+      </annotation>
+      <annotation id="85">
+        <infon key="score">0.91149104</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="3257" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="86">
+        <infon key="score">0.9992151</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3300" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="87">
+        <infon key="score">0.9987693</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:20:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3351" length="17"/>
+        <text>crystal structure</text>
+      </annotation>
+      <annotation id="1065">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:48Z</infon>
+        <location offset="3432" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="1160">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
+        <location offset="3441" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="88">
+        <infon key="score">0.9989596</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:06:51Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="3460" length="19"/>
+        <text>second binding site</text>
+      </annotation>
+      <annotation id="89">
+        <infon key="score">0.9989705</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:18:43Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3486" length="19"/>
+        <text>double-stranded RNA</text>
+      </annotation>
+      <annotation id="90">
+        <infon key="score">0.99912053</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:18:48Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="3507" length="5"/>
+        <text>dsRNA</text>
+      </annotation>
+      <annotation id="91">
+        <infon key="score">0.9981774</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:19:31Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="3524" length="8"/>
+        <text>extended</text>
+      </annotation>
+      <annotation id="92">
+        <infon key="score">0.9983943</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="3533" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+      <annotation id="1066">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:48Z</infon>
+        <location offset="3570" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="93">
+        <infon key="score">0.9977644</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:19:10Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3593" length="8"/>
+        <text>Roquin-2</text>
+      </annotation>
+      <annotation id="94">
+        <infon key="score">0.9992968</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="3602" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
+      <offset>3652</offset>
+      <text>We found that the posttranscriptional activity of Roquin-1 and Roquin-2 is regulated through cleavage by the paracaspase MALT1 (refs). Enhanced MALT1-dependent cleavage and inactivation of Roquin, and thus less effective repression of target genes, result from increased strength of antigen recognition in T cells. These findings suggest that dependent on the strength of cognate antigen recognition differential gene expression and cell fate decisions can be established in naive T cells by a graded cleavage and inactivation of Roquin. In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology.</text>
+      <annotation id="95">
+        <infon key="score">0.99875194</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:35Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3702" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+      <annotation id="96">
+        <infon key="score">0.99869066</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:19:11Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3715" length="8"/>
+        <text>Roquin-2</text>
+      </annotation>
+      <annotation id="97">
+        <infon key="score">0.9988292</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:19:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="3761" length="11"/>
+        <text>paracaspase</text>
+      </annotation>
+      <annotation id="98">
+        <infon key="score">0.9991509</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:19:18Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3773" length="5"/>
+        <text>MALT1</text>
+      </annotation>
+      <annotation id="99">
+        <infon key="score">0.998901</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:19:18Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3796" length="5"/>
+        <text>MALT1</text>
+      </annotation>
+      <annotation id="100">
+        <infon key="score">0.999226</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="3841" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="101">
+        <infon key="score">0.99925715</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4182" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="102">
+        <infon key="score">0.9953003</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:58:31Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="4241" length="16"/>
+        <text>binding affinity</text>
+      </annotation>
+      <annotation id="1161">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
+        <location offset="4276" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="103">
+        <infon key="score">0.99862415</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="4295" length="7"/>
+        <text>3′-UTRs</text>
+      </annotation>
+      <annotation id="104">
+        <infon key="score">0.9908908</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:19:47Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="4313" length="5"/>
+        <text>mRNAs</text>
+      </annotation>
+      <annotation id="105">
+        <infon key="score">0.9993056</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4390" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="1162">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
+        <location offset="4411" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="1238">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:52Z</infon>
+        <location offset="4414" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="106">
+        <infon key="score">0.9992386</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4452" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="107">
+        <infon key="score">0.99929154</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="4546" length="6"/>
+        <text>Roquin</text>
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+    <passage>
+      <infon key="section_type">INTRO</infon>
+      <infon key="type">paragraph</infon>
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+      <text>Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain. We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments. Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells.</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:48Z</infon>
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+        <text>CDE</text>
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+        <text>hexaloop</text>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>SELEX</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>X-ray crystallographic</text>
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+        <infon key="identifier">MESH:</infon>
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+        <text>NMR</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>biochemical and functional data</text>
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+        <text>mutational analysis</text>
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+        <text>triloop</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:22:53Z</infon>
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+        <text>RNAs</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:55Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="5254" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_1</infon>
+      <offset>5275</offset>
+      <text>Results</text>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
+      <offset>5283</offset>
+      <text>SELEX identifies novel RNA ligands of Roquin-1</text>
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+        <infon key="identifier">MESH:</infon>
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+        <text>SELEX</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>RNA</text>
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+      <annotation id="128">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <text>Roquin-1</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>5330</offset>
+      <text>We set out to identify Roquin-bound RNA motifs in an unbiased manner by performing SELEX experiments. A biotinylated amino-terminal protein fragment of Roquin-1 (residues 2–440) was used to enrich RNAs from a library containing 47 random nucleotides over three sequential selection rounds. Next-generation sequencing (NGS) of the RNA before and after each selection round revealed that the starting pool represented about 99.6% unique reads in ∼4.2 × 106 sequences. Bioinformatic analysis of NGS data sets derived from the starting pool and enriched selection rounds revealed that the complexity was reduced to 78.6% unique reads in 3.7 × 106 sequences that were analysed after 3 rounds of selection and enrichment. For NGS data analysis, the COMPAS software (AptaIT, Munich, Germany) was applied. Enriched sequences were clustered into so-called patterns with highly homologous sequences. Hereby, the algorithm at first identified frequent motifs of five to eight nucleotides length and subsequently used iterative cycles of proto-pattern formation to cluster sequences bearing two of such frequent motifs. A final aptamer pattern was built up by sequences bearing two frequent motifs and, at the same time, having high similarities also in other sequence parts. Based on this so-called co-occurrence approach, patterns on the basis of frequent motifs were generated and were searched for prominent hexamer sequences (Supplementary Fig. 1a). We identified 5′-CGTTTT-3′, 5′-GCGTTT-3′, 5′-TGCGTT-3′ and 5′-GTTTTA-3′ motifs that were also reconfirmed in an independent experiment (Supplementary Fig. 1a) and are located within highly similar sequences (Fig. 1a and Supplementary Fig. 1b). Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b). Notably, our SELEX approach did not reveal the previously identified CDE sequence. We assume that the region of sequence identity in the CDE is too short for our sequence clustering algorithm. Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5–8 nt stem, with one base in the stem not being paired (Supplementary Fig. 1c). Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b). Importantly, this motif is present across species in the 3′-UTRs of respective mRNAs and showed highest conservation in the loop and the upper stem sequences with a drop of conservation towards the boundaries of the motif (Fig. 1c,d). The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d).</text>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="identifier">MESH:</infon>
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+      <text>NMR analysis of Roquin-bound SL RNAs</text>
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+      <text>We used NMR to analyse the secondary structure of Roquin-1-binding motifs derived from SELEX. Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs. Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson–Crick G–G base pair at this position (Fig. 2a). Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red). No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. In contrast, all expected base pairs are observed for the Ox40 CDE-like SL RNA (Fig. 2c; see also Supplementary Notes).</text>
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+        <text>SL</text>
+      </annotation>
+      <annotation id="1276">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:23:25Z</infon>
+        <location offset="10052" length="4"/>
+        <text>RNAs</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>10057</offset>
+      <text>To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements. The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively. In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b). The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e). As may be expected, the recognition of the hexaloop is significantly different from the triloop in the CDE RNA (Fig. 3b,c and Supplementary Fig. 2b). Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e). The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution.</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10074" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="1182">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
+        <location offset="10105" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="208">
+        <infon key="score">0.9976331</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:10:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10135" length="5"/>
+        <text>SELEX</text>
+      </annotation>
+      <annotation id="209">
+        <infon key="score">0.88738066</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:56:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="10154" length="6"/>
+        <text>solved</text>
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+      <annotation id="210">
+        <infon key="score">0.9981201</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:58:35Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10161" length="18"/>
+        <text>crystal structures</text>
+      </annotation>
+      <annotation id="211">
+        <infon key="score">0.99574417</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:35Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10187" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+      <annotation id="212">
+        <infon key="score">0.99937755</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10196" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+      <annotation id="213">
+        <infon key="score">0.9989785</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:16:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10207" length="8"/>
+        <text>bound to</text>
+      </annotation>
+      <annotation id="214">
+        <infon key="score">0.9867727</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10236" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="215">
+        <infon key="score">0.99827456</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:35:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10254" length="10"/>
+        <text>structures</text>
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+        <infon key="score">0.99751127</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10268" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+      <annotation id="217">
+        <infon key="score">0.9990022</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:16:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10272" length="8"/>
+        <text>bound to</text>
+      </annotation>
+      <annotation id="218">
+        <infon key="score">0.9742184</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10292" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="219">
+        <infon key="score">0.8739459</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10296" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="220">
+        <infon key="score">0.9306097</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:57Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10341" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="1109">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
+        <location offset="10346" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="1183">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
+        <location offset="10355" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="1277">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:23:25Z</infon>
+        <location offset="10358" length="4"/>
+        <text>RNAs</text>
+      </annotation>
+      <annotation id="221">
+        <infon key="score">0.99819595</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:58:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="10442" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="222">
+        <infon key="score">0.99550533</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10457" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="1184">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
+        <location offset="10471" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="223">
+        <infon key="score">0.9942965</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:36:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10490" length="8"/>
+        <text>hexaloop</text>
+      </annotation>
+      <annotation id="224">
+        <infon key="score">0.99924064</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10557" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+      <annotation id="225">
+        <infon key="score">0.9994573</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:36:23Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10561" length="5"/>
+        <text>helix</text>
+      </annotation>
+      <annotation id="226">
+        <infon key="score">0.99950755</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:36:28Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10567" length="2"/>
+        <text>α4</text>
+      </annotation>
+      <annotation id="227">
+        <infon key="score">0.9995253</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:36:34Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10597" length="2"/>
+        <text>β3</text>
+      </annotation>
+      <annotation id="228">
+        <infon key="score">0.99905974</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:18:49Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10645" length="5"/>
+        <text>dsRNA</text>
+      </annotation>
+      <annotation id="229">
+        <infon key="score">0.8476505</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:36:50Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10651" length="4"/>
+        <text>stem</text>
+      </annotation>
+      <annotation id="230">
+        <infon key="score">0.54066336</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:03Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="10717" length="3"/>
+        <text>Tnf</text>
+      </annotation>
+      <annotation id="231">
+        <infon key="score">0.938291</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10721" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="232">
+        <infon key="score">0.6966292</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10725" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="233">
+        <infon key="score">0.96888745</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10728" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="234">
+        <infon key="score">0.99511886</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:49:50Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10802" length="8"/>
+        <text>hexaloop</text>
+      </annotation>
+      <annotation id="235">
+        <infon key="score">0.99347335</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:37:18Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10847" length="7"/>
+        <text>triloop</text>
+      </annotation>
+      <annotation id="236">
+        <infon key="score">0.9789211</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10862" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="237">
+        <infon key="score">0.873177</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="10866" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="238">
+        <infon key="score">0.9607949</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10954" length="3"/>
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+      <annotation id="239">
+        <infon key="score">0.7767095</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10958" length="2"/>
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+      </annotation>
+      <annotation id="240">
+        <infon key="score">0.8281227</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="10965" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="1185">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
+        <location offset="10974" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="1278">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:23:25Z</infon>
+        <location offset="10977" length="4"/>
+        <text>RNAs</text>
+      </annotation>
+      <annotation id="241">
+        <infon key="score">0.9970162</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:35:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11009" length="10"/>
+        <text>structures</text>
+      </annotation>
+      <annotation id="1239">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:53Z</infon>
+        <location offset="11032" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="242">
+        <infon key="score">0.99493295</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:03:50Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11125" length="3"/>
+        <text>C19</text>
+      </annotation>
+      <annotation id="243">
+        <infon key="score">0.5360428</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:35:23Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11129" length="5"/>
+        <text>bulge</text>
+      </annotation>
+      <annotation id="1304">
+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:35:44Z</infon>
+        <location offset="11140" length="16"/>
+        <text>non-Watson–Crick</text>
+      </annotation>
+      <annotation id="244">
+        <infon key="score">0.99560696</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:03:54Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11157" length="2"/>
+        <text>G6</text>
+      </annotation>
+      <annotation id="245">
+        <infon key="score">0.96903014</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:41:45Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11160" length="3"/>
+        <text>G15</text>
+      </annotation>
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+        <infon key="type">bond_interaction</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:35:56Z</infon>
+        <location offset="11164" length="9"/>
+        <text>base pair</text>
+      </annotation>
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+        <infon key="score">0.9992611</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:03:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11197" length="2"/>
+        <text>U1</text>
+      </annotation>
+      <annotation id="247">
+        <infon key="score">0.9995658</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:35:14Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11205" length="6"/>
+        <text>Trp184</text>
+      </annotation>
+      <annotation id="248">
+        <infon key="score">0.99956375</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:35:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11216" length="6"/>
+        <text>Phe194</text>
+      </annotation>
+      <annotation id="249">
+        <infon key="score">0.7797527</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11230" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="1186">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
+        <location offset="11239" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="1240">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:53Z</infon>
+        <location offset="11242" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="250">
+        <infon key="score">0.99824846</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:35:09Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11357" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="251">
+        <infon key="score">0.8127755</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:57Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11374" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="1110">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
+        <location offset="11379" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="1187">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
+        <location offset="11388" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="1241">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:53Z</infon>
+        <location offset="11391" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="252">
+        <infon key="score">0.68048805</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:57Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11426" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="253">
+        <infon key="score">0.9942849</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:55Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11431" length="6"/>
+        <text>3′-UTR</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>11492</offset>
+      <text>The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop. Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts. For example, the main chain atoms of Phe255 form two hydrogen bonds with the Watson–Crick face of the U11 base (Fig. 3d). Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). In addition, the Tyr250 main-chain carbonyl interacts with U13 imino proton (Fig. 3d,e). Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop. The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f). The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA). Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b). The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex. This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e). The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f). This is possible by exposing the base C12 of the Ox-40 ADE-like SL towards the solvent, which accordingly does not show any contacts to the protein. In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts. However, these involve an extended binding surface on the ROQ domain with a number of additional residues compared with the triloop RNA.</text>
+      <annotation id="254">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:49:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11539" length="20"/>
+        <text>double-stranded stem</text>
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+      <annotation id="255">
+        <infon key="score">0.68550116</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:57Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11567" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="256">
+        <infon key="score">0.7644723</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11572" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="1188">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
+        <location offset="11581" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="257">
+        <infon key="score">0.98503137</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11602" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="258">
+        <infon key="score">0.9581188</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:37:44Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11606" length="7"/>
+        <text>triloop</text>
+      </annotation>
+      <annotation id="1306">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:38:04Z</infon>
+        <location offset="11628" length="15"/>
+        <text>U-rich hexaloop</text>
+      </annotation>
+      <annotation id="259">
+        <infon key="score">0.52676684</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:57Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11651" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="260">
+        <infon key="score">0.64240104</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11656" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="1189">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
+        <location offset="11665" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="261">
+        <infon key="score">0.65158087</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="11668" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="262">
+        <infon key="score">0.9272881</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:06:59Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11693" length="7"/>
+        <text>surface</text>
+      </annotation>
+      <annotation id="1266">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:53Z</infon>
+        <location offset="11708" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+      <annotation id="263">
+        <infon key="score">0.98314255</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11750" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="264">
+        <infon key="score">0.9670553</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:38:07Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11754" length="7"/>
+        <text>triloop</text>
+      </annotation>
+      <annotation id="265">
+        <infon key="score">0.9995376</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:39:10Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11860" length="6"/>
+        <text>Phe255</text>
+      </annotation>
+      <annotation id="266">
+        <infon key="score">0.9970485</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:38:12Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="11876" length="14"/>
+        <text>hydrogen bonds</text>
+      </annotation>
+      <annotation id="267">
+        <infon key="score">0.99941254</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:39:20Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11925" length="3"/>
+        <text>U11</text>
+      </annotation>
+      <annotation id="268">
+        <infon key="score">0.9979755</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:58:43Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11961" length="9"/>
+        <text>structure</text>
+      </annotation>
+      <annotation id="269">
+        <infon key="score">0.98815536</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:03Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="11978" length="3"/>
+        <text>Tnf</text>
+      </annotation>
+      <annotation id="270">
+        <infon key="score">0.95641446</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11982" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="271">
+        <infon key="score">0.95013833</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:39:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="11986" length="7"/>
+        <text>triloop</text>
+      </annotation>
+      <annotation id="272">
+        <infon key="score">0.9995419</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:07:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="11998" length="6"/>
+        <text>Tyr250</text>
+      </annotation>
+      <annotation id="273">
+        <infon key="score">0.99712324</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:38:50Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12033" length="13"/>
+        <text>hydrogen bond</text>
+      </annotation>
+      <annotation id="274">
+        <infon key="score">0.9995309</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:39:24Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12073" length="3"/>
+        <text>G12</text>
+      </annotation>
+      <annotation id="275">
+        <infon key="score">0.94750226</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:39:03Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12128" length="8"/>
+        <text>hexaloop</text>
+      </annotation>
+      <annotation id="276">
+        <infon key="score">0.9995185</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:07:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12177" length="6"/>
+        <text>Tyr250</text>
+      </annotation>
+      <annotation id="277">
+        <infon key="score">0.9994367</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:39:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12216" length="3"/>
+        <text>U11</text>
+      </annotation>
+      <annotation id="278">
+        <infon key="score">0.988433</infon>
+        <infon key="type">bond_interaction</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:38:37Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="12286" length="21"/>
+        <text>stacking interactions</text>
+      </annotation>
+      <annotation id="279">
+        <infon key="score">0.999371</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:39:40Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12317" length="3"/>
+        <text>U10</text>
+      </annotation>
+      <annotation id="280">
+        <infon key="score">0.99931526</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:39:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12325" length="3"/>
+        <text>U11</text>
+      </annotation>
+      <annotation id="281">
+        <infon key="score">0.9995111</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:07:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12393" length="6"/>
+        <text>Tyr250</text>
+      </annotation>
+      <annotation id="282">
+        <infon key="score">0.99948704</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:39:46Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12435" length="3"/>
+        <text>U13</text>
+      </annotation>
+      <annotation id="283">
+        <infon key="score">0.9995566</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:39:51Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12465" length="6"/>
+        <text>Val257</text>
+      </annotation>
+      <annotation id="284">
+        <infon key="score">0.99952316</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:39:56Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="12476" length="6"/>
+        <text>Lys259</text>
+      </annotation>
+      <annotation id="285">
+        <infon key="score">0.97526985</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:50:01Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12486" length="6"/>
+        <text>strand</text>
+      </annotation>
+      <annotation id="286">
+        <infon key="score">0.9988292</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:36:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12493" length="2"/>
+        <text>β3</text>
+      </annotation>
+      <annotation id="287">
+        <infon key="score">0.63233346</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:03:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12523" length="3"/>
+        <text>UGU</text>
+      </annotation>
+      <annotation id="288">
+        <infon key="score">0.862775</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:40:04Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12527" length="7"/>
+        <text>triloop</text>
+      </annotation>
+      <annotation id="289">
+        <infon key="score">0.8768791</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:03Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="12542" length="3"/>
+        <text>Tnf</text>
+      </annotation>
+      <annotation id="290">
+        <infon key="score">0.85423577</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="12546" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="291">
+        <infon key="score">0.82655025</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="12550" length="3"/>
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+      <text>We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution. CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). This is fully consistent with the interactions observed in the crystal structure (Supplementary Fig. 2c–e) and indicates a similar binding surface. However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d). On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs. This is consistent with the very similar structural features and mode of RNA recognition of the ROQ domain with these RNAs (Supplementary Fig. 2a,d,e).</text>
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+      <text>To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4). Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2). We next tested a set of mutants (Supplementary Fig. 4), which were designed based on contacts observed in the crystal structure (Fig. 3) and the NMR CSPs (Fig. 4a,b). In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine. We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a). This underlines the central role of Tyr250 for stabilization of the hexaloop structure and recognition by stacking interactions (Fig. 3b,e). Mutation of Ser253, which shows large CSPs in the NMR titrations (Fig. 4a,b), does not significantly impair complex formation (Supplementary Fig. 4). The large chemical shift change is probably caused by ring current effects induced by the close proximity of the U11 and U13 bases. Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4). This indicates that replacement by Tyr does not strongly affect the RNA interaction, and that some conformational variations are tolerated. Thus, the mutational analysis is fully consistent with the recognition of the hexaloop observed in our crystal structures.</text>
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+        <infon key="identifier">GO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:31:39Z</infon>
+        <location offset="15706" length="11"/>
+        <text>ROQ-Tnf CDE</text>
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+        <infon key="updated_at">2023-09-15T12:44:48Z</infon>
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+        <location offset="15912" length="7"/>
+        <text>alanine</text>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="15979" length="5"/>
+        <text>Y250A</text>
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+        <text>hexaloop</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
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+        <infon key="updated_at">2023-09-15T12:17:53Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="updated_at">2023-09-15T13:56:06Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16219" length="8"/>
+        <text>Mutation</text>
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+      <annotation id="391">
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+        <infon key="type">residue_name_number</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="updated_at">2023-09-15T12:31:48Z</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="updated_at">2023-09-15T13:59:07Z</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+      <annotation id="394">
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+        <infon key="type">residue_name_number</infon>
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+        <infon key="updated_at">2023-09-15T12:39:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16482" length="3"/>
+        <text>U11</text>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:39:47Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16490" length="3"/>
+        <text>U13</text>
+      </annotation>
+      <annotation id="396">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:45:52Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16512" length="6"/>
+        <text>mutant</text>
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+      <annotation id="397">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="16526" length="4"/>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="16538" length="3"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:03Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="16653" length="3"/>
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+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="16657" length="3"/>
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+      </annotation>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:45:59Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16705" length="11"/>
+        <text>replacement</text>
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+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:45:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="16720" length="3"/>
+        <text>Tyr</text>
+      </annotation>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="16753" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="405">
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:23:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="16835" length="19"/>
+        <text>mutational analysis</text>
+      </annotation>
+      <annotation id="406">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:46:05Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="16903" length="8"/>
+        <text>hexaloop</text>
+      </annotation>
+      <annotation id="407">
+        <infon key="score">0.99865377</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:59:12Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="16928" length="18"/>
+        <text>crystal structures</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>16950</offset>
+      <text>To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells. Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion. The cells were then transduced with doxycycline-inducible retroviral vectors to reconstitute Roquin-1 expression (Fig. 5b). Depletion of Roquin proteins on tamoxifen treatment (Supplementary Fig. 5a) strongly increased surface expression of Ox40 and Icos (Fig. 5c). This increase in surface expression of both costimulatory receptors was partially corrected by the doxycycline-induced reconstitution with Roquin-1 WT protein (Fig. 5c left panels). Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels). The observed partial rescue may relate to the low, close to endogenous expression of these constructs (Supplementary Fig. 5b). However, it is also possible that continuous overexpression of targets following Roquin deletion induces a hyperactivated state in the T cells. This hyperactivation, compared with the actual posttranscriptional derepression, may contribute even stronger to the increased Icos and Ox40 expression levels. Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos.</text>
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+        <infon key="identifier">CHEBI:</infon>
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+        <text>retroviral reconstitution system</text>
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+        <text>Roquin</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>2</text>
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+        <text>reverse tetracycline-controlled transactivator</text>
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+        <text>4-hydroxy tamoxifen</text>
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+        <infon key="identifier">CHEBI:</infon>
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+        <text>doxycycline</text>
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+        <infon key="identifier">PR:</infon>
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+        <text>Roquin-1</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <text>Roquin</text>
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+        <text>tamoxifen</text>
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+        <infon key="identifier">PR:</infon>
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+        <text>Ox40</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:50:11Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17662" length="4"/>
+        <text>Icos</text>
+      </annotation>
+      <annotation id="425">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:13:35Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17722" length="23"/>
+        <text>costimulatory receptors</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:52:22Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="17777" length="11"/>
+        <text>doxycycline</text>
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+        <infon key="identifier">PR:</infon>
+        <location offset="17817" length="8"/>
+        <text>Roquin-1</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:46:59Z</infon>
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+        <text>WT</text>
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+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:47:15Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17917" length="5"/>
+        <text>Y250A</text>
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+      <annotation id="430">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:47:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>mutant</text>
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+        <infon key="updated_at">2023-09-15T12:07:36Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="17933" length="8"/>
+        <text>Roquin-1</text>
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+      <annotation id="432">
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+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:47:26Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17949" length="5"/>
+        <text>K220A</text>
+      </annotation>
+      <annotation id="433">
+        <infon key="score">0.99901557</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:47:31Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17956" length="5"/>
+        <text>K239A</text>
+      </annotation>
+      <annotation id="434">
+        <infon key="score">0.9989231</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:47:36Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="17966" length="4"/>
+        <text>R260</text>
+      </annotation>
+      <annotation id="435">
+        <infon key="score">0.99922574</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:47:04Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="17971" length="6"/>
+        <text>mutant</text>
+      </annotation>
+      <annotation id="436">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18009" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="437">
+        <infon key="score">0.65221393</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18013" length="2"/>
+        <text>SL</text>
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+      <annotation id="1310">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:50:32Z</infon>
+        <location offset="18236" length="14"/>
+        <text>overexpression</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18272" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="439">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:50:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18462" length="4"/>
+        <text>Icos</text>
+      </annotation>
+      <annotation id="440">
+        <infon key="score">0.9990062</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:57Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18471" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="441">
+        <infon key="score">0.99869114</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:47:11Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="18506" length="27"/>
+        <text>structure–function analyses</text>
+      </annotation>
+      <annotation id="442">
+        <infon key="score">0.99956244</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:50:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18561" length="4"/>
+        <text>Y250</text>
+      </annotation>
+      <annotation id="443">
+        <infon key="score">0.99811304</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18591" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="444">
+        <infon key="score">0.9992256</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:57Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18628" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="445">
+        <infon key="score">0.98053783</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18665" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="446">
+        <infon key="score">0.9991659</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:50:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18688" length="4"/>
+        <text>Icos</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>18696</offset>
+      <text>We also investigated the role of individual nucleotides in the Ox40 ADE-like SL for complex formation with the ROQ domain. We designed four mutants (Mut1–4, see Supplementary Fig. 6) that were expected to disrupt key interactions with the protein according to our co-crystal structure (Fig. 3d–f and Supplementary Fig. 2). NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6). We next used surface plasmon resonance experiments to determine dissociation constants for the ROQ-RNA interaction (Table 2 and Supplementary Fig. 7). Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. As intended, the mutation Mut 1 allows the formation of an additional base pair and thus leads to the formation of a tetraloop with a new G-C closing base pair (Supplementary Fig. 6a). Consistent with the structural analysis, we assume that this variant alters the hexaloop conformation and thus reduces the interaction with ROQ. Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7). No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d). Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ. Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7).</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:57Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="18759" length="4"/>
+        <text>Ox40</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
+        <location offset="18764" length="3"/>
+        <text>ADE</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:14Z</infon>
+        <location offset="18773" length="2"/>
+        <text>SL</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="18807" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+      <annotation id="449">
+        <infon key="score">0.9972644</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:52:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="18960" length="20"/>
+        <text>co-crystal structure</text>
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+        <text>wild-type</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+    <passage>
+      <infon key="section_type">RESULTS</infon>
+      <infon key="type">title_2</infon>
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+      <text>Roquin binding to different SLs in the Ox40 3′-UTR</text>
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+        <infon key="annotator">cleaner0</infon>
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+      <text>We have recently shown that Roquin-1 binds to a CDE-like motif in the 3′-UTR of Ox40 mRNA (Figs 1d and 4c). We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR. The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2). The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2). This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR. We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a). These RNAs were then tested in EMSAs with the Roquin-1 N terminus (residues 2–440) (Fig. 6b). Gel shift assays show that binding to the wild-type 3′-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively. Consistent with this, both bands are strongly reduced when mutations are introduced that interfere with the formation of both SLs. Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL. We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR.</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:48Z</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:57:39Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>2–440</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>ROQ</text>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:05:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:57:52Z</infon>
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+        <infon key="type">protein</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+      </annotation>
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+        <text>titrations</text>
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <text>3′-UTR</text>
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+      <text>Regulation of Ox40 expression via two motifs in its 3′-UTR</text>
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+      <text>To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells. Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts. Doxycycline treatment of cells from this cell line enabled ectopic Roquin-1 and co-translational mCherry expression due to the stable integration of an inducible lentiviral vector (Supplementary Fig. 8c). The expression of Ox40 in cells with and without doxycycline treatment was then quantified by flow cytometry (Supplementary Fig. 8c). Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c). In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40. In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c).</text>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
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+        <infon key="type">protein</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:51:08Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>retroviruses</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:36Z</infon>
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+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="identifier">PR:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:00:57Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>mouse</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:52:23Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>lentiviral</text>
+      </annotation>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="identifier">PR:</infon>
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+      <text>To further analyse the functional consequences of Roquin binding to the 3′-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d). Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min). A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min). Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively. These findings underscore the importance of both structural motifs and reveal that they have an additive effect on the regulation of Ox40 mRNA expression in cells.</text>
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+        <text>Ox40</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
+        <location offset="25123" length="4"/>
+        <text>mRNA</text>
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+      <infon key="type">title_1</infon>
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+      <text>Discussion</text>
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+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>25176</offset>
+      <text>Recent structural and functional studies have provided first insight into the RNA binding of Roquin. Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. Considering that the CDE RNA recognition is mostly structure specific and not sequence dependent, a wide spectrum of target mRNA might be recognized by Roquin. Some evidence for this is provided by a recent study by Landthaler and colleagues.</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>structural and functional studies</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:56Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <text>Roquin</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <text>Structures</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25291" length="6"/>
+        <text>Roquin</text>
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+        <infon key="score">0.9990138</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:16:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <text>bound to</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="25307" length="3"/>
+        <text>CDE</text>
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+      <annotation id="1212">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
+        <location offset="25311" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="1287">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:23:25Z</infon>
+        <location offset="25314" length="4"/>
+        <text>RNAs</text>
+      </annotation>
+      <annotation id="634">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25361" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="635">
+        <infon key="score">0.93589884</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="25364" length="3"/>
+        <text>RNA</text>
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+      <annotation id="636">
+        <infon key="score">0.9987804</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:07:11Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25385" length="6"/>
+        <text>A-site</text>
+      </annotation>
+      <annotation id="637">
+        <infon key="score">0.997312</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:51:26Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25399" length="17"/>
+        <text>N-terminal region</text>
+      </annotation>
+      <annotation id="638">
+        <infon key="score">0.99929726</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25424" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="639">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-21T18:27:48Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25498" length="36"/>
+        <text>pyrimidine–purine–pyrimidine triloop</text>
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+        <infon key="score">0.60207105</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:48Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>CDE</text>
+      </annotation>
+      <annotation id="641">
+        <infon key="score">0.97175974</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:53Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="25561" length="3"/>
+        <text>RNA</text>
+      </annotation>
+      <annotation id="642">
+        <infon key="score">0.99886143</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:59:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="25660" length="4"/>
+        <text>mRNA</text>
+      </annotation>
+      <annotation id="643">
+        <infon key="score">0.99937844</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25688" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>25783</offset>
+      <text>Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes. Our experiments show that this SELEX-derived ADE shows functional activity comparable to the previously established CDE motif. The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop. Notably, the recognition of the respective RNA-helical stem regions by the ROQ domain is identical for the triloop and hexaloop motifs. However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). Of particular importance for the hexaloop recognition is Tyr250, which acts as a stabilizing element for the integrity of a defined loop conformation. It stacks with nucleotides in the hexaloop but not the CDE triloop (Fig. 3b,c). The functional role of Tyr250 for ADE-mediated mRNA regulation by Roquin-1 is thus explained by our experiments (Fig. 5b,c). The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL. Consistent with this, loss of ROQ binding is observed on replacement of U11 and U13 by other bases (Table 2). In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively. A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c). Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops.</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:05:30Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="25801" length="12"/>
+        <text>SELEX assays</text>
+      </annotation>
+      <annotation id="645">
+        <infon key="score">0.9923244</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:05:39Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25834" length="21"/>
+        <text>RNA recognition motif</text>
+      </annotation>
+      <annotation id="646">
+        <infon key="score">0.99919313</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:36Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25859" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+      <annotation id="647">
+        <infon key="score">0.88746005</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:58Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="25893" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="648">
+        <infon key="score">0.9967925</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25898" length="6"/>
+        <text>3′-UTR</text>
+      </annotation>
+      <annotation id="649">
+        <infon key="score">0.9957634</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="25949" length="7"/>
+        <text>3′-UTRs</text>
+      </annotation>
+      <annotation id="650">
+        <infon key="score">0.99643964</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:10:46Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="26009" length="5"/>
+        <text>SELEX</text>
+      </annotation>
+      <annotation id="651">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26023" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="1086">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:49Z</infon>
+        <location offset="26094" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="652">
+        <infon key="score">0.9281349</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26109" length="3"/>
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+      </annotation>
+      <annotation id="653">
+        <infon key="score">0.87787974</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:58Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="26117" length="4"/>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26122" length="3"/>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
+        <location offset="26131" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="1288">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:23:25Z</infon>
+        <location offset="26134" length="4"/>
+        <text>RNAs</text>
+      </annotation>
+      <annotation id="1214">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
+        <location offset="26145" length="2"/>
+        <text>SL</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:07:36Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26161" length="8"/>
+        <text>hexaloop</text>
+      </annotation>
+      <annotation id="656">
+        <infon key="score">0.9957638</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:51:30Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26183" length="7"/>
+        <text>triloop</text>
+      </annotation>
+      <annotation id="657">
+        <infon key="score">0.9988192</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:06:12Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26235" length="24"/>
+        <text>RNA-helical stem regions</text>
+      </annotation>
+      <annotation id="658">
+        <infon key="score">0.8417624</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:53Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26267" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+      <annotation id="659">
+        <infon key="score">0.99877614</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:06:17Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26299" length="7"/>
+        <text>triloop</text>
+      </annotation>
+      <annotation id="660">
+        <infon key="score">0.9985618</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:06:19Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26311" length="8"/>
+        <text>hexaloop</text>
+      </annotation>
+      <annotation id="1323">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:06:37Z</infon>
+        <location offset="26341" length="16"/>
+        <text>U-rich hexaloops</text>
+      </annotation>
+      <annotation id="661">
+        <infon key="score">0.6006369</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26365" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="1131">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
+        <location offset="26373" length="3"/>
+        <text>ADE</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
+        <location offset="26382" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="662">
+        <infon key="score">0.9911668</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:23:25Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="26385" length="4"/>
+        <text>RNAs</text>
+      </annotation>
+      <annotation id="663">
+        <infon key="score">0.9992685</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:36:24Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26439" length="5"/>
+        <text>helix</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:36:29Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26445" length="2"/>
+        <text>α4</text>
+      </annotation>
+      <annotation id="665">
+        <infon key="score">0.9989827</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:51:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26452" length="6"/>
+        <text>strand</text>
+      </annotation>
+      <annotation id="666">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:36:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="26459" length="2"/>
+        <text>β3</text>
+      </annotation>
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+        <infon key="score">0.93937474</infon>
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+      <text>Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work. Most probably, the experimental setup of Murakawa et al. revealed both high- and low-affinity target motifs for Roquin, whereas our structural study reports on a high-affinity binding motif. Notably, Murakawa et al. neither found the Roquin-regulated Ox40 nor the Tnf 3′-UTRs, as both genes are not expressed in HEK 293 cells. However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement. It remains to be seen which exact features govern the recognition of the A20 SL by Roquin.</text>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:56Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <location offset="28322" length="6"/>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:58Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="28339" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="715">
+        <infon key="score">0.99075</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:03Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="28352" length="3"/>
+        <text>Tnf</text>
+      </annotation>
+      <annotation id="716">
+        <infon key="score">0.9986799</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28356" length="7"/>
+        <text>3′-UTRs</text>
+      </annotation>
+      <annotation id="1217">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
+        <location offset="28461" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="717">
+        <infon key="score">0.9981084</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28475" length="6"/>
+        <text>3′-UTR</text>
+      </annotation>
+      <annotation id="718">
+        <infon key="score">0.9981975</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:54:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="28485" length="3"/>
+        <text>A20</text>
+      </annotation>
+      <annotation id="719">
+        <infon key="score">0.99777573</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:59:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="28489" length="4"/>
+        <text>mRNA</text>
+      </annotation>
+      <annotation id="720">
+        <infon key="score">0.99930227</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="28523" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="721">
+        <infon key="score">0.9984925</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:49Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28592" length="3"/>
+        <text>CDE</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:09:32Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28596" length="7"/>
+        <text>triloop</text>
+      </annotation>
+      <annotation id="723">
+        <infon key="score">0.99866307</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:54:13Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="28690" length="3"/>
+        <text>A20</text>
+      </annotation>
+      <annotation id="724">
+        <infon key="score">0.9991019</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28694" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="725">
+        <infon key="score">0.999321</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="28700" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>28712</offset>
+      <text>The regulatory cis RNA elements in 3′-UTRs may also be targeted by additional trans-acting factors. We have recently identified the endonuclease Regnase-1 as a cofactor of Roquin function that shares an overlapping set of target mRNAs. In another study, the overlap in targets was confirmed, but a mutually exclusive regulation was proposed based on studies in lipopolysaccharide (LPS)-stimulated myeloid cells. In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target. In CD4+ T cells, Ox40 does not show derepression in individual knockouts of Roquin-1 or Roquin-2 encoding genes, but is strongly induced upon combined deficiency of both genes. In addition, conditional deletion of the Regnase-1-encoding gene induced Ox40 expression in these cells. Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes. However, in cultures of CD4+ T cells, Ox40 is translated on day 4–5 and is expressed much higher in T cells with combined deficiency of Roquin-1 and Roquin-2. At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c).</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:53:15Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28727" length="16"/>
+        <text>cis RNA elements</text>
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+      <annotation id="727">
+        <infon key="score">0.9985352</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:35Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="28747" length="7"/>
+        <text>3′-UTRs</text>
+      </annotation>
+      <annotation id="728">
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+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:10:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="28844" length="12"/>
+        <text>endonuclease</text>
+      </annotation>
+      <annotation id="729">
+        <infon key="score">0.9992896</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:10:21Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="28857" length="9"/>
+        <text>Regnase-1</text>
+      </annotation>
+      <annotation id="730">
+        <infon key="score">0.99783343</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="28884" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="731">
+        <infon key="score">0.9970878</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:19:47Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="28941" length="5"/>
+        <text>mRNAs</text>
+      </annotation>
+      <annotation id="1326">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:10:42Z</infon>
+        <location offset="29073" length="18"/>
+        <text>lipopolysaccharide</text>
+      </annotation>
+      <annotation id="1327">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:10:51Z</infon>
+        <location offset="29093" length="3"/>
+        <text>LPS</text>
+      </annotation>
+      <annotation id="732">
+        <infon key="score">0.9987564</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29140" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="733">
+        <infon key="score">0.9633093</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:59:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="29155" length="4"/>
+        <text>mRNA</text>
+      </annotation>
+      <annotation id="734">
+        <infon key="score">0.62592363</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:05:58Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="29191" length="8"/>
+        <text>inactive</text>
+      </annotation>
+      <annotation id="735">
+        <infon key="score">0.9974112</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:19:47Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="29200" length="5"/>
+        <text>mRNAs</text>
+      </annotation>
+      <annotation id="736">
+        <infon key="score">0.9992381</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:10:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29213" length="9"/>
+        <text>Regnase-1</text>
+      </annotation>
+      <annotation id="737">
+        <infon key="score">0.9640897</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:59:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="29231" length="4"/>
+        <text>mRNA</text>
+      </annotation>
+      <annotation id="738">
+        <infon key="score">0.9966534</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:58Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29305" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="739">
+        <infon key="score">0.9888611</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:36Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29364" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+      <annotation id="740">
+        <infon key="score">0.9852724</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:19:11Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29376" length="8"/>
+        <text>Roquin-2</text>
+      </annotation>
+      <annotation id="741">
+        <infon key="score">0.8447384</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:57:33Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="29439" length="10"/>
+        <text>deficiency</text>
+      </annotation>
+      <annotation id="1158">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:16:54Z</infon>
+        <location offset="29490" length="11"/>
+        <text>deletion of</text>
+      </annotation>
+      <annotation id="742">
+        <infon key="score">0.9980139</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:10:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29506" length="9"/>
+        <text>Regnase-1</text>
+      </annotation>
+      <annotation id="743">
+        <infon key="score">0.9953726</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:58Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29538" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="744">
+        <infon key="score">0.9960139</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:58Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29595" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="745">
+        <infon key="score">0.9970066</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:59:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="29600" length="4"/>
+        <text>mRNA</text>
+      </annotation>
+      <annotation id="746">
+        <infon key="score">0.99847347</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:56Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29608" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="747">
+        <infon key="score">0.99569356</infon>
+        <infon key="type">protein_type</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:55:11Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="29618" length="7"/>
+        <text>Regnase</text>
+      </annotation>
+      <annotation id="1351">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:57:52Z</infon>
+        <location offset="29813" length="27"/>
+        <text>double and triple knockouts</text>
+      </annotation>
+      <annotation id="748">
+        <infon key="score">0.99602485</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:58Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29895" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="749">
+        <infon key="score">0.99506897</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:36Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="29993" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+      <annotation id="750">
+        <infon key="score">0.992296</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:19:11Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30006" length="8"/>
+        <text>Roquin-2</text>
+      </annotation>
+      <annotation id="751">
+        <infon key="score">0.5500128</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:57:57Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30061" length="14"/>
+        <text>reconstitution</text>
+      </annotation>
+      <annotation id="752">
+        <infon key="score">0.99912375</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:47:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="30081" length="2"/>
+        <text>WT</text>
+      </annotation>
+      <annotation id="753">
+        <infon key="score">0.9981454</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:36Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30084" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+      <annotation id="754">
+        <infon key="score">0.9952649</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:58Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30118" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="755">
+        <infon key="score">0.54457563</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:06:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="30185" length="6"/>
+        <text>active</text>
+      </annotation>
+      <annotation id="756">
+        <infon key="score">0.99797314</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:59:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="30192" length="4"/>
+        <text>mRNA</text>
+      </annotation>
+      <annotation id="757">
+        <infon key="score">0.99881476</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:36Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30200" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>30235</offset>
+      <text>Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays. Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3′-UTR by both proteins. In contrast, structural details on how Regnase-1 can interact with these SL RNAs are currently missing. Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene. Both were required for Regnase-1-mediated repression. These findings therefore raise the possibility that Regnase-1 interacts with ADE-like hexaloop structures either in a direct or indirect manner.</text>
+      <annotation id="758">
+        <infon key="score">0.9991494</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:36Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30279" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+      <annotation id="759">
+        <infon key="score">0.999046</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:19:11Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30291" length="8"/>
+        <text>Roquin-2</text>
+      </annotation>
+      <annotation id="760">
+        <infon key="score">0.8295006</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:58Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:56Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
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+        <text>filter binding assays</text>
+      </annotation>
+      <annotation id="1256">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="30550" length="3"/>
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+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:49Z</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:10:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="30645" length="9"/>
+        <text>Regnase-1</text>
+      </annotation>
+      <annotation id="1218">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
+        <location offset="30679" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="1290">
+        <infon key="type">chemical</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:23:25Z</infon>
+        <location offset="30682" length="4"/>
+        <text>RNAs</text>
+      </annotation>
+      <annotation id="770">
+        <infon key="score">0.9973216</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:12:13Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>Regnase-1-binding sites</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:12:17Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="30781" length="48"/>
+        <text>crosslinking and immunoprecipitation experiments</text>
+      </annotation>
+      <annotation id="772">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:12:42Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="30850" length="7"/>
+        <text>triloop</text>
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+      <annotation id="773">
+        <infon key="score">0.973413</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:12:31Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>pyrimidine–purine–pyrimidine loops</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <text>stems</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:12:33Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <infon key="type">gene</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:54:30Z</infon>
+        <infon key="identifier">GENE:</infon>
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+        <text>Ptgs2</text>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:10:22Z</infon>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:10:22Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="31098" length="9"/>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="31132" length="8"/>
+        <text>hexaloop</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
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+      <text>Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation. The novel 3′-UTR loop motif that we have identified as a bona fide target of Roquin now expands this multilayer mode of co-regulation. We suggest that differential regulation of mRNA expression is not only achieved through multiple regulators with individual preferences for a given motif or variants thereof, but that regulators may also identify and use distinct motifs, as long as they exhibit some basic features regarding shape, size and sequence.</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:51:43Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="31245" length="12"/>
+        <text>cis-elements</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:58:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="31292" length="3"/>
+        <text>SLs</text>
+      </annotation>
+      <annotation id="783">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:58Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="31302" length="4"/>
+        <text>Ox40</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:50:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="31310" length="4"/>
+        <text>Icos</text>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:59:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="31430" length="4"/>
+        <text>mRNA</text>
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+      <annotation id="786">
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="31513" length="6"/>
+        <text>3′-UTR</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:52:06Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="31520" length="10"/>
+        <text>loop motif</text>
+      </annotation>
+      <annotation id="788">
+        <infon key="score">0.99890745</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:06:57Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="31580" length="6"/>
+        <text>Roquin</text>
+      </annotation>
+      <annotation id="789">
+        <infon key="score">0.99247086</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:59:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="31681" length="4"/>
+        <text>mRNA</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="section_type">DISCUSS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>31958</offset>
+      <text>The presence of distinct motifs in 3′-UTRs offers a broader variability for gene regulation by RNA cis elements. Their accessibility can be modulated by trans-acting factors that may bind regulatory motifs, unfold higher-order structures in the RNA or maintain a preference for duplex structures as was shown recently for mRNAs that are recognized by Staufen-1 (ref.). In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain. The exact stoichiometry of Roquin bound to the Ox40 3′-UTR is unknown. The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs. In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site. This opens the possibility that one Roquin molecule may cluster two motifs in a given 3′-UTR and/or cluster motifs from distinct 3′-UTRs to enhance downstream processing. Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA. We therefore hypothesize that the combination of multiple binding sites may be more commonly used to enhance the functional activity of Roquin. At the same time, the combination of cis elements may be important for differential gene regulation, as composite cis elements with lower affinity may be less sensitive to Roquin. This will lead to less effective repression in T cells when antigen recognition is of moderate signal strength and only incomplete cleavage of Roquin by MALT1 occurs. For understanding the intricate complexity of 3′-UTR regulation, future work will be necessary by combining large-scale approaches, such as cross-linking and immunoprecipitation experiments to identify RNA-binding sites, and structural biology to dissect the underlying molecular mechanisms.</text>
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+        <infon key="identifier">SO:</infon>
+        <location offset="31993" length="7"/>
+        <text>3′-UTRs</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="updated_at">2023-09-15T13:52:01Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:54:45Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="32309" length="9"/>
+        <text>Staufen-1</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32334" length="6"/>
+        <text>3′-UTR</text>
+      </annotation>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:58Z</infon>
+        <infon key="identifier">PR:</infon>
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+        <text>Ox40</text>
+      </annotation>
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+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:59:32Z</infon>
+        <infon key="identifier">CHEBI:</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:49Z</infon>
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+      </annotation>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32397" length="2"/>
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+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:53Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <text>ROQ</text>
+      </annotation>
+      <annotation id="800">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <text>Roquin</text>
+      </annotation>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:16:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="32475" length="8"/>
+        <text>bound to</text>
+      </annotation>
+      <annotation id="802">
+        <infon key="score">0.9245208</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:58Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="32488" length="4"/>
+        <text>Ox40</text>
+      </annotation>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32493" length="6"/>
+        <text>3′-UTR</text>
+      </annotation>
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+        <infon key="score">0.9989626</infon>
+        <infon key="type">site</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:13:23Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="32536" length="22"/>
+        <text>secondary binding site</text>
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+        <text>cross-linking and immunoprecipitation experiments</text>
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+        <text>structural biology</text>
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+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_1</infon>
+      <offset>34078</offset>
+      <text>Methods</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>34086</offset>
+      <text>SELEX experiments</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>34104</offset>
+      <text>Selection of Roquin-1-bound RNAs from a random RNA library was performed in three rounds of selection with increased stringency of washing (3 × 100 μl, 4 × 100 μl and 5 × 100 μl washing steps) and with decreased protein concentrations (250, 150 and 50 nM). Before selection, 100 μg recombinant Roquin-1 and Roquin-1 M199R N-terminal protein (residues 2–440) were biotinylated: proteins were incubated for 30 min on ice with 10 × molar excess of EZ-link PEG4-NHS-Biotin (Pierce) in PBS (0.1 mg ml−1). Subsequently, the biotinylated protein was purified via gel filtration (MicroSpin column P6, BioRad) and the loss of protein during the biotinylation procedure was estimated by SDS–PAGE and Coomassie staining. The efficiency of the biotinylation reaction was evaluated after spotting of unlabelled and labelled proteins onto a nitrocellulose membrane. After blocking the membrane with 1% BSA in PBS, it was incubated in streptavidin–PE (R-Phycoerythrin) diluted 1:1,000 in PBS for 30 min at room temperature (RT). Subsequently, the membrane was washed three times with PBS and fluorescence intensity of PE bound to biotinylated protein was determined by fluoroimaging (Raytest, FLA5000, 473 nm, Y510 filter).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>35349</offset>
+      <text>The RNA startpool containing the 47-nt random sequence as well as the RNA pools for the second and third selection rounds were transcribed in vitro from double-stranded PCR DNA, and protein-bound RNA was isolated and reverse transcribed before PCR amplification, as previously described. Following transcription, the samples were separated on an 8% PAGE, the bands excised and RNA purified. Every round of selection started by combining the RNA pool (400 pmol) with biotinylated protein and incubating the mix for 30 min at 37 °C. Subsequently, binding buffer-equilibrated streptavidin-magnetic beads were added and incubated (10 min, 37 °C) to bind the protein–RNA complexes, followed by washes. By boiling the beads in 0.2 mM EDTA in water for 3 min, protein and RNA molecules were released. After removal of beads, the solution served as template for reverse transcription (One-Step RT-PCR Kit, Qiagen) and from the obtained complementary DNA the RNA pool of the next round of selection was transcribed. The cDNAs from every selection round (startpool, round 1, round 2 and round 3) were used for Index-PCRs to analyse the pool composition at every stage during selection. Comparable amounts of the PCR products were combined to one cDNA library and analysed by Solexa Illumina sequencing.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>36660</offset>
+      <text>Sequence motif and structural analysis</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>36699</offset>
+      <text>To identify sequence motifs to which Roquin specifically binds, we counted the number of occurrences of each hexamer (46=4,096 motifs) in the sequences obtained by SELEX. We then generated a data set of randomized sequences of the same nucleotide composition as the SELEX-derived sequences, by permuting the SELEX-derived sequences with a custom script. Finally, we counted the number of occurrences of each hexamer in the set of randomized sequences and computed the log2 ratio of the number of occurrences of each motif in the real and randomized sequence sets. To identify a shared sequence motif in the SELEX patterns that showed the strongest enrichment in our selection experiments, the top 100 patterns were analysed with the Motif-based sequence analysis tool MEME (http://meme-suite.org) using the default settings. This analysis revealed three sequence motifs of which the first is shown in Fig. 1b. For the construction of sequence logos, we screened the obtained nucleotide sequences from SELEX replicate 1 and extracted the nucleotide sequences including the 7-nt flanking regions. Sequence logos were constructed with WebLogo 2.8.2 (http://weblogo.berkeley.edu/).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>37877</offset>
+      <text>For the Ox40 3′-UTR sequence alignment, we extracted Multiz alignments of 60 Vertebrates from the UCSC mouse GRCm38/mm10 assembly for the genomic region chr4:156,016,498–156,016,520. For each species contained in the alignment, we extracted genomic coordinates of the aligned sequence, extended the coordinates by 10 nt upstream and downstream, and retrieved the extended sequences from the corresponding genome assemblies. Finally, sequences were aligned with ClustalW 2.1 with standard settings and the alignment was visualized using Jalview.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>38428</offset>
+      <text>To evaluate the structural context the inferred motif is located in, we first appended to the nucleotide sequences obtained from the SELEX experiment the SELEX primers 5′-GGAGAGATGTGAACTT-3′ and 5′-AGTTTCGTGGATGCCAC-3′ to the 5′- and 3′-end, respectively. Next, we screened for sequences that contained the inferred motif and performed secondary structure prediction on those sequences with RNAfold from the ViennaRNA package version 1.8 with parameters '-p -d2'. Next, we used a custom Perl script to parse the base-pairing probability file generated by RNAfold and to calculate an average base-pair probability over all sequences that contained the inferred motif.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>39107</offset>
+      <text>Production of proteins</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>39130</offset>
+      <text>Cloning of expression vectors for Roquin-1 ROQ (residues 147–326), ROQ (residues 171–326) and Roquin-1 N-term (residues 2–440) was carried out by standard procedures as described previously. Briefly, PCR-amplified fragments were put into pETM11 and pETTrx1a vectors based on pET24d as provided by the Protein Expression and Purification Facility at Helmholtz Zentrum München. All vectors contained tobacco etch virus (TEV) protease recognition sites for subsequent proteolytic removal of the tags. All length-variable Roquin-1 expression constructs were designed and cloned via restriction sites NcoI (5′) and XhoI (3′). ROQ domain RNA-binding mutants were cloned by Quick change PCR with high-fidelity Phusion DNA polymerase and subsequent treatment with DnpI. Alternatively, we used conventional cloning with a two-step PCR protocol and enzymatic restriction.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>40003</offset>
+      <text>The Roquin-1 fragments (147–326) and (171–326) were expressed as N-terminal His6-thioredoxin fusion proteins as recently described. Isotope-labelled protein for NMR studies was expressed in M9 minimal medium supplemented with 0.5 g l−1 15N ammonium chloride and 2 g l−1unlabelled or [U-13C] glucose. For the preparation of deuterated proteins, cells were adapted and grown as described previously. Briefly, we used a protocol with stepwise adaptation of cells to deuterium changing buffer from no D2O, low glucose to 50% D2O, low glucose and finally 99.5% D2O with deuterated glucose. The Roquin-1 N-terminal domain (residues 2–440) was expressed and purified essentially as described above for the ROQ domain, but no thioredoxin tag was used. For Roquin-1 N-terminal domain, all expression media and the final buffer contained 100 or 25 μM of zinc chloride, respectively.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>40897</offset>
+      <text>RNA preparation</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>40913</offset>
+      <text>RNAs were synthesized and purchased from IBA GmbH (Göttingen, Germany), purified via PAGE followed by two steps of desalting. No major impurities were seen in NMR spectra. Complex formation for crystallography and NMR experiments was achieved by dissolving the lyophilized RNA in water or NMR buffer. This stock solution was snap-cooled by boiling at 95 °C for 5 min and transferred to an ice-cold bath for 10 min before aliquoting. All RNAs were stored at −80 °C, to avoid degradation and thermodynamically favoured duplex formation.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>41461</offset>
+      <text>Full length and fragments of Ox40 3′-UTR mRNA were produced by in vitro transcription (IVT) from DNA templates harbouring a T7 promoter site either with direct incorporation of α-32P-labelled UTP or subsequent 3′-labelling of purified RNA with γ-32P-labelled ATP. DNA templates were cloned by primer extension PCR. For IVT, 50–150 nM of DNA were incubated with 11 mM magnesium chloride, 8% (w/v) PEG8000, 1.25 mM of each NTP and 0.05 mg ml−1 of T7 polymerase in 1 × standard reaction buffer for 3–5 h at 37 °C. Labelled RNAs were produced in 50 μl reactions and purified via spin columns and directly subjected to EMSA assays. Unlabelled RNAs were produced in reactions of 500–5,000 μl. After IVT, the reactions were separated on 8% denaturing SDS–PAGEs, RNA of interest excised and eluted from the gel using the Elutrap kit (GE Healthcare). After elution, RNAs were dialysed against water and lyophilized. Subsequently, RNAs were dissolved in water and stocks generated by boiling them at 95 °C for 5 min and transferred to an ice-cold bath for 10 min before aliquoting. Labelling for EMSA assays was carried out as for short motifs and described recently. As a modification, dephosphorylation was performed for 30 min and 3′-phosphorylation with γ-32P-labelled ATP and T4 polynucleotide kinase for 90 min for higher efficiency, respectively.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>42857</offset>
+      <text>NMR spectroscopy</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>42874</offset>
+      <text>NMR measurements of Roquin-1 ROQ (147–326) and ROQ (171–326) were performed in buffers as described, mixed with 10% D2O. Backbone chemical shift assignments of ROQ (171–326) with 1.1- to 1.2-fold excess of the Ox40 ADE-like SL motif or consensus ADE SL RNAs were recorded with protein concentrations of 350–400 μM. HNCA, HNCACB, HNCO, HNcaCO and 3D 15N-edited NOESY spectra were acquired at 298K on Bruker Avance III spectrometers at field strengths corresponding to 600 and 800 MHz proton Larmor frequency, equipped with TCI cryogenic probe heads. Spectra of ROQ in complex with Ox40 CDE-like SL RNA and the RNA alone have been reported before. Spectra were processed with Topspin3.2 and analysed with CCPNMR Analysis and Sparky. For RNA motifs, one- and two-dimensional imino NOESY spectra with water-flip-back WATERGATE were recorded at 600–900 MHz, at 278 and 298 K at 150–350 μM RNA concentrations. Sequential assignments were guided by secondary structure predictions with mfold and supported by 15N chemical shifts from natural abundance SOFAST-HMQC experiments.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>43968</offset>
+      <text>Electrophoretic mobility shift assays</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>44006</offset>
+      <text>The EMSAs with ROQ domain and individual motifs were performed as described previously. In short, for the binding reaction a mastermix containing transfer RNA, 32P-labelled SL RNA and reaction buffer was prepared and then mixed with dilutions of the recombinant proteins to achieve the indicated protein concentrations. The binding was performed for 10 min at RT or 20 min on ice in 20 μl reaction volume in the presence of 2.5 μg μl−1 tRNA from baker’s yeast (Sigma), 500 pM 32P-labelled RNA, 20 mM HEPES (pH 7.4), 50 mM NaCl, 1 mM MgCl2, 1 mM dithiothreitol and 1 μg μl−1 BSA. For the binding reaction of Roquin-1 N-terminal with full-length Ox40 3′-UTRs or fragments thereof, ∼1 pmol of RNA was incubated with protein concentrations between 0 and 1,000 μM in a volume of 20 μl. RNP complexes were resolved by PAGE (6% polyacrylamide, 5% glycerol, 0.5 × TBE) at 120 V for 40 min at RT. Gels were then fixed, dried and exposed to a phosphor imager screen.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>45015</offset>
+      <text>X-ray crystallography</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>45037</offset>
+      <text>The crystallization experiments for ROQ–RNA complexes were performed at the X-ray Crystallography Platform at Helmholtz Zentrum München. The crystals of both, Roquin-1 ROQ (171–326) with Ox40 ADE-like SL motif (22mer, 5′-UCCACACCGUUCUAGGUGCUGG-3′) and with the consensus SELEX-derived ADE SL motif (20mer, 5′-UGACUGCGUUUUAGGAGUUA-3′) were obtained from the same condition: 100 mM Bis-Tris buffer pH 5.5, 200 mM sodium chloride and 25% (v/w) PEG 3350. Crystallization was performed using the sitting-drop vapour-diffusion method at 292 K in 24-well plates and a protein concentration of 12 mg ml−1. The crystals appeared after 1 day. For the X-ray diffraction experiments, the crystals of both co-complexes were mounted in a nylon fibre loop and flash cooled to 100 K in liquid nitrogen. The cryoprotection was performed for 2 s in reservoir solution complemented with 20% (v/v) ethylene glycol. Diffraction data for ROQ Ox40 ADE-like motif was collected on the ID29 beamline (ESRF, Grenoble, France) using a Pilatus 6M at a wavelength of 1.25363 Å. Diffraction data for the ROQ-ADE complex were collected using Pilatus 2M detector at 1.00003 Å wavelength at PXIII beamline at SLS (Villigen, Switzerland). All data sets were indexed and integrated using XDS and scaled using SCALA. Intensities were converted to structure–factor amplitudes using the programme TRUNCATE. Table 1 summarizes data collection and processing statistics for both data sets.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>46524</offset>
+      <text>Structure determination and refinement</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>46563</offset>
+      <text>The structure of both ROQ-Ox40 ADE-like SL and ROQ-ADE SL were solved by molecular replacement using the native Roquin-1 ROQ (147–326) structure as a search model (PDB: 4QI0 (ref.)). Model building was performed in COOT. RNA molecules were modelled manually. The refinement of both structures was done in REFMAC5 (ref.) using the maximum-likelihood target function including translation, libration and screw-rotation displacements of a pseudo-rigid body (TLS). For the ROQ-ADE SL structure, non-crystallographic symmetry (NCS) averaging was implemented. The final models are characterized by R and Rfree factors of 21.8 and 25.7% for ROQ-Ox40 ADE-like SL, and 18.6 and 23.4% for ROQ-ADE SL (Table 1), respecively. The stereochemical analysis of both final models was done in PROCHECK and MolProbity. It indicates that there are no residues with generously allowed or unfavourable backbone dihedral angles, and that 99.4% (for ROQ-Ox40 ADE-like SL structure) and 92.3% (for ROQ-ADE SL structure) of all residues are in the core region of the Ramachandran plot.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>47625</offset>
+      <text>Functional assays</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>47643</offset>
+      <text>Functional assays determining the Roquin-mediated regulation of Ox40 with different 3′-UTR variants were performed as described previously. In brief, Rc3h1/2−/− mouse embryonic fibroblast (MEF) cells, stably transduced with a doxycycline-inducible Roquin-1-p2A-mCherry construct, were retrovirally infected with Ox40 constructs of different 3′-UTR length or mutation, which led to the expression of Ox40 on the cell surface (CDE-like mutation changing nt 14–16 GCA to CGT, ADE-like mutation changing nt 15–17 from GGT to CCA). Forty-eight hours after infection, the cells were split and one half of the cells was treated with doxycycline (1 μg ml−1), to induce expression of Roquin-1 and mCherry, connected via the self-cleaving peptide p2A. Thus, Roquin-expressing cells were marked by mCherry expression. Sixteen to 20 h after induction, the cells were harvested, stained with allophycocyanin (APC)-conjugated anti-Ox40 and analysed by flow cytometry. To compare the Ox40 expression levels achieved by different constructs, the relative Ox40 mean fluorescence intensity (MFI) was determined by dividing the MFI of treated (mCherry+) cells by the MFI of untreated (mCherry−) cells.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>48849</offset>
+      <text>Mouse experiments</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>48867</offset>
+      <text>Compound mutant mice with the Rc3h1fl/fl (ref.) and Rc3h2fl/fl (ref.) (denoted Rc3h1/2fl/fl), as well as Cd4-Cre-ERT2 (ref.) and Gt(ROSA)26Sortm1(rtTA*M2)Jae alleles were maintained on a C57BL/6 genetic background. All animals were housed in a pathogen-free barrier facility in accordance with the Ludwig-Maximilians-University München institutional, state and federal guidelines.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>49249</offset>
+      <text>Generation of overexpression vectors</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>49286</offset>
+      <text>Expression constructs of Roquin-1 and Ox40 were cloned into a modified pRetroX-Tight vector (Clontech). The puromycine-resistance cassette was removed and a cassette containing attR1-ccdB-attR2 was inserted, to generate a Gateway destination vector. Roquin-1 and Ox40 constructs were inserted by LR reaction (Invitrogen). Any mutants thereof were generated by site-directed mutagenesis.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>49673</offset>
+      <text>Virus production</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>49690</offset>
+      <text>Replication-deficient retrovirus production and T-cell transduction was performed as previously described. Briefly, retroviral and packaging plasmids were introduced into HEK293T cells by calcium-phosphate transfection. Forty-eight hours after transfection, cell culture supernatants containing the retrovirus particles were harvested, passed through 0.45-μm filters and stored at −80 °C.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>50085</offset>
+      <text>Cell isolation and culture</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>50112</offset>
+      <text>Splenocytes were isolated from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice. CD4+ T cells were isolated by negative selection with magnetic beads according to the manufacturer’s instructions (Stem Cell Technologies). CD4+ T cells were cultured in DMEM medium supplemented with 10% (vol/vol) fetal bovine serum, 1 × nonessential amino acids (Lonza), 10 mM HEPES pH 7.4 (Invitrogen), 50 μM β-mercaptoethanol (Invitrogen) and 100 U ml−1 penicillin–streptomycin (Invitrogen). Rc3h1/2fl/fl deletion was induced by addition of 4′OH-Tamoxifen (0.3 μM) for 24 h. For TH1 differentiation, CD4+ T cells were cultured in six-well plates pre-coated with goat anti-hamster IgG (MP Biochemicals) and DMEM medium further supplemented with anti-CD3 (0,25 μg ml−1), anti-CD28 (2,5 μg ml−1), IL-12 (10 ng ml−1) and anti-IL-4 (10 μg ml−1) for 40 h. Cells were then infected with retroviral constructs, allowing reconstitution with either Roquin-1, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A, and cultured in IL-2 containing media (20 U ml−1). Forty-eight hours after transduction, the cells were split and one half of cells was treated with doxycycline (1 μg ml−1), to induce expression of Roquin-1 WT and Roquin-1 mutants. Twenty-four hours after induction, the cells were harvested for analysis by immunoblot and flow cytometry with the indicated antibodies (1:200 anti-mouse Icos-biotin clone 7E–17G9 (eBioscience); 1:200 Streptavidin-PerCP (Becton Dickinson); 1:200 anti-mouse Ox40-PE clone OX-86 (eBioscience)).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>51678</offset>
+      <text>Immunoblot analysis</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>51698</offset>
+      <text>CD4+ T cells were incubated for 15 min on ice with lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.25% (vol/vol) Nonidet-P40, 1.5 mM MgCl2 and protease inhibitor mix without EDTA (Roche) and 1 mM dithiothreitol). Lysate was cleared by centrifugation (10 min, 10 000 g, 4 °C). Immunoblotting was performed by standard protocols with hybridoma supernatants containing monoclonal antibody recognizing Roquin-1 and Roquin-2 (anti-roquin, clone 3F12).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>52168</offset>
+      <text>mRNA decay experiments</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>52191</offset>
+      <text>Hela Tet-Off Advanced Cells (Clontech 631156) were stably transduced with retroviruses expressing different Ox40 constructs. FACS analysis 41 h post transduction revealed similar Ox40 surface expression levels on all five cell samples. After transduction, the cell lines were initially cultured for at least 48 h without doxycycline, to ensure high Ox40-expression levels. For each time point, 400 000 cells were spread on one well in a six-well plate. To switch off Ox40-transcription, doxycycline was supplied with the medium at time point 0. After one washing step with PBS, cells were directly harvested from each well with Trizol before Dox application (0 h), as well as 2, 3 and 4 h after Dox application. RNA was isolated using standard Trizol protocols. Reverse transcription was performed with the Qiagen Quantitect Reverse Transcription Kit following the manufacturer’s protocols. Quantitative PCR was carried out on a Roche Light Cycler 480 using the Light Cycler 480 Probes Master Mix and primer-/probe-combinations from Roches Universal Probe Library. Relative mRNA expression levels were calculated by normalization to the housekeeper gene ywhaz.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_2</infon>
+      <offset>53364</offset>
+      <text>Surface plasmon resonance</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>53390</offset>
+      <text>ROQ–RNA binding experiments were performed on a BIACORE 3000 instrument (Biacore Inc.). ROQ domain was diluted to a final concentration of 35 μg ml−1 in 10 mM HEPES pH 7.0 and chemically immobilized (amine coupling) onto CM5 sensor chips (Biacore Inc.). The RNA samples were diluted in the running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 2 mM MgCl2 and 0.005% Tween 20) to the final concentration of 31.25, 62.5, 125, 250 and 500 nM, and 1 and 2 μM, and injected over the sensor chip surface at 30 μl min−1 at 10 °C. The RNA samples were injected onto the sensor chip from the lowest to the highest concentration. Each RNA-type sample was tested three times with the exception of Mut1–3 two times. Injection of 250 nM RNA was always performed in duplicate within each experiment. To subtract any background noise from each data set, all samples were also run over an unmodified sensor chip surface. Data were analysed using BIAevaluation programme (Biacore Inc.) (Supplementary Fig. 7). For each measurement, the equilibrium dissociation constant was calculated (KD) from steady state binding. The KD from three independent experiments were used to calculate the mean values of these variables and the s.e.m. The results for all tested RNA samples are compared in Table 2.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">title_1</infon>
+      <offset>54700</offset>
+      <text>Additional information</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>54723</offset>
+      <text>Accession codes: Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5F5H and 5F5F for the ROQ-Ox40 ADE-like SL and ROQ-ADE SL, respectively. Chemical shifts of the ROQ-Ox40 ADE-like SL and ROQ-ADE SL have been deposited in the Biological Magnetic Resonance Data Bank under accession codes 26587 and 26588, respectively.</text>
+    </passage>
+    <passage>
+      <infon key="section_type">METHODS</infon>
+      <infon key="type">paragraph</infon>
+      <offset>55100</offset>
+      <text>How to cite this article: Janowski, R. et al. Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40. Nat. Commun. 7:11032 doi: 10.1038/ncomms11032 (2016).</text>
+    </passage>
+    <passage>
+      <infon key="section_type">SUPPL</infon>
+      <infon key="type">title_1</infon>
+      <offset>55278</offset>
+      <text>Supplementary Material</text>
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+      <infon key="type">footnote</infon>
+      <offset>59039</offset>
+      <text>Author contributions A.S. carried out cloning, protein expression and purification, and NMR experiments. R.J. performed crystallization and structure determination. G.A.H. carried out EMSA assays and SELEX experiments, and functional studies were performed by G.A.H, N.W. and S.B. M.B. and R.B. helped setting up the SELEX experiments and identified patterns from NGS data, on the basis of which A.G. and M.Z. analysed the motifs and secondary structures. T.B. contributed unpublished reagents and advice. A.S., G.A.H., R.J., V.H., D.N. and M.S. designed the project and wrote the paper. All authors discussed the results and commented on the manuscript.</text>
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+      <text>SELEX identifies a novel SL RNA ligand of Roquin-1.</text>
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+      <text>(a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences. (c) Conservation of the motif found in Ox40 3′-UTRs for various species as indicated. The labels correspond to the versions of the genome assemblies in the UCSC server (see Method section). rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL. The broken line between the G–G base pair in the ADE SL indicates a putative non-Watson–Crick pairing. The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison. See also Supplementary Fig. 1.</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">protein</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
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+      <infon key="section_type">FIG</infon>
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+      <offset>60600</offset>
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+        <infon key="annotator">cleaner0</infon>
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+      <text>Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). The respective SL RNAs and their base pairs are indicated. Red asterisks indicate NMR signals of the protein. Black asterisks in a indicate a second conformation (see Supplementary Notes). Green lines in the secondary structure schemes on the left refer to visible imino NMR signals and thus experimental confirmation of the base pairs indicated. Red nucleotides indicate significant chemical shift changes observed. The dotted green line between G6 and G15 in a highlights a G–G base pair.</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:49Z</infon>
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+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
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+      <text>Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA.</text>
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+      <text>(a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta). Selected RNA bases and protein secondary structure elements are labelled. (b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green). Only RNA-interacting residues that are different in both structures are shown. Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA. The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257). The protein chain is shown in turquoise and the RNA is shown in grey. Atoms are colour coded according to charge. (e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop. U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247. The side chain of Tyr250 makes hydrophobic interactions with the pyrimidine side chain of U10 on one side and U11 on the other side. Lys259 interacts with the phosphate groups of U10 and U11. (f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11. In d – f, amino acids are shown in turquoise and blue, nucleotides in grey colour. See also Supplementary Notes and Supplementary Fig. 2.</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">GO:</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <infon key="type">residue_name_number</infon>
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+        <infon key="updated_at">2023-09-15T12:39:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">residue_name_number</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="62118" length="3"/>
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:58Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="62129" length="4"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
+        <location offset="62143" length="2"/>
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+        <infon key="identifier">CHEBI:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:54Z</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:36:24Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="62203" length="5"/>
+        <text>helix</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:36:29Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="62209" length="2"/>
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+        <infon key="score">0.9995265</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:07:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="62213" length="6"/>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:32:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="62224" length="6"/>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:32:19Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="62259" length="6"/>
+        <text>strand</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:36:35Z</infon>
+        <infon key="identifier">SO:</infon>
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+        <infon key="type">residue_name_number</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="identifier">CHEBI:</infon>
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+        <text>RNA</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
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+        <text>RNA</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="62505" length="8"/>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="62557" length="5"/>
+        <text>helix</text>
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+        <infon key="score">0.9994973</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">SO:</infon>
+        <location offset="62563" length="2"/>
+        <text>α4</text>
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+        <infon key="score">0.999548</infon>
+        <infon key="type">residue_name_number</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="62576" length="6"/>
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+        <infon key="type">residue_name_number</infon>
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+        <infon key="identifier">DUMMY:</infon>
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+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="62626" length="24"/>
+        <text>hydrophobic interactions</text>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="62812" length="23"/>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="62844" length="6"/>
+        <text>Val257</text>
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+        <infon key="type">residue_name_number</infon>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:38:51Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="62882" length="13"/>
+        <text>hydrogen bond</text>
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+      <annotation id="941">
+        <infon key="score">0.9994991</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="62899" length="6"/>
+        <text>Lys259</text>
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+      <annotation id="942">
+        <infon key="score">0.9987135</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">DUMMY:</infon>
+        <location offset="62931" length="3"/>
+        <text>U10</text>
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+      <annotation id="943">
+        <infon key="score">0.99837035</infon>
+        <infon key="type">residue_name_number</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:39:21Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="62939" length="3"/>
+        <text>U11</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">ncomms11032-f4.jpg</infon>
+      <infon key="id">f4</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>63093</offset>
+      <text>NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA.</text>
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+        <infon key="score">0.99871564</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:11:01Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="63093" length="3"/>
+        <text>NMR</text>
+      </annotation>
+      <annotation id="945">
+        <infon key="score">0.99909353</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="63109" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+      <annotation id="946">
+        <infon key="score">0.31922945</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="63142" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="1140">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:20Z</infon>
+        <location offset="63147" length="3"/>
+        <text>ADE</text>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:32:58Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="63156" length="8"/>
+        <text>hexaloop</text>
+      </annotation>
+      <annotation id="948">
+        <infon key="score">0.99889934</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:54Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="63165" length="3"/>
+        <text>RNA</text>
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+    </passage>
+    <passage>
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+      <infon key="id">f4</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>63170</offset>
+      <text>(a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red). Selected shifts of amide resonances are indicated. (b) Plot of chemical shift change versus residue number in the ROQ domain (residues 171–326) from a. Grey negative bars indicate missing assignments in one of the spectra. Gaps indicate prolines. (c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green). See also Supplementary Notes and Supplementary Fig. 3.</text>
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+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:33:09Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="63174" length="7"/>
+        <text>Overlay</text>
+      </annotation>
+      <annotation id="950">
+        <infon key="score">0.9984504</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
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+        <infon key="identifier">MESH:</infon>
+        <location offset="63185" length="11"/>
+        <text>1H,15N HSQC</text>
+      </annotation>
+      <annotation id="951">
+        <infon key="score">0.9905441</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:30:44Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="63197" length="7"/>
+        <text>spectra</text>
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+      <annotation id="952">
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:33:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="63219" length="4"/>
+        <text>free</text>
+      </annotation>
+      <annotation id="953">
+        <infon key="score">0.99527836</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="63224" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+      <annotation id="954">
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+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:33:02Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="63236" length="7"/>
+        <text>171–326</text>
+      </annotation>
+      <annotation id="955">
+        <infon key="score">0.998635</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:33:34Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="63255" length="15"/>
+        <text>in complex with</text>
+      </annotation>
+      <annotation id="956">
+        <infon key="score">0.9983432</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="63301" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="1141">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:21Z</infon>
+        <location offset="63306" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="1227">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
+        <location offset="63315" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="957">
+        <infon key="score">0.8958258</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:33:18Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="63388" length="21"/>
+        <text>chemical shift change</text>
+      </annotation>
+      <annotation id="958">
+        <infon key="score">0.9957028</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="63439" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+      <annotation id="959">
+        <infon key="score">0.99748224</infon>
+        <infon key="type">residue_range</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:33:29Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="63460" length="7"/>
+        <text>171–326</text>
+      </annotation>
+      <annotation id="1299">
+        <infon key="type">evidence</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:30:44Z</infon>
+        <location offset="63539" length="7"/>
+        <text>spectra</text>
+      </annotation>
+      <annotation id="960">
+        <infon key="score">0.9977138</infon>
+        <infon key="type">residue_name</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:33:52Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="63562" length="8"/>
+        <text>prolines</text>
+      </annotation>
+      <annotation id="961">
+        <infon key="score">0.99569786</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:58:02Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="63576" length="7"/>
+        <text>Overlay</text>
+      </annotation>
+      <annotation id="962">
+        <infon key="score">0.9927193</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="63591" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+      <annotation id="963">
+        <infon key="score">0.9894419</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:33:38Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="63602" length="5"/>
+        <text>alone</text>
+      </annotation>
+      <annotation id="964">
+        <infon key="score">0.99848765</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:33:35Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="63619" length="15"/>
+        <text>in complex with</text>
+      </annotation>
+      <annotation id="965">
+        <infon key="score">0.9983423</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="63639" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="1142">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:21Z</infon>
+        <location offset="63644" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="1228">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
+        <location offset="63653" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="966">
+        <infon key="score">0.9987739</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="63669" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="1092">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:49Z</infon>
+        <location offset="63674" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="1229">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
+        <location offset="63683" length="2"/>
+        <text>SL</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">ncomms11032-f5.jpg</infon>
+      <infon key="id">f5</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>63754</offset>
+      <text>Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR.</text>
+      <annotation id="967">
+        <infon key="score">0.99873173</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:23:49Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="63754" length="19"/>
+        <text>Mutational analysis</text>
+      </annotation>
+      <annotation id="968">
+        <infon key="score">0.9989117</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:37Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="63777" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+      <annotation id="969">
+        <infon key="score">0.9989034</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="63804" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="1143">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:21Z</infon>
+        <location offset="63809" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="1230">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
+        <location offset="63818" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="970">
+        <infon key="score">0.99886</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="63825" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="971">
+        <infon key="score">0.99926394</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:56Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="63830" length="6"/>
+        <text>3′-UTR</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">ncomms11032-f5.jpg</infon>
+      <infon key="id">f5</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>63840</offset>
+      <text>(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).</text>
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+        <infon key="score">0.9986687</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:34:27Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="63844" length="10"/>
+        <text>EMSA assay</text>
+      </annotation>
+      <annotation id="973">
+        <infon key="score">0.99906</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:56:48Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="63880" length="9"/>
+        <text>wild-type</text>
+      </annotation>
+      <annotation id="974">
+        <infon key="score">0.9991366</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:47:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="63901" length="5"/>
+        <text>Y250A</text>
+      </annotation>
+      <annotation id="975">
+        <infon key="score">0.9991787</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:47:05Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="63907" length="6"/>
+        <text>mutant</text>
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+      <annotation id="976">
+        <infon key="score">0.99912864</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="63914" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+      <annotation id="977">
+        <infon key="score">0.9992931</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="63944" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="1144">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:21Z</infon>
+        <location offset="63949" length="3"/>
+        <text>ADE</text>
+      </annotation>
+      <annotation id="1231">
+        <infon key="type">structure_element</infon>
+        <infon key="identifier">SO:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
+        <location offset="63958" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="978">
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+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:03Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="63996" length="3"/>
+        <text>Tnf</text>
+      </annotation>
+      <annotation id="979">
+        <infon key="score">0.9992507</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:08:49Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="64000" length="3"/>
+        <text>CDE</text>
+      </annotation>
+      <annotation id="980">
+        <infon key="score">0.9995161</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:17:15Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="64004" length="2"/>
+        <text>SL</text>
+      </annotation>
+      <annotation id="981">
+        <infon key="score">0.99809736</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:58:08Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="64176" length="14"/>
+        <text>Flow cytometry</text>
+      </annotation>
+      <annotation id="982">
+        <infon key="score">0.9991708</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="64194" length="4"/>
+        <text>Ox40</text>
+      </annotation>
+      <annotation id="983">
+        <infon key="score">0.99922466</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:50:12Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="64203" length="4"/>
+        <text>Icos</text>
+      </annotation>
+      <annotation id="1332">
+        <infon key="type">gene</infon>
+        <infon key="identifier">GENE:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:04:32Z</infon>
+        <location offset="64250" length="5"/>
+        <text>Rc3h1</text>
+      </annotation>
+      <annotation id="1336">
+        <infon key="type">gene</infon>
+        <infon key="identifier">GENE:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:40:24Z</infon>
+        <location offset="64256" length="3"/>
+        <text>2fl</text>
+      </annotation>
+      <annotation id="1337">
+        <infon key="type">gene</infon>
+        <infon key="identifier">GENE:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:40:33Z</infon>
+        <location offset="64260" length="2"/>
+        <text>fl</text>
+      </annotation>
+      <annotation id="1155">
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:12:08Z</infon>
+        <location offset="64283" length="4"/>
+        <text>mice</text>
+      </annotation>
+      <annotation id="984">
+        <infon key="score">0.99808097</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:52:07Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="64301" length="9"/>
+        <text>tamoxifen</text>
+      </annotation>
+      <annotation id="1333">
+        <infon key="type">gene</infon>
+        <infon key="identifier">GENE:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:38:23Z</infon>
+        <location offset="64328" length="5"/>
+        <text>Rc3h1</text>
+      </annotation>
+      <annotation id="1338">
+        <infon key="type">gene</infon>
+        <infon key="identifier">GENE:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:40:55Z</infon>
+        <location offset="64334" length="3"/>
+        <text>2fl</text>
+      </annotation>
+      <annotation id="1339">
+        <infon key="type">gene</infon>
+        <infon key="identifier">GENE:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:41:04Z</infon>
+        <location offset="64338" length="2"/>
+        <text>fl</text>
+      </annotation>
+      <annotation id="1340">
+        <infon key="type">experimental_method</infon>
+        <infon key="identifier">MESH:</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:41:12Z</infon>
+        <location offset="64341" length="8"/>
+        <text>deletion</text>
+      </annotation>
+      <annotation id="985">
+        <infon key="score">0.8627631</infon>
+        <infon key="type">taxonomy_domain</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:04:28Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="64451" length="10"/>
+        <text>retrovirus</text>
+      </annotation>
+      <annotation id="986">
+        <infon key="score">0.9809732</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:52:23Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="64475" length="11"/>
+        <text>doxycycline</text>
+      </annotation>
+      <annotation id="987">
+        <infon key="score">0.99905056</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:37Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="64518" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+      <annotation id="988">
+        <infon key="score">0.999236</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:47:00Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="64527" length="2"/>
+        <text>WT</text>
+      </annotation>
+      <annotation id="989">
+        <infon key="score">0.99354047</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:37Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="64531" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+      <annotation id="990">
+        <infon key="score">0.99891853</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:47:16Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="64540" length="5"/>
+        <text>Y250A</text>
+      </annotation>
+      <annotation id="991">
+        <infon key="score">0.9958498</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:37Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="64549" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+      <annotation id="992">
+        <infon key="score">0.9989888</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:47:27Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="64558" length="5"/>
+        <text>K220A</text>
+      </annotation>
+      <annotation id="993">
+        <infon key="score">0.99904567</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:47:32Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="64565" length="5"/>
+        <text>K239A</text>
+      </annotation>
+      <annotation id="994">
+        <infon key="score">0.9991043</infon>
+        <infon key="type">mutant</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:08:08Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="64575" length="5"/>
+        <text>R260A</text>
+      </annotation>
+      <annotation id="995">
+        <infon key="score">0.9720185</infon>
+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:06:07Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="64581" length="7"/>
+        <text>mutants</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">ncomms11032-f6.jpg</infon>
+      <infon key="id">f6</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_title_caption</infon>
+      <offset>64624</offset>
+      <text>Functional importance of Roquin-1 target motifs in cells.</text>
+      <annotation id="996">
+        <infon key="score">0.99915963</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:37Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="64649" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+    </passage>
+    <passage>
+      <infon key="file">ncomms11032-f6.jpg</infon>
+      <infon key="id">f6</infon>
+      <infon key="section_type">FIG</infon>
+      <infon key="type">fig_caption</infon>
+      <offset>64682</offset>
+      <text>(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). Arrows indicate the individual binding events to either motif. It is noteworthy that the higher bands observed at large protein concentrations are probably additional nonspecific, lower-affinity interactions of Roquin-1 with the 3′-UTR or protein aggregates. (c) Relative Ox40 MFI normalized to expression levels from the Ox40 CDS construct. Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments. Statistical significance was calculated by one-way analysis of variance (ANOVA) Kruskal–Wallis test followed by Dunn’s multiple comparison test (**P&lt;0.01). (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). Error bars represent the mean of technical duplicates in one experiment. mRNA half-life times were calculated with Graph Pad Prism. Data are representative of two experiments with similar results.</text>
+      <annotation id="997">
+        <infon key="score">0.99841917</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:59Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="64702" length="4"/>
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+        <text>mutated</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <text>mut</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:59Z</infon>
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+        <location offset="66053" length="4"/>
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+        <infon key="type">protein_state</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:41:57Z</infon>
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+        <text>full length</text>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:09:21Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="66083" length="3"/>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="annotator">cleaner0</infon>
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+      <infon key="section_type">TABLE</infon>
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+        <infon key="annotator">cleaner0</infon>
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+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table</infon>
+      <infon key="xml">&lt;?xml version="1.0" encoding="UTF-8"?&gt;
+&lt;table frame="hsides" rules="groups" border="1"&gt;&lt;colgroup&gt;&lt;col align="left"/&gt;&lt;col align="center"/&gt;&lt;col align="center"/&gt;&lt;/colgroup&gt;&lt;thead valign="bottom"&gt;&lt;tr&gt;&lt;th align="left" valign="top" charoff="50"&gt; &lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;&lt;bold&gt;ROQ-&lt;/bold&gt;&lt;italic&gt;&lt;bold&gt;Ox40&lt;/bold&gt;&lt;/italic&gt;
+&lt;bold&gt;ADE-like SL&lt;/bold&gt;&lt;/th&gt;&lt;th align="center" valign="top" charoff="50"&gt;&lt;bold&gt;ROQ-ADE SL&lt;/bold&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign="top"&gt;&lt;tr&gt;&lt;td colspan="3" align="center" valign="top" charoff="50"&gt;&lt;italic&gt;Data collection&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; space group&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&lt;italic&gt;P&lt;/italic&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;&lt;italic&gt;P&lt;/italic&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="3" align="center" valign="top" charoff="50"&gt; Cell dimensions&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; &lt;italic&gt;a&lt;/italic&gt;, &lt;italic&gt;b&lt;/italic&gt;, &lt;italic&gt;c&lt;/italic&gt; (Å)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;89.66, 115.79, 42.61&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;72.90, 89.30, 144.70&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; &lt;italic&gt;α, β, γ&lt;/italic&gt; (°)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;90, 90, 90&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;90, 90, 90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Resolution (Å)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;50–2.23 (2.29–2.23)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;50–3.0 (3.08–3.00)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;merge&lt;/sub&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;5.9 (68.3)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;14.8 (93.8)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; &lt;italic&gt;I&lt;/italic&gt;/σ&lt;italic&gt;I&lt;/italic&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;14.9 (2.1)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;16.7 (3.1)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Completeness (%)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;98.7 (97.7)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;99.9 (99.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Redundancy&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;3.9 (3.7)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;13.2 (12.7)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="3" align="center" valign="top" charoff="50"&gt;&lt;italic&gt;Refinement&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Resolution (Å)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;2.23&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;3.00&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; No. reflections&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;21,018&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;18,598&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;work&lt;/sub&gt;/&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt;&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;21.8/25.7&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;18.6/23.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="3" align="center" valign="top" charoff="50"&gt; No. atoms&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Protein&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;2,404&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;4,820&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Ligand/ion&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;894&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;1,708&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Water&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;99&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;49&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt;&lt;italic&gt; B&lt;/italic&gt;-factor overall&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;47.2&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;60.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="3" align="center" valign="top" charoff="50"&gt;&lt;italic&gt;Root mean squared deviations&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Bond lengths (Å)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;0.006&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;0.014&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Bond angles (°)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;1.07&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;1.77&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan="3" align="center" valign="top" charoff="50"&gt;Ramachandran plot&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Most favoured (%)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;98.6&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;99.8&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align="left" valign="top" charoff="50"&gt; Additional allowed (%)&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;1.4&lt;/td&gt;&lt;td align="center" valign="top" charoff="50"&gt;0.2&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon>
+      <offset>66389</offset>
+      <text> 	ROQ-Ox40ADE-like SL	ROQ-ADE SL	 	Data collection	 	 space group	P21212	P212121	 	 	 	 	 	 Cell dimensions	 	 a, b, c (Å)	89.66, 115.79, 42.61	72.90, 89.30, 144.70	 	 α, β, γ (°)	90, 90, 90	90, 90, 90	 	 Resolution (Å)	50–2.23 (2.29–2.23)	50–3.0 (3.08–3.00)	 	 Rmerge	5.9 (68.3)	14.8 (93.8)	 	 I/σI	14.9 (2.1)	16.7 (3.1)	 	 Completeness (%)	98.7 (97.7)	99.9 (99.9)	 	 Redundancy	3.9 (3.7)	13.2 (12.7)	 	 	 	 	 	Refinement	 	 Resolution (Å)	2.23	3.00	 	 No. reflections	21,018	18,598	 	 Rwork/Rfree	21.8/25.7	18.6/23.4	 	 	 	 	 	 No. atoms	 	 Protein	2,404	4,820	 	 Ligand/ion	894	1,708	 	 Water	99	49	 	 B-factor overall	47.2	60.4	 	 	 	 	 	Root mean squared deviations	 	 Bond lengths (Å)	0.006	0.014	 	 Bond angles (°)	1.07	1.77	 	 	 	 	 	Ramachandran plot	 	 Most favoured (%)	98.6	99.8	 	 Additional allowed (%)	1.4	0.2	 	</text>
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+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:54Z</infon>
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+        <infon key="identifier">PR:</infon>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T14:02:09Z</infon>
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+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_footnote</infon>
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+      <text>ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop.</text>
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+        <infon key="type">structure_element</infon>
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+        <infon key="type">structure_element</infon>
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+        <infon key="updated_at">2023-09-15T12:08:49Z</infon>
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+        <infon key="type">structure_element</infon>
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+        <infon key="annotator">cleaner0</infon>
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+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:48:26Z</infon>
+        <infon key="identifier">SO:</infon>
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+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_footnote</infon>
+      <offset>67373</offset>
+      <text>For each data set, only one crystal has been used.</text>
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+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:48:24Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="67401" length="7"/>
+        <text>crystal</text>
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+      <infon key="id">t1</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_footnote</infon>
+      <offset>67424</offset>
+      <text>*Values in parentheses are for highest-resolution shell.</text>
+    </passage>
+    <passage>
+      <infon key="file"></infon>
+      <infon key="id">t2</infon>
+      <infon key="section_type">TABLE</infon>
+      <infon key="type">table_title_caption</infon>
+      <offset>67481</offset>
+      <text>KD for selected RNAs obtained from SPR measurements with immobilized ROQ domain of Roquin-1.</text>
+      <annotation id="1055">
+        <infon key="score">0.9948697</infon>
+        <infon key="type">evidence</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:48:44Z</infon>
+        <infon key="identifier">DUMMY:</infon>
+        <location offset="67481" length="2"/>
+        <text>KD</text>
+      </annotation>
+      <annotation id="1056">
+        <infon key="score">0.9986085</infon>
+        <infon key="type">chemical</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:23:25Z</infon>
+        <infon key="identifier">CHEBI:</infon>
+        <location offset="67497" length="4"/>
+        <text>RNAs</text>
+      </annotation>
+      <annotation id="1057">
+        <infon key="score">0.99467736</infon>
+        <infon key="type">experimental_method</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T13:48:41Z</infon>
+        <infon key="identifier">MESH:</infon>
+        <location offset="67516" length="16"/>
+        <text>SPR measurements</text>
+      </annotation>
+      <annotation id="1058">
+        <infon key="score">0.8360924</infon>
+        <infon key="type">structure_element</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:21:54Z</infon>
+        <infon key="identifier">SO:</infon>
+        <location offset="67550" length="3"/>
+        <text>ROQ</text>
+      </annotation>
+      <annotation id="1059">
+        <infon key="score">0.9990565</infon>
+        <infon key="type">protein</infon>
+        <infon key="annotator">cleaner0</infon>
+        <infon key="updated_at">2023-09-15T12:07:37Z</infon>
+        <infon key="identifier">PR:</infon>
+        <location offset="67564" length="8"/>
+        <text>Roquin-1</text>
+      </annotation>
+    </passage>
+  </document>
+</collection>
diff --git a/annotation_CSV/PMC4806292.csv b/annotation_CSV/PMC4806292.csv
new file mode 100644
index 0000000000000000000000000000000000000000..1dcb7207dd98eb6dcc92c3e1eaf6a5338cca2fd7
--- /dev/null
+++ b/annotation_CSV/PMC4806292.csv
@@ -0,0 +1,761 @@
+anno_start	anno_end	anno_text	entity_type	sentence	section
+24	47	in vitro reconstitution	experimental_method	Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein	TITLE
+88	93	human	species	Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein	TITLE
+94	99	PI4KB	protein	Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein	TITLE
+107	112	ACBD3	protein	Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein	TITLE
+0	34	Phosphatidylinositol 4-kinase beta	protein	Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells.	ABSTRACT
+36	41	PI4KB	protein	Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells.	ABSTRACT
+58	63	human	species	Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells.	ABSTRACT
+64	68	PI4K	protein_type	Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells.	ABSTRACT
+91	123	phosphatidylinositol 4-phosphate	chemical	Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells.	ABSTRACT
+125	129	PI4P	chemical	Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells.	ABSTRACT
+184	194	eukaryotic	taxonomy_domain	Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells.	ABSTRACT
+35	40	PI4Ks	protein_type	To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment.	ABSTRACT
+0	5	PI4KB	protein	PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown.	ABSTRACT
+81	85	PI4P	chemical	PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown.	ABSTRACT
+136	141	PI4KB	protein	PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown.	ABSTRACT
+20	23	NMR	experimental_method	Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3).	ABSTRACT
+24	33	structure	evidence	Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3).	ABSTRACT
+52	57	PI4KB	protein	Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3).	ABSTRACT
+87	108	Golgi adaptor protein	protein_type	Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3).	ABSTRACT
+109	160	acyl-coenzyme A binding domain containing protein 3	protein	Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3).	ABSTRACT
+162	167	ACBD3	protein	Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3).	ABSTRACT
+13	18	ACBD3	protein	We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.	ABSTRACT
+44	49	PI4KB	protein	We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.	ABSTRACT
+123	128	PI4KB	protein	We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.	ABSTRACT
+132	137	ACBD3	protein	We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.	ABSTRACT
+152	170	enzymatic activity	evidence	We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.	ABSTRACT
+184	195	ACBD3:PI4KB	complex_assembly	We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.	ABSTRACT
+0	34	Phosphatidylinositol 4-kinase beta	protein	Phosphatidylinositol 4-kinase beta (PI4KB, also known as PI4K IIIβ) is a soluble cytosolic protein yet its function is to phosphorylate membrane lipids.	INTRO
+36	41	PI4KB	protein	Phosphatidylinositol 4-kinase beta (PI4KB, also known as PI4K IIIβ) is a soluble cytosolic protein yet its function is to phosphorylate membrane lipids.	INTRO
+57	66	PI4K IIIβ	protein	Phosphatidylinositol 4-kinase beta (PI4KB, also known as PI4K IIIβ) is a soluble cytosolic protein yet its function is to phosphorylate membrane lipids.	INTRO
+18	23	human	species	It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P).	INTRO
+24	28	PI4K	protein_type	It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P).	INTRO
+56	76	phosphatidylinositol	chemical	It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P).	INTRO
+78	80	PI	chemical	It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P).	INTRO
+94	126	phosphatidylinositol 4-phosphate	chemical	It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P).	INTRO
+128	132	PI4P	chemical	It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P).	INTRO
+0	4	PI4P	chemical	PI4P is an essential lipid found in various membrane compartments including the Golgi and trans-Golgi network (TGN), the plasma membrane and the endocytic compartments.	INTRO
+20	24	PI4P	chemical	In these locations, PI4P plays an important role in cell signaling and lipid transport, and serves as a precursor for higher phosphoinositides or as a docking site for clathrin adaptor or lipid transfer proteins.	INTRO
+125	142	phosphoinositides	chemical	In these locations, PI4P plays an important role in cell signaling and lipid transport, and serves as a precursor for higher phosphoinositides or as a docking site for clathrin adaptor or lipid transfer proteins.	INTRO
+168	176	clathrin	protein_type	In these locations, PI4P plays an important role in cell signaling and lipid transport, and serves as a precursor for higher phosphoinositides or as a docking site for clathrin adaptor or lipid transfer proteins.	INTRO
+16	58	positive-sense single-stranded RNA viruses	taxonomy_domain	A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories.	INTRO
+60	72	+RNA viruses	taxonomy_domain	A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories.	INTRO
+109	114	human	species	A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories.	INTRO
+133	138	human	species	A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories.	INTRO
+139	144	PI4KA	protein	A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories.	INTRO
+148	153	PI4KB	protein	A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories.	INTRO
+183	187	PI4P	chemical	A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories.	INTRO
+6	16	structures	evidence	These structures are essential for effective viral replication.	INTRO
+45	50	viral	taxonomy_domain	These structures are essential for effective viral replication.	INTRO
+26	31	PI4KB	protein	Recently, highly specific PI4KB inhibitors were developed as potential antivirals.	INTRO
+0	4	PI4K	protein_type	PI4K kinases must be recruited to the correct membrane type to fulfill their enzymatic functions.	INTRO
+5	12	kinases	protein_type	PI4K kinases must be recruited to the correct membrane type to fulfill their enzymatic functions.	INTRO
+0	13	Type II PI4Ks	protein_type	Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins.	INTRO
+15	21	PI4K2A	protein	Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins.	INTRO
+26	32	PI4K2B	protein	Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins.	INTRO
+38	59	heavily palmitoylated	protein_state	Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins.	INTRO
+79	96	membrane proteins	protein	Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins.	INTRO
+13	27	type III PI4Ks	protein_type	In contrast, type III PI4Ks (PI4KA and PI4KB) are soluble cytosolic proteins that are recruited to appropriate membranes indirectly via protein-protein interactions.	INTRO
+29	34	PI4KA	protein	In contrast, type III PI4Ks (PI4KA and PI4KB) are soluble cytosolic proteins that are recruited to appropriate membranes indirectly via protein-protein interactions.	INTRO
+39	44	PI4KB	protein	In contrast, type III PI4Ks (PI4KA and PI4KB) are soluble cytosolic proteins that are recruited to appropriate membranes indirectly via protein-protein interactions.	INTRO
+19	24	PI4KA	protein	The recruitment of PI4KA to the plasma membrane by EFR3 and TTC7 is relatively well understood even at the structural level, but, the actual molecular mechanism of PI4KB recruitment to the Golgi is still poorly understood.	INTRO
+51	55	EFR3	protein	The recruitment of PI4KA to the plasma membrane by EFR3 and TTC7 is relatively well understood even at the structural level, but, the actual molecular mechanism of PI4KB recruitment to the Golgi is still poorly understood.	INTRO
+60	64	TTC7	protein	The recruitment of PI4KA to the plasma membrane by EFR3 and TTC7 is relatively well understood even at the structural level, but, the actual molecular mechanism of PI4KB recruitment to the Golgi is still poorly understood.	INTRO
+164	169	PI4KB	protein	The recruitment of PI4KA to the plasma membrane by EFR3 and TTC7 is relatively well understood even at the structural level, but, the actual molecular mechanism of PI4KB recruitment to the Golgi is still poorly understood.	INTRO
+0	51	Acyl-coenzyme A binding domain containing protein 3	protein	Acyl-coenzyme A binding domain containing protein 3 (ACBD3, also known as GCP60 and PAP7) is a Golgi resident protein.	INTRO
+53	58	ACBD3	protein	Acyl-coenzyme A binding domain containing protein 3 (ACBD3, also known as GCP60 and PAP7) is a Golgi resident protein.	INTRO
+74	79	GCP60	protein	Acyl-coenzyme A binding domain containing protein 3 (ACBD3, also known as GCP60 and PAP7) is a Golgi resident protein.	INTRO
+84	88	PAP7	protein	Acyl-coenzyme A binding domain containing protein 3 (ACBD3, also known as GCP60 and PAP7) is a Golgi resident protein.	INTRO
+89	98	golgin B1	protein	Its membrane localization is mediated by the interaction with the Golgi integral protein golgin B1/giantin.	INTRO
+99	106	giantin	protein	Its membrane localization is mediated by the interaction with the Golgi integral protein golgin B1/giantin.	INTRO
+0	5	ACBD3	protein	ACBD3 functions as an adaptor protein and signaling hub across cellular signaling pathways.	INTRO
+0	5	ACBD3	protein	ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis.	INTRO
+55	64	golgin A3	protein	ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis.	INTRO
+65	75	golgin-160	protein	ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis.	INTRO
+99	112	Numb proteins	protein_type	ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis.	INTRO
+179	196	metal transporter	protein_type	ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis.	INTRO
+197	201	DMT1	protein	ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis.	INTRO
+206	215	monomeric	oligomeric_state	ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis.	INTRO
+216	225	G protein	protein_type	ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis.	INTRO
+226	233	Dexras1	protein	ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis.	INTRO
+246	250	iron	chemical	ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis.	INTRO
+272	284	lipid kinase	protein_type	ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis.	INTRO
+285	290	PI4KB	protein	ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis.	INTRO
+0	5	ACBD3	protein	ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2.	INTRO
+138	158	polyglutamine repeat	structure_element	ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2.	INTRO
+170	176	mutant	protein_state	ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2.	INTRO
+177	187	huntingtin	protein	ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2.	INTRO
+215	224	monomeric	oligomeric_state	ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2.	INTRO
+225	234	G protein	protein_type	ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2.	INTRO
+235	239	Rhes	protein	ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2.	INTRO
+240	247	Dexras2	protein	ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2.	INTRO
+0	5	ACBD3	protein	ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus.	INTRO
+30	35	viral	taxonomy_domain	ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus.	INTRO
+36	62	non-structural 3A proteins	protein_type	ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus.	INTRO
+92	106	picornaviruses	taxonomy_domain	ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus.	INTRO
+117	127	poliovirus	taxonomy_domain	ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus.	INTRO
+129	146	coxsackievirus B3	taxonomy_domain	ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus.	INTRO
+152	163	Aichi virus	taxonomy_domain	ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus.	INTRO
+13	56	biochemical and structural characterization	experimental_method	We present a biochemical and structural characterization of the molecular complex composed of the ACBD3 protein and the PI4KB enzyme.	INTRO
+98	103	ACBD3	protein	We present a biochemical and structural characterization of the molecular complex composed of the ACBD3 protein and the PI4KB enzyme.	INTRO
+120	125	PI4KB	protein	We present a biochemical and structural characterization of the molecular complex composed of the ACBD3 protein and the PI4KB enzyme.	INTRO
+13	18	ACBD3	protein	We show that ACBD3 can recruit PI4KB to model membranes as well as redirect PI4KB to cellular membranes where it is not naturally found.	INTRO
+31	36	PI4KB	protein	We show that ACBD3 can recruit PI4KB to model membranes as well as redirect PI4KB to cellular membranes where it is not naturally found.	INTRO
+76	81	PI4KB	protein	We show that ACBD3 can recruit PI4KB to model membranes as well as redirect PI4KB to cellular membranes where it is not naturally found.	INTRO
+24	29	ACBD3	protein	Our data also show that ACBD3 regulates the enzymatic activity of PI4KB kinase through membrane recruitment rather than allostery.	INTRO
+44	62	enzymatic activity	evidence	Our data also show that ACBD3 regulates the enzymatic activity of PI4KB kinase through membrane recruitment rather than allostery.	INTRO
+66	71	PI4KB	protein	Our data also show that ACBD3 regulates the enzymatic activity of PI4KB kinase through membrane recruitment rather than allostery.	INTRO
+72	78	kinase	protein_type	Our data also show that ACBD3 regulates the enzymatic activity of PI4KB kinase through membrane recruitment rather than allostery.	INTRO
+0	5	ACBD3	protein	ACBD3 and PI4KB interact with 1:1 stoichiometry with submicromolar affinity	RESULTS
+10	15	PI4KB	protein	ACBD3 and PI4KB interact with 1:1 stoichiometry with submicromolar affinity	RESULTS
+44	49	ACBD3	protein	In order to verify the interactions between ACBD3 and PI4KB we expressed and purified both proteins.	RESULTS
+54	59	PI4KB	protein	In order to verify the interactions between ACBD3 and PI4KB we expressed and purified both proteins.	RESULTS
+63	85	expressed and purified	experimental_method	In order to verify the interactions between ACBD3 and PI4KB we expressed and purified both proteins.	RESULTS
+22	42	bacterial expression	experimental_method	To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423–522) was removed (Fig. 1A).	RESULTS
+47	78	intrinsically disordered region	structure_element	To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423–522) was removed (Fig. 1A).	RESULTS
+82	87	PI4KB	protein	To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423–522) was removed (Fig. 1A).	RESULTS
+98	105	423–522	residue_range	To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423–522) was removed (Fig. 1A).	RESULTS
+111	118	removed	experimental_method	To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423–522) was removed (Fig. 1A).	RESULTS
+14	22	deletion	experimental_method	This internal deletion does not significantly affect the kinase activity(SI Fig. 1A) or interaction with ACBD3 (SI Fig. 1B,C).	RESULTS
+57	63	kinase	protein_type	This internal deletion does not significantly affect the kinase activity(SI Fig. 1A) or interaction with ACBD3 (SI Fig. 1B,C).	RESULTS
+105	110	ACBD3	protein	This internal deletion does not significantly affect the kinase activity(SI Fig. 1A) or interaction with ACBD3 (SI Fig. 1B,C).	RESULTS
+6	28	in vitro binding assay	experimental_method	In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction.	RESULTS
+30	35	ACBD3	protein	In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction.	RESULTS
+36	74	co-purified with the NiNTA-immobilized	experimental_method	In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction.	RESULTS
+86	100	His6GB1-tagged	protein_state	In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction.	RESULTS
+101	106	PI4KB	protein	In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction.	RESULTS
+8	34	mammalian two-hybrid assay	experimental_method	Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain.	RESULTS
+60	69	localized	evidence	Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain.	RESULTS
+94	102	Q domain	structure_element	Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain.	RESULTS
+106	111	ACBD3	protein	Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain.	RESULTS
+152	161	glutamine	residue_name	Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain.	RESULTS
+180	197	N-terminal region	structure_element	Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain.	RESULTS
+201	206	PI4KB	protein	Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain.	RESULTS
+221	235	helical domain	structure_element	Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain.	RESULTS
+3	12	expressed	experimental_method	We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel).	RESULTS
+17	25	Q domain	structure_element	We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel).	RESULTS
+29	34	ACBD3	protein	We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel).	RESULTS
+45	52	241–308	residue_range	We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel).	RESULTS
+62	79	N-terminal region	structure_element	We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel).	RESULTS
+83	88	PI4KB	protein	We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel).	RESULTS
+99	103	1–68	residue_range	We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel).	RESULTS
+108	115	E. coli	species	We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel).	RESULTS
+34	39	ACBD3	protein	Because it has been reported that ACBD3 can dimerize in a mammalian two-hybrid assay, we were interested in determining the stoichiometry of the ACBD3:PI4KB protein complex.	RESULTS
+44	52	dimerize	oligomeric_state	Because it has been reported that ACBD3 can dimerize in a mammalian two-hybrid assay, we were interested in determining the stoichiometry of the ACBD3:PI4KB protein complex.	RESULTS
+58	84	mammalian two-hybrid assay	experimental_method	Because it has been reported that ACBD3 can dimerize in a mammalian two-hybrid assay, we were interested in determining the stoichiometry of the ACBD3:PI4KB protein complex.	RESULTS
+145	156	ACBD3:PI4KB	complex_assembly	Because it has been reported that ACBD3 can dimerize in a mammalian two-hybrid assay, we were interested in determining the stoichiometry of the ACBD3:PI4KB protein complex.	RESULTS
+4	30	sedimentation coefficients	evidence	The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively.	RESULTS
+34	39	ACBD3	protein	The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively.	RESULTS
+44	49	PI4KB	protein	The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively.	RESULTS
+50	55	alone	protein_state	The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively.	RESULTS
+60	71	ACBD3:PI4KB	complex_assembly	The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively.	RESULTS
+99	129	analytical ultracentrifugation	experimental_method	The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively.	RESULTS
+198	215	molecular weights	evidence	The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively.	RESULTS
+44	53	monomeric	oligomeric_state	This result suggests that both proteins are monomeric and the stoichiometry of the ACBD3: PI4KB protein complex is 1:1 (Fig. 1C, left panel).	RESULTS
+83	95	ACBD3: PI4KB	complex_assembly	This result suggests that both proteins are monomeric and the stoichiometry of the ACBD3: PI4KB protein complex is 1:1 (Fig. 1C, left panel).	RESULTS
+53	61	Q domain	structure_element	Similar results were obtained for the complex of the Q domain of ACBD3 and the N-terminal region of PI4KB (Fig. 1C, right panel).	RESULTS
+65	70	ACBD3	protein	Similar results were obtained for the complex of the Q domain of ACBD3 and the N-terminal region of PI4KB (Fig. 1C, right panel).	RESULTS
+79	96	N-terminal region	structure_element	Similar results were obtained for the complex of the Q domain of ACBD3 and the N-terminal region of PI4KB (Fig. 1C, right panel).	RESULTS
+100	105	PI4KB	protein	Similar results were obtained for the complex of the Q domain of ACBD3 and the N-terminal region of PI4KB (Fig. 1C, right panel).	RESULTS
+71	82	full length	protein_state	We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR).	RESULTS
+83	88	ACBD3	protein	We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR).	RESULTS
+93	98	PI4KB	protein	We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR).	RESULTS
+105	130	surface plasmon resonance	experimental_method	We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR).	RESULTS
+132	135	SPR	experimental_method	We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR).	RESULTS
+0	3	SPR	experimental_method	SPR measurements revealed a strong interaction with a Kd value of 320 +/−130 nM (Fig. 1D, SI Fig. 1D).	RESULTS
+54	56	Kd	evidence	SPR measurements revealed a strong interaction with a Kd value of 320 +/−130 nM (Fig. 1D, SI Fig. 1D).	RESULTS
+18	23	ACBD3	protein	We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range.	RESULTS
+28	33	PI4KB	protein	We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range.	RESULTS
+64	72	Q domain	structure_element	We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range.	RESULTS
+76	81	ACBD3	protein	We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range.	RESULTS
+90	107	N-terminal region	structure_element	We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range.	RESULTS
+111	116	PI4KB	protein	We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range.	RESULTS
+146	167	dissociation constant	evidence	We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range.	RESULTS
+0	19	Structural analysis	experimental_method	Structural analysis of the ACBD3:PI4KB complex	RESULTS
+27	38	ACBD3:PI4KB	complex_assembly	Structural analysis of the ACBD3:PI4KB complex	RESULTS
+0	11	Full length	protein_state	Full length ACBD3 and PI4KB both contain large intrinsically disordered regions that impede crystallization.	RESULTS
+12	17	ACBD3	protein	Full length ACBD3 and PI4KB both contain large intrinsically disordered regions that impede crystallization.	RESULTS
+22	27	PI4KB	protein	Full length ACBD3 and PI4KB both contain large intrinsically disordered regions that impede crystallization.	RESULTS
+47	79	intrinsically disordered regions	structure_element	Full length ACBD3 and PI4KB both contain large intrinsically disordered regions that impede crystallization.	RESULTS
+8	53	hydrogen-deuterium exchange mass spectrometry	experimental_method	We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of the complex to determine which parts of the complex are well folded (SI Fig. 2).	RESULTS
+55	61	HDX-MS	experimental_method	We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of the complex to determine which parts of the complex are well folded (SI Fig. 2).	RESULTS
+131	142	well folded	protein_state	We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of the complex to determine which parts of the complex are well folded (SI Fig. 2).	RESULTS
+34	42	crystals	evidence	However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB.	RESULTS
+73	82	truncated	protein_state	However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB.	RESULTS
+117	122	ACBD3	protein	However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB.	RESULTS
+123	131	Q domain	structure_element	However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB.	RESULTS
+140	157	N-terminal region	structure_element	However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB.	RESULTS
+161	166	PI4KB	protein	However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB.	RESULTS
+32	52	isotopically labeled	protein_state	For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization.	RESULTS
+53	58	ACBD3	protein	For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization.	RESULTS
+59	67	Q domain	structure_element	For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization.	RESULTS
+72	92	isotopically labeled	protein_state	For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization.	RESULTS
+93	98	ACBD3	protein	For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization.	RESULTS
+99	107	Q domain	structure_element	For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization.	RESULTS
+108	113	PI4KB	protein	For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization.	RESULTS
+114	131	N-terminal region	structure_element	For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization.	RESULTS
+157	173	NMR spectroscopy	experimental_method	For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization.	RESULTS
+7	24	N-terminal region	structure_element	As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules.	RESULTS
+57	70	co-expression	experimental_method	As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules.	RESULTS
+152	155	15N	chemical	As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules.	RESULTS
+156	159	13C	chemical	As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules.	RESULTS
+160	168	labelled	protein_state	As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules.	RESULTS
+68	72	free	protein_state	Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously.	RESULTS
+73	78	ACBD3	protein	Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously.	RESULTS
+79	87	Q domain	structure_element	Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously.	RESULTS
+127	141	2D 15N/1H HSQC	experimental_method	Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously.	RESULTS
+142	149	spectra	evidence	Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously.	RESULTS
+215	243	triple-resonance experiments	experimental_method	Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously.	RESULTS
+24	27	15N	chemical	Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44.	RESULTS
+32	34	1H	chemical	Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44.	RESULTS
+44	48	free	protein_state	Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44.	RESULTS
+49	54	ACBD3	protein	Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44.	RESULTS
+55	63	Q domain	structure_element	Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44.	RESULTS
+143	152	Met1-Lys4	residue_range	Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44.	RESULTS
+158	163	Gln44	residue_name_number	Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44.	RESULTS
+70	74	free	protein_state	Over 93% of non-exchangeable side-chain signals were assigned for the free ACBD3 Q domain.	RESULTS
+75	80	ACBD3	protein	Over 93% of non-exchangeable side-chain signals were assigned for the free ACBD3 Q domain.	RESULTS
+81	89	Q domain	structure_element	Over 93% of non-exchangeable side-chain signals were assigned for the free ACBD3 Q domain.	RESULTS
+85	88	Gln	residue_name	Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances.	RESULTS
+96	99	Gln	residue_name	Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances.	RESULTS
+112	117	Gln44	residue_name_number	Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances.	RESULTS
+133	138	Gln48	residue_name_number	Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances.	RESULTS
+234	243	glutamine	residue_name	Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances.	RESULTS
+53	60	spectra	evidence	The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB).	RESULTS
+155	158	Gln	residue_name	The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB).	RESULTS
+160	165	ACBD3	protein	The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB).	RESULTS
+171	175	Ala2	residue_name_number	The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB).	RESULTS
+177	182	PI4KB	protein	The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB).	RESULTS
+25	28	15N	chemical	The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation.	RESULTS
+30	33	13C	chemical	The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation.	RESULTS
+38	40	1H	chemical	The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation.	RESULTS
+99	103	NOEs	evidence	The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation.	RESULTS
+122	142	3D 15N/1H NOESY-HSQC	experimental_method	The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation.	RESULTS
+147	164	13C/1H HMQC-NOESY	experimental_method	The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation.	RESULTS
+165	172	spectra	evidence	The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation.	RESULTS
+204	226	structural calculation	experimental_method	The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation.	RESULTS
+0	21	Structural statistics	evidence	Structural statistics for the final water-refined sets of structures are shown in SI Table 1.	RESULTS
+58	68	structures	evidence	Structural statistics for the final water-refined sets of structures are shown in SI Table 1.	RESULTS
+5	14	structure	evidence	This structure revealed that the Q domain forms a two helix hairpin.	RESULTS
+33	41	Q domain	structure_element	This structure revealed that the Q domain forms a two helix hairpin.	RESULTS
+50	67	two helix hairpin	structure_element	This structure revealed that the Q domain forms a two helix hairpin.	RESULTS
+10	15	helix	structure_element	The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A).	RESULTS
+46	51	helix	structure_element	The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A).	RESULTS
+84	102	three helix bundle	structure_element	The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A).	RESULTS
+133	138	helix	structure_element	The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A).	RESULTS
+146	151	PI4KB	protein	The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A).	RESULTS
+173	178	44–64	residue_range	The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A).	RESULTS
+218	230	kinase helix	structure_element	The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A).	RESULTS
+14	26	kinase helix	structure_element	Preceding the kinase helix are three ordered residues (Val42, Ile43, and Asp44) that also contribute to the interaction (Fig. 2B).	RESULTS
+55	60	Val42	residue_name_number	Preceding the kinase helix are three ordered residues (Val42, Ile43, and Asp44) that also contribute to the interaction (Fig. 2B).	RESULTS
+62	67	Ile43	residue_name_number	Preceding the kinase helix are three ordered residues (Val42, Ile43, and Asp44) that also contribute to the interaction (Fig. 2B).	RESULTS
+73	78	Asp44	residue_name_number	Preceding the kinase helix are three ordered residues (Val42, Ile43, and Asp44) that also contribute to the interaction (Fig. 2B).	RESULTS
+26	31	PI4KB	protein	The remaining part of the PI4KB N-termini, however, is disordered (SI Fig. 5).	RESULTS
+18	29	PI4KB:ACBD3	complex_assembly	Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B).	RESULTS
+30	58	interactions are hydrophobic	bond_interaction	Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B).	RESULTS
+81	95	hydrogen bonds	bond_interaction	Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B).	RESULTS
+123	128	ACBD3	protein	Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B).	RESULTS
+129	135	Tyr261	residue_name_number	Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B).	RESULTS
+140	145	PI4KB	protein	Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B).	RESULTS
+146	151	His63	residue_name_number	Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B).	RESULTS
+182	187	ACBD3	protein	Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B).	RESULTS
+188	194	Tyr288	residue_name_number	Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B).	RESULTS
+203	208	PI4KB	protein	Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B).	RESULTS
+219	224	Asp44	residue_name_number	Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B).	RESULTS
+33	38	PI4KB	protein	Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C).	RESULTS
+39	44	helix	structure_element	Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C).	RESULTS
+48	59	amphipathic	protein_state	Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C).	RESULTS
+68	87	hydrophobic surface	site	Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C).	RESULTS
+101	109	Q domain	structure_element	Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C).	RESULTS
+19	34	structural data	evidence	To corroborate the structural data, we introduced a number of point mutations and validated their effect on complex formation using an in vitro pull-down assay (Fig. 2D).	RESULTS
+39	49	introduced	experimental_method	To corroborate the structural data, we introduced a number of point mutations and validated their effect on complex formation using an in vitro pull-down assay (Fig. 2D).	RESULTS
+62	77	point mutations	experimental_method	To corroborate the structural data, we introduced a number of point mutations and validated their effect on complex formation using an in vitro pull-down assay (Fig. 2D).	RESULTS
+135	159	in vitro pull-down assay	experimental_method	To corroborate the structural data, we introduced a number of point mutations and validated their effect on complex formation using an in vitro pull-down assay (Fig. 2D).	RESULTS
+0	9	Wild type	protein_state	Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).	RESULTS
+10	15	ACBD3	protein	Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).	RESULTS
+24	35	co-purified	experimental_method	Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).	RESULTS
+72	83	His6-tagged	protein_state	Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).	RESULTS
+84	93	wild type	protein_state	Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).	RESULTS
+94	99	PI4KB	protein	Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).	RESULTS
+120	125	PI4KB	protein	Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).	RESULTS
+126	130	V42A	mutant	Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).	RESULTS
+135	139	V47A	mutant	Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).	RESULTS
+140	147	mutants	protein_state	Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).	RESULTS
+162	169	mutants	protein_state	Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).	RESULTS
+190	207	binding interface	site	Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).	RESULTS
+209	213	I43A	mutant	Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).	RESULTS
+215	219	V55A	mutant	Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).	RESULTS
+221	225	L56A	mutant	Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).	RESULTS
+14	23	wild type	protein_state	As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).	RESULTS
+24	29	PI4KB	protein	As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).	RESULTS
+50	55	ACBD3	protein	As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).	RESULTS
+56	61	Y266A	mutant	As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).	RESULTS
+62	68	mutant	protein_state	As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).	RESULTS
+91	96	Y285A	mutant	As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).	RESULTS
+97	103	mutant	protein_state	As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).	RESULTS
+122	127	F258A	mutant	As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).	RESULTS
+129	134	H284A	mutant	As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).	RESULTS
+140	145	Y288A	mutant	As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).	RESULTS
+146	153	mutants	protein_state	As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).	RESULTS
+0	5	ACBD3	protein	ACBD3 efficiently recruits the PI4KB enzyme to membranes	RESULTS
+31	36	PI4KB	protein	ACBD3 efficiently recruits the PI4KB enzyme to membranes	RESULTS
+35	46	ACBD3:PI4KB	complex_assembly	We next sought to determine if the ACBD3:PI4KB interaction drives membrane localization of the PI4KB enzyme.	RESULTS
+95	100	PI4KB	protein	We next sought to determine if the ACBD3:PI4KB interaction drives membrane localization of the PI4KB enzyme.	RESULTS
+36	72	in vitro membrane recruitment system	experimental_method	To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate – the PI lipid.	RESULTS
+79	105	Giant Unilamellar Vesicles	experimental_method	To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate – the PI lipid.	RESULTS
+107	111	GUVs	experimental_method	To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate – the PI lipid.	RESULTS
+128	133	PI4KB	protein	To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate – the PI lipid.	RESULTS
+150	152	PI	chemical	To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate – the PI lipid.	RESULTS
+17	22	PI4KB	protein	We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A).	RESULTS
+23	29	kinase	protein_type	We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A).	RESULTS
+47	56	localized	evidence	We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A).	RESULTS
+75	79	GUVs	experimental_method	We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A).	RESULTS
+139	144	ACBD3	protein	We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A).	RESULTS
+167	171	GUVs	experimental_method	We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A).	RESULTS
+195	200	ACBD3	protein	We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A).	RESULTS
+209	227	DGS-NTA (Ni) lipid	chemical	We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A).	RESULTS
+246	251	PI4KB	protein	We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A).	RESULTS
+24	29	ACBD3	protein	We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel).	RESULTS
+55	75	localization signals	evidence	We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel).	RESULTS
+80	85	PI4KB	protein	We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel).	RESULTS
+87	101	overexpression	experimental_method	We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel).	RESULTS
+109	117	Q domain	structure_element	We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel).	RESULTS
+163	169	kinase	protein_type	We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel).	RESULTS
+224	229	PI4KB	protein	We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel).	RESULTS
+259	273	overexpressing	experimental_method	We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel).	RESULTS
+278	281	GFP	experimental_method	We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel).	RESULTS
+284	292	Q domain	structure_element	We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel).	RESULTS
+25	31	signal	evidence	We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B).	RESULTS
+67	73	kinase	protein_type	We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B).	RESULTS
+179	185	signal	evidence	We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B).	RESULTS
+194	208	overexpression	experimental_method	We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B).	RESULTS
+212	215	GFP	experimental_method	We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B).	RESULTS
+227	238	non-binding	protein_state	We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B).	RESULTS
+239	247	Q domain	structure_element	We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B).	RESULTS
+248	254	mutant	protein_state	We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B).	RESULTS
+276	288	localization	evidence	We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B).	RESULTS
+307	312	PI4KB	protein	We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B).	RESULTS
+19	33	overexpression	experimental_method	Given this result, overexpression of the Q domain should also interfere with the PI4KB dependent Golgi functions.	RESULTS
+41	49	Q domain	structure_element	Given this result, overexpression of the Q domain should also interfere with the PI4KB dependent Golgi functions.	RESULTS
+81	86	PI4KB	protein	Given this result, overexpression of the Q domain should also interfere with the PI4KB dependent Golgi functions.	RESULTS
+0	8	Ceramide	chemical	Ceramide transport and accumulation in Golgi is a well-known PI4KB dependent process.	RESULTS
+61	66	PI4KB	protein	Ceramide transport and accumulation in Golgi is a well-known PI4KB dependent process.	RESULTS
+13	34	fluorescently labeled	protein_state	We have used fluorescently labeled ceramide and analyzed its trafficking in non-transfected cells and cell overexpressing the Q domain.	RESULTS
+35	43	ceramide	chemical	We have used fluorescently labeled ceramide and analyzed its trafficking in non-transfected cells and cell overexpressing the Q domain.	RESULTS
+107	121	overexpressing	experimental_method	We have used fluorescently labeled ceramide and analyzed its trafficking in non-transfected cells and cell overexpressing the Q domain.	RESULTS
+126	134	Q domain	structure_element	We have used fluorescently labeled ceramide and analyzed its trafficking in non-transfected cells and cell overexpressing the Q domain.	RESULTS
+39	47	ceramide	chemical	As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi.	RESULTS
+74	84	expressing	experimental_method	As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi.	RESULTS
+89	91	wt	protein_state	As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi.	RESULTS
+92	100	Q domain	structure_element	As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi.	RESULTS
+124	127	RFP	experimental_method	As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi.	RESULTS
+135	141	mutant	protein_state	As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi.	RESULTS
+142	150	Q domain	structure_element	As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi.	RESULTS
+163	171	ceramide	chemical	As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi.	RESULTS
+207	218	ACBD3:PI4KB	complex_assembly	As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi.	RESULTS
+36	47	ACBD3:PI4KB	complex_assembly	We further analyzed the function of ACBD3:PI4KB interaction in membrane recruitment of PI4KB in living cells using fluorescently tagged proteins.	RESULTS
+87	92	PI4KB	protein	We further analyzed the function of ACBD3:PI4KB interaction in membrane recruitment of PI4KB in living cells using fluorescently tagged proteins.	RESULTS
+115	135	fluorescently tagged	protein_state	We further analyzed the function of ACBD3:PI4KB interaction in membrane recruitment of PI4KB in living cells using fluorescently tagged proteins.	RESULTS
+12	21	rapamycin	chemical	We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system.	RESULTS
+52	58	FKBP12	protein	We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system.	RESULTS
+60	84	FK506 binding protein 12	protein	We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system.	RESULTS
+90	93	FRB	structure_element	We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system.	RESULTS
+95	103	fragment	structure_element	We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system.	RESULTS
+107	111	mTOR	protein	We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system.	RESULTS
+123	132	rapamycin	chemical	We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system.	RESULTS
+3	8	fused	experimental_method	We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP.	RESULTS
+13	16	FRB	structure_element	We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP.	RESULTS
+29	34	34–63	residue_range	We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP.	RESULTS
+42	75	mitochondrial localization signal	structure_element	We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP.	RESULTS
+81	120	mitochondrial A-kinase anchor protein 1	protein	We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP.	RESULTS
+122	127	AKAP1	protein	We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP.	RESULTS
+133	136	CFP	experimental_method	We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP.	RESULTS
+4	9	ACBD3	protein	The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D).	RESULTS
+10	18	Q domain	structure_element	The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D).	RESULTS
+28	36	fused to	experimental_method	The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D).	RESULTS
+37	43	FKBP12	protein	The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D).	RESULTS
+48	52	mRFP	experimental_method	The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D).	RESULTS
+12	24	localization	evidence	We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin.	RESULTS
+32	37	ACBD3	protein	We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin.	RESULTS
+38	46	Q domain	structure_element	We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin.	RESULTS
+51	54	GFP	experimental_method	We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin.	RESULTS
+57	62	PI4KB	protein	We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin.	RESULTS
+96	105	rapamycin	chemical	We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin.	RESULTS
+21	26	H284A	mutant	As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase.	RESULTS
+27	33	mutant	protein_state	As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase.	RESULTS
+41	46	ACBD3	protein	As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase.	RESULTS
+47	55	Q domain	structure_element	As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase.	RESULTS
+89	94	PI4KB	protein	As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase.	RESULTS
+95	101	kinase	protein_type	As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase.	RESULTS
+18	23	ACDB3	protein	In every case the ACDB3 Q domain was rapidly (within 5 minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2).	RESULTS
+24	32	Q domain	structure_element	In every case the ACDB3 Q domain was rapidly (within 5 minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2).	RESULTS
+121	130	rapamycin	chemical	In every case the ACDB3 Q domain was rapidly (within 5 minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2).	RESULTS
+145	154	wild-type	protein_state	In every case the ACDB3 Q domain was rapidly (within 5 minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2).	RESULTS
+188	194	kinase	protein_type	In every case the ACDB3 Q domain was rapidly (within 5 minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2).	RESULTS
+35	38	GFP	experimental_method	Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel).	RESULTS
+39	44	PI4KB	protein	Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel).	RESULTS
+45	51	kinase	protein_type	Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel).	RESULTS
+55	67	co-expressed	experimental_method	Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel).	RESULTS
+77	86	wild-type	protein_state	Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel).	RESULTS
+87	92	ACDB3	protein	Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel).	RESULTS
+93	101	Q domain	structure_element	Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel).	RESULTS
+129	141	localization	evidence	Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel).	RESULTS
+9	14	PI4KB	protein	However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel).	RESULTS
+32	44	localization	evidence	However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel).	RESULTS
+50	62	co-expressed	experimental_method	However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel).	RESULTS
+72	87	non-interacting	protein_state	However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel).	RESULTS
+88	96	Q domain	structure_element	However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel).	RESULTS
+97	103	mutant	protein_state	However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel).	RESULTS
+0	5	ACBD3	protein	ACBD3 increases PI4KB enzymatic activity by recruiting PI4KB to close vicinity of its substrate	RESULTS
+16	21	PI4KB	protein	ACBD3 increases PI4KB enzymatic activity by recruiting PI4KB to close vicinity of its substrate	RESULTS
+22	40	enzymatic activity	evidence	ACBD3 increases PI4KB enzymatic activity by recruiting PI4KB to close vicinity of its substrate	RESULTS
+55	60	PI4KB	protein	ACBD3 increases PI4KB enzymatic activity by recruiting PI4KB to close vicinity of its substrate	RESULTS
+16	21	ACBD3	protein	To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate.	RESULTS
+36	41	PI4KB	protein	To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate.	RESULTS
+42	48	kinase	protein_type	To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate.	RESULTS
+49	67	enzymatic activity	evidence	To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate.	RESULTS
+92	116	luminescent kinase assay	experimental_method	To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate.	RESULTS
+123	125	PI	chemical	To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate.	RESULTS
+29	35	kinase	protein_type	We observed no effect on the kinase activity of PI4KB (Fig. 4A) suggesting that ACBD3 does not directly affect the enzyme (e.g. induction of a conformation change).	RESULTS
+48	53	PI4KB	protein	We observed no effect on the kinase activity of PI4KB (Fig. 4A) suggesting that ACBD3 does not directly affect the enzyme (e.g. induction of a conformation change).	RESULTS
+80	85	ACBD3	protein	We observed no effect on the kinase activity of PI4KB (Fig. 4A) suggesting that ACBD3 does not directly affect the enzyme (e.g. induction of a conformation change).	RESULTS
+17	22	ACBD3	protein	However, in vivo ACBD3 is located at the Golgi membranes, whereas in this experiment, ACBD3 was located in the solution and PI is provided as micelles.	RESULTS
+86	91	ACBD3	protein	However, in vivo ACBD3 is located at the Golgi membranes, whereas in this experiment, ACBD3 was located in the solution and PI is provided as micelles.	RESULTS
+124	126	PI	chemical	However, in vivo ACBD3 is located at the Golgi membranes, whereas in this experiment, ACBD3 was located in the solution and PI is provided as micelles.	RESULTS
+33	36	GUV	experimental_method	For this, we again turned to the GUV system with ACBD3 localized to the GUV membrane.	RESULTS
+49	54	ACBD3	protein	For this, we again turned to the GUV system with ACBD3 localized to the GUV membrane.	RESULTS
+55	64	localized	evidence	For this, we again turned to the GUV system with ACBD3 localized to the GUV membrane.	RESULTS
+72	75	GUV	experimental_method	For this, we again turned to the GUV system with ACBD3 localized to the GUV membrane.	RESULTS
+4	8	GUVs	experimental_method	The GUVs contained 10% PI to serve as a substrate for PI4KB kinase.	RESULTS
+23	25	PI	chemical	The GUVs contained 10% PI to serve as a substrate for PI4KB kinase.	RESULTS
+54	59	PI4KB	protein	The GUVs contained 10% PI to serve as a substrate for PI4KB kinase.	RESULTS
+60	66	kinase	protein_type	The GUVs contained 10% PI to serve as a substrate for PI4KB kinase.	RESULTS
+26	29	CFP	experimental_method	The buffer also contained CFP-SidC, which binds to PI4P with nanomolar affinity.	RESULTS
+30	34	SidC	protein	The buffer also contained CFP-SidC, which binds to PI4P with nanomolar affinity.	RESULTS
+51	55	PI4P	chemical	The buffer also contained CFP-SidC, which binds to PI4P with nanomolar affinity.	RESULTS
+34	40	kinase	protein_type	This enabled visualization of the kinase reaction using a confocal microscope.	RESULTS
+58	77	confocal microscope	experimental_method	This enabled visualization of the kinase reaction using a confocal microscope.	RESULTS
+34	49	phosphorylation	ptm	We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3.	RESULTS
+66	72	kinase	protein_type	We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3.	RESULTS
+73	78	alone	protein_state	We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3.	RESULTS
+92	98	kinase	protein_type	We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3.	RESULTS
+131	135	GUVs	experimental_method	We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3.	RESULTS
+139	144	ACBD3	protein	We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3.	RESULTS
+35	45	absence of	protein_state	Reaction was also performed in the absence of ATP as a negative control (Fig. 4B).	RESULTS
+46	49	ATP	chemical	Reaction was also performed in the absence of ATP as a negative control (Fig. 4B).	RESULTS
+30	35	PI4KB	protein	These experiments showed that PI4KB enzymatic activity increases when ACBD3 is membrane localized (Fig. 4C, SI Fig. 6).	RESULTS
+36	54	enzymatic activity	evidence	These experiments showed that PI4KB enzymatic activity increases when ACBD3 is membrane localized (Fig. 4C, SI Fig. 6).	RESULTS
+70	75	ACBD3	protein	These experiments showed that PI4KB enzymatic activity increases when ACBD3 is membrane localized (Fig. 4C, SI Fig. 6).	RESULTS
+24	29	PI4KB	protein	Membrane recruitment of PI4KB enzyme is crucial to ensure its proper function at the Golgi and TGN.	DISCUSS
+58	63	PI4KB	protein	However, the molecular mechanism and structural basis for PI4KB interaction with the membrane is poorly understood.	DISCUSS
+45	50	PI4KB	protein	In principle, any of the binding partners of PI4KB could play a role in membrane recruitment.	DISCUSS
+17	22	PI4KB	protein	To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.	DISCUSS
+78	91	small GTPases	protein_type	To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.	DISCUSS
+92	97	Rab11	protein	To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.	DISCUSS
+102	106	Arf1	protein	To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.	DISCUSS
+127	163	acyl-CoA binding domain containing 3	protein	To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.	DISCUSS
+165	170	ACBD3	protein	To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.	DISCUSS
+181	206	neuronal calcium sensor-1	protein	To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.	DISCUSS
+208	213	NCS-1	protein	To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.	DISCUSS
+228	237	frequenin	protein	To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.	DISCUSS
+241	246	yeast	taxonomy_domain	To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.	DISCUSS
+256	271	14-3-3 proteins	protein_type	To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.	DISCUSS
+4	13	monomeric	oligomeric_state	The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase.	DISCUSS
+14	23	G protein	protein_type	The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase.	DISCUSS
+24	29	Rab11	protein	The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase.	DISCUSS
+36	45	mammalian	taxonomy_domain	The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase.	DISCUSS
+46	51	PI4KB	protein	The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase.	DISCUSS
+64	78	helical domain	structure_element	The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase.	DISCUSS
+86	92	kinase	protein_type	The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase.	DISCUSS
+9	14	Rab11	protein	Although Rab11 does not appear to be required for recruitment of PI4KB to the Golgi, PI4KB is required for Golgi recruitment of Rab11.	DISCUSS
+65	70	PI4KB	protein	Although Rab11 does not appear to be required for recruitment of PI4KB to the Golgi, PI4KB is required for Golgi recruitment of Rab11.	DISCUSS
+85	90	PI4KB	protein	Although Rab11 does not appear to be required for recruitment of PI4KB to the Golgi, PI4KB is required for Golgi recruitment of Rab11.	DISCUSS
+128	133	Rab11	protein	Although Rab11 does not appear to be required for recruitment of PI4KB to the Golgi, PI4KB is required for Golgi recruitment of Rab11.	DISCUSS
+0	4	Arf1	protein	Arf1, the other small GTP binding protein, is known to influence the activity and localization of PI4KB, but it does not appear to interact directly with PI4KB (our unpublished data).	DISCUSS
+16	41	small GTP binding protein	protein_type	Arf1, the other small GTP binding protein, is known to influence the activity and localization of PI4KB, but it does not appear to interact directly with PI4KB (our unpublished data).	DISCUSS
+98	103	PI4KB	protein	Arf1, the other small GTP binding protein, is known to influence the activity and localization of PI4KB, but it does not appear to interact directly with PI4KB (our unpublished data).	DISCUSS
+154	159	PI4KB	protein	Arf1, the other small GTP binding protein, is known to influence the activity and localization of PI4KB, but it does not appear to interact directly with PI4KB (our unpublished data).	DISCUSS
+4	9	yeast	taxonomy_domain	The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear.	DISCUSS
+23	27	NCS1	protein	The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear.	DISCUSS
+35	44	frequenin	protein	The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear.	DISCUSS
+77	82	Pik1p	protein	The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear.	DISCUSS
+88	93	yeast	taxonomy_domain	The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear.	DISCUSS
+108	113	PI4KB	protein	The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear.	DISCUSS
+194	199	NCS-1	protein	The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear.	DISCUSS
+203	208	PI4KB	protein	The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear.	DISCUSS
+224	233	mammalian	taxonomy_domain	The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear.	DISCUSS
+0	5	NCS-1	protein	NCS-1 is an N-terminally myristoylated protein that participates in exocytosis.	DISCUSS
+25	38	myristoylated	protein_state	NCS-1 is an N-terminally myristoylated protein that participates in exocytosis.	DISCUSS
+81	86	PI4KB	protein	It is expressed only in certain cell types, suggesting that if it contributes to PI4KB membrane recruitment, it does so in a tissues specific manner.	DISCUSS
+19	24	PI4KB	protein	The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation.	DISCUSS
+30	45	14-3-3 proteins	protein_type	The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation.	DISCUSS
+59	74	phosphorylation	ptm	The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation.	DISCUSS
+78	83	PI4KB	protein	The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation.	DISCUSS
+87	103	protein kinase D	protein	The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation.	DISCUSS
+132	137	PI4KB	protein	The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation.	DISCUSS
+157	163	active	protein_state	The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation.	DISCUSS
+9	24	14-3-3 proteins	protein_type	However, 14-3-3 proteins do not appear to interfere with membrane recruitment of this kinase.	DISCUSS
+86	92	kinase	protein_type	However, 14-3-3 proteins do not appear to interfere with membrane recruitment of this kinase.	DISCUSS
+0	5	ACBD3	protein	ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase.	DISCUSS
+35	44	conserved	protein_state	ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase.	DISCUSS
+51	62	vertebrates	taxonomy_domain	ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase.	DISCUSS
+105	110	PI4KB	protein	ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase.	DISCUSS
+228	234	kinase	protein_type	ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase.	DISCUSS
+55	60	PI4KB	protein	For these reasons we focused on the interaction of the PI4KB enzyme with the Golgi resident ACBD3 protein in this study.	DISCUSS
+92	97	ACBD3	protein	For these reasons we focused on the interaction of the PI4KB enzyme with the Golgi resident ACBD3 protein in this study.	DISCUSS
+58	63	PI4KB	protein	Here we present the mechanism for membrane recruitment of PI4KB by the Golgi resident ACBD3 protein.	DISCUSS
+86	91	ACBD3	protein	Here we present the mechanism for membrane recruitment of PI4KB by the Golgi resident ACBD3 protein.	DISCUSS
+53	55	Kd	evidence	We show that these proteins interact directly with a Kd value in the submicromolar range.	DISCUSS
+41	46	PI4KB	protein	The interaction is sufficient to recruit PI4KB to model membranes in vitro as well as to the mitochondria where PI4KB is never naturally found.	DISCUSS
+112	117	PI4KB	protein	The interaction is sufficient to recruit PI4KB to model membranes in vitro as well as to the mitochondria where PI4KB is never naturally found.	DISCUSS
+50	56	solved	experimental_method	To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain.	DISCUSS
+61	79	solution structure	evidence	To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain.	DISCUSS
+83	94	ACBD3:PI4KB	complex_assembly	To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain.	DISCUSS
+136	141	PI4KB	protein	To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain.	DISCUSS
+142	159	N-terminal region	structure_element	To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain.	DISCUSS
+171	193	short amphipatic helix	structure_element	To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain.	DISCUSS
+204	209	44–64	residue_range	To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain.	DISCUSS
+226	231	ACBD3	protein	To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain.	DISCUSS
+232	240	Q domain	structure_element	To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain.	DISCUSS
+4	12	Q domain	structure_element	The Q domain adopts a helical hairpin fold that is further stabilized upon binding the kinase helix (Fig. 2A).	DISCUSS
+22	42	helical hairpin fold	structure_element	The Q domain adopts a helical hairpin fold that is further stabilized upon binding the kinase helix (Fig. 2A).	DISCUSS
+87	99	kinase helix	structure_element	The Q domain adopts a helical hairpin fold that is further stabilized upon binding the kinase helix (Fig. 2A).	DISCUSS
+115	121	kinase	protein_type	Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase.	DISCUSS
+173	178	ACBD3	protein	Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase.	DISCUSS
+214	220	kinase	protein_type	Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase.	DISCUSS
+271	277	kinase	protein_type	Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase.	DISCUSS
+377	383	kinase	protein_type	Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase.	DISCUSS
+38	48	structures	evidence	Based on our and previously published structures we built a pseudoatomic model of PI4KB multi-protein assembly on the membrane (Fig. 5) that illustrates how the enzyme is recruited and positioned towards its lipidic substrate and how it in turn recruits Rab11.	DISCUSS
+60	78	pseudoatomic model	evidence	Based on our and previously published structures we built a pseudoatomic model of PI4KB multi-protein assembly on the membrane (Fig. 5) that illustrates how the enzyme is recruited and positioned towards its lipidic substrate and how it in turn recruits Rab11.	DISCUSS
+82	87	PI4KB	protein	Based on our and previously published structures we built a pseudoatomic model of PI4KB multi-protein assembly on the membrane (Fig. 5) that illustrates how the enzyme is recruited and positioned towards its lipidic substrate and how it in turn recruits Rab11.	DISCUSS
+254	259	Rab11	protein	Based on our and previously published structures we built a pseudoatomic model of PI4KB multi-protein assembly on the membrane (Fig. 5) that illustrates how the enzyme is recruited and positioned towards its lipidic substrate and how it in turn recruits Rab11.	DISCUSS
+0	12	+RNA viruses	taxonomy_domain	+RNA viruses replicate at specific PI4P-enriched membranous compartments.	DISCUSS
+35	39	PI4P	chemical	+RNA viruses replicate at specific PI4P-enriched membranous compartments.	DISCUSS
+61	66	viral	taxonomy_domain	These are called replication factories (because they enhance viral replication) or membranous webs (because of their appearance under the electron microscope).	DISCUSS
+35	42	viruses	taxonomy_domain	To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid.	DISCUSS
+85	89	PI4K	protein_type	To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid.	DISCUSS
+90	97	kinases	protein_type	To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid.	DISCUSS
+128	132	PI4P	chemical	To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid.	DISCUSS
+133	138	lipid	chemical	To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid.	DISCUSS
+0	26	Non-structural 3A proteins	protein_type	Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3.	DISCUSS
+37	51	picornaviruses	taxonomy_domain	Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3.	DISCUSS
+61	72	Enterovirus	taxonomy_domain	Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3.	DISCUSS
+79	89	poliovirus	species	Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3.	DISCUSS
+91	108	coxsackievirus-B3	species	Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3.	DISCUSS
+110	123	rhinovirus-14	species	Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3.	DISCUSS
+129	138	Kobuvirus	taxonomy_domain	Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3.	DISCUSS
+145	158	Aichi virus-1	species	Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3.	DISCUSS
+190	195	ACBD3	protein	Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3.	DISCUSS
+45	59	3A:ACBD3:PI4KB	complex_assembly	Our data suggest that they could do this via 3A:ACBD3:PI4KB complex formation.	DISCUSS
+4	13	structure	evidence	The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication.	DISCUSS
+21	26	ACBD3	protein	The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication.	DISCUSS
+27	35	Q domain	structure_element	The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication.	DISCUSS
+44	56	kinase helix	structure_element	The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication.	DISCUSS
+137	142	ACBD3	protein	The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication.	DISCUSS
+144	149	PI4KB	protein	The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication.	DISCUSS
+159	170	ACBD3:PI4KB	complex_assembly	The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication.	DISCUSS
+186	198	picornaviral	taxonomy_domain	The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication.	DISCUSS
+79	93	picornaviruses	taxonomy_domain	This could eventually have implications for therapeutic intervention to combat picornaviruses-mediated diseases ranging from polio to the common cold.	DISCUSS
+0	28	Biochemical characterization	experimental_method	Biochemical characterization of the ACBD3:PI4KB complex.	FIG
+36	47	ACBD3:PI4KB	complex_assembly	Biochemical characterization of the ACBD3:PI4KB complex.	FIG
+36	41	ACBD3	protein	(A) Schematic representation of the ACBD3 and PI4KB constructs used for the experiments.	FIG
+46	51	PI4KB	protein	(A) Schematic representation of the ACBD3 and PI4KB constructs used for the experiments.	FIG
+0	5	ACBD3	protein	ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD).	FIG
+19	42	acyl-CoA binding domain	structure_element	ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD).	FIG
+44	48	ACBD	structure_element	ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD).	FIG
+51	77	charged amino acids region	structure_element	ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD).	FIG
+79	82	CAR	structure_element	ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD).	FIG
+85	106	glutamine rich region	structure_element	ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD).	FIG
+108	109	Q	structure_element	ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD).	FIG
+116	137	Golgi dynamics domain	structure_element	ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD).	FIG
+139	143	GOLD	structure_element	ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD).	FIG
+0	5	PI4KB	protein	PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes.	FIG
+25	42	N-terminal region	structure_element	PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes.	FIG
+44	58	helical domain	structure_element	PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes.	FIG
+64	77	kinase domain	structure_element	PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes.	FIG
+104	127	N- and C-terminal lobes	structure_element	PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes.	FIG
+4	28	In vitro pull-down assay	experimental_method	(B) In vitro pull-down assay.	FIG
+0	16	Pull-down assays	experimental_method	Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3.	FIG
+67	81	His6GB1-tagged	protein_state	Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3.	FIG
+108	116	untagged	protein_state	Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3.	FIG
+117	128	full-length	protein_state	Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3.	FIG
+129	134	PI4KB	protein	Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3.	FIG
+138	143	ACBD3	protein	Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3.	FIG
+47	55	SDS gels	experimental_method	The inputs and bound proteins were analyzed on SDS gels stained with Coomassie Blue.	FIG
+35	46	full-length	protein_state	Please, see SI Fig. 9 for original full-length gels. (C) Analytical Ultracentrifugation.	FIG
+57	87	Analytical Ultracentrifugation	experimental_method	Please, see SI Fig. 9 for original full-length gels. (C) Analytical Ultracentrifugation.	FIG
+0	3	AUC	experimental_method	AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance.	FIG
+20	31	ACBD3:PI4KB	complex_assembly	AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance.	FIG
+32	43	full-length	protein_state	AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance.	FIG
+113	152	ACBD3 Q domain: PI4KB N terminal region	complex_assembly	AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance.	FIG
+225	250	Surface plasmon resonance	experimental_method	AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance.	FIG
+0	3	SPR	experimental_method	SPR analysis of the PI4KB binding to immobilized ACBD3.	FIG
+20	25	PI4KB	protein	SPR analysis of the PI4KB binding to immobilized ACBD3.	FIG
+49	54	ACBD3	protein	SPR analysis of the PI4KB binding to immobilized ACBD3.	FIG
+0	11	Sensorgrams	evidence	Sensorgrams for four concentrations of PI4KB are shown.	FIG
+39	44	PI4KB	protein	Sensorgrams for four concentrations of PI4KB are shown.	FIG
+0	19	Structural analysis	experimental_method	Structural analysis of the ACBD3:PI4KB complex.	FIG
+27	38	ACBD3:PI4KB	complex_assembly	Structural analysis of the ACBD3:PI4KB complex.	FIG
+12	21	structure	evidence	(A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region.	FIG
+29	34	ACBD3	protein	(A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region.	FIG
+35	43	Q domain	structure_element	(A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region.	FIG
+58	73	in complex with	protein_state	(A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region.	FIG
+78	83	PI4KB	protein	(A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region.	FIG
+84	101	N-terminal region	structure_element	(A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region.	FIG
+0	13	Superposition	experimental_method	Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex.	FIG
+34	44	structures	evidence	Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex.	FIG
+62	70	Q domain	structure_element	Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex.	FIG
+98	108	structures	evidence	Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex.	FIG
+158	164	folded	protein_state	Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex.	FIG
+173	178	PI4KB	protein	Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex.	FIG
+43	57	hydrogen bonds	bond_interaction	The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain.	FIG
+59	64	ACBD3	protein	The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain.	FIG
+65	71	Tyr261	residue_name_number	The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain.	FIG
+73	78	PI4KB	protein	The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain.	FIG
+79	84	His63	residue_name_number	The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain.	FIG
+89	94	ACBD3	protein	The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain.	FIG
+95	101	Tyr288	residue_name_number	The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain.	FIG
+103	108	PI4KB	protein	The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain.	FIG
+109	114	Asp44	residue_name_number	The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain.	FIG
+127	146	hydrophobic surface	site	The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain.	FIG
+154	166	kinase helix	structure_element	The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain.	FIG
+180	185	ACBD3	protein	The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain.	FIG
+186	194	Q domain	structure_element	The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain.	FIG
+0	5	ACBD3	protein	ACBD3 is shown in magenta and PI4KB in orange.	FIG
+30	35	PI4KB	protein	ACBD3 is shown in magenta and PI4KB in orange.	FIG
+20	32	kinase helix	structure_element	(C) Top view of the kinase helix.	FIG
+4	16	kinase helix	structure_element	The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta).	FIG
+20	31	amphipathic	protein_state	The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta).	FIG
+40	59	hydrophobic surface	site	The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta).	FIG
+78	83	ACBD3	protein	The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta).	FIG
+84	99	binding surface	site	The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta).	FIG
+160	175	Pull-down assay	experimental_method	Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein.	FIG
+214	228	His6GB1-tagged	protein_state	Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein.	FIG
+229	234	PI4KB	protein	Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein.	FIG
+235	241	kinase	protein_type	Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein.	FIG
+246	254	untagged	protein_state	Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein.	FIG
+255	260	ACBD3	protein	Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein.	FIG
+0	9	Wild type	protein_state	Wild type proteins and selected point mutants of both PI4KB and ACBD3 were used.	FIG
+38	45	mutants	protein_state	Wild type proteins and selected point mutants of both PI4KB and ACBD3 were used.	FIG
+54	59	PI4KB	protein	Wild type proteins and selected point mutants of both PI4KB and ACBD3 were used.	FIG
+64	69	ACBD3	protein	Wild type proteins and selected point mutants of both PI4KB and ACBD3 were used.	FIG
+35	46	full-length	protein_state	Please, see SI Fig. 9 for original full-length gels.	FIG
+0	5	ACBD3	protein	ACBD3 is sufficient to recruit the PI4KB kinase to membranes.	FIG
+35	40	PI4KB	protein	ACBD3 is sufficient to recruit the PI4KB kinase to membranes.	FIG
+41	47	kinase	protein_type	ACBD3 is sufficient to recruit the PI4KB kinase to membranes.	FIG
+4	26	GUVs recruitment assay	experimental_method	(A) GUVs recruitment assay.	FIG
+34	40	kinase	protein_type	Top – Virtually no membrane bound kinase was observed when 600 nM PI4KB was added to the GUVs.	FIG
+66	71	PI4KB	protein	Top – Virtually no membrane bound kinase was observed when 600 nM PI4KB was added to the GUVs.	FIG
+89	93	GUVs	experimental_method	Top – Virtually no membrane bound kinase was observed when 600 nM PI4KB was added to the GUVs.	FIG
+16	27	presence of	protein_state	Bottom – in the presence of 600 nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs.	FIG
+35	47	GUV tethered	protein_state	Bottom – in the presence of 600 nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs.	FIG
+48	53	ACBD3	protein	Bottom – in the presence of 600 nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs.	FIG
+82	88	kinase	protein_type	Bottom – in the presence of 600 nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs.	FIG
+119	123	GUVs	experimental_method	Bottom – in the presence of 600 nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs.	FIG
+4	33	Golgi displacement experiment	experimental_method	(B) Golgi displacement experiment.	FIG
+13	18	ACBD3	protein	Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained.	FIG
+19	27	Q domain	structure_element	Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained.	FIG
+37	40	GFP	experimental_method	Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained.	FIG
+45	58	overexpressed	experimental_method	Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained.	FIG
+78	83	PI4KB	protein	Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained.	FIG
+88	101	immunostained	experimental_method	Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained.	FIG
+49	52	GFP	experimental_method	Middle panel: The same experiment performed with GFP alone.	FIG
+48	54	mutant	protein_state	Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min.	FIG
+55	63	Q domain	structure_element	Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min.	FIG
+65	70	F258A	mutant	Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min.	FIG
+72	77	H284A	mutant	Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min.	FIG
+79	84	Y288A	mutant	Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min.	FIG
+109	114	PI4KB	protein	Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min.	FIG
+120	125	ACBD3	protein	Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min.	FIG
+126	134	Q domain	structure_element	Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min.	FIG
+135	149	overexpression	experimental_method	Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min.	FIG
+159	167	ceramide	chemical	Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min.	FIG
+218	227	wild-type	protein_state	Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min.	FIG
+228	233	ACBD3	protein	Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min.	FIG
+234	242	Q domain	structure_element	Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min.	FIG
+243	247	FKBP	protein	Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min.	FIG
+248	252	mRFP	experimental_method	Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min.	FIG
+278	296	Bodipy FL-Ceramide	chemical	Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min.	FIG
+50	54	mRFP	experimental_method	Middle panel – The same experiment performed with mRFP-FKBP alone.	FIG
+55	59	FKBP	protein	Middle panel – The same experiment performed with mRFP-FKBP alone.	FIG
+49	55	mutant	protein_state	Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment.	FIG
+56	64	Q domain	structure_element	Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment.	FIG
+66	71	F258A	mutant	Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment.	FIG
+73	78	H284A	mutant	Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment.	FIG
+80	85	Y288A	mutant	Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment.	FIG
+110	115	PI4KB	protein	Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment.	FIG
+135	170	mitochondria recruitment experiment	experimental_method	Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment.	FIG
+6	11	AKAP1	protein	– The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex.	FIG
+12	15	FRB	structure_element	– The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex.	FIG
+16	19	CFP	experimental_method	– The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex.	FIG
+33	42	localized	evidence	– The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex.	FIG
+90	93	GFP	experimental_method	– The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex.	FIG
+94	99	PI4KB	protein	– The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex.	FIG
+104	112	Q domain	structure_element	– The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex.	FIG
+113	117	FKBP	protein	– The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex.	FIG
+118	122	mRFP	experimental_method	– The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex.	FIG
+138	147	localized	evidence	– The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex.	FIG
+17	26	rapamycin	chemical	Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment.	FIG
+31	39	Q domain	structure_element	Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment.	FIG
+40	44	FKBP	protein	Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment.	FIG
+45	49	mRFP	experimental_method	Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment.	FIG
+103	106	GFP	experimental_method	Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment.	FIG
+107	112	PI4KB	protein	Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment.	FIG
+126	161	Mitochondria recruitment experiment	experimental_method	Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment.	FIG
+30	35	AKAP1	protein	Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin.	FIG
+36	39	FRB	structure_element	Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin.	FIG
+40	43	CFP	experimental_method	Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin.	FIG
+45	48	GFP	experimental_method	Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin.	FIG
+49	54	PI4KB	protein	Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin.	FIG
+59	68	wild-type	protein_state	Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin.	FIG
+69	77	Q domain	structure_element	Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin.	FIG
+78	82	FKBP	protein	Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin.	FIG
+83	87	mRFP	experimental_method	Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin.	FIG
+141	150	rapamycin	chemical	Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin.	FIG
+48	53	H264A	mutant	Right – The same experiment performed using the H264A Q domain mutant.	FIG
+54	62	Q domain	structure_element	Right – The same experiment performed using the H264A Q domain mutant.	FIG
+63	69	mutant	protein_state	Right – The same experiment performed using the H264A Q domain mutant.	FIG
+0	5	ACBD3	protein	ACBD3 indirectly increases the activity of PI4KB.	FIG
+43	48	PI4KB	protein	ACBD3 indirectly increases the activity of PI4KB.	FIG
+4	31	Micelles-based kinase assay	experimental_method	(A) Micelles-based kinase assay – PI in TX100 micelles was used in a luminescent kinase assay and the production of PI4P was measured.	FIG
+34	36	PI	chemical	(A) Micelles-based kinase assay – PI in TX100 micelles was used in a luminescent kinase assay and the production of PI4P was measured.	FIG
+69	93	luminescent kinase assay	experimental_method	(A) Micelles-based kinase assay – PI in TX100 micelles was used in a luminescent kinase assay and the production of PI4P was measured.	FIG
+116	120	PI4P	chemical	(A) Micelles-based kinase assay – PI in TX100 micelles was used in a luminescent kinase assay and the production of PI4P was measured.	FIG
+38	42	PI4P	chemical	Bar graph presents the mean values of PI4P generated in the presence of the proteins as indicated, normalized to the amount of PI4P generated by PI4KB alone.	FIG
+60	71	presence of	protein_state	Bar graph presents the mean values of PI4P generated in the presence of the proteins as indicated, normalized to the amount of PI4P generated by PI4KB alone.	FIG
+127	131	PI4P	chemical	Bar graph presents the mean values of PI4P generated in the presence of the proteins as indicated, normalized to the amount of PI4P generated by PI4KB alone.	FIG
+145	150	PI4KB	protein	Bar graph presents the mean values of PI4P generated in the presence of the proteins as indicated, normalized to the amount of PI4P generated by PI4KB alone.	FIG
+15	42	standard errors of the mean	evidence	Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor.	FIG
+44	47	SEM	evidence	Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor.	FIG
+93	124	GUV-based phosphorylation assay	experimental_method	Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor.	FIG
+127	131	GUVs	experimental_method	Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor.	FIG
+147	149	PI	chemical	Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor.	FIG
+197	201	PI4P	chemical	Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor.	FIG
+225	243	CFP-SidC biosensor	experimental_method	Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor.	FIG
+26	51	GUV phosphorylation assay	experimental_method	(C)–Quantification of the GUV phosphorylation assay – Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions.	FIG
+54	90	Mean membrane fluorescence intensity	evidence	(C)–Quantification of the GUV phosphorylation assay – Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions.	FIG
+98	102	PI4P	chemical	(C)–Quantification of the GUV phosphorylation assay – Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions.	FIG
+113	117	SidC	protein	(C)–Quantification of the GUV phosphorylation assay – Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions.	FIG
+149	152	ATP	chemical	(C)–Quantification of the GUV phosphorylation assay – Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions.	FIG
+4	27	mean membrane intensity	evidence	The mean membrane intensity value is relative to the background signal and the difference between the membrane and background signal in the reference system lacking ATP.	FIG
+165	168	ATP	chemical	The mean membrane intensity value is relative to the background signal and the difference between the membrane and background signal in the reference system lacking ATP.	FIG
+25	28	SEM	evidence	The error bars stand for SEM based on three independent experiments (also SI Fig. 6).	FIG
+0	18	Pseudoatomic model	evidence	Pseudoatomic model of the PI4KB multiprotein complex assembly.	FIG
+26	31	PI4KB	protein	Pseudoatomic model of the PI4KB multiprotein complex assembly.	FIG
+0	5	PI4KB	protein	PI4KB in orange, Rab11 in purple, ACBD3 in blue.	FIG
+17	22	Rab11	protein	PI4KB in orange, Rab11 in purple, ACBD3 in blue.	FIG
+34	39	ACBD3	protein	PI4KB in orange, Rab11 in purple, ACBD3 in blue.	FIG
+26	29	NMR	experimental_method	The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server.	FIG
+30	39	structure	evidence	The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server.	FIG
+67	84	crystal structure	evidence	The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server.	FIG
+88	99	PI4KB:Rab11	complex_assembly	The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server.	FIG
+125	129	ACBD	structure_element	The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server.	FIG
+134	138	GOLD	structure_element	The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server.	FIG
+151	167	homology modeled	experimental_method	The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server.	FIG
+200	210	structures	evidence	The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server.	FIG
+227	233	Phyre2	experimental_method	The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server.	FIG
+4	8	GOLD	structure_element	The GOLD domain is tethered to the membrane by GolginB1 (also known as Giantin) which is not shown for clarity.	FIG
+47	55	GolginB1	protein	The GOLD domain is tethered to the membrane by GolginB1 (also known as Giantin) which is not shown for clarity.	FIG
+71	78	Giantin	protein	The GOLD domain is tethered to the membrane by GolginB1 (also known as Giantin) which is not shown for clarity.	FIG
+0	32	Intrinsically disordered linkers	structure_element	Intrinsically disordered linkers are modeled in an arbitrary but physically plausible conformation.	FIG
diff --git a/annotation_CSV/PMC4817029.csv b/annotation_CSV/PMC4817029.csv
new file mode 100644
index 0000000000000000000000000000000000000000..4a337810d88157831cf8ce72d88c41be6d52cbc1
--- /dev/null
+++ b/annotation_CSV/PMC4817029.csv
@@ -0,0 +1,898 @@
+anno_start	anno_end	anno_text	entity_type	sentence	section
+32	60	family 5 glycoside hydrolase	protein_type	Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism	TITLE
+0	20	Glycoside hydrolases	protein_type	Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials.	ABSTRACT
+22	25	GHs	protein_type	Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials.	ABSTRACT
+20	31	full-length	protein_state	Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity.	ABSTRACT
+32	41	structure	evidence	Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity.	ABSTRACT
+47	56	cellulase	protein_type	Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity.	ABSTRACT
+62	84	Bacillus licheniformis	species	Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity.	ABSTRACT
+86	93	BlCel5B	protein	Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity.	ABSTRACT
+112	127	GH5 subfamily 4	protein_type	Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity.	ABSTRACT
+166	183	ancillary modules	structure_element	Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity.	ABSTRACT
+185	199	Ig-like module	structure_element	Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity.	ABSTRACT
+204	209	CBM46	structure_element	Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity.	ABSTRACT
+6	27	X-ray crystallography	experimental_method	Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms.	ABSTRACT
+29	57	small-angle X-ray scattering	experimental_method	Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms.	ABSTRACT
+62	92	molecular dynamics simulations	experimental_method	Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms.	ABSTRACT
+125	130	CBM46	structure_element	Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms.	ABSTRACT
+158	174	catalytic domain	structure_element	Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms.	ABSTRACT
+176	178	CD	structure_element	Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms.	ABSTRACT
+195	211	fully functional	protein_state	Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms.	ABSTRACT
+212	223	active site	site	Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms.	ABSTRACT
+4	18	Ig-like module	structure_element	The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite.	ABSTRACT
+68	73	CBM46	structure_element	The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite.	ABSTRACT
+86	88	CD	structure_element	The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite.	ABSTRACT
+112	127	binding subsite	site	The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite.	ABSTRACT
+43	45	GH	protein_type	This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.	ABSTRACT
+88	93	CBM46	structure_element	This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.	ABSTRACT
+114	137	catalytically competent	protein_state	This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.	ABSTRACT
+0	5	Plant	taxonomy_domain	Plant biomass-the most abundant source of carbohydrates on Earth-is primarily composed of cellulose microfibrils surrounded by a hydrated heteropolymeric matrix of hemicellulose and lignin.	INTRO
+42	55	carbohydrates	chemical	Plant biomass-the most abundant source of carbohydrates on Earth-is primarily composed of cellulose microfibrils surrounded by a hydrated heteropolymeric matrix of hemicellulose and lignin.	INTRO
+90	99	cellulose	chemical	Plant biomass-the most abundant source of carbohydrates on Earth-is primarily composed of cellulose microfibrils surrounded by a hydrated heteropolymeric matrix of hemicellulose and lignin.	INTRO
+164	177	hemicellulose	chemical	Plant biomass-the most abundant source of carbohydrates on Earth-is primarily composed of cellulose microfibrils surrounded by a hydrated heteropolymeric matrix of hemicellulose and lignin.	INTRO
+182	188	lignin	chemical	Plant biomass-the most abundant source of carbohydrates on Earth-is primarily composed of cellulose microfibrils surrounded by a hydrated heteropolymeric matrix of hemicellulose and lignin.	INTRO
+0	5	Plant	taxonomy_domain	Plant biomass may be subjected to thermo-chemical pretreatments and enzymatic reactions to produce soluble fermentable sugars.	INTRO
+119	125	sugars	chemical	Plant biomass may be subjected to thermo-chemical pretreatments and enzymatic reactions to produce soluble fermentable sugars.	INTRO
+49	58	cellulose	chemical	The canonical model of hydrolytic degradation of cellulose requires at least three classes of enzymes.	INTRO
+0	18	Cellobiohydrolases	protein_type	Cellobiohydrolases (CBHs) processively cleave the glycosidic bonds at the reducing and non-reducing ends of cellulose chains in crystalline regions to produce cellobiose.	INTRO
+20	24	CBHs	protein_type	Cellobiohydrolases (CBHs) processively cleave the glycosidic bonds at the reducing and non-reducing ends of cellulose chains in crystalline regions to produce cellobiose.	INTRO
+108	117	cellulose	chemical	Cellobiohydrolases (CBHs) processively cleave the glycosidic bonds at the reducing and non-reducing ends of cellulose chains in crystalline regions to produce cellobiose.	INTRO
+159	169	cellobiose	chemical	Cellobiohydrolases (CBHs) processively cleave the glycosidic bonds at the reducing and non-reducing ends of cellulose chains in crystalline regions to produce cellobiose.	INTRO
+0	14	Endoglucanases	protein_type	Endoglucanases (EGs) introduce random cuts in the amorphous regions of cellulose and create new chain extremities for CBH attack; thus, these enzymes act synergistically.	INTRO
+16	19	EGs	protein_type	Endoglucanases (EGs) introduce random cuts in the amorphous regions of cellulose and create new chain extremities for CBH attack; thus, these enzymes act synergistically.	INTRO
+71	80	cellulose	chemical	Endoglucanases (EGs) introduce random cuts in the amorphous regions of cellulose and create new chain extremities for CBH attack; thus, these enzymes act synergistically.	INTRO
+118	121	CBH	protein_type	Endoglucanases (EGs) introduce random cuts in the amorphous regions of cellulose and create new chain extremities for CBH attack; thus, these enzymes act synergistically.	INTRO
+13	23	cellobiose	chemical	The released cellobiose molecules are then enzymatically converted into glucose by β-glucosidases.	INTRO
+72	79	glucose	chemical	The released cellobiose molecules are then enzymatically converted into glucose by β-glucosidases.	INTRO
+83	97	β-glucosidases	protein_type	The released cellobiose molecules are then enzymatically converted into glucose by β-glucosidases.	INTRO
+30	50	glycoside hydrolases	protein_type	The molecular architecture of glycoside hydrolases (GHs) frequently consists of a catalytic domain (CD), where hydrolysis occurs, and one or more ancillary modules (AMs), which are usually connected by less structured linkers.	INTRO
+52	55	GHs	protein_type	The molecular architecture of glycoside hydrolases (GHs) frequently consists of a catalytic domain (CD), where hydrolysis occurs, and one or more ancillary modules (AMs), which are usually connected by less structured linkers.	INTRO
+82	98	catalytic domain	structure_element	The molecular architecture of glycoside hydrolases (GHs) frequently consists of a catalytic domain (CD), where hydrolysis occurs, and one or more ancillary modules (AMs), which are usually connected by less structured linkers.	INTRO
+100	102	CD	structure_element	The molecular architecture of glycoside hydrolases (GHs) frequently consists of a catalytic domain (CD), where hydrolysis occurs, and one or more ancillary modules (AMs), which are usually connected by less structured linkers.	INTRO
+146	163	ancillary modules	structure_element	The molecular architecture of glycoside hydrolases (GHs) frequently consists of a catalytic domain (CD), where hydrolysis occurs, and one or more ancillary modules (AMs), which are usually connected by less structured linkers.	INTRO
+165	168	AMs	structure_element	The molecular architecture of glycoside hydrolases (GHs) frequently consists of a catalytic domain (CD), where hydrolysis occurs, and one or more ancillary modules (AMs), which are usually connected by less structured linkers.	INTRO
+202	217	less structured	protein_state	The molecular architecture of glycoside hydrolases (GHs) frequently consists of a catalytic domain (CD), where hydrolysis occurs, and one or more ancillary modules (AMs), which are usually connected by less structured linkers.	INTRO
+218	225	linkers	structure_element	The molecular architecture of glycoside hydrolases (GHs) frequently consists of a catalytic domain (CD), where hydrolysis occurs, and one or more ancillary modules (AMs), which are usually connected by less structured linkers.	INTRO
+24	27	AMs	structure_element	The most common type of AMs are carbohydrate-binding modules (CBMs), which are able to recognize and bind specific carbohydrate chains.	INTRO
+32	60	carbohydrate-binding modules	structure_element	The most common type of AMs are carbohydrate-binding modules (CBMs), which are able to recognize and bind specific carbohydrate chains.	INTRO
+62	66	CBMs	structure_element	The most common type of AMs are carbohydrate-binding modules (CBMs), which are able to recognize and bind specific carbohydrate chains.	INTRO
+115	127	carbohydrate	chemical	The most common type of AMs are carbohydrate-binding modules (CBMs), which are able to recognize and bind specific carbohydrate chains.	INTRO
+59	63	CBMs	structure_element	Generally distinct and independent structural domains, the CBMs facilitate carbohydrate hydrolysis by increasing the local concentration of enzymes at the surface of insoluble substrates, thereby targeting the CD component to its cognate ligands.	INTRO
+75	87	carbohydrate	chemical	Generally distinct and independent structural domains, the CBMs facilitate carbohydrate hydrolysis by increasing the local concentration of enzymes at the surface of insoluble substrates, thereby targeting the CD component to its cognate ligands.	INTRO
+210	212	CD	structure_element	Generally distinct and independent structural domains, the CBMs facilitate carbohydrate hydrolysis by increasing the local concentration of enzymes at the surface of insoluble substrates, thereby targeting the CD component to its cognate ligands.	INTRO
+0	4	CBMs	structure_element	CBMs might also disrupt the crystalline structure of cellulose microfibrils, although the underlying mechanism remains poorly understood.	INTRO
+53	62	cellulose	chemical	CBMs might also disrupt the crystalline structure of cellulose microfibrils, although the underlying mechanism remains poorly understood.	INTRO
+6	10	CBMs	structure_element	Thus, CBMs enhance the accessibility of CDs to carbohydrate chains to improve enzymatic activity, making them important candidates for the development of effective biomass-degrading enzymes in industrial settings.	INTRO
+40	43	CDs	structure_element	Thus, CBMs enhance the accessibility of CDs to carbohydrate chains to improve enzymatic activity, making them important candidates for the development of effective biomass-degrading enzymes in industrial settings.	INTRO
+47	59	carbohydrate	chemical	Thus, CBMs enhance the accessibility of CDs to carbohydrate chains to improve enzymatic activity, making them important candidates for the development of effective biomass-degrading enzymes in industrial settings.	INTRO
+31	37	active	protein_state	Although there are examples of active GHs that lack AMs, the majority of the enzymes depend on AMs for activity.	INTRO
+38	41	GHs	protein_type	Although there are examples of active GHs that lack AMs, the majority of the enzymes depend on AMs for activity.	INTRO
+47	51	lack	protein_state	Although there are examples of active GHs that lack AMs, the majority of the enzymes depend on AMs for activity.	INTRO
+52	55	AMs	structure_element	Although there are examples of active GHs that lack AMs, the majority of the enzymes depend on AMs for activity.	INTRO
+95	98	AMs	structure_element	Although there are examples of active GHs that lack AMs, the majority of the enzymes depend on AMs for activity.	INTRO
+18	22	CBMs	structure_element	In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).	INTRO
+63	65	CD	structure_element	In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).	INTRO
+66	88	substrate-binding site	site	In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).	INTRO
+105	132	carbohydrate-active enzymes	protein_type	In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).	INTRO
+142	159	endo/exocellulase	protein_type	In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).	INTRO
+160	162	E4	protein	In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).	INTRO
+168	186	Thermobifida fusca	species	In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).	INTRO
+188	199	chitinase B	protein	In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).	INTRO
+205	224	Serratia marcescens	species	In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).	INTRO
+228	246	starch phosphatase	protein_type	In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).	INTRO
+252	272	Arabidopsis thaliana	species	In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).	INTRO
+279	294	GH5 subfamily 4	protein_type	In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).	INTRO
+296	301	GH5_4	protein_type	In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).	INTRO
+303	316	endoglucanase	protein_type	In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).	INTRO
+322	341	Bacillus halodurans	species	In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).	INTRO
+343	350	BhCel5B	protein	In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).	INTRO
+79	82	GH9	protein_type	A pioneer work of Sakon et al. revealed that rigid structural extension of the GH9 CD by a type C CBM3 imprints a processive mode of action to this endoglucanase.	INTRO
+83	85	CD	structure_element	A pioneer work of Sakon et al. revealed that rigid structural extension of the GH9 CD by a type C CBM3 imprints a processive mode of action to this endoglucanase.	INTRO
+91	102	type C CBM3	structure_element	A pioneer work of Sakon et al. revealed that rigid structural extension of the GH9 CD by a type C CBM3 imprints a processive mode of action to this endoglucanase.	INTRO
+148	161	endoglucanase	protein_type	A pioneer work of Sakon et al. revealed that rigid structural extension of the GH9 CD by a type C CBM3 imprints a processive mode of action to this endoglucanase.	INTRO
+33	36	CBM	structure_element	Further publications showed that CBM-based structural extensions of the active site are important for substrate engagement and recognition.	INTRO
+72	83	active site	site	Further publications showed that CBM-based structural extensions of the active site are important for substrate engagement and recognition.	INTRO
+39	54	X-ray structure	evidence	Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module.	INTRO
+62	73	tri-modular	structure_element	Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module.	INTRO
+74	79	GH5_4	protein_type	Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module.	INTRO
+80	93	endoglucanase	protein_type	Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module.	INTRO
+99	118	Bacillus halodurans	species	Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module.	INTRO
+145	152	BlCel5B	protein	Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module.	INTRO
+164	169	CBM46	structure_element	Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module.	INTRO
+187	198	active site	site	Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module.	INTRO
+215	217	CD	structure_element	Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module.	INTRO
+225	256	immunoglobulin (Ig)-like module	structure_element	Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module.	INTRO
+0	10	Removal of	experimental_method	Removal of the CBM46 caused a ~60-fold reduction of the activity of the enzyme against β-glucans, but showed little or no effect against xyloglucan hydrolysis.	INTRO
+15	20	CBM46	structure_element	Removal of the CBM46 caused a ~60-fold reduction of the activity of the enzyme against β-glucans, but showed little or no effect against xyloglucan hydrolysis.	INTRO
+87	96	β-glucans	chemical	Removal of the CBM46 caused a ~60-fold reduction of the activity of the enzyme against β-glucans, but showed little or no effect against xyloglucan hydrolysis.	INTRO
+137	147	xyloglucan	chemical	Removal of the CBM46 caused a ~60-fold reduction of the activity of the enzyme against β-glucans, but showed little or no effect against xyloglucan hydrolysis.	INTRO
+14	19	CBM46	structure_element	Moreover, the CBM46 mediated a significant increase in the BhCel5B activity in plant cell wall settings.	INTRO
+59	66	BhCel5B	protein	Moreover, the CBM46 mediated a significant increase in the BhCel5B activity in plant cell wall settings.	INTRO
+79	84	plant	taxonomy_domain	Moreover, the CBM46 mediated a significant increase in the BhCel5B activity in plant cell wall settings.	INTRO
+0	8	Modeling	experimental_method	Modeling of cellotriose in the negative subsites of the active site of BhCel5B demonstrated the structural conservation of the -1 position, but provided little information about direct interactions between CBM46 and the substrate.	INTRO
+12	23	cellotriose	chemical	Modeling of cellotriose in the negative subsites of the active site of BhCel5B demonstrated the structural conservation of the -1 position, but provided little information about direct interactions between CBM46 and the substrate.	INTRO
+31	48	negative subsites	site	Modeling of cellotriose in the negative subsites of the active site of BhCel5B demonstrated the structural conservation of the -1 position, but provided little information about direct interactions between CBM46 and the substrate.	INTRO
+56	67	active site	site	Modeling of cellotriose in the negative subsites of the active site of BhCel5B demonstrated the structural conservation of the -1 position, but provided little information about direct interactions between CBM46 and the substrate.	INTRO
+71	78	BhCel5B	protein	Modeling of cellotriose in the negative subsites of the active site of BhCel5B demonstrated the structural conservation of the -1 position, but provided little information about direct interactions between CBM46 and the substrate.	INTRO
+96	119	structural conservation	protein_state	Modeling of cellotriose in the negative subsites of the active site of BhCel5B demonstrated the structural conservation of the -1 position, but provided little information about direct interactions between CBM46 and the substrate.	INTRO
+127	129	-1	residue_number	Modeling of cellotriose in the negative subsites of the active site of BhCel5B demonstrated the structural conservation of the -1 position, but provided little information about direct interactions between CBM46 and the substrate.	INTRO
+206	211	CBM46	structure_element	Modeling of cellotriose in the negative subsites of the active site of BhCel5B demonstrated the structural conservation of the -1 position, but provided little information about direct interactions between CBM46 and the substrate.	INTRO
+41	49	β-glucan	chemical	It was speculated that β-1,3 kink of the β-glucan might allow the ligand to reach for the CBM46, whereas pure β-1,4 linkages in the backbone of xyloglucan chains would restrict binding to the CD, thus explaining the lack of influence of the CBM46 on the enzymatic activity of BhCel5B against xyloglucans in solution.	INTRO
+90	95	CBM46	structure_element	It was speculated that β-1,3 kink of the β-glucan might allow the ligand to reach for the CBM46, whereas pure β-1,4 linkages in the backbone of xyloglucan chains would restrict binding to the CD, thus explaining the lack of influence of the CBM46 on the enzymatic activity of BhCel5B against xyloglucans in solution.	INTRO
+144	154	xyloglucan	chemical	It was speculated that β-1,3 kink of the β-glucan might allow the ligand to reach for the CBM46, whereas pure β-1,4 linkages in the backbone of xyloglucan chains would restrict binding to the CD, thus explaining the lack of influence of the CBM46 on the enzymatic activity of BhCel5B against xyloglucans in solution.	INTRO
+192	194	CD	structure_element	It was speculated that β-1,3 kink of the β-glucan might allow the ligand to reach for the CBM46, whereas pure β-1,4 linkages in the backbone of xyloglucan chains would restrict binding to the CD, thus explaining the lack of influence of the CBM46 on the enzymatic activity of BhCel5B against xyloglucans in solution.	INTRO
+241	246	CBM46	structure_element	It was speculated that β-1,3 kink of the β-glucan might allow the ligand to reach for the CBM46, whereas pure β-1,4 linkages in the backbone of xyloglucan chains would restrict binding to the CD, thus explaining the lack of influence of the CBM46 on the enzymatic activity of BhCel5B against xyloglucans in solution.	INTRO
+276	283	BhCel5B	protein	It was speculated that β-1,3 kink of the β-glucan might allow the ligand to reach for the CBM46, whereas pure β-1,4 linkages in the backbone of xyloglucan chains would restrict binding to the CD, thus explaining the lack of influence of the CBM46 on the enzymatic activity of BhCel5B against xyloglucans in solution.	INTRO
+292	303	xyloglucans	chemical	It was speculated that β-1,3 kink of the β-glucan might allow the ligand to reach for the CBM46, whereas pure β-1,4 linkages in the backbone of xyloglucan chains would restrict binding to the CD, thus explaining the lack of influence of the CBM46 on the enzymatic activity of BhCel5B against xyloglucans in solution.	INTRO
+28	33	CBM46	structure_element	It was also argued that the CBM46 could potentialize the activity by driving BhCel5B towards xyloglucan-rich regions in the context of the plant cell walls, but no large-scale conformational adjustments of the AMs have been shown to occur or suggested to take part in the enzymatic activity.	INTRO
+77	84	BhCel5B	protein	It was also argued that the CBM46 could potentialize the activity by driving BhCel5B towards xyloglucan-rich regions in the context of the plant cell walls, but no large-scale conformational adjustments of the AMs have been shown to occur or suggested to take part in the enzymatic activity.	INTRO
+93	116	xyloglucan-rich regions	structure_element	It was also argued that the CBM46 could potentialize the activity by driving BhCel5B towards xyloglucan-rich regions in the context of the plant cell walls, but no large-scale conformational adjustments of the AMs have been shown to occur or suggested to take part in the enzymatic activity.	INTRO
+139	144	plant	taxonomy_domain	It was also argued that the CBM46 could potentialize the activity by driving BhCel5B towards xyloglucan-rich regions in the context of the plant cell walls, but no large-scale conformational adjustments of the AMs have been shown to occur or suggested to take part in the enzymatic activity.	INTRO
+210	213	AMs	structure_element	It was also argued that the CBM46 could potentialize the activity by driving BhCel5B towards xyloglucan-rich regions in the context of the plant cell walls, but no large-scale conformational adjustments of the AMs have been shown to occur or suggested to take part in the enzymatic activity.	INTRO
+145	153	extended	protein_state	Although initially introduced as contradictory theories, these two limiting cases can be unified considering the flux description concept or the extended conformational selection model.	INTRO
+77	104	carbohydrate-active enzymes	protein_type	While local ligand-induced conformational adjustments have been reported for carbohydrate-active enzymes, cognate ligands recognition and hydrolysis mediated by a large-scale conformational mobility of distinct domains in multidomain settings is uncommon for endoglucanases.	INTRO
+259	273	endoglucanases	protein_type	While local ligand-induced conformational adjustments have been reported for carbohydrate-active enzymes, cognate ligands recognition and hydrolysis mediated by a large-scale conformational mobility of distinct domains in multidomain settings is uncommon for endoglucanases.	INTRO
+20	37	crystal structure	evidence	Here, we report the crystal structure of a full-length GH5_4 enzyme from Bacillus licheniformis (BlCel5B) that exhibits two AMs (Ig-like module and CBM46) appended to the CD.	INTRO
+43	54	full-length	protein_state	Here, we report the crystal structure of a full-length GH5_4 enzyme from Bacillus licheniformis (BlCel5B) that exhibits two AMs (Ig-like module and CBM46) appended to the CD.	INTRO
+55	60	GH5_4	protein_type	Here, we report the crystal structure of a full-length GH5_4 enzyme from Bacillus licheniformis (BlCel5B) that exhibits two AMs (Ig-like module and CBM46) appended to the CD.	INTRO
+73	95	Bacillus licheniformis	species	Here, we report the crystal structure of a full-length GH5_4 enzyme from Bacillus licheniformis (BlCel5B) that exhibits two AMs (Ig-like module and CBM46) appended to the CD.	INTRO
+97	104	BlCel5B	protein	Here, we report the crystal structure of a full-length GH5_4 enzyme from Bacillus licheniformis (BlCel5B) that exhibits two AMs (Ig-like module and CBM46) appended to the CD.	INTRO
+124	127	AMs	structure_element	Here, we report the crystal structure of a full-length GH5_4 enzyme from Bacillus licheniformis (BlCel5B) that exhibits two AMs (Ig-like module and CBM46) appended to the CD.	INTRO
+129	143	Ig-like module	structure_element	Here, we report the crystal structure of a full-length GH5_4 enzyme from Bacillus licheniformis (BlCel5B) that exhibits two AMs (Ig-like module and CBM46) appended to the CD.	INTRO
+148	153	CBM46	structure_element	Here, we report the crystal structure of a full-length GH5_4 enzyme from Bacillus licheniformis (BlCel5B) that exhibits two AMs (Ig-like module and CBM46) appended to the CD.	INTRO
+171	173	CD	structure_element	Here, we report the crystal structure of a full-length GH5_4 enzyme from Bacillus licheniformis (BlCel5B) that exhibits two AMs (Ig-like module and CBM46) appended to the CD.	INTRO
+3	45	structurally and functionally characterize	experimental_method	We structurally and functionally characterize the enzyme using a combination of protein crystallography, small-angle X-ray scattering (SAXS), molecular dynamics computer simulations and site-directed mutagenesis, and show that the AMs and their conformational mobility are essential for the enzymatic activity of BlCel5B.	INTRO
+80	103	protein crystallography	experimental_method	We structurally and functionally characterize the enzyme using a combination of protein crystallography, small-angle X-ray scattering (SAXS), molecular dynamics computer simulations and site-directed mutagenesis, and show that the AMs and their conformational mobility are essential for the enzymatic activity of BlCel5B.	INTRO
+105	133	small-angle X-ray scattering	experimental_method	We structurally and functionally characterize the enzyme using a combination of protein crystallography, small-angle X-ray scattering (SAXS), molecular dynamics computer simulations and site-directed mutagenesis, and show that the AMs and their conformational mobility are essential for the enzymatic activity of BlCel5B.	INTRO
+135	139	SAXS	experimental_method	We structurally and functionally characterize the enzyme using a combination of protein crystallography, small-angle X-ray scattering (SAXS), molecular dynamics computer simulations and site-directed mutagenesis, and show that the AMs and their conformational mobility are essential for the enzymatic activity of BlCel5B.	INTRO
+142	181	molecular dynamics computer simulations	experimental_method	We structurally and functionally characterize the enzyme using a combination of protein crystallography, small-angle X-ray scattering (SAXS), molecular dynamics computer simulations and site-directed mutagenesis, and show that the AMs and their conformational mobility are essential for the enzymatic activity of BlCel5B.	INTRO
+186	211	site-directed mutagenesis	experimental_method	We structurally and functionally characterize the enzyme using a combination of protein crystallography, small-angle X-ray scattering (SAXS), molecular dynamics computer simulations and site-directed mutagenesis, and show that the AMs and their conformational mobility are essential for the enzymatic activity of BlCel5B.	INTRO
+231	234	AMs	structure_element	We structurally and functionally characterize the enzyme using a combination of protein crystallography, small-angle X-ray scattering (SAXS), molecular dynamics computer simulations and site-directed mutagenesis, and show that the AMs and their conformational mobility are essential for the enzymatic activity of BlCel5B.	INTRO
+313	320	BlCel5B	protein	We structurally and functionally characterize the enzyme using a combination of protein crystallography, small-angle X-ray scattering (SAXS), molecular dynamics computer simulations and site-directed mutagenesis, and show that the AMs and their conformational mobility are essential for the enzymatic activity of BlCel5B.	INTRO
+70	75	CBM46	structure_element	We find that the large-scale conformational adjustments of the distal CBM46 mediated by the Ig-like hinge domain are crucial in active-site assembly for optimal substrate binding and hydrolysis.	INTRO
+92	112	Ig-like hinge domain	structure_element	We find that the large-scale conformational adjustments of the distal CBM46 mediated by the Ig-like hinge domain are crucial in active-site assembly for optimal substrate binding and hydrolysis.	INTRO
+128	139	active-site	site	We find that the large-scale conformational adjustments of the distal CBM46 mediated by the Ig-like hinge domain are crucial in active-site assembly for optimal substrate binding and hydrolysis.	INTRO
+20	27	BlCel5B	protein	We propose that the BlCel5B conformational selection/induced-fit mechanism of hydrolysis represents a novel paradigm that applies to several GH5_4 members and, possibly, to a number of other multidomain GHs.	INTRO
+141	146	GH5_4	protein_type	We propose that the BlCel5B conformational selection/induced-fit mechanism of hydrolysis represents a novel paradigm that applies to several GH5_4 members and, possibly, to a number of other multidomain GHs.	INTRO
+203	206	GHs	protein_type	We propose that the BlCel5B conformational selection/induced-fit mechanism of hydrolysis represents a novel paradigm that applies to several GH5_4 members and, possibly, to a number of other multidomain GHs.	INTRO
+0	7	BlCel5B	protein	BlCel5B Crystal Structure	RESULTS
+8	25	Crystal Structure	evidence	BlCel5B Crystal Structure	RESULTS
+0	7	BlCel5B	protein	BlCel5B crystals in the substrate-free form and complexed with cellopentaose (C5) were obtained and diffracted to 1.7 Å and 1.75 Å resolutions, respectively (Supplementary Table 1).	RESULTS
+8	16	crystals	evidence	BlCel5B crystals in the substrate-free form and complexed with cellopentaose (C5) were obtained and diffracted to 1.7 Å and 1.75 Å resolutions, respectively (Supplementary Table 1).	RESULTS
+24	38	substrate-free	protein_state	BlCel5B crystals in the substrate-free form and complexed with cellopentaose (C5) were obtained and diffracted to 1.7 Å and 1.75 Å resolutions, respectively (Supplementary Table 1).	RESULTS
+48	62	complexed with	protein_state	BlCel5B crystals in the substrate-free form and complexed with cellopentaose (C5) were obtained and diffracted to 1.7 Å and 1.75 Å resolutions, respectively (Supplementary Table 1).	RESULTS
+63	76	cellopentaose	chemical	BlCel5B crystals in the substrate-free form and complexed with cellopentaose (C5) were obtained and diffracted to 1.7 Å and 1.75 Å resolutions, respectively (Supplementary Table 1).	RESULTS
+78	80	C5	chemical	BlCel5B crystals in the substrate-free form and complexed with cellopentaose (C5) were obtained and diffracted to 1.7 Å and 1.75 Å resolutions, respectively (Supplementary Table 1).	RESULTS
+4	18	substrate-free	protein_state	The substrate-free and complexed structures exhibited no substantial conformational differences (with the exception of the substrate).	RESULTS
+23	32	complexed	protein_state	The substrate-free and complexed structures exhibited no substantial conformational differences (with the exception of the substrate).	RESULTS
+33	43	structures	evidence	The substrate-free and complexed structures exhibited no substantial conformational differences (with the exception of the substrate).	RESULTS
+35	40	loops	structure_element	Because of minor variations in the loops located distal to the substrate-binding site, a root mean squared deviation (rmsd) of 0.33 Å between the complexed and substrate-free structures was observed.	RESULTS
+63	85	substrate-binding site	site	Because of minor variations in the loops located distal to the substrate-binding site, a root mean squared deviation (rmsd) of 0.33 Å between the complexed and substrate-free structures was observed.	RESULTS
+89	116	root mean squared deviation	evidence	Because of minor variations in the loops located distal to the substrate-binding site, a root mean squared deviation (rmsd) of 0.33 Å between the complexed and substrate-free structures was observed.	RESULTS
+118	122	rmsd	evidence	Because of minor variations in the loops located distal to the substrate-binding site, a root mean squared deviation (rmsd) of 0.33 Å between the complexed and substrate-free structures was observed.	RESULTS
+146	155	complexed	protein_state	Because of minor variations in the loops located distal to the substrate-binding site, a root mean squared deviation (rmsd) of 0.33 Å between the complexed and substrate-free structures was observed.	RESULTS
+160	174	substrate-free	protein_state	Because of minor variations in the loops located distal to the substrate-binding site, a root mean squared deviation (rmsd) of 0.33 Å between the complexed and substrate-free structures was observed.	RESULTS
+175	185	structures	evidence	Because of minor variations in the loops located distal to the substrate-binding site, a root mean squared deviation (rmsd) of 0.33 Å between the complexed and substrate-free structures was observed.	RESULTS
+116	133	first 17 residues	residue_range	A single protein chain occupies the asymmetric unit, and most of the residues were built, with the exception of the first 17 residues and those in the loop between L398 and P405 due to weak electron density.	RESULTS
+151	155	loop	structure_element	A single protein chain occupies the asymmetric unit, and most of the residues were built, with the exception of the first 17 residues and those in the loop between L398 and P405 due to weak electron density.	RESULTS
+164	168	L398	residue_name_number	A single protein chain occupies the asymmetric unit, and most of the residues were built, with the exception of the first 17 residues and those in the loop between L398 and P405 due to weak electron density.	RESULTS
+173	177	P405	residue_name_number	A single protein chain occupies the asymmetric unit, and most of the residues were built, with the exception of the first 17 residues and those in the loop between L398 and P405 due to weak electron density.	RESULTS
+190	206	electron density	evidence	A single protein chain occupies the asymmetric unit, and most of the residues were built, with the exception of the first 17 residues and those in the loop between L398 and P405 due to weak electron density.	RESULTS
+4	11	BlCel5B	protein	The BlCel5B structure comprises three distinct domains: an N-terminal CD (residues 18 to 330), an Ig-like module (residues 335 to 428) and a family 46 CBM (residues 432 to 533) (Fig. 1A,B).	RESULTS
+12	21	structure	evidence	The BlCel5B structure comprises three distinct domains: an N-terminal CD (residues 18 to 330), an Ig-like module (residues 335 to 428) and a family 46 CBM (residues 432 to 533) (Fig. 1A,B).	RESULTS
+70	72	CD	structure_element	The BlCel5B structure comprises three distinct domains: an N-terminal CD (residues 18 to 330), an Ig-like module (residues 335 to 428) and a family 46 CBM (residues 432 to 533) (Fig. 1A,B).	RESULTS
+83	92	18 to 330	residue_range	The BlCel5B structure comprises three distinct domains: an N-terminal CD (residues 18 to 330), an Ig-like module (residues 335 to 428) and a family 46 CBM (residues 432 to 533) (Fig. 1A,B).	RESULTS
+98	112	Ig-like module	structure_element	The BlCel5B structure comprises three distinct domains: an N-terminal CD (residues 18 to 330), an Ig-like module (residues 335 to 428) and a family 46 CBM (residues 432 to 533) (Fig. 1A,B).	RESULTS
+123	133	335 to 428	residue_range	The BlCel5B structure comprises three distinct domains: an N-terminal CD (residues 18 to 330), an Ig-like module (residues 335 to 428) and a family 46 CBM (residues 432 to 533) (Fig. 1A,B).	RESULTS
+141	154	family 46 CBM	structure_element	The BlCel5B structure comprises three distinct domains: an N-terminal CD (residues 18 to 330), an Ig-like module (residues 335 to 428) and a family 46 CBM (residues 432 to 533) (Fig. 1A,B).	RESULTS
+165	175	432 to 533	residue_range	The BlCel5B structure comprises three distinct domains: an N-terminal CD (residues 18 to 330), an Ig-like module (residues 335 to 428) and a family 46 CBM (residues 432 to 533) (Fig. 1A,B).	RESULTS
+34	37	GH5	protein_type	Similarly to other members of the GH5 family, the CD of BlCel5B has a typical TIM barrel fold with eight inner β-strands and eight outer α helices that are interconnected by loops and three short α helices.	RESULTS
+50	52	CD	structure_element	Similarly to other members of the GH5 family, the CD of BlCel5B has a typical TIM barrel fold with eight inner β-strands and eight outer α helices that are interconnected by loops and three short α helices.	RESULTS
+56	63	BlCel5B	protein	Similarly to other members of the GH5 family, the CD of BlCel5B has a typical TIM barrel fold with eight inner β-strands and eight outer α helices that are interconnected by loops and three short α helices.	RESULTS
+78	93	TIM barrel fold	structure_element	Similarly to other members of the GH5 family, the CD of BlCel5B has a typical TIM barrel fold with eight inner β-strands and eight outer α helices that are interconnected by loops and three short α helices.	RESULTS
+111	120	β-strands	structure_element	Similarly to other members of the GH5 family, the CD of BlCel5B has a typical TIM barrel fold with eight inner β-strands and eight outer α helices that are interconnected by loops and three short α helices.	RESULTS
+137	146	α helices	structure_element	Similarly to other members of the GH5 family, the CD of BlCel5B has a typical TIM barrel fold with eight inner β-strands and eight outer α helices that are interconnected by loops and three short α helices.	RESULTS
+174	179	loops	structure_element	Similarly to other members of the GH5 family, the CD of BlCel5B has a typical TIM barrel fold with eight inner β-strands and eight outer α helices that are interconnected by loops and three short α helices.	RESULTS
+196	205	α helices	structure_element	Similarly to other members of the GH5 family, the CD of BlCel5B has a typical TIM barrel fold with eight inner β-strands and eight outer α helices that are interconnected by loops and three short α helices.	RESULTS
+11	18	linkers	structure_element	Very short linkers, D429-D430-P431 and V331-P332-N333-A334, connect the CBM46 to the Ig-like module and the Ig-like module to the CD, respectively.	RESULTS
+20	34	D429-D430-P431	structure_element	Very short linkers, D429-D430-P431 and V331-P332-N333-A334, connect the CBM46 to the Ig-like module and the Ig-like module to the CD, respectively.	RESULTS
+39	58	V331-P332-N333-A334	structure_element	Very short linkers, D429-D430-P431 and V331-P332-N333-A334, connect the CBM46 to the Ig-like module and the Ig-like module to the CD, respectively.	RESULTS
+72	77	CBM46	structure_element	Very short linkers, D429-D430-P431 and V331-P332-N333-A334, connect the CBM46 to the Ig-like module and the Ig-like module to the CD, respectively.	RESULTS
+85	99	Ig-like module	structure_element	Very short linkers, D429-D430-P431 and V331-P332-N333-A334, connect the CBM46 to the Ig-like module and the Ig-like module to the CD, respectively.	RESULTS
+108	122	Ig-like module	structure_element	Very short linkers, D429-D430-P431 and V331-P332-N333-A334, connect the CBM46 to the Ig-like module and the Ig-like module to the CD, respectively.	RESULTS
+130	132	CD	structure_element	Very short linkers, D429-D430-P431 and V331-P332-N333-A334, connect the CBM46 to the Ig-like module and the Ig-like module to the CD, respectively.	RESULTS
+5	19	Ig-like module	structure_element	Both Ig-like module and CBM46 have a β-sandwich fold composed of two β-sheets of four and three antiparallel β-strands interconnected by loops and a short α helix between strands β3 and β4 (Fig. 1C).	RESULTS
+24	29	CBM46	structure_element	Both Ig-like module and CBM46 have a β-sandwich fold composed of two β-sheets of four and three antiparallel β-strands interconnected by loops and a short α helix between strands β3 and β4 (Fig. 1C).	RESULTS
+37	52	β-sandwich fold	structure_element	Both Ig-like module and CBM46 have a β-sandwich fold composed of two β-sheets of four and three antiparallel β-strands interconnected by loops and a short α helix between strands β3 and β4 (Fig. 1C).	RESULTS
+69	77	β-sheets	structure_element	Both Ig-like module and CBM46 have a β-sandwich fold composed of two β-sheets of four and three antiparallel β-strands interconnected by loops and a short α helix between strands β3 and β4 (Fig. 1C).	RESULTS
+96	118	antiparallel β-strands	structure_element	Both Ig-like module and CBM46 have a β-sandwich fold composed of two β-sheets of four and three antiparallel β-strands interconnected by loops and a short α helix between strands β3 and β4 (Fig. 1C).	RESULTS
+137	142	loops	structure_element	Both Ig-like module and CBM46 have a β-sandwich fold composed of two β-sheets of four and three antiparallel β-strands interconnected by loops and a short α helix between strands β3 and β4 (Fig. 1C).	RESULTS
+155	162	α helix	structure_element	Both Ig-like module and CBM46 have a β-sandwich fold composed of two β-sheets of four and three antiparallel β-strands interconnected by loops and a short α helix between strands β3 and β4 (Fig. 1C).	RESULTS
+171	178	strands	structure_element	Both Ig-like module and CBM46 have a β-sandwich fold composed of two β-sheets of four and three antiparallel β-strands interconnected by loops and a short α helix between strands β3 and β4 (Fig. 1C).	RESULTS
+179	181	β3	structure_element	Both Ig-like module and CBM46 have a β-sandwich fold composed of two β-sheets of four and three antiparallel β-strands interconnected by loops and a short α helix between strands β3 and β4 (Fig. 1C).	RESULTS
+186	188	β4	structure_element	Both Ig-like module and CBM46 have a β-sandwich fold composed of two β-sheets of four and three antiparallel β-strands interconnected by loops and a short α helix between strands β3 and β4 (Fig. 1C).	RESULTS
+2	23	structural comparison	experimental_method	A structural comparison between the Ig-like module and the CBM46 using the Dali server yielded an rmsd of 2.3 Å and a Z-score of 10.2.	RESULTS
+36	50	Ig-like module	structure_element	A structural comparison between the Ig-like module and the CBM46 using the Dali server yielded an rmsd of 2.3 Å and a Z-score of 10.2.	RESULTS
+59	64	CBM46	structure_element	A structural comparison between the Ig-like module and the CBM46 using the Dali server yielded an rmsd of 2.3 Å and a Z-score of 10.2.	RESULTS
+75	86	Dali server	experimental_method	A structural comparison between the Ig-like module and the CBM46 using the Dali server yielded an rmsd of 2.3 Å and a Z-score of 10.2.	RESULTS
+98	102	rmsd	evidence	A structural comparison between the Ig-like module and the CBM46 using the Dali server yielded an rmsd of 2.3 Å and a Z-score of 10.2.	RESULTS
+118	125	Z-score	evidence	A structural comparison between the Ig-like module and the CBM46 using the Dali server yielded an rmsd of 2.3 Å and a Z-score of 10.2.	RESULTS
+2	24	structure-based search	experimental_method	A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd = 1.3 Å and Z-score = 15.3.	RESULTS
+73	87	Ig-like module	structure_element	A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd = 1.3 Å and Z-score = 15.3.	RESULTS
+106	120	Ig-like module	structure_element	A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd = 1.3 Å and Z-score = 15.3.	RESULTS
+137	143	solved	experimental_method	A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd = 1.3 Å and Z-score = 15.3.	RESULTS
+144	161	crystal structure	evidence	A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd = 1.3 Å and Z-score = 15.3.	RESULTS
+167	178	tri-modular	structure_element	A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd = 1.3 Å and Z-score = 15.3.	RESULTS
+179	184	GH5_4	protein_type	A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd = 1.3 Å and Z-score = 15.3.	RESULTS
+197	216	Bacillus halodurans	species	A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd = 1.3 Å and Z-score = 15.3.	RESULTS
+218	225	BhCel5B	protein	A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd = 1.3 Å and Z-score = 15.3.	RESULTS
+232	236	rmsd	evidence	A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd = 1.3 Å and Z-score = 15.3.	RESULTS
+249	256	Z-score	evidence	A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd = 1.3 Å and Z-score = 15.3.	RESULTS
+4	9	CBM46	structure_element	The CBM46 from BhCel5B is the most structurally similar to BlCel5B CBM46, with rmsd = 1.6 Å and Z-score = 12.4.	RESULTS
+15	22	BhCel5B	protein	The CBM46 from BhCel5B is the most structurally similar to BlCel5B CBM46, with rmsd = 1.6 Å and Z-score = 12.4.	RESULTS
+59	66	BlCel5B	protein	The CBM46 from BhCel5B is the most structurally similar to BlCel5B CBM46, with rmsd = 1.6 Å and Z-score = 12.4.	RESULTS
+67	72	CBM46	structure_element	The CBM46 from BhCel5B is the most structurally similar to BlCel5B CBM46, with rmsd = 1.6 Å and Z-score = 12.4.	RESULTS
+79	83	rmsd	evidence	The CBM46 from BhCel5B is the most structurally similar to BlCel5B CBM46, with rmsd = 1.6 Å and Z-score = 12.4.	RESULTS
+96	103	Z-score	evidence	The CBM46 from BhCel5B is the most structurally similar to BlCel5B CBM46, with rmsd = 1.6 Å and Z-score = 12.4.	RESULTS
+34	41	BhCel5B	protein	The sequence identity relative to BhCel5B, however, is low (28% for Ig-like and 25% for CBM46).	RESULTS
+68	75	Ig-like	structure_element	The sequence identity relative to BhCel5B, however, is low (28% for Ig-like and 25% for CBM46).	RESULTS
+88	93	CBM46	structure_element	The sequence identity relative to BhCel5B, however, is low (28% for Ig-like and 25% for CBM46).	RESULTS
+4	18	Ig-like module	structure_element	The Ig-like module, adjacent to the CD, contains only one tyrosine (Y367) exposed to solvent and no tryptophan residues.	RESULTS
+36	38	CD	structure_element	The Ig-like module, adjacent to the CD, contains only one tyrosine (Y367) exposed to solvent and no tryptophan residues.	RESULTS
+58	66	tyrosine	residue_name	The Ig-like module, adjacent to the CD, contains only one tyrosine (Y367) exposed to solvent and no tryptophan residues.	RESULTS
+68	72	Y367	residue_name_number	The Ig-like module, adjacent to the CD, contains only one tyrosine (Y367) exposed to solvent and no tryptophan residues.	RESULTS
+100	110	tryptophan	residue_name	The Ig-like module, adjacent to the CD, contains only one tyrosine (Y367) exposed to solvent and no tryptophan residues.	RESULTS
+47	54	glucose	chemical	Because aromatic residues play a major role in glucose recognition, this observation suggests that substrate binding may not be the primary function of Ig-like module.	RESULTS
+152	166	Ig-like module	structure_element	Because aromatic residues play a major role in glucose recognition, this observation suggests that substrate binding may not be the primary function of Ig-like module.	RESULTS
+17	22	CBM46	structure_element	In contrast, the CBM46 has three tryptophan residues, two of which face the CD substrate binding site (Fig. 1A), indicating that it may be actively engaged in the carbohydrate binding.	RESULTS
+33	43	tryptophan	residue_name	In contrast, the CBM46 has three tryptophan residues, two of which face the CD substrate binding site (Fig. 1A), indicating that it may be actively engaged in the carbohydrate binding.	RESULTS
+76	78	CD	structure_element	In contrast, the CBM46 has three tryptophan residues, two of which face the CD substrate binding site (Fig. 1A), indicating that it may be actively engaged in the carbohydrate binding.	RESULTS
+79	101	substrate binding site	site	In contrast, the CBM46 has three tryptophan residues, two of which face the CD substrate binding site (Fig. 1A), indicating that it may be actively engaged in the carbohydrate binding.	RESULTS
+163	175	carbohydrate	chemical	In contrast, the CBM46 has three tryptophan residues, two of which face the CD substrate binding site (Fig. 1A), indicating that it may be actively engaged in the carbohydrate binding.	RESULTS
+0	21	Electron density maps	evidence	Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule.	RESULTS
+41	52	presence of	protein_state	Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule.	RESULTS
+55	68	cellotetraose	chemical	Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule.	RESULTS
+70	72	C4	chemical	Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule.	RESULTS
+91	104	cellopentaose	chemical	Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule.	RESULTS
+106	108	C5	chemical	Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule.	RESULTS
+117	119	CD	structure_element	Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule.	RESULTS
+120	155	negative substrate-binding subsites	site	Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule.	RESULTS
+183	190	BlCel5B	protein	Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule.	RESULTS
+194	214	catalytically active	protein_state	Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule.	RESULTS
+257	259	C5	chemical	Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule.	RESULTS
+4	28	lack of electron density	evidence	The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end.	RESULTS
+42	52	absence of	protein_state	The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end.	RESULTS
+57	62	fifth	residue_number	The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end.	RESULTS
+63	70	glucose	chemical	The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end.	RESULTS
+94	96	C5	chemical	The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end.	RESULTS
+129	138	structure	evidence	The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end.	RESULTS
+158	169	presence of	protein_state	The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end.	RESULTS
+172	177	fifth	residue_number	The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end.	RESULTS
+178	185	glucose	chemical	The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end.	RESULTS
+227	245	catalytic residues	site	The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end.	RESULTS
+281	285	R234	residue_name_number	The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end.	RESULTS
+15	22	BlCel5B	protein	The ability of BlCel5B to cleave C5 into glucose and C4 molecules in solution was demonstrated by enzymatic product profile mass spectrometry analysis (Fig. 2A).	RESULTS
+33	35	C5	chemical	The ability of BlCel5B to cleave C5 into glucose and C4 molecules in solution was demonstrated by enzymatic product profile mass spectrometry analysis (Fig. 2A).	RESULTS
+41	48	glucose	chemical	The ability of BlCel5B to cleave C5 into glucose and C4 molecules in solution was demonstrated by enzymatic product profile mass spectrometry analysis (Fig. 2A).	RESULTS
+53	55	C4	chemical	The ability of BlCel5B to cleave C5 into glucose and C4 molecules in solution was demonstrated by enzymatic product profile mass spectrometry analysis (Fig. 2A).	RESULTS
+98	141	enzymatic product profile mass spectrometry	experimental_method	The ability of BlCel5B to cleave C5 into glucose and C4 molecules in solution was demonstrated by enzymatic product profile mass spectrometry analysis (Fig. 2A).	RESULTS
+4	6	C4	chemical	The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D).	RESULTS
+23	30	BlCel5B	protein	The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D).	RESULTS
+31	43	binding site	site	The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D).	RESULTS
+47	58	coordinated	bond_interaction	The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D).	RESULTS
+62	76	hydrogen bonds	bond_interaction	The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D).	RESULTS
+89	92	N36	residue_name_number	The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D).	RESULTS
+94	98	H113	residue_name_number	The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D).	RESULTS
+100	104	H114	residue_name_number	The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D).	RESULTS
+106	110	N158	residue_name_number	The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D).	RESULTS
+112	116	W301	residue_name_number	The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D).	RESULTS
+122	126	N303	residue_name_number	The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D).	RESULTS
+136	152	CH-π interaction	bond_interaction	The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D).	RESULTS
+166	169	W47	residue_name_number	The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D).	RESULTS
+29	31	CD	structure_element	These residues belong to the CD and are conserved in the GH5 family.	RESULTS
+40	49	conserved	protein_state	These residues belong to the CD and are conserved in the GH5 family.	RESULTS
+57	60	GH5	protein_type	These residues belong to the CD and are conserved in the GH5 family.	RESULTS
+0	7	BlCel5B	protein	BlCel5B enzymatic activity	RESULTS
+0	7	BlCel5B	protein	BlCel5B exhibits optimum activity toward carboxymethylcellulose (CMC; 8.7 U/mg) at a pH of 4.0 and 55 °C and retains approximately half of its maximum activity at 80 °C, demonstrating considerable thermal stability (Fig. 2B,C).	RESULTS
+41	63	carboxymethylcellulose	chemical	BlCel5B exhibits optimum activity toward carboxymethylcellulose (CMC; 8.7 U/mg) at a pH of 4.0 and 55 °C and retains approximately half of its maximum activity at 80 °C, demonstrating considerable thermal stability (Fig. 2B,C).	RESULTS
+65	68	CMC	chemical	BlCel5B exhibits optimum activity toward carboxymethylcellulose (CMC; 8.7 U/mg) at a pH of 4.0 and 55 °C and retains approximately half of its maximum activity at 80 °C, demonstrating considerable thermal stability (Fig. 2B,C).	RESULTS
+0	7	BlCel5B	protein	BlCel5B is also active on β-glucan (34 U/mg), lichenan (17.8 U/mg) and xyloglucan (15.7 U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-β-mannan or the insoluble substrate Azo-Avicel.	RESULTS
+16	22	active	protein_state	BlCel5B is also active on β-glucan (34 U/mg), lichenan (17.8 U/mg) and xyloglucan (15.7 U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-β-mannan or the insoluble substrate Azo-Avicel.	RESULTS
+26	34	β-glucan	chemical	BlCel5B is also active on β-glucan (34 U/mg), lichenan (17.8 U/mg) and xyloglucan (15.7 U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-β-mannan or the insoluble substrate Azo-Avicel.	RESULTS
+46	54	lichenan	chemical	BlCel5B is also active on β-glucan (34 U/mg), lichenan (17.8 U/mg) and xyloglucan (15.7 U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-β-mannan or the insoluble substrate Azo-Avicel.	RESULTS
+71	81	xyloglucan	chemical	BlCel5B is also active on β-glucan (34 U/mg), lichenan (17.8 U/mg) and xyloglucan (15.7 U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-β-mannan or the insoluble substrate Azo-Avicel.	RESULTS
+152	165	galactomannan	chemical	BlCel5B is also active on β-glucan (34 U/mg), lichenan (17.8 U/mg) and xyloglucan (15.7 U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-β-mannan or the insoluble substrate Azo-Avicel.	RESULTS
+167	170	rye	taxonomy_domain	BlCel5B is also active on β-glucan (34 U/mg), lichenan (17.8 U/mg) and xyloglucan (15.7 U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-β-mannan or the insoluble substrate Azo-Avicel.	RESULTS
+171	183	arabinoxylan	chemical	BlCel5B is also active on β-glucan (34 U/mg), lichenan (17.8 U/mg) and xyloglucan (15.7 U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-β-mannan or the insoluble substrate Azo-Avicel.	RESULTS
+185	197	1,4-β-mannan	chemical	BlCel5B is also active on β-glucan (34 U/mg), lichenan (17.8 U/mg) and xyloglucan (15.7 U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-β-mannan or the insoluble substrate Azo-Avicel.	RESULTS
+225	235	Azo-Avicel	chemical	BlCel5B is also active on β-glucan (34 U/mg), lichenan (17.8 U/mg) and xyloglucan (15.7 U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-β-mannan or the insoluble substrate Azo-Avicel.	RESULTS
+44	69	Michaelis-Menten behavior	experimental_method	Kinetic parameters were calculated assuming Michaelis-Menten behavior with CMC as substrate: KM = 1.78 g L−1 and Vmax = 1.41 × 10−4 g s−1 mg protein−1 (Fig. 2D).	RESULTS
+75	78	CMC	chemical	Kinetic parameters were calculated assuming Michaelis-Menten behavior with CMC as substrate: KM = 1.78 g L−1 and Vmax = 1.41 × 10−4 g s−1 mg protein−1 (Fig. 2D).	RESULTS
+93	95	KM	evidence	Kinetic parameters were calculated assuming Michaelis-Menten behavior with CMC as substrate: KM = 1.78 g L−1 and Vmax = 1.41 × 10−4 g s−1 mg protein−1 (Fig. 2D).	RESULTS
+113	117	Vmax	evidence	Kinetic parameters were calculated assuming Michaelis-Menten behavior with CMC as substrate: KM = 1.78 g L−1 and Vmax = 1.41 × 10−4 g s−1 mg protein−1 (Fig. 2D).	RESULTS
+9	16	BlCel5B	protein	Although BlCel5B is not a highly active enzyme against one specific substrate as compared to others GH5_4, it has the advantage of being active against different substrates with β-1,3 and/or β-1,4 glycosidic linkages.	RESULTS
+33	39	active	protein_state	Although BlCel5B is not a highly active enzyme against one specific substrate as compared to others GH5_4, it has the advantage of being active against different substrates with β-1,3 and/or β-1,4 glycosidic linkages.	RESULTS
+100	105	GH5_4	protein_type	Although BlCel5B is not a highly active enzyme against one specific substrate as compared to others GH5_4, it has the advantage of being active against different substrates with β-1,3 and/or β-1,4 glycosidic linkages.	RESULTS
+137	143	active	protein_state	Although BlCel5B is not a highly active enzyme against one specific substrate as compared to others GH5_4, it has the advantage of being active against different substrates with β-1,3 and/or β-1,4 glycosidic linkages.	RESULTS
+36	53	ancillary modules	structure_element	To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.	RESULTS
+58	65	BlCel5B	protein	To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.	RESULTS
+76	92	enzymatic assays	experimental_method	To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.	RESULTS
+128	135	mutants	protein_state	To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.	RESULTS
+139	144	CBM46	structure_element	To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.	RESULTS
+145	153	deletion	experimental_method	To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.	RESULTS
+155	161	ΔCBM46	mutant	To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.	RESULTS
+170	177	Ig-like	structure_element	To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.	RESULTS
+180	185	CBM46	structure_element	To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.	RESULTS
+186	194	deletion	experimental_method	To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.	RESULTS
+196	205	ΔIg-CBM46	mutant	To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.	RESULTS
+218	233	point mutations	experimental_method	To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.	RESULTS
+241	246	CBM46	structure_element	To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.	RESULTS
+270	275	W479A	mutant	To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.	RESULTS
+280	285	W481A	mutant	To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.	RESULTS
+6	13	mutants	protein_state	These mutants were expressed and purified as described for the wild-type enzyme.	RESULTS
+19	41	expressed and purified	experimental_method	These mutants were expressed and purified as described for the wild-type enzyme.	RESULTS
+63	72	wild-type	protein_state	These mutants were expressed and purified as described for the wild-type enzyme.	RESULTS
+27	44	deletion variants	protein_state	Strikingly, neither of the deletion variants exhibited detectable activity toward any of the substrates tested using full-length BlCel5B (Table 1), demonstrating that the Ig-like module and the CBM46 are essential for BlCel5B activity.	RESULTS
+117	128	full-length	protein_state	Strikingly, neither of the deletion variants exhibited detectable activity toward any of the substrates tested using full-length BlCel5B (Table 1), demonstrating that the Ig-like module and the CBM46 are essential for BlCel5B activity.	RESULTS
+129	136	BlCel5B	protein	Strikingly, neither of the deletion variants exhibited detectable activity toward any of the substrates tested using full-length BlCel5B (Table 1), demonstrating that the Ig-like module and the CBM46 are essential for BlCel5B activity.	RESULTS
+171	185	Ig-like module	structure_element	Strikingly, neither of the deletion variants exhibited detectable activity toward any of the substrates tested using full-length BlCel5B (Table 1), demonstrating that the Ig-like module and the CBM46 are essential for BlCel5B activity.	RESULTS
+194	199	CBM46	structure_element	Strikingly, neither of the deletion variants exhibited detectable activity toward any of the substrates tested using full-length BlCel5B (Table 1), demonstrating that the Ig-like module and the CBM46 are essential for BlCel5B activity.	RESULTS
+218	225	BlCel5B	protein	Strikingly, neither of the deletion variants exhibited detectable activity toward any of the substrates tested using full-length BlCel5B (Table 1), demonstrating that the Ig-like module and the CBM46 are essential for BlCel5B activity.	RESULTS
+0	20	Thermal shift assays	experimental_method	Thermal shift assays were conducted to confirm structural stability of the mutants (Supplementary Fig. 1).	RESULTS
+75	82	mutants	protein_state	Thermal shift assays were conducted to confirm structural stability of the mutants (Supplementary Fig. 1).	RESULTS
+37	57	melting temperatures	evidence	All of the constructs showed similar melting temperatures: 62 °C for BlCel5B, 58 °C for BlCel5BΔCBM46, 56 °C for BlCel5BΔIg-CBM46, 65 °C for BlCel5BW479A and 59 °C for BlCel5BW479A, thus confirming their proper overall fold.	RESULTS
+69	76	BlCel5B	protein	All of the constructs showed similar melting temperatures: 62 °C for BlCel5B, 58 °C for BlCel5BΔCBM46, 56 °C for BlCel5BΔIg-CBM46, 65 °C for BlCel5BW479A and 59 °C for BlCel5BW479A, thus confirming their proper overall fold.	RESULTS
+88	101	BlCel5BΔCBM46	mutant	All of the constructs showed similar melting temperatures: 62 °C for BlCel5B, 58 °C for BlCel5BΔCBM46, 56 °C for BlCel5BΔIg-CBM46, 65 °C for BlCel5BW479A and 59 °C for BlCel5BW479A, thus confirming their proper overall fold.	RESULTS
+113	129	BlCel5BΔIg-CBM46	mutant	All of the constructs showed similar melting temperatures: 62 °C for BlCel5B, 58 °C for BlCel5BΔCBM46, 56 °C for BlCel5BΔIg-CBM46, 65 °C for BlCel5BW479A and 59 °C for BlCel5BW479A, thus confirming their proper overall fold.	RESULTS
+141	153	BlCel5BW479A	mutant	All of the constructs showed similar melting temperatures: 62 °C for BlCel5B, 58 °C for BlCel5BΔCBM46, 56 °C for BlCel5BΔIg-CBM46, 65 °C for BlCel5BW479A and 59 °C for BlCel5BW479A, thus confirming their proper overall fold.	RESULTS
+168	180	BlCel5BW479A	mutant	All of the constructs showed similar melting temperatures: 62 °C for BlCel5B, 58 °C for BlCel5BΔCBM46, 56 °C for BlCel5BΔIg-CBM46, 65 °C for BlCel5BW479A and 59 °C for BlCel5BW479A, thus confirming their proper overall fold.	RESULTS
+37	42	CBM46	structure_element	We also examined the function of the CBM46 inner surface residues W479 and W481 (Fig. 1A) in BlCel5B activity by performing enzymatic assays with W479A and W481A mutants.	RESULTS
+49	56	surface	site	We also examined the function of the CBM46 inner surface residues W479 and W481 (Fig. 1A) in BlCel5B activity by performing enzymatic assays with W479A and W481A mutants.	RESULTS
+66	70	W479	residue_name_number	We also examined the function of the CBM46 inner surface residues W479 and W481 (Fig. 1A) in BlCel5B activity by performing enzymatic assays with W479A and W481A mutants.	RESULTS
+75	79	W481	residue_name_number	We also examined the function of the CBM46 inner surface residues W479 and W481 (Fig. 1A) in BlCel5B activity by performing enzymatic assays with W479A and W481A mutants.	RESULTS
+93	100	BlCel5B	protein	We also examined the function of the CBM46 inner surface residues W479 and W481 (Fig. 1A) in BlCel5B activity by performing enzymatic assays with W479A and W481A mutants.	RESULTS
+124	140	enzymatic assays	experimental_method	We also examined the function of the CBM46 inner surface residues W479 and W481 (Fig. 1A) in BlCel5B activity by performing enzymatic assays with W479A and W481A mutants.	RESULTS
+146	151	W479A	mutant	We also examined the function of the CBM46 inner surface residues W479 and W481 (Fig. 1A) in BlCel5B activity by performing enzymatic assays with W479A and W481A mutants.	RESULTS
+156	161	W481A	mutant	We also examined the function of the CBM46 inner surface residues W479 and W481 (Fig. 1A) in BlCel5B activity by performing enzymatic assays with W479A and W481A mutants.	RESULTS
+162	169	mutants	protein_state	We also examined the function of the CBM46 inner surface residues W479 and W481 (Fig. 1A) in BlCel5B activity by performing enzymatic assays with W479A and W481A mutants.	RESULTS
+5	14	mutations	experimental_method	Both mutations reduced enzymatic activity toward all tested substrates (Table 1), with W481A having a stronger effect than W479A (~64% vs. 79% activity relative to wt BlCel5B using β-glucan and ~10% vs. 50% using CMC).	RESULTS
+87	92	W481A	mutant	Both mutations reduced enzymatic activity toward all tested substrates (Table 1), with W481A having a stronger effect than W479A (~64% vs. 79% activity relative to wt BlCel5B using β-glucan and ~10% vs. 50% using CMC).	RESULTS
+123	128	W479A	mutant	Both mutations reduced enzymatic activity toward all tested substrates (Table 1), with W481A having a stronger effect than W479A (~64% vs. 79% activity relative to wt BlCel5B using β-glucan and ~10% vs. 50% using CMC).	RESULTS
+164	166	wt	protein_state	Both mutations reduced enzymatic activity toward all tested substrates (Table 1), with W481A having a stronger effect than W479A (~64% vs. 79% activity relative to wt BlCel5B using β-glucan and ~10% vs. 50% using CMC).	RESULTS
+167	174	BlCel5B	protein	Both mutations reduced enzymatic activity toward all tested substrates (Table 1), with W481A having a stronger effect than W479A (~64% vs. 79% activity relative to wt BlCel5B using β-glucan and ~10% vs. 50% using CMC).	RESULTS
+181	189	β-glucan	chemical	Both mutations reduced enzymatic activity toward all tested substrates (Table 1), with W481A having a stronger effect than W479A (~64% vs. 79% activity relative to wt BlCel5B using β-glucan and ~10% vs. 50% using CMC).	RESULTS
+213	216	CMC	chemical	Both mutations reduced enzymatic activity toward all tested substrates (Table 1), with W481A having a stronger effect than W479A (~64% vs. 79% activity relative to wt BlCel5B using β-glucan and ~10% vs. 50% using CMC).	RESULTS
+20	25	CBM46	structure_element	This indicates that CBM46 must interact with the substrate via residues W479 and W481.	RESULTS
+72	76	W479	residue_name_number	This indicates that CBM46 must interact with the substrate via residues W479 and W481.	RESULTS
+81	85	W481	residue_name_number	This indicates that CBM46 must interact with the substrate via residues W479 and W481.	RESULTS
+19	26	BlCel5B	protein	However, since the BlCel5B crystal structure exhibits no close contact between these residues and the substrate, these results suggest the existence of large-amplitude interdomain motions that may enable direct interactions between CBM46 and the carbohydrate.	RESULTS
+27	44	crystal structure	evidence	However, since the BlCel5B crystal structure exhibits no close contact between these residues and the substrate, these results suggest the existence of large-amplitude interdomain motions that may enable direct interactions between CBM46 and the carbohydrate.	RESULTS
+57	62	close	protein_state	However, since the BlCel5B crystal structure exhibits no close contact between these residues and the substrate, these results suggest the existence of large-amplitude interdomain motions that may enable direct interactions between CBM46 and the carbohydrate.	RESULTS
+232	237	CBM46	structure_element	However, since the BlCel5B crystal structure exhibits no close contact between these residues and the substrate, these results suggest the existence of large-amplitude interdomain motions that may enable direct interactions between CBM46 and the carbohydrate.	RESULTS
+246	258	carbohydrate	chemical	However, since the BlCel5B crystal structure exhibits no close contact between these residues and the substrate, these results suggest the existence of large-amplitude interdomain motions that may enable direct interactions between CBM46 and the carbohydrate.	RESULTS
+0	7	BlCelB5	protein	BlCelB5 dynamics and binding-site architecture	RESULTS
+21	33	binding-site	site	BlCelB5 dynamics and binding-site architecture	RESULTS
+0	18	Molecular dynamics	experimental_method	Molecular dynamics (MD) simulations were performed to investigate the conformational mobility of BlCel5B.	RESULTS
+20	22	MD	experimental_method	Molecular dynamics (MD) simulations were performed to investigate the conformational mobility of BlCel5B.	RESULTS
+24	35	simulations	experimental_method	Molecular dynamics (MD) simulations were performed to investigate the conformational mobility of BlCel5B.	RESULTS
+97	104	BlCel5B	protein	Molecular dynamics (MD) simulations were performed to investigate the conformational mobility of BlCel5B.	RESULTS
+7	18	simulations	experimental_method	In the simulations of the crystal structure for BlCel5B bound to C4, the substrate dissociates from the protein within the first 100 ns of the simulation time (Supplementary Fig. 2A).	RESULTS
+26	43	crystal structure	evidence	In the simulations of the crystal structure for BlCel5B bound to C4, the substrate dissociates from the protein within the first 100 ns of the simulation time (Supplementary Fig. 2A).	RESULTS
+48	55	BlCel5B	protein	In the simulations of the crystal structure for BlCel5B bound to C4, the substrate dissociates from the protein within the first 100 ns of the simulation time (Supplementary Fig. 2A).	RESULTS
+56	64	bound to	protein_state	In the simulations of the crystal structure for BlCel5B bound to C4, the substrate dissociates from the protein within the first 100 ns of the simulation time (Supplementary Fig. 2A).	RESULTS
+65	67	C4	chemical	In the simulations of the crystal structure for BlCel5B bound to C4, the substrate dissociates from the protein within the first 100 ns of the simulation time (Supplementary Fig. 2A).	RESULTS
+143	153	simulation	experimental_method	In the simulations of the crystal structure for BlCel5B bound to C4, the substrate dissociates from the protein within the first 100 ns of the simulation time (Supplementary Fig. 2A).	RESULTS
+31	44	cellotetraose	chemical	This observation suggests that cellotetraose does not exhibit detectable affinity for this specific BlCel5B conformation in solution, as one might otherwise expect for a reaction product.	RESULTS
+100	107	BlCel5B	protein	This observation suggests that cellotetraose does not exhibit detectable affinity for this specific BlCel5B conformation in solution, as one might otherwise expect for a reaction product.	RESULTS
+71	78	BlCel5B	protein	No changes beyond local fluctuations were observed in any of the three BlCel5B domains within the time scale of these runs (400 ns; Supplementary Fig. 2B).	RESULTS
+13	18	CBM46	structure_element	However, the CBM46 and Ig-like domains did exhibit rigid body-like motions relative to the CD, with rmsd values around 2.3 Å and 1.8 Å, respectively, suggesting that BlCel5B may execute large-amplitude interdomain motions over longer time scales (Supplementary Fig. 2B,C).	RESULTS
+23	38	Ig-like domains	structure_element	However, the CBM46 and Ig-like domains did exhibit rigid body-like motions relative to the CD, with rmsd values around 2.3 Å and 1.8 Å, respectively, suggesting that BlCel5B may execute large-amplitude interdomain motions over longer time scales (Supplementary Fig. 2B,C).	RESULTS
+91	93	CD	structure_element	However, the CBM46 and Ig-like domains did exhibit rigid body-like motions relative to the CD, with rmsd values around 2.3 Å and 1.8 Å, respectively, suggesting that BlCel5B may execute large-amplitude interdomain motions over longer time scales (Supplementary Fig. 2B,C).	RESULTS
+100	104	rmsd	evidence	However, the CBM46 and Ig-like domains did exhibit rigid body-like motions relative to the CD, with rmsd values around 2.3 Å and 1.8 Å, respectively, suggesting that BlCel5B may execute large-amplitude interdomain motions over longer time scales (Supplementary Fig. 2B,C).	RESULTS
+166	173	BlCel5B	protein	However, the CBM46 and Ig-like domains did exhibit rigid body-like motions relative to the CD, with rmsd values around 2.3 Å and 1.8 Å, respectively, suggesting that BlCel5B may execute large-amplitude interdomain motions over longer time scales (Supplementary Fig. 2B,C).	RESULTS
+13	24	simulations	experimental_method	Accordingly, simulations were then performed using accelerated molecular dynamics (aMD) techniques to probe BlCel5B interdomain motions.	RESULTS
+51	81	accelerated molecular dynamics	experimental_method	Accordingly, simulations were then performed using accelerated molecular dynamics (aMD) techniques to probe BlCel5B interdomain motions.	RESULTS
+83	86	aMD	experimental_method	Accordingly, simulations were then performed using accelerated molecular dynamics (aMD) techniques to probe BlCel5B interdomain motions.	RESULTS
+108	115	BlCel5B	protein	Accordingly, simulations were then performed using accelerated molecular dynamics (aMD) techniques to probe BlCel5B interdomain motions.	RESULTS
+0	3	aMD	experimental_method	aMD enhances conformational sampling by raising the basins of the dihedral potential energy surface without affecting the general form of the atomistic potential, thereby increasing transition rates between different local minima.	RESULTS
+66	99	dihedral potential energy surface	evidence	aMD enhances conformational sampling by raising the basins of the dihedral potential energy surface without affecting the general form of the atomistic potential, thereby increasing transition rates between different local minima.	RESULTS
+0	3	aMD	experimental_method	aMD trajectories corresponding to more than 1.0 μs of conventional MD runs were generated.	RESULTS
+4	16	trajectories	evidence	aMD trajectories corresponding to more than 1.0 μs of conventional MD runs were generated.	RESULTS
+67	69	MD	experimental_method	aMD trajectories corresponding to more than 1.0 μs of conventional MD runs were generated.	RESULTS
+13	24	simulations	experimental_method	During these simulations, we observed occlusive conformations between CBM46 and CD that resulted in a rearrangement of the enzyme’s architecture around the active site (Video S1).	RESULTS
+70	75	CBM46	structure_element	During these simulations, we observed occlusive conformations between CBM46 and CD that resulted in a rearrangement of the enzyme’s architecture around the active site (Video S1).	RESULTS
+80	82	CD	structure_element	During these simulations, we observed occlusive conformations between CBM46 and CD that resulted in a rearrangement of the enzyme’s architecture around the active site (Video S1).	RESULTS
+156	167	active site	site	During these simulations, we observed occlusive conformations between CBM46 and CD that resulted in a rearrangement of the enzyme’s architecture around the active site (Video S1).	RESULTS
+16	23	BlCel5B	protein	Figure 3A shows BlCel5B in the crystallographic conformation (red) and in a selected configuration obtained with aMD (blue) in the absence of the substrate.	RESULTS
+31	47	crystallographic	experimental_method	Figure 3A shows BlCel5B in the crystallographic conformation (red) and in a selected configuration obtained with aMD (blue) in the absence of the substrate.	RESULTS
+113	116	aMD	experimental_method	Figure 3A shows BlCel5B in the crystallographic conformation (red) and in a selected configuration obtained with aMD (blue) in the absence of the substrate.	RESULTS
+131	141	absence of	protein_state	Figure 3A shows BlCel5B in the crystallographic conformation (red) and in a selected configuration obtained with aMD (blue) in the absence of the substrate.	RESULTS
+61	69	distance	evidence	Interdomain motions were gauged by the time evolution of the distance between the α carbons of residues I120 and E477 (represented as spheres in Fig. 3A), belonging to the CD and CBM46, respectively.	RESULTS
+104	108	I120	residue_name_number	Interdomain motions were gauged by the time evolution of the distance between the α carbons of residues I120 and E477 (represented as spheres in Fig. 3A), belonging to the CD and CBM46, respectively.	RESULTS
+113	117	E477	residue_name_number	Interdomain motions were gauged by the time evolution of the distance between the α carbons of residues I120 and E477 (represented as spheres in Fig. 3A), belonging to the CD and CBM46, respectively.	RESULTS
+172	174	CD	structure_element	Interdomain motions were gauged by the time evolution of the distance between the α carbons of residues I120 and E477 (represented as spheres in Fig. 3A), belonging to the CD and CBM46, respectively.	RESULTS
+179	184	CBM46	structure_element	Interdomain motions were gauged by the time evolution of the distance between the α carbons of residues I120 and E477 (represented as spheres in Fig. 3A), belonging to the CD and CBM46, respectively.	RESULTS
+25	29	I120	residue_name_number	Figure 3C shows that the I120-E477 distance (red curve) gradually decreases from ~35 Å to ~7 Å within the first half of the 1.0 μs aMD trajectory, indicating a transition between the semi-open (crystallographic) and occluded (aMD sampled) configurations.	RESULTS
+30	34	E477	residue_name_number	Figure 3C shows that the I120-E477 distance (red curve) gradually decreases from ~35 Å to ~7 Å within the first half of the 1.0 μs aMD trajectory, indicating a transition between the semi-open (crystallographic) and occluded (aMD sampled) configurations.	RESULTS
+35	43	distance	evidence	Figure 3C shows that the I120-E477 distance (red curve) gradually decreases from ~35 Å to ~7 Å within the first half of the 1.0 μs aMD trajectory, indicating a transition between the semi-open (crystallographic) and occluded (aMD sampled) configurations.	RESULTS
+131	134	aMD	experimental_method	Figure 3C shows that the I120-E477 distance (red curve) gradually decreases from ~35 Å to ~7 Å within the first half of the 1.0 μs aMD trajectory, indicating a transition between the semi-open (crystallographic) and occluded (aMD sampled) configurations.	RESULTS
+135	145	trajectory	evidence	Figure 3C shows that the I120-E477 distance (red curve) gradually decreases from ~35 Å to ~7 Å within the first half of the 1.0 μs aMD trajectory, indicating a transition between the semi-open (crystallographic) and occluded (aMD sampled) configurations.	RESULTS
+183	192	semi-open	protein_state	Figure 3C shows that the I120-E477 distance (red curve) gradually decreases from ~35 Å to ~7 Å within the first half of the 1.0 μs aMD trajectory, indicating a transition between the semi-open (crystallographic) and occluded (aMD sampled) configurations.	RESULTS
+194	210	crystallographic	experimental_method	Figure 3C shows that the I120-E477 distance (red curve) gradually decreases from ~35 Å to ~7 Å within the first half of the 1.0 μs aMD trajectory, indicating a transition between the semi-open (crystallographic) and occluded (aMD sampled) configurations.	RESULTS
+216	224	occluded	protein_state	Figure 3C shows that the I120-E477 distance (red curve) gradually decreases from ~35 Å to ~7 Å within the first half of the 1.0 μs aMD trajectory, indicating a transition between the semi-open (crystallographic) and occluded (aMD sampled) configurations.	RESULTS
+226	229	aMD	experimental_method	Figure 3C shows that the I120-E477 distance (red curve) gradually decreases from ~35 Å to ~7 Å within the first half of the 1.0 μs aMD trajectory, indicating a transition between the semi-open (crystallographic) and occluded (aMD sampled) configurations.	RESULTS
+30	44	aMD simulation	experimental_method	During the second half of the aMD simulation, the full-length enzyme remained in the closed conformation, with the CBM46 covering the carbohydrate-binding site.	RESULTS
+50	61	full-length	protein_state	During the second half of the aMD simulation, the full-length enzyme remained in the closed conformation, with the CBM46 covering the carbohydrate-binding site.	RESULTS
+85	91	closed	protein_state	During the second half of the aMD simulation, the full-length enzyme remained in the closed conformation, with the CBM46 covering the carbohydrate-binding site.	RESULTS
+115	120	CBM46	structure_element	During the second half of the aMD simulation, the full-length enzyme remained in the closed conformation, with the CBM46 covering the carbohydrate-binding site.	RESULTS
+134	159	carbohydrate-binding site	site	During the second half of the aMD simulation, the full-length enzyme remained in the closed conformation, with the CBM46 covering the carbohydrate-binding site.	RESULTS
+27	34	BlCel5B	protein	These results suggest that BlCel5B undergoes large-scale interdomain movements that enable interactions between CBM46 and the substrate bound to the CD.	RESULTS
+112	117	CBM46	structure_element	These results suggest that BlCel5B undergoes large-scale interdomain movements that enable interactions between CBM46 and the substrate bound to the CD.	RESULTS
+136	144	bound to	protein_state	These results suggest that BlCel5B undergoes large-scale interdomain movements that enable interactions between CBM46 and the substrate bound to the CD.	RESULTS
+149	151	CD	structure_element	These results suggest that BlCel5B undergoes large-scale interdomain movements that enable interactions between CBM46 and the substrate bound to the CD.	RESULTS
+29	36	BlCel5B	protein	To study the interactions of BlCel5B with a non-hydrolyzed glucan chain, we built a model structure with a cellooctaose (C8) chain spanning the entire positive (+1 to +4) and negative (−4 to −1) subsites of the enzyme.	RESULTS
+59	65	glucan	chemical	To study the interactions of BlCel5B with a non-hydrolyzed glucan chain, we built a model structure with a cellooctaose (C8) chain spanning the entire positive (+1 to +4) and negative (−4 to −1) subsites of the enzyme.	RESULTS
+90	99	structure	evidence	To study the interactions of BlCel5B with a non-hydrolyzed glucan chain, we built a model structure with a cellooctaose (C8) chain spanning the entire positive (+1 to +4) and negative (−4 to −1) subsites of the enzyme.	RESULTS
+107	119	cellooctaose	chemical	To study the interactions of BlCel5B with a non-hydrolyzed glucan chain, we built a model structure with a cellooctaose (C8) chain spanning the entire positive (+1 to +4) and negative (−4 to −1) subsites of the enzyme.	RESULTS
+121	123	C8	chemical	To study the interactions of BlCel5B with a non-hydrolyzed glucan chain, we built a model structure with a cellooctaose (C8) chain spanning the entire positive (+1 to +4) and negative (−4 to −1) subsites of the enzyme.	RESULTS
+151	170	positive (+1 to +4)	site	To study the interactions of BlCel5B with a non-hydrolyzed glucan chain, we built a model structure with a cellooctaose (C8) chain spanning the entire positive (+1 to +4) and negative (−4 to −1) subsites of the enzyme.	RESULTS
+175	194	negative (−4 to −1)	site	To study the interactions of BlCel5B with a non-hydrolyzed glucan chain, we built a model structure with a cellooctaose (C8) chain spanning the entire positive (+1 to +4) and negative (−4 to −1) subsites of the enzyme.	RESULTS
+195	203	subsites	site	To study the interactions of BlCel5B with a non-hydrolyzed glucan chain, we built a model structure with a cellooctaose (C8) chain spanning the entire positive (+1 to +4) and negative (−4 to −1) subsites of the enzyme.	RESULTS
+35	42	BlCel5B	protein	Starting from the crystallographic BlCel5B conformation, the C8 molecule deviated significantly from the active site and assumed a non-productive binding mode (Supplementary Fig. 2D).	RESULTS
+61	63	C8	chemical	Starting from the crystallographic BlCel5B conformation, the C8 molecule deviated significantly from the active site and assumed a non-productive binding mode (Supplementary Fig. 2D).	RESULTS
+105	116	active site	site	Starting from the crystallographic BlCel5B conformation, the C8 molecule deviated significantly from the active site and assumed a non-productive binding mode (Supplementary Fig. 2D).	RESULTS
+35	39	open	protein_state	This observation suggests that the open conformation of BlCel5B is not able to hold the substrate in a position suitable for hydrolysis (Supplementary Fig. 2E).	RESULTS
+56	63	BlCel5B	protein	This observation suggests that the open conformation of BlCel5B is not able to hold the substrate in a position suitable for hydrolysis (Supplementary Fig. 2E).	RESULTS
+30	40	BlCel5B-C8	complex_assembly	However, after subjecting the BlCel5B-C8 complex to a 0.5 μs aMD simulation with harmonic restraints on the C8 chain to prevent it from deviating from the productive binding mode, the CBM46 readily closed over the CD and trapped the C8 chain in position for hydrolysis (Fig. 3B).	RESULTS
+61	75	aMD simulation	experimental_method	However, after subjecting the BlCel5B-C8 complex to a 0.5 μs aMD simulation with harmonic restraints on the C8 chain to prevent it from deviating from the productive binding mode, the CBM46 readily closed over the CD and trapped the C8 chain in position for hydrolysis (Fig. 3B).	RESULTS
+108	110	C8	chemical	However, after subjecting the BlCel5B-C8 complex to a 0.5 μs aMD simulation with harmonic restraints on the C8 chain to prevent it from deviating from the productive binding mode, the CBM46 readily closed over the CD and trapped the C8 chain in position for hydrolysis (Fig. 3B).	RESULTS
+184	189	CBM46	structure_element	However, after subjecting the BlCel5B-C8 complex to a 0.5 μs aMD simulation with harmonic restraints on the C8 chain to prevent it from deviating from the productive binding mode, the CBM46 readily closed over the CD and trapped the C8 chain in position for hydrolysis (Fig. 3B).	RESULTS
+198	204	closed	protein_state	However, after subjecting the BlCel5B-C8 complex to a 0.5 μs aMD simulation with harmonic restraints on the C8 chain to prevent it from deviating from the productive binding mode, the CBM46 readily closed over the CD and trapped the C8 chain in position for hydrolysis (Fig. 3B).	RESULTS
+214	216	CD	structure_element	However, after subjecting the BlCel5B-C8 complex to a 0.5 μs aMD simulation with harmonic restraints on the C8 chain to prevent it from deviating from the productive binding mode, the CBM46 readily closed over the CD and trapped the C8 chain in position for hydrolysis (Fig. 3B).	RESULTS
+233	235	C8	chemical	However, after subjecting the BlCel5B-C8 complex to a 0.5 μs aMD simulation with harmonic restraints on the C8 chain to prevent it from deviating from the productive binding mode, the CBM46 readily closed over the CD and trapped the C8 chain in position for hydrolysis (Fig. 3B).	RESULTS
+7	18	presence of	protein_state	In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C).	RESULTS
+34	39	CBM46	structure_element	In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C).	RESULTS
+93	119	crystallographic structure	evidence	In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C).	RESULTS
+145	159	substrate-free	protein_state	In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C).	RESULTS
+160	167	BlCel5B	protein	In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C).	RESULTS
+168	183	aMD simulations	experimental_method	In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C).	RESULTS
+212	216	I120	residue_name_number	In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C).	RESULTS
+217	221	E477	residue_name_number	In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C).	RESULTS
+222	230	distance	evidence	In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C).	RESULTS
+266	272	closed	protein_state	In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C).	RESULTS
+302	304	C8	chemical	In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C).	RESULTS
+339	353	substrate-free	protein_state	In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C).	RESULTS
+354	361	BlCel5B	protein	In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C).	RESULTS
+5	15	BlCel5B-C8	complex_assembly	This BlCel5B-C8 configuration remains stable over an additional 500 ns of conventional MD simulation with no restraints (Fig. 3C cyan line, Supplementary Fig. 2E,F).	RESULTS
+87	100	MD simulation	experimental_method	This BlCel5B-C8 configuration remains stable over an additional 500 ns of conventional MD simulation with no restraints (Fig. 3C cyan line, Supplementary Fig. 2E,F).	RESULTS
+81	92	simulations	experimental_method	A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D.	RESULTS
+110	115	CBM46	structure_element	A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D.	RESULTS
+116	126	tryptophan	residue_name	A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D.	RESULTS
+136	140	W479	residue_name_number	A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D.	RESULTS
+145	149	W481	residue_name_number	A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D.	RESULTS
+162	164	CD	structure_element	A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D.	RESULTS
+165	175	tryptophan	residue_name	A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D.	RESULTS
+210	222	carbohydrate	chemical	A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D.	RESULTS
+265	271	tunnel	site	A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D.	RESULTS
+296	303	BlCel5B	protein	A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D.	RESULTS
+304	317	binding cleft	site	A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D.	RESULTS
+38	43	CBM46	structure_element	Together, these results indicate that CBM46 is a key component of the catalytic active complex, providing an explanation as to why CBM46 is essential for the enzymatic activity of BlCel5B.	RESULTS
+70	86	catalytic active	protein_state	Together, these results indicate that CBM46 is a key component of the catalytic active complex, providing an explanation as to why CBM46 is essential for the enzymatic activity of BlCel5B.	RESULTS
+131	136	CBM46	structure_element	Together, these results indicate that CBM46 is a key component of the catalytic active complex, providing an explanation as to why CBM46 is essential for the enzymatic activity of BlCel5B.	RESULTS
+180	187	BlCel5B	protein	Together, these results indicate that CBM46 is a key component of the catalytic active complex, providing an explanation as to why CBM46 is essential for the enzymatic activity of BlCel5B.	RESULTS
+55	76	atomistic simulations	experimental_method	To enable substantially longer time scales compared to atomistic simulations, we further explored the dynamics of BlCel5B using coarse-grained MD (CG-MD) simulations.	RESULTS
+114	121	BlCel5B	protein	To enable substantially longer time scales compared to atomistic simulations, we further explored the dynamics of BlCel5B using coarse-grained MD (CG-MD) simulations.	RESULTS
+128	145	coarse-grained MD	experimental_method	To enable substantially longer time scales compared to atomistic simulations, we further explored the dynamics of BlCel5B using coarse-grained MD (CG-MD) simulations.	RESULTS
+147	152	CG-MD	experimental_method	To enable substantially longer time scales compared to atomistic simulations, we further explored the dynamics of BlCel5B using coarse-grained MD (CG-MD) simulations.	RESULTS
+154	165	simulations	experimental_method	To enable substantially longer time scales compared to atomistic simulations, we further explored the dynamics of BlCel5B using coarse-grained MD (CG-MD) simulations.	RESULTS
+39	56	CG-MD simulations	experimental_method	We performed three independent ~120 μs CG-MD simulations, for a total of approximately 360 μs of sampling.	RESULTS
+4	12	distance	evidence	The distance between the α carbons of two residues centrally positioned in the CD and CBM46 (Fig. 4A) was monitored, and the results shown in Fig. 4B indicate that the wide-amplitude events described above frequently appear in this time scale.	RESULTS
+79	81	CD	structure_element	The distance between the α carbons of two residues centrally positioned in the CD and CBM46 (Fig. 4A) was monitored, and the results shown in Fig. 4B indicate that the wide-amplitude events described above frequently appear in this time scale.	RESULTS
+86	91	CBM46	structure_element	The distance between the α carbons of two residues centrally positioned in the CD and CBM46 (Fig. 4A) was monitored, and the results shown in Fig. 4B indicate that the wide-amplitude events described above frequently appear in this time scale.	RESULTS
+4	34	computed distance distribution	evidence	The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.	RESULTS
+115	121	closed	protein_state	The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.	RESULTS
+172	186	substrate-free	protein_state	The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.	RESULTS
+187	202	aMD simulations	experimental_method	The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.	RESULTS
+213	218	CBM46	structure_element	The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.	RESULTS
+238	240	CD	structure_element	The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.	RESULTS
+254	276	substrate binding site	site	The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.	RESULTS
+286	295	semi-open	protein_state	The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.	RESULTS
+325	351	crystallographic structure	evidence	The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.	RESULTS
+363	371	extended	protein_state	The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.	RESULTS
+372	379	BlCel5B	protein	The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.	RESULTS
+407	409	CD	structure_element	The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.	RESULTS
+414	419	CBM46	structure_element	The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.	RESULTS
+455	472	crystal structure	evidence	The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.	RESULTS
+0	7	BlCel5B	protein	BlCel5B conformers fit the SAXS envelope	RESULTS
+27	31	SAXS	experimental_method	BlCel5B conformers fit the SAXS envelope	RESULTS
+32	40	envelope	evidence	BlCel5B conformers fit the SAXS envelope	RESULTS
+0	4	SAXS	experimental_method	SAXS experiments were conducted to assess BlCel5B conformational states in solution, and the results revealed the enzyme in its monomeric form, with average values of Rg = 27.17 Å and Dmax = 87.59 Å (Supplementary Table 2).	RESULTS
+42	49	BlCel5B	protein	SAXS experiments were conducted to assess BlCel5B conformational states in solution, and the results revealed the enzyme in its monomeric form, with average values of Rg = 27.17 Å and Dmax = 87.59 Å (Supplementary Table 2).	RESULTS
+128	137	monomeric	oligomeric_state	SAXS experiments were conducted to assess BlCel5B conformational states in solution, and the results revealed the enzyme in its monomeric form, with average values of Rg = 27.17 Å and Dmax = 87.59 Å (Supplementary Table 2).	RESULTS
+167	169	Rg	evidence	SAXS experiments were conducted to assess BlCel5B conformational states in solution, and the results revealed the enzyme in its monomeric form, with average values of Rg = 27.17 Å and Dmax = 87.59 Å (Supplementary Table 2).	RESULTS
+184	188	Dmax	evidence	SAXS experiments were conducted to assess BlCel5B conformational states in solution, and the results revealed the enzyme in its monomeric form, with average values of Rg = 27.17 Å and Dmax = 87.59 Å (Supplementary Table 2).	RESULTS
+4	30	ab initio dummy atom model	experimental_method	The ab initio dummy atom model (DAM) demonstrated that the SAXS-derived BlCel5B molecular envelope could not be single-handedly filled by any of the main conformational states encountered in the simulations (Fig. 4D).	RESULTS
+32	35	DAM	experimental_method	The ab initio dummy atom model (DAM) demonstrated that the SAXS-derived BlCel5B molecular envelope could not be single-handedly filled by any of the main conformational states encountered in the simulations (Fig. 4D).	RESULTS
+59	63	SAXS	experimental_method	The ab initio dummy atom model (DAM) demonstrated that the SAXS-derived BlCel5B molecular envelope could not be single-handedly filled by any of the main conformational states encountered in the simulations (Fig. 4D).	RESULTS
+72	79	BlCel5B	protein	The ab initio dummy atom model (DAM) demonstrated that the SAXS-derived BlCel5B molecular envelope could not be single-handedly filled by any of the main conformational states encountered in the simulations (Fig. 4D).	RESULTS
+90	98	envelope	evidence	The ab initio dummy atom model (DAM) demonstrated that the SAXS-derived BlCel5B molecular envelope could not be single-handedly filled by any of the main conformational states encountered in the simulations (Fig. 4D).	RESULTS
+195	206	simulations	experimental_method	The ab initio dummy atom model (DAM) demonstrated that the SAXS-derived BlCel5B molecular envelope could not be single-handedly filled by any of the main conformational states encountered in the simulations (Fig. 4D).	RESULTS
+19	30	Kratky plot	evidence	It is known that a Kratky plot exhibits a peak with an elevated baseline at high q for a monodisperse system composed of multi-domain particles with flexible extensions.	RESULTS
+85	92	BlCel5B	protein	Indeed, an elevation of the baseline toward a hyperbolic-like curve was observed for BlCel5B, indicating a considerable degree of molecular mobility in solution (Supplementary Fig. 3).	RESULTS
+151	182	crystallographic and MD studies	experimental_method	Thus, the conformational heterogeneity of the enzyme can be decomposed in structural terms as a combination of conformational states identified in our crystallographic and MD studies.	RESULTS
+18	22	SAXS	experimental_method	We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.	RESULTS
+23	31	envelope	evidence	We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.	RESULTS
+75	90	superimposition	experimental_method	We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.	RESULTS
+152	159	BlCel5B	protein	We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.	RESULTS
+173	179	closed	protein_state	We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.	RESULTS
+183	188	CBM46	structure_element	We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.	RESULTS
+189	191	CD	structure_element	We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.	RESULTS
+192	200	occluded	protein_state	We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.	RESULTS
+233	244	simulations	experimental_method	We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.	RESULTS
+278	287	semi-open	protein_state	We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.	RESULTS
+320	337	crystal structure	evidence	We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.	RESULTS
+367	375	extended	protein_state	We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.	RESULTS
+398	409	simulations	experimental_method	We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.	RESULTS
+445	449	SAXS	experimental_method	We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.	RESULTS
+450	458	envelope	evidence	We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.	RESULTS
+14	38	average scattering curve	evidence	The resulting average scattering curve from this model fits the experimental protein scattering intensity, with χ = 1.89 (Supplementary Fig. 3).	RESULTS
+85	105	scattering intensity	evidence	The resulting average scattering curve from this model fits the experimental protein scattering intensity, with χ = 1.89 (Supplementary Fig. 3).	RESULTS
+112	113	χ	evidence	The resulting average scattering curve from this model fits the experimental protein scattering intensity, with χ = 1.89 (Supplementary Fig. 3).	RESULTS
+0	5	GH5_4	protein_type	GH5_4 phylogenetic analysis	RESULTS
+6	27	phylogenetic analysis	experimental_method	GH5_4 phylogenetic analysis	RESULTS
+150	161	277 and 400	residue_range	After the exclusion of partial sequences and the suppression of highly identical members (higher than 90% identity), 144 sequences containing between 277 and 400 residues were aligned and used to construct a phylogenetic tree (Supplementary Fig. 4A).	RESULTS
+176	183	aligned	experimental_method	After the exclusion of partial sequences and the suppression of highly identical members (higher than 90% identity), 144 sequences containing between 277 and 400 residues were aligned and used to construct a phylogenetic tree (Supplementary Fig. 4A).	RESULTS
+208	225	phylogenetic tree	evidence	After the exclusion of partial sequences and the suppression of highly identical members (higher than 90% identity), 144 sequences containing between 277 and 400 residues were aligned and used to construct a phylogenetic tree (Supplementary Fig. 4A).	RESULTS
+64	67	GH5	protein_type	According to PFAM database conserved domain classification, 128 GH5 enzymes have an architecture consisting of an N-terminal catalytic module, a CBM_X2 module and an unknown module of approximately 100 residues at the C-terminus (Supplementary Fig. 4B).	RESULTS
+125	141	catalytic module	structure_element	According to PFAM database conserved domain classification, 128 GH5 enzymes have an architecture consisting of an N-terminal catalytic module, a CBM_X2 module and an unknown module of approximately 100 residues at the C-terminus (Supplementary Fig. 4B).	RESULTS
+145	151	CBM_X2	structure_element	According to PFAM database conserved domain classification, 128 GH5 enzymes have an architecture consisting of an N-terminal catalytic module, a CBM_X2 module and an unknown module of approximately 100 residues at the C-terminus (Supplementary Fig. 4B).	RESULTS
+40	44	CBM1	structure_element	Of these, 12 enzymes have an additional CBM1, and 5 have a CBM2 at the N-terminal region.	RESULTS
+59	63	CBM2	structure_element	Of these, 12 enzymes have an additional CBM1, and 5 have a CBM2 at the N-terminal region.	RESULTS
+98	105	BlCel5B	protein	Based on this PFAM architecture and CAZy subfamily classification, all the 144 enzymes (including BlCel5B) belong to the GH5_4 subfamily and group together in the same branch of the phylogenetic tree, evidencing a common ancestor.	RESULTS
+121	126	GH5_4	protein_type	Based on this PFAM architecture and CAZy subfamily classification, all the 144 enzymes (including BlCel5B) belong to the GH5_4 subfamily and group together in the same branch of the phylogenetic tree, evidencing a common ancestor.	RESULTS
+182	199	phylogenetic tree	evidence	Based on this PFAM architecture and CAZy subfamily classification, all the 144 enzymes (including BlCel5B) belong to the GH5_4 subfamily and group together in the same branch of the phylogenetic tree, evidencing a common ancestor.	RESULTS
+217	222	CBM46	structure_element	These results support the hypothesis that the enzymes may employ the same mechanism by which ligand binding is mediated by an extensive conformational breathing of the enzyme that involves the large-scale movement of CBM46 around the Ig-like module (CBM_X2) as a structural hinge.	RESULTS
+234	248	Ig-like module	structure_element	These results support the hypothesis that the enzymes may employ the same mechanism by which ligand binding is mediated by an extensive conformational breathing of the enzyme that involves the large-scale movement of CBM46 around the Ig-like module (CBM_X2) as a structural hinge.	RESULTS
+250	256	CBM_X2	structure_element	These results support the hypothesis that the enzymes may employ the same mechanism by which ligand binding is mediated by an extensive conformational breathing of the enzyme that involves the large-scale movement of CBM46 around the Ig-like module (CBM_X2) as a structural hinge.	RESULTS
+263	279	structural hinge	structure_element	These results support the hypothesis that the enzymes may employ the same mechanism by which ligand binding is mediated by an extensive conformational breathing of the enzyme that involves the large-scale movement of CBM46 around the Ig-like module (CBM_X2) as a structural hinge.	RESULTS
+23	33	trimodular	protein_state	Here, we elucidate the trimodular molecular architecture of the full-length BlCel5B, a member of the GH5_4 subfamily, for which large-scale conformational dynamics appears to play a central role in its enzymatic activity.	DISCUSS
+64	75	full-length	protein_state	Here, we elucidate the trimodular molecular architecture of the full-length BlCel5B, a member of the GH5_4 subfamily, for which large-scale conformational dynamics appears to play a central role in its enzymatic activity.	DISCUSS
+76	83	BlCel5B	protein	Here, we elucidate the trimodular molecular architecture of the full-length BlCel5B, a member of the GH5_4 subfamily, for which large-scale conformational dynamics appears to play a central role in its enzymatic activity.	DISCUSS
+101	106	GH5_4	protein_type	Here, we elucidate the trimodular molecular architecture of the full-length BlCel5B, a member of the GH5_4 subfamily, for which large-scale conformational dynamics appears to play a central role in its enzymatic activity.	DISCUSS
+0	11	Full-length	protein_state	Full-length BlCel5B is active on both cellulosic and hemicellulosic substrates and auxiliary modules are crucial for its activity.	DISCUSS
+12	19	BlCel5B	protein	Full-length BlCel5B is active on both cellulosic and hemicellulosic substrates and auxiliary modules are crucial for its activity.	DISCUSS
+23	29	active	protein_state	Full-length BlCel5B is active on both cellulosic and hemicellulosic substrates and auxiliary modules are crucial for its activity.	DISCUSS
+38	48	cellulosic	chemical	Full-length BlCel5B is active on both cellulosic and hemicellulosic substrates and auxiliary modules are crucial for its activity.	DISCUSS
+53	67	hemicellulosic	chemical	Full-length BlCel5B is active on both cellulosic and hemicellulosic substrates and auxiliary modules are crucial for its activity.	DISCUSS
+5	32	carbohydrate-active enzymes	protein_type	Most carbohydrate-active enzymes are modular and consist of a catalytic domain appended to one or more separate AMs.	DISCUSS
+62	78	catalytic domain	structure_element	Most carbohydrate-active enzymes are modular and consist of a catalytic domain appended to one or more separate AMs.	DISCUSS
+112	115	AMs	structure_element	Most carbohydrate-active enzymes are modular and consist of a catalytic domain appended to one or more separate AMs.	DISCUSS
+0	3	AMs	structure_element	AMs, such as CBMs, typically recognize carbohydrates and target their cognate catalytic domains toward the substrate.	DISCUSS
+13	17	CBMs	structure_element	AMs, such as CBMs, typically recognize carbohydrates and target their cognate catalytic domains toward the substrate.	DISCUSS
+39	52	carbohydrates	chemical	AMs, such as CBMs, typically recognize carbohydrates and target their cognate catalytic domains toward the substrate.	DISCUSS
+78	95	catalytic domains	structure_element	AMs, such as CBMs, typically recognize carbohydrates and target their cognate catalytic domains toward the substrate.	DISCUSS
+12	31	structural analysis	experimental_method	Because the structural analysis of the protein is challenging if the linkers connecting the structural subunits of the enzyme are long and flexible, the standard approach is to study the domains separately.	DISCUSS
+69	76	linkers	structure_element	Because the structural analysis of the protein is challenging if the linkers connecting the structural subunits of the enzyme are long and flexible, the standard approach is to study the domains separately.	DISCUSS
+31	54	protein crystallography	experimental_method	In this work, a combination of protein crystallography, computational molecular dynamics, and SAXS analyses enabled the identification of a new conformational selection-based molecular mechanism that involves GH5 catalytic domain and two AMs in full-length BlCel5B.	DISCUSS
+56	88	computational molecular dynamics	experimental_method	In this work, a combination of protein crystallography, computational molecular dynamics, and SAXS analyses enabled the identification of a new conformational selection-based molecular mechanism that involves GH5 catalytic domain and two AMs in full-length BlCel5B.	DISCUSS
+94	98	SAXS	experimental_method	In this work, a combination of protein crystallography, computational molecular dynamics, and SAXS analyses enabled the identification of a new conformational selection-based molecular mechanism that involves GH5 catalytic domain and two AMs in full-length BlCel5B.	DISCUSS
+209	212	GH5	protein_type	In this work, a combination of protein crystallography, computational molecular dynamics, and SAXS analyses enabled the identification of a new conformational selection-based molecular mechanism that involves GH5 catalytic domain and two AMs in full-length BlCel5B.	DISCUSS
+213	229	catalytic domain	structure_element	In this work, a combination of protein crystallography, computational molecular dynamics, and SAXS analyses enabled the identification of a new conformational selection-based molecular mechanism that involves GH5 catalytic domain and two AMs in full-length BlCel5B.	DISCUSS
+238	241	AMs	structure_element	In this work, a combination of protein crystallography, computational molecular dynamics, and SAXS analyses enabled the identification of a new conformational selection-based molecular mechanism that involves GH5 catalytic domain and two AMs in full-length BlCel5B.	DISCUSS
+245	256	full-length	protein_state	In this work, a combination of protein crystallography, computational molecular dynamics, and SAXS analyses enabled the identification of a new conformational selection-based molecular mechanism that involves GH5 catalytic domain and two AMs in full-length BlCel5B.	DISCUSS
+257	264	BlCel5B	protein	In this work, a combination of protein crystallography, computational molecular dynamics, and SAXS analyses enabled the identification of a new conformational selection-based molecular mechanism that involves GH5 catalytic domain and two AMs in full-length BlCel5B.	DISCUSS
+21	28	BlCel5B	protein	We observed that the BlCel5B distal CBM46 is directly involved in shaping the local architecture of the substrate-binding site.	DISCUSS
+36	41	CBM46	structure_element	We observed that the BlCel5B distal CBM46 is directly involved in shaping the local architecture of the substrate-binding site.	DISCUSS
+104	126	substrate-binding site	site	We observed that the BlCel5B distal CBM46 is directly involved in shaping the local architecture of the substrate-binding site.	DISCUSS
+13	15	CD	structure_element	Although the CD alone appears unable to bind the substrate for catalysis, the AMs exhibit open-close motions that allow the substrate to be captured in a suitable position for hydrolysis.	DISCUSS
+16	21	alone	protein_state	Although the CD alone appears unable to bind the substrate for catalysis, the AMs exhibit open-close motions that allow the substrate to be captured in a suitable position for hydrolysis.	DISCUSS
+78	81	AMs	structure_element	Although the CD alone appears unable to bind the substrate for catalysis, the AMs exhibit open-close motions that allow the substrate to be captured in a suitable position for hydrolysis.	DISCUSS
+90	94	open	protein_state	Although the CD alone appears unable to bind the substrate for catalysis, the AMs exhibit open-close motions that allow the substrate to be captured in a suitable position for hydrolysis.	DISCUSS
+95	100	close	protein_state	Although the CD alone appears unable to bind the substrate for catalysis, the AMs exhibit open-close motions that allow the substrate to be captured in a suitable position for hydrolysis.	DISCUSS
+50	53	AMs	structure_element	Here, we advocate that large-amplitude motions of AMs are crucial for assembling the enzyme into its active conformation, highlighting a new function of CBMs.	DISCUSS
+101	107	active	protein_state	Here, we advocate that large-amplitude motions of AMs are crucial for assembling the enzyme into its active conformation, highlighting a new function of CBMs.	DISCUSS
+153	157	CBMs	structure_element	Here, we advocate that large-amplitude motions of AMs are crucial for assembling the enzyme into its active conformation, highlighting a new function of CBMs.	DISCUSS
+58	66	extended	protein_state	This mechanism of substrate binding closely resembles the extended conformational selection model, with the induced-fit mechanism of reaction as its limiting case.	DISCUSS
+96	98	GH	protein_type	To the best of our knowledge, this enzymatic mechanism has not been proposed previously for any GH.	DISCUSS
+4	19	CD binding site	site	The CD binding site of BlCel5B is open and relatively flat and is thus barely able to properly hold the substrate in position for catalysis without assistance from the CBM46.	DISCUSS
+23	30	BlCel5B	protein	The CD binding site of BlCel5B is open and relatively flat and is thus barely able to properly hold the substrate in position for catalysis without assistance from the CBM46.	DISCUSS
+168	173	CBM46	structure_element	The CD binding site of BlCel5B is open and relatively flat and is thus barely able to properly hold the substrate in position for catalysis without assistance from the CBM46.	DISCUSS
+19	23	GH5s	protein_type	In contrast, other GH5s belonging to subfamily 4 listed in the Protein Data Bank exhibit a deep binding cleft or tunnel that can effectively entrap the substrate for catalysis (Fig. 5).	DISCUSS
+96	109	binding cleft	site	In contrast, other GH5s belonging to subfamily 4 listed in the Protein Data Bank exhibit a deep binding cleft or tunnel that can effectively entrap the substrate for catalysis (Fig. 5).	DISCUSS
+113	119	tunnel	site	In contrast, other GH5s belonging to subfamily 4 listed in the Protein Data Bank exhibit a deep binding cleft or tunnel that can effectively entrap the substrate for catalysis (Fig. 5).	DISCUSS
+75	86	simulations	experimental_method	Due to the marked interdomain conformational rearrangement observed in our simulations, the CBM46 generates a confined binding site in BlCel5B that resembles the binding site architecture of the other GH5 enzymes that lack AMs.	DISCUSS
+92	97	CBM46	structure_element	Due to the marked interdomain conformational rearrangement observed in our simulations, the CBM46 generates a confined binding site in BlCel5B that resembles the binding site architecture of the other GH5 enzymes that lack AMs.	DISCUSS
+119	131	binding site	site	Due to the marked interdomain conformational rearrangement observed in our simulations, the CBM46 generates a confined binding site in BlCel5B that resembles the binding site architecture of the other GH5 enzymes that lack AMs.	DISCUSS
+135	142	BlCel5B	protein	Due to the marked interdomain conformational rearrangement observed in our simulations, the CBM46 generates a confined binding site in BlCel5B that resembles the binding site architecture of the other GH5 enzymes that lack AMs.	DISCUSS
+162	174	binding site	site	Due to the marked interdomain conformational rearrangement observed in our simulations, the CBM46 generates a confined binding site in BlCel5B that resembles the binding site architecture of the other GH5 enzymes that lack AMs.	DISCUSS
+201	204	GH5	protein_type	Due to the marked interdomain conformational rearrangement observed in our simulations, the CBM46 generates a confined binding site in BlCel5B that resembles the binding site architecture of the other GH5 enzymes that lack AMs.	DISCUSS
+218	222	lack	protein_state	Due to the marked interdomain conformational rearrangement observed in our simulations, the CBM46 generates a confined binding site in BlCel5B that resembles the binding site architecture of the other GH5 enzymes that lack AMs.	DISCUSS
+223	226	AMs	structure_element	Due to the marked interdomain conformational rearrangement observed in our simulations, the CBM46 generates a confined binding site in BlCel5B that resembles the binding site architecture of the other GH5 enzymes that lack AMs.	DISCUSS
+6	13	BlCel5B	protein	Thus, BlCel5B appears to have adopted a strategy of CBM46-mediated interactions for proper functioning.	DISCUSS
+52	57	CBM46	structure_element	Thus, BlCel5B appears to have adopted a strategy of CBM46-mediated interactions for proper functioning.	DISCUSS
+24	31	BhCel5B	protein	Although the homologous BhCel5B has the same domain architecture of BlCel5B and belongs to the same subfamily (a comparison of the sequence and structure of BlCel5B and BhCel5B is presented in Supplementary Fig. 5), its binding site exhibits important differences that may impact the catalytic mechanism.	DISCUSS
+68	75	BlCel5B	protein	Although the homologous BhCel5B has the same domain architecture of BlCel5B and belongs to the same subfamily (a comparison of the sequence and structure of BlCel5B and BhCel5B is presented in Supplementary Fig. 5), its binding site exhibits important differences that may impact the catalytic mechanism.	DISCUSS
+144	153	structure	evidence	Although the homologous BhCel5B has the same domain architecture of BlCel5B and belongs to the same subfamily (a comparison of the sequence and structure of BlCel5B and BhCel5B is presented in Supplementary Fig. 5), its binding site exhibits important differences that may impact the catalytic mechanism.	DISCUSS
+157	164	BlCel5B	protein	Although the homologous BhCel5B has the same domain architecture of BlCel5B and belongs to the same subfamily (a comparison of the sequence and structure of BlCel5B and BhCel5B is presented in Supplementary Fig. 5), its binding site exhibits important differences that may impact the catalytic mechanism.	DISCUSS
+169	176	BhCel5B	protein	Although the homologous BhCel5B has the same domain architecture of BlCel5B and belongs to the same subfamily (a comparison of the sequence and structure of BlCel5B and BhCel5B is presented in Supplementary Fig. 5), its binding site exhibits important differences that may impact the catalytic mechanism.	DISCUSS
+220	232	binding site	site	Although the homologous BhCel5B has the same domain architecture of BlCel5B and belongs to the same subfamily (a comparison of the sequence and structure of BlCel5B and BhCel5B is presented in Supplementary Fig. 5), its binding site exhibits important differences that may impact the catalytic mechanism.	DISCUSS
+4	11	BhCel5B	protein	The BhCel5B binding site is V-shaped and deeper than the BlCel5B binding site (Figs 5 and 6).	DISCUSS
+12	24	binding site	site	The BhCel5B binding site is V-shaped and deeper than the BlCel5B binding site (Figs 5 and 6).	DISCUSS
+28	36	V-shaped	protein_state	The BhCel5B binding site is V-shaped and deeper than the BlCel5B binding site (Figs 5 and 6).	DISCUSS
+57	64	BlCel5B	protein	The BhCel5B binding site is V-shaped and deeper than the BlCel5B binding site (Figs 5 and 6).	DISCUSS
+65	77	binding site	site	The BhCel5B binding site is V-shaped and deeper than the BlCel5B binding site (Figs 5 and 6).	DISCUSS
+19	23	loop	structure_element	This is due to the loop between residues F177 and R185 from BhCel5B (absent in the BlCel5B), which contains residue W181 that forms part of the binding cleft (Fig. 6).	DISCUSS
+41	45	F177	residue_name_number	This is due to the loop between residues F177 and R185 from BhCel5B (absent in the BlCel5B), which contains residue W181 that forms part of the binding cleft (Fig. 6).	DISCUSS
+50	54	R185	residue_name_number	This is due to the loop between residues F177 and R185 from BhCel5B (absent in the BlCel5B), which contains residue W181 that forms part of the binding cleft (Fig. 6).	DISCUSS
+60	67	BhCel5B	protein	This is due to the loop between residues F177 and R185 from BhCel5B (absent in the BlCel5B), which contains residue W181 that forms part of the binding cleft (Fig. 6).	DISCUSS
+69	75	absent	protein_state	This is due to the loop between residues F177 and R185 from BhCel5B (absent in the BlCel5B), which contains residue W181 that forms part of the binding cleft (Fig. 6).	DISCUSS
+83	90	BlCel5B	protein	This is due to the loop between residues F177 and R185 from BhCel5B (absent in the BlCel5B), which contains residue W181 that forms part of the binding cleft (Fig. 6).	DISCUSS
+116	120	W181	residue_name_number	This is due to the loop between residues F177 and R185 from BhCel5B (absent in the BlCel5B), which contains residue W181 that forms part of the binding cleft (Fig. 6).	DISCUSS
+144	157	binding cleft	site	This is due to the loop between residues F177 and R185 from BhCel5B (absent in the BlCel5B), which contains residue W181 that forms part of the binding cleft (Fig. 6).	DISCUSS
+23	30	BhCel5B	protein	Consistently, although BhCel5B CBM46 is important for β-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates.	DISCUSS
+31	36	CBM46	structure_element	Consistently, although BhCel5B CBM46 is important for β-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates.	DISCUSS
+54	70	β-1,3-1,4-glucan	chemical	Consistently, although BhCel5B CBM46 is important for β-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates.	DISCUSS
+83	90	BhCel5B	protein	Consistently, although BhCel5B CBM46 is important for β-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates.	DISCUSS
+113	119	active	protein_state	Consistently, although BhCel5B CBM46 is important for β-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates.	DISCUSS
+120	127	without	protein_state	Consistently, although BhCel5B CBM46 is important for β-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates.	DISCUSS
+128	133	CBM46	structure_element	Consistently, although BhCel5B CBM46 is important for β-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates.	DISCUSS
+140	149	truncated	protein_state	Consistently, although BhCel5B CBM46 is important for β-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates.	DISCUSS
+171	177	active	protein_state	Consistently, although BhCel5B CBM46 is important for β-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates.	DISCUSS
+186	196	xyloglucan	chemical	Consistently, although BhCel5B CBM46 is important for β-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates.	DISCUSS
+218	223	CBM46	structure_element	Consistently, although BhCel5B CBM46 is important for β-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates.	DISCUSS
+38	59	phylogenetic analysis	experimental_method	A closer inspection of results of the phylogenetic analysis, more specifically of the clade composed by GH5_4 enzymes with trimodular architecture (Supplementary Fig. 4C), reveals subclades whose main characteristic is the varying length of the loop located between residues 161 and 163 (BlCel5B residue numbering).	DISCUSS
+104	109	GH5_4	protein_type	A closer inspection of results of the phylogenetic analysis, more specifically of the clade composed by GH5_4 enzymes with trimodular architecture (Supplementary Fig. 4C), reveals subclades whose main characteristic is the varying length of the loop located between residues 161 and 163 (BlCel5B residue numbering).	DISCUSS
+123	133	trimodular	protein_state	A closer inspection of results of the phylogenetic analysis, more specifically of the clade composed by GH5_4 enzymes with trimodular architecture (Supplementary Fig. 4C), reveals subclades whose main characteristic is the varying length of the loop located between residues 161 and 163 (BlCel5B residue numbering).	DISCUSS
+245	249	loop	structure_element	A closer inspection of results of the phylogenetic analysis, more specifically of the clade composed by GH5_4 enzymes with trimodular architecture (Supplementary Fig. 4C), reveals subclades whose main characteristic is the varying length of the loop located between residues 161 and 163 (BlCel5B residue numbering).	DISCUSS
+275	286	161 and 163	residue_range	A closer inspection of results of the phylogenetic analysis, more specifically of the clade composed by GH5_4 enzymes with trimodular architecture (Supplementary Fig. 4C), reveals subclades whose main characteristic is the varying length of the loop located between residues 161 and 163 (BlCel5B residue numbering).	DISCUSS
+288	295	BlCel5B	protein	A closer inspection of results of the phylogenetic analysis, more specifically of the clade composed by GH5_4 enzymes with trimodular architecture (Supplementary Fig. 4C), reveals subclades whose main characteristic is the varying length of the loop located between residues 161 and 163 (BlCel5B residue numbering).	DISCUSS
+33	40	BlCel5B	protein	Therefore, our results show that BlCel5B represents a smaller group of enzymes that are completely dependent on its AMs for hydrolysis of plant cell wall polysaccharides, and that the underlying mechanism may rely on large-scale interdomain motions.	DISCUSS
+116	119	AMs	structure_element	Therefore, our results show that BlCel5B represents a smaller group of enzymes that are completely dependent on its AMs for hydrolysis of plant cell wall polysaccharides, and that the underlying mechanism may rely on large-scale interdomain motions.	DISCUSS
+138	143	plant	taxonomy_domain	Therefore, our results show that BlCel5B represents a smaller group of enzymes that are completely dependent on its AMs for hydrolysis of plant cell wall polysaccharides, and that the underlying mechanism may rely on large-scale interdomain motions.	DISCUSS
+154	169	polysaccharides	chemical	Therefore, our results show that BlCel5B represents a smaller group of enzymes that are completely dependent on its AMs for hydrolysis of plant cell wall polysaccharides, and that the underlying mechanism may rely on large-scale interdomain motions.	DISCUSS
+31	38	BlCel5B	protein	The amino acid sequence of the BlCel5B Ig-like module is recognized by BLASTP as belonging to CBM_X2, a poorly described group that has been compared with CBM-like accessory modules without a defined function.	DISCUSS
+39	53	Ig-like module	structure_element	The amino acid sequence of the BlCel5B Ig-like module is recognized by BLASTP as belonging to CBM_X2, a poorly described group that has been compared with CBM-like accessory modules without a defined function.	DISCUSS
+71	77	BLASTP	experimental_method	The amino acid sequence of the BlCel5B Ig-like module is recognized by BLASTP as belonging to CBM_X2, a poorly described group that has been compared with CBM-like accessory modules without a defined function.	DISCUSS
+94	100	CBM_X2	structure_element	The amino acid sequence of the BlCel5B Ig-like module is recognized by BLASTP as belonging to CBM_X2, a poorly described group that has been compared with CBM-like accessory modules without a defined function.	DISCUSS
+155	181	CBM-like accessory modules	structure_element	The amino acid sequence of the BlCel5B Ig-like module is recognized by BLASTP as belonging to CBM_X2, a poorly described group that has been compared with CBM-like accessory modules without a defined function.	DISCUSS
+26	33	BlCel5B	protein	Despite the similarity of BlCel5B Ig-like module to CBMs, it lacks an identifiable aromatic residue-rich carbohydrate-binding site.	DISCUSS
+34	48	Ig-like module	structure_element	Despite the similarity of BlCel5B Ig-like module to CBMs, it lacks an identifiable aromatic residue-rich carbohydrate-binding site.	DISCUSS
+52	56	CBMs	structure_element	Despite the similarity of BlCel5B Ig-like module to CBMs, it lacks an identifiable aromatic residue-rich carbohydrate-binding site.	DISCUSS
+105	130	carbohydrate-binding site	site	Despite the similarity of BlCel5B Ig-like module to CBMs, it lacks an identifiable aromatic residue-rich carbohydrate-binding site.	DISCUSS
+43	57	Ig-like module	structure_element	Nonetheless, according to our results, the Ig-like module seems to play an important function as a structural hinge, dynamically holding the CBM46 and CD in positions that are appropriate for enzymatic activity.	DISCUSS
+99	115	structural hinge	structure_element	Nonetheless, according to our results, the Ig-like module seems to play an important function as a structural hinge, dynamically holding the CBM46 and CD in positions that are appropriate for enzymatic activity.	DISCUSS
+141	146	CBM46	structure_element	Nonetheless, according to our results, the Ig-like module seems to play an important function as a structural hinge, dynamically holding the CBM46 and CD in positions that are appropriate for enzymatic activity.	DISCUSS
+151	153	CD	structure_element	Nonetheless, according to our results, the Ig-like module seems to play an important function as a structural hinge, dynamically holding the CBM46 and CD in positions that are appropriate for enzymatic activity.	DISCUSS
+28	65	crystallographic, computer simulation	experimental_method	Based on the results of our crystallographic, computer simulation, and SAXS structural analyses, as well as site-directed mutagenesis and activity assays, we propose a molecular mechanism for BlCel5B substrate binding, which might apply to other GH5_4 subfamily enzymes that share this tri-modular architecture.	DISCUSS
+71	95	SAXS structural analyses	experimental_method	Based on the results of our crystallographic, computer simulation, and SAXS structural analyses, as well as site-directed mutagenesis and activity assays, we propose a molecular mechanism for BlCel5B substrate binding, which might apply to other GH5_4 subfamily enzymes that share this tri-modular architecture.	DISCUSS
+108	133	site-directed mutagenesis	experimental_method	Based on the results of our crystallographic, computer simulation, and SAXS structural analyses, as well as site-directed mutagenesis and activity assays, we propose a molecular mechanism for BlCel5B substrate binding, which might apply to other GH5_4 subfamily enzymes that share this tri-modular architecture.	DISCUSS
+138	153	activity assays	experimental_method	Based on the results of our crystallographic, computer simulation, and SAXS structural analyses, as well as site-directed mutagenesis and activity assays, we propose a molecular mechanism for BlCel5B substrate binding, which might apply to other GH5_4 subfamily enzymes that share this tri-modular architecture.	DISCUSS
+192	199	BlCel5B	protein	Based on the results of our crystallographic, computer simulation, and SAXS structural analyses, as well as site-directed mutagenesis and activity assays, we propose a molecular mechanism for BlCel5B substrate binding, which might apply to other GH5_4 subfamily enzymes that share this tri-modular architecture.	DISCUSS
+246	251	GH5_4	protein_type	Based on the results of our crystallographic, computer simulation, and SAXS structural analyses, as well as site-directed mutagenesis and activity assays, we propose a molecular mechanism for BlCel5B substrate binding, which might apply to other GH5_4 subfamily enzymes that share this tri-modular architecture.	DISCUSS
+286	297	tri-modular	structure_element	Based on the results of our crystallographic, computer simulation, and SAXS structural analyses, as well as site-directed mutagenesis and activity assays, we propose a molecular mechanism for BlCel5B substrate binding, which might apply to other GH5_4 subfamily enzymes that share this tri-modular architecture.	DISCUSS
+0	7	BlCel5B	protein	BlCel5B can be found in several different conformational states ranging from CBM46/CD closed (or occluded) to extended conformations (Fig. 7).	DISCUSS
+77	82	CBM46	structure_element	BlCel5B can be found in several different conformational states ranging from CBM46/CD closed (or occluded) to extended conformations (Fig. 7).	DISCUSS
+83	85	CD	structure_element	BlCel5B can be found in several different conformational states ranging from CBM46/CD closed (or occluded) to extended conformations (Fig. 7).	DISCUSS
+86	92	closed	protein_state	BlCel5B can be found in several different conformational states ranging from CBM46/CD closed (or occluded) to extended conformations (Fig. 7).	DISCUSS
+97	105	occluded	protein_state	BlCel5B can be found in several different conformational states ranging from CBM46/CD closed (or occluded) to extended conformations (Fig. 7).	DISCUSS
+110	118	extended	protein_state	BlCel5B can be found in several different conformational states ranging from CBM46/CD closed (or occluded) to extended conformations (Fig. 7).	DISCUSS
+3	11	extended	protein_state	In extended configurations, the substrate may dock at the shallow substrate binding site of CD in one of the semi-closed conformations of the enzyme; however, its binding is properly stabilized for hydrolysis only with the aid of induced-fit repositioning mediated by CBM46.	DISCUSS
+66	88	substrate binding site	site	In extended configurations, the substrate may dock at the shallow substrate binding site of CD in one of the semi-closed conformations of the enzyme; however, its binding is properly stabilized for hydrolysis only with the aid of induced-fit repositioning mediated by CBM46.	DISCUSS
+92	94	CD	structure_element	In extended configurations, the substrate may dock at the shallow substrate binding site of CD in one of the semi-closed conformations of the enzyme; however, its binding is properly stabilized for hydrolysis only with the aid of induced-fit repositioning mediated by CBM46.	DISCUSS
+109	120	semi-closed	protein_state	In extended configurations, the substrate may dock at the shallow substrate binding site of CD in one of the semi-closed conformations of the enzyme; however, its binding is properly stabilized for hydrolysis only with the aid of induced-fit repositioning mediated by CBM46.	DISCUSS
+268	273	CBM46	structure_element	In extended configurations, the substrate may dock at the shallow substrate binding site of CD in one of the semi-closed conformations of the enzyme; however, its binding is properly stabilized for hydrolysis only with the aid of induced-fit repositioning mediated by CBM46.	DISCUSS
+42	49	BlCel5B	protein	After cleavage, the intrinsic dynamics of BlCel5B would eventually allow the opening of the active site for product release.	DISCUSS
+92	103	active site	site	After cleavage, the intrinsic dynamics of BlCel5B would eventually allow the opening of the active site for product release.	DISCUSS
+46	87	mutagenesis and enzymatic activity assays	experimental_method	The proposed mechanism is consistent with our mutagenesis and enzymatic activity assays, which show that the Ig-like module and CBM46 are indispensable for BlCel5B catalytic activity and, together with the CD, form the unique catalytic domain of the enzyme.	DISCUSS
+109	123	Ig-like module	structure_element	The proposed mechanism is consistent with our mutagenesis and enzymatic activity assays, which show that the Ig-like module and CBM46 are indispensable for BlCel5B catalytic activity and, together with the CD, form the unique catalytic domain of the enzyme.	DISCUSS
+128	133	CBM46	structure_element	The proposed mechanism is consistent with our mutagenesis and enzymatic activity assays, which show that the Ig-like module and CBM46 are indispensable for BlCel5B catalytic activity and, together with the CD, form the unique catalytic domain of the enzyme.	DISCUSS
+156	163	BlCel5B	protein	The proposed mechanism is consistent with our mutagenesis and enzymatic activity assays, which show that the Ig-like module and CBM46 are indispensable for BlCel5B catalytic activity and, together with the CD, form the unique catalytic domain of the enzyme.	DISCUSS
+206	208	CD	structure_element	The proposed mechanism is consistent with our mutagenesis and enzymatic activity assays, which show that the Ig-like module and CBM46 are indispensable for BlCel5B catalytic activity and, together with the CD, form the unique catalytic domain of the enzyme.	DISCUSS
+219	225	unique	protein_state	The proposed mechanism is consistent with our mutagenesis and enzymatic activity assays, which show that the Ig-like module and CBM46 are indispensable for BlCel5B catalytic activity and, together with the CD, form the unique catalytic domain of the enzyme.	DISCUSS
+226	242	catalytic domain	structure_element	The proposed mechanism is consistent with our mutagenesis and enzymatic activity assays, which show that the Ig-like module and CBM46 are indispensable for BlCel5B catalytic activity and, together with the CD, form the unique catalytic domain of the enzyme.	DISCUSS
+46	50	CBMs	structure_element	These experiments reveal a novel function for CBMs in which they are intimately involved in the assembly of the active site and catalytic process.	DISCUSS
+112	123	active site	site	These experiments reveal a novel function for CBMs in which they are intimately involved in the assembly of the active site and catalytic process.	DISCUSS
+0	20	Computer simulations	experimental_method	Computer simulations suggest that large-scale motions of the CBM46 and Ig-like domains mediate conformational selection and final induced-fit adjustments to trap the substrate at the active site and promote hydrolysis.	DISCUSS
+61	66	CBM46	structure_element	Computer simulations suggest that large-scale motions of the CBM46 and Ig-like domains mediate conformational selection and final induced-fit adjustments to trap the substrate at the active site and promote hydrolysis.	DISCUSS
+71	86	Ig-like domains	structure_element	Computer simulations suggest that large-scale motions of the CBM46 and Ig-like domains mediate conformational selection and final induced-fit adjustments to trap the substrate at the active site and promote hydrolysis.	DISCUSS
+183	194	active site	site	Computer simulations suggest that large-scale motions of the CBM46 and Ig-like domains mediate conformational selection and final induced-fit adjustments to trap the substrate at the active site and promote hydrolysis.	DISCUSS
+0	4	SAXS	experimental_method	SAXS data support the modeling results, providing compelling evidence for highly mobile domains in solution.	DISCUSS
+22	30	modeling	experimental_method	SAXS data support the modeling results, providing compelling evidence for highly mobile domains in solution.	DISCUSS
+74	87	highly mobile	protein_state	SAXS data support the modeling results, providing compelling evidence for highly mobile domains in solution.	DISCUSS
+9	17	spectrum	evidence	A single spectrum was obtained by averaging four independent spectra generated by 300 laser shots at 60% potency.	METHODS
+61	68	spectra	evidence	A single spectrum was obtained by averaging four independent spectra generated by 300 laser shots at 60% potency.	METHODS
+41	44	apo	protein_state	The missing residues were taken from the apo BlCel5B structure after structural alignment using the LovoAlign server.	METHODS
+0	20	BlCel5B-cellooctaose	complex_assembly	BlCel5B-cellooctaose	METHODS
+22	42	BlCel5B-cellooctaose	complex_assembly	To get a model of the BlCel5B-cellooctaose complex in the closed conformation, we took the configuration after 80 ns of the restrained 200-ns MD simulation as the starting point for a 500-ns-long restrained aMD simulation, in which the CBM46 moved towards the CD in the presence of the harmonically-restrained cellooctaose chain.	METHODS
+75	95	BlCel5B-cellooctaose	complex_assembly	After this procedure, we released the restraints and propagated the closed BlCel5B-cellooctaose complex for additional 500 ns of conventional, restraint-free MD simulation.	METHODS
+0	14	Crystal models	evidence	Crystal models of BlCel5B.	FIG
+18	25	BlCel5B	protein	Crystal models of BlCel5B.	FIG
+9	18	structure	evidence	Complete structure is shown as a cartoon illustration in (a) and a van der Waals surface in (b).	FIG
+4	6	CD	structure_element	The CD module (red) has a typical TIM-barrel fold, and its substrate-binding site is adjacent to CBM46 (blue).	FIG
+34	49	TIM-barrel fold	structure_element	The CD module (red) has a typical TIM-barrel fold, and its substrate-binding site is adjacent to CBM46 (blue).	FIG
+59	81	substrate-binding site	site	The CD module (red) has a typical TIM-barrel fold, and its substrate-binding site is adjacent to CBM46 (blue).	FIG
+97	102	CBM46	structure_element	The CD module (red) has a typical TIM-barrel fold, and its substrate-binding site is adjacent to CBM46 (blue).	FIG
+29	41	binding site	site	Despite the proximity of the binding site in the crystallographic model, the CBM46 residues W479 and W481 are distant from the substrate cellotetraose (yellow).	FIG
+77	82	CBM46	structure_element	Despite the proximity of the binding site in the crystallographic model, the CBM46 residues W479 and W481 are distant from the substrate cellotetraose (yellow).	FIG
+92	96	W479	residue_name_number	Despite the proximity of the binding site in the crystallographic model, the CBM46 residues W479 and W481 are distant from the substrate cellotetraose (yellow).	FIG
+101	105	W481	residue_name_number	Despite the proximity of the binding site in the crystallographic model, the CBM46 residues W479 and W481 are distant from the substrate cellotetraose (yellow).	FIG
+137	150	cellotetraose	chemical	Despite the proximity of the binding site in the crystallographic model, the CBM46 residues W479 and W481 are distant from the substrate cellotetraose (yellow).	FIG
+4	18	Ig-like domain	structure_element	The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray.	FIG
+86	88	CD	structure_element	The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray.	FIG
+93	98	CBM46	structure_element	The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray.	FIG
+106	119	superposition	experimental_method	The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray.	FIG
+127	141	Ig-like domain	structure_element	The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray.	FIG
+146	151	CBM46	structure_element	The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray.	FIG
+248	252	loop	structure_element	The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray.	FIG
+286	299	Cellotetraose	chemical	The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray.	FIG
+309	326	subsites -1 to -3	site	The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray.	FIG
+344	355	coordinated	bond_interaction	The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray.	FIG
+0	7	BlCel5B	protein	BlCel5B enzymatic activity characterization.	FIG
+8	43	enzymatic activity characterization	experimental_method	BlCel5B enzymatic activity characterization.	FIG
+4	16	MALDI/TOF-MS	experimental_method	(a) MALDI/TOF-MS spectra of the products released after incubation of BlCel5B and its two deletion constructs (ΔCBM46 and ΔIg-CBM46) with the substrate cellopentaose (C5).	FIG
+17	24	spectra	evidence	(a) MALDI/TOF-MS spectra of the products released after incubation of BlCel5B and its two deletion constructs (ΔCBM46 and ΔIg-CBM46) with the substrate cellopentaose (C5).	FIG
+70	77	BlCel5B	protein	(a) MALDI/TOF-MS spectra of the products released after incubation of BlCel5B and its two deletion constructs (ΔCBM46 and ΔIg-CBM46) with the substrate cellopentaose (C5).	FIG
+90	109	deletion constructs	experimental_method	(a) MALDI/TOF-MS spectra of the products released after incubation of BlCel5B and its two deletion constructs (ΔCBM46 and ΔIg-CBM46) with the substrate cellopentaose (C5).	FIG
+111	117	ΔCBM46	mutant	(a) MALDI/TOF-MS spectra of the products released after incubation of BlCel5B and its two deletion constructs (ΔCBM46 and ΔIg-CBM46) with the substrate cellopentaose (C5).	FIG
+122	131	ΔIg-CBM46	mutant	(a) MALDI/TOF-MS spectra of the products released after incubation of BlCel5B and its two deletion constructs (ΔCBM46 and ΔIg-CBM46) with the substrate cellopentaose (C5).	FIG
+152	165	cellopentaose	chemical	(a) MALDI/TOF-MS spectra of the products released after incubation of BlCel5B and its two deletion constructs (ΔCBM46 and ΔIg-CBM46) with the substrate cellopentaose (C5).	FIG
+167	169	C5	chemical	(a) MALDI/TOF-MS spectra of the products released after incubation of BlCel5B and its two deletion constructs (ΔCBM46 and ΔIg-CBM46) with the substrate cellopentaose (C5).	FIG
+16	23	spectra	evidence	The first three spectra show the substrate, enzyme and buffer controls.	FIG
+10	18	spectrum	evidence	The forth spectrum reveals that full length BlCel5B is capable of enzymatic hydrolysis of C5 into smaller oligosaccharides such as C4, C3 and C2.	FIG
+32	43	full length	protein_state	The forth spectrum reveals that full length BlCel5B is capable of enzymatic hydrolysis of C5 into smaller oligosaccharides such as C4, C3 and C2.	FIG
+44	51	BlCel5B	protein	The forth spectrum reveals that full length BlCel5B is capable of enzymatic hydrolysis of C5 into smaller oligosaccharides such as C4, C3 and C2.	FIG
+90	92	C5	chemical	The forth spectrum reveals that full length BlCel5B is capable of enzymatic hydrolysis of C5 into smaller oligosaccharides such as C4, C3 and C2.	FIG
+106	122	oligosaccharides	chemical	The forth spectrum reveals that full length BlCel5B is capable of enzymatic hydrolysis of C5 into smaller oligosaccharides such as C4, C3 and C2.	FIG
+131	133	C4	chemical	The forth spectrum reveals that full length BlCel5B is capable of enzymatic hydrolysis of C5 into smaller oligosaccharides such as C4, C3 and C2.	FIG
+135	137	C3	chemical	The forth spectrum reveals that full length BlCel5B is capable of enzymatic hydrolysis of C5 into smaller oligosaccharides such as C4, C3 and C2.	FIG
+142	144	C2	chemical	The forth spectrum reveals that full length BlCel5B is capable of enzymatic hydrolysis of C5 into smaller oligosaccharides such as C4, C3 and C2.	FIG
+13	20	spectra	evidence	The last two spectra show that the C-terminal deletions eliminate the enzyme activity.	FIG
+56	85	eliminate the enzyme activity	protein_state	The last two spectra show that the C-terminal deletions eliminate the enzyme activity.	FIG
+0	7	BlCel5B	protein	BlCel5B activities on CMC as functions of pH and temperature are shown in (b) and (c), respectively.	FIG
+22	25	CMC	chemical	BlCel5B activities on CMC as functions of pH and temperature are shown in (b) and (c), respectively.	FIG
+4	26	Michaelis-Menten curve	evidence	(d) Michaelis-Menten curve using CMC as a substrate.	FIG
+33	36	CMC	chemical	(d) Michaelis-Menten curve using CMC as a substrate.	FIG
+0	4	Open	protein_state	Open-close transitions of BlCel5B.	FIG
+5	10	close	protein_state	Open-close transitions of BlCel5B.	FIG
+26	33	BlCel5B	protein	Open-close transitions of BlCel5B.	FIG
+4	11	BlCel5B	protein	(a) BlCel5B in the absence of substrate and (b) in the presence of cellooctaose, as observed in our aMD simulations.	FIG
+19	29	absence of	protein_state	(a) BlCel5B in the absence of substrate and (b) in the presence of cellooctaose, as observed in our aMD simulations.	FIG
+55	66	presence of	protein_state	(a) BlCel5B in the absence of substrate and (b) in the presence of cellooctaose, as observed in our aMD simulations.	FIG
+67	79	cellooctaose	chemical	(a) BlCel5B in the absence of substrate and (b) in the presence of cellooctaose, as observed in our aMD simulations.	FIG
+100	115	aMD simulations	experimental_method	(a) BlCel5B in the absence of substrate and (b) in the presence of cellooctaose, as observed in our aMD simulations.	FIG
+4	12	distance	evidence	The distance between the α carbon of residues I120 (CD) and E477 (CBM46), illustrated as spheres in (a), is plotted in (c), revealing a transition by the decrease in the distance from 40 Å to 7 Å (substrate-free) or 20 Å (in presence of cellooctaose).	FIG
+46	50	I120	residue_name_number	The distance between the α carbon of residues I120 (CD) and E477 (CBM46), illustrated as spheres in (a), is plotted in (c), revealing a transition by the decrease in the distance from 40 Å to 7 Å (substrate-free) or 20 Å (in presence of cellooctaose).	FIG
+52	54	CD	structure_element	The distance between the α carbon of residues I120 (CD) and E477 (CBM46), illustrated as spheres in (a), is plotted in (c), revealing a transition by the decrease in the distance from 40 Å to 7 Å (substrate-free) or 20 Å (in presence of cellooctaose).	FIG
+60	64	E477	residue_name_number	The distance between the α carbon of residues I120 (CD) and E477 (CBM46), illustrated as spheres in (a), is plotted in (c), revealing a transition by the decrease in the distance from 40 Å to 7 Å (substrate-free) or 20 Å (in presence of cellooctaose).	FIG
+66	71	CBM46	structure_element	The distance between the α carbon of residues I120 (CD) and E477 (CBM46), illustrated as spheres in (a), is plotted in (c), revealing a transition by the decrease in the distance from 40 Å to 7 Å (substrate-free) or 20 Å (in presence of cellooctaose).	FIG
+170	178	distance	evidence	The distance between the α carbon of residues I120 (CD) and E477 (CBM46), illustrated as spheres in (a), is plotted in (c), revealing a transition by the decrease in the distance from 40 Å to 7 Å (substrate-free) or 20 Å (in presence of cellooctaose).	FIG
+197	211	substrate-free	protein_state	The distance between the α carbon of residues I120 (CD) and E477 (CBM46), illustrated as spheres in (a), is plotted in (c), revealing a transition by the decrease in the distance from 40 Å to 7 Å (substrate-free) or 20 Å (in presence of cellooctaose).	FIG
+225	236	presence of	protein_state	The distance between the α carbon of residues I120 (CD) and E477 (CBM46), illustrated as spheres in (a), is plotted in (c), revealing a transition by the decrease in the distance from 40 Å to 7 Å (substrate-free) or 20 Å (in presence of cellooctaose).	FIG
+237	249	cellooctaose	chemical	The distance between the α carbon of residues I120 (CD) and E477 (CBM46), illustrated as spheres in (a), is plotted in (c), revealing a transition by the decrease in the distance from 40 Å to 7 Å (substrate-free) or 20 Å (in presence of cellooctaose).	FIG
+8	22	substrate-free	protein_state	For the substrate-free enzyme, the red line refers to a 1 μs-long aMD; for the BlCel5B-cellooctaose complex, the first 500 ns refers to aMD (in blue) and the second 500 ns to conventional MD (in turquoise).	FIG
+66	69	aMD	experimental_method	For the substrate-free enzyme, the red line refers to a 1 μs-long aMD; for the BlCel5B-cellooctaose complex, the first 500 ns refers to aMD (in blue) and the second 500 ns to conventional MD (in turquoise).	FIG
+79	99	BlCel5B-cellooctaose	complex_assembly	For the substrate-free enzyme, the red line refers to a 1 μs-long aMD; for the BlCel5B-cellooctaose complex, the first 500 ns refers to aMD (in blue) and the second 500 ns to conventional MD (in turquoise).	FIG
+136	139	aMD	experimental_method	For the substrate-free enzyme, the red line refers to a 1 μs-long aMD; for the BlCel5B-cellooctaose complex, the first 500 ns refers to aMD (in blue) and the second 500 ns to conventional MD (in turquoise).	FIG
+188	190	MD	experimental_method	For the substrate-free enzyme, the red line refers to a 1 μs-long aMD; for the BlCel5B-cellooctaose complex, the first 500 ns refers to aMD (in blue) and the second 500 ns to conventional MD (in turquoise).	FIG
+22	42	BlCel5B-cellooctaose	complex_assembly	(d) A snapshot of the BlCel5B-cellooctaose complex, highlighting the tryptophan residues that interact with the glucan chain in subsites −4 to +4.	FIG
+69	79	tryptophan	residue_name	(d) A snapshot of the BlCel5B-cellooctaose complex, highlighting the tryptophan residues that interact with the glucan chain in subsites −4 to +4.	FIG
+112	118	glucan	chemical	(d) A snapshot of the BlCel5B-cellooctaose complex, highlighting the tryptophan residues that interact with the glucan chain in subsites −4 to +4.	FIG
+128	145	subsites −4 to +4	site	(d) A snapshot of the BlCel5B-cellooctaose complex, highlighting the tryptophan residues that interact with the glucan chain in subsites −4 to +4.	FIG
+9	13	W479	residue_name_number	Residues W479 and W481 belong to CBM46 and only become available for substrate interactions in the closed configuration of BlCel5B.	FIG
+18	22	W481	residue_name_number	Residues W479 and W481 belong to CBM46 and only become available for substrate interactions in the closed configuration of BlCel5B.	FIG
+33	38	CBM46	structure_element	Residues W479 and W481 belong to CBM46 and only become available for substrate interactions in the closed configuration of BlCel5B.	FIG
+99	105	closed	protein_state	Residues W479 and W481 belong to CBM46 and only become available for substrate interactions in the closed configuration of BlCel5B.	FIG
+123	130	BlCel5B	protein	Residues W479 and W481 belong to CBM46 and only become available for substrate interactions in the closed configuration of BlCel5B.	FIG
+25	32	BlCel5B	protein	Large-scale movements of BlCel5B modules and superposition of their representative conformations with the SAXS envelope.	FIG
+45	58	superposition	experimental_method	Large-scale movements of BlCel5B modules and superposition of their representative conformations with the SAXS envelope.	FIG
+106	110	SAXS	experimental_method	Large-scale movements of BlCel5B modules and superposition of their representative conformations with the SAXS envelope.	FIG
+111	119	envelope	evidence	Large-scale movements of BlCel5B modules and superposition of their representative conformations with the SAXS envelope.	FIG
+4	11	BlCel5B	protein	(a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations.	FIG
+12	21	structure	evidence	(a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations.	FIG
+34	42	distance	evidence	(a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations.	FIG
+82	86	I120	residue_name_number	(a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations.	FIG
+91	95	E477	residue_name_number	(a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations.	FIG
+128	130	CD	structure_element	(a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations.	FIG
+135	140	CBM46	structure_element	(a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations.	FIG
+246	250	I120	residue_name_number	(a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations.	FIG
+251	255	E477	residue_name_number	(a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations.	FIG
+256	264	distance	evidence	(a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations.	FIG
+280	297	CG-MD simulations	experimental_method	(a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations.	FIG
+71	82	simulations	experimental_method	Different colors separated by vertical lines correspond to independent simulations of approximately 120 μs. (c) The distance distribution indicates three major peaks: closed or occluded CBM46/CD conformations (I); semi-open (II), which is similar to the crystallographic structure; and extended conformers (III).	FIG
+116	137	distance distribution	evidence	Different colors separated by vertical lines correspond to independent simulations of approximately 120 μs. (c) The distance distribution indicates three major peaks: closed or occluded CBM46/CD conformations (I); semi-open (II), which is similar to the crystallographic structure; and extended conformers (III).	FIG
+167	173	closed	protein_state	Different colors separated by vertical lines correspond to independent simulations of approximately 120 μs. (c) The distance distribution indicates three major peaks: closed or occluded CBM46/CD conformations (I); semi-open (II), which is similar to the crystallographic structure; and extended conformers (III).	FIG
+177	185	occluded	protein_state	Different colors separated by vertical lines correspond to independent simulations of approximately 120 μs. (c) The distance distribution indicates three major peaks: closed or occluded CBM46/CD conformations (I); semi-open (II), which is similar to the crystallographic structure; and extended conformers (III).	FIG
+186	191	CBM46	structure_element	Different colors separated by vertical lines correspond to independent simulations of approximately 120 μs. (c) The distance distribution indicates three major peaks: closed or occluded CBM46/CD conformations (I); semi-open (II), which is similar to the crystallographic structure; and extended conformers (III).	FIG
+192	194	CD	structure_element	Different colors separated by vertical lines correspond to independent simulations of approximately 120 μs. (c) The distance distribution indicates three major peaks: closed or occluded CBM46/CD conformations (I); semi-open (II), which is similar to the crystallographic structure; and extended conformers (III).	FIG
+214	223	semi-open	protein_state	Different colors separated by vertical lines correspond to independent simulations of approximately 120 μs. (c) The distance distribution indicates three major peaks: closed or occluded CBM46/CD conformations (I); semi-open (II), which is similar to the crystallographic structure; and extended conformers (III).	FIG
+254	280	crystallographic structure	evidence	Different colors separated by vertical lines correspond to independent simulations of approximately 120 μs. (c) The distance distribution indicates three major peaks: closed or occluded CBM46/CD conformations (I); semi-open (II), which is similar to the crystallographic structure; and extended conformers (III).	FIG
+286	294	extended	protein_state	Different colors separated by vertical lines correspond to independent simulations of approximately 120 μs. (c) The distance distribution indicates three major peaks: closed or occluded CBM46/CD conformations (I); semi-open (II), which is similar to the crystallographic structure; and extended conformers (III).	FIG
+4	19	Superimposition	experimental_method	(d) Superimposition of the three representative molecular conformations of BlCel5B with the SAXS model. (e) Average structures obtained from the simulation segments corresponding to population groups I-III, which are individually superposed on the SAXS envelope.	FIG
+75	82	BlCel5B	protein	(d) Superimposition of the three representative molecular conformations of BlCel5B with the SAXS model. (e) Average structures obtained from the simulation segments corresponding to population groups I-III, which are individually superposed on the SAXS envelope.	FIG
+92	96	SAXS	experimental_method	(d) Superimposition of the three representative molecular conformations of BlCel5B with the SAXS model. (e) Average structures obtained from the simulation segments corresponding to population groups I-III, which are individually superposed on the SAXS envelope.	FIG
+97	102	model	evidence	(d) Superimposition of the three representative molecular conformations of BlCel5B with the SAXS model. (e) Average structures obtained from the simulation segments corresponding to population groups I-III, which are individually superposed on the SAXS envelope.	FIG
+116	126	structures	evidence	(d) Superimposition of the three representative molecular conformations of BlCel5B with the SAXS model. (e) Average structures obtained from the simulation segments corresponding to population groups I-III, which are individually superposed on the SAXS envelope.	FIG
+145	155	simulation	experimental_method	(d) Superimposition of the three representative molecular conformations of BlCel5B with the SAXS model. (e) Average structures obtained from the simulation segments corresponding to population groups I-III, which are individually superposed on the SAXS envelope.	FIG
+230	240	superposed	experimental_method	(d) Superimposition of the three representative molecular conformations of BlCel5B with the SAXS model. (e) Average structures obtained from the simulation segments corresponding to population groups I-III, which are individually superposed on the SAXS envelope.	FIG
+248	252	SAXS	experimental_method	(d) Superimposition of the three representative molecular conformations of BlCel5B with the SAXS model. (e) Average structures obtained from the simulation segments corresponding to population groups I-III, which are individually superposed on the SAXS envelope.	FIG
+253	261	envelope	evidence	(d) Superimposition of the three representative molecular conformations of BlCel5B with the SAXS model. (e) Average structures obtained from the simulation segments corresponding to population groups I-III, which are individually superposed on the SAXS envelope.	FIG
+0	10	Comparison	experimental_method	Comparison of the binding site shape of GH5_4 enzymes available on the Protein Data Bank.	FIG
+18	30	binding site	site	Comparison of the binding site shape of GH5_4 enzymes available on the Protein Data Bank.	FIG
+40	45	GH5_4	protein_type	Comparison of the binding site shape of GH5_4 enzymes available on the Protein Data Bank.	FIG
+4	11	BlCel5B	protein	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+19	35	crystallographic	experimental_method	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+40	46	closed	protein_state	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+66	85	Bacillus halodurans	species	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+86	91	Cel5B	protein	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+93	100	BhCel5B	protein	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+121	142	Piromyces rhizinflata	species	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+143	146	GH5	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+147	160	endoglucanase	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+181	207	Clostridium cellulolyticum	species	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+208	211	GH5	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+212	225	endoglucanase	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+246	271	Clostridium cellulovorans	species	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+272	275	GH5	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+276	289	endoglucanase	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+310	328	Bacteroides ovatus	species	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+329	332	GH5	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+333	346	xyloglucanase	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+367	387	Paenibacillus pabuli	species	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+388	391	GH5	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+392	405	xyloglucanase	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+426	445	Prevotella bryantii	species	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+446	449	GH5	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+450	463	endoglucanase	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+484	514	Ruminiclostridium thermocellum	species	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+531	534	GH5	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+535	544	cellulase	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+546	554	xylanase	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+559	566	mannase	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+587	610	Bacteroidetes bacterium	taxonomy_domain	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+611	615	AC2a	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+616	629	endocellulase	protein_type	(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).	FIG
+0	10	Comparison	experimental_method	Comparison of the binding cleft of the BlCel5B and BhCel5B.	FIG
+18	31	binding cleft	site	Comparison of the binding cleft of the BlCel5B and BhCel5B.	FIG
+39	46	BlCel5B	protein	Comparison of the binding cleft of the BlCel5B and BhCel5B.	FIG
+51	58	BhCel5B	protein	Comparison of the binding cleft of the BlCel5B and BhCel5B.	FIG
+28	35	BlCel5B	protein	The main difference between BlCel5B and BhCel5B is that the latter exhibits a deeper cleft due to the presence of residue W181 in the loop between F177 and R185.	FIG
+40	47	BhCel5B	protein	The main difference between BlCel5B and BhCel5B is that the latter exhibits a deeper cleft due to the presence of residue W181 in the loop between F177 and R185.	FIG
+85	90	cleft	site	The main difference between BlCel5B and BhCel5B is that the latter exhibits a deeper cleft due to the presence of residue W181 in the loop between F177 and R185.	FIG
+102	113	presence of	protein_state	The main difference between BlCel5B and BhCel5B is that the latter exhibits a deeper cleft due to the presence of residue W181 in the loop between F177 and R185.	FIG
+122	126	W181	residue_name_number	The main difference between BlCel5B and BhCel5B is that the latter exhibits a deeper cleft due to the presence of residue W181 in the loop between F177 and R185.	FIG
+134	138	loop	structure_element	The main difference between BlCel5B and BhCel5B is that the latter exhibits a deeper cleft due to the presence of residue W181 in the loop between F177 and R185.	FIG
+147	151	F177	residue_name_number	The main difference between BlCel5B and BhCel5B is that the latter exhibits a deeper cleft due to the presence of residue W181 in the loop between F177 and R185.	FIG
+156	160	R185	residue_name_number	The main difference between BlCel5B and BhCel5B is that the latter exhibits a deeper cleft due to the presence of residue W181 in the loop between F177 and R185.	FIG
+42	54	binding site	site	We conjecture that this difference in the binding site architecture relates to the importance that the CBM46 plays in the BlCel5B enzymatic mechanism.	FIG
+103	108	CBM46	structure_element	We conjecture that this difference in the binding site architecture relates to the importance that the CBM46 plays in the BlCel5B enzymatic mechanism.	FIG
+122	129	BlCel5B	protein	We conjecture that this difference in the binding site architecture relates to the importance that the CBM46 plays in the BlCel5B enzymatic mechanism.	FIG
+32	39	BlCel5B	protein	Proposed molecular mechanism of BlCel5B conformational selection.	FIG
+20	31	simulations	experimental_method	As suggested by the simulations and SAXS data, BlCel5B spans multiple conformations ranging from closed to extended CBM46/CD states.	FIG
+36	40	SAXS	experimental_method	As suggested by the simulations and SAXS data, BlCel5B spans multiple conformations ranging from closed to extended CBM46/CD states.	FIG
+47	54	BlCel5B	protein	As suggested by the simulations and SAXS data, BlCel5B spans multiple conformations ranging from closed to extended CBM46/CD states.	FIG
+97	103	closed	protein_state	As suggested by the simulations and SAXS data, BlCel5B spans multiple conformations ranging from closed to extended CBM46/CD states.	FIG
+107	115	extended	protein_state	As suggested by the simulations and SAXS data, BlCel5B spans multiple conformations ranging from closed to extended CBM46/CD states.	FIG
+116	121	CBM46	structure_element	As suggested by the simulations and SAXS data, BlCel5B spans multiple conformations ranging from closed to extended CBM46/CD states.	FIG
+122	124	CD	structure_element	As suggested by the simulations and SAXS data, BlCel5B spans multiple conformations ranging from closed to extended CBM46/CD states.	FIG
+11	15	open	protein_state	In a given open state, the substrate may reach the active site and become entrapped by the capping of CBM46 onto CD and induced-fit conformational adjustments.	FIG
+51	62	active site	site	In a given open state, the substrate may reach the active site and become entrapped by the capping of CBM46 onto CD and induced-fit conformational adjustments.	FIG
+102	107	CBM46	structure_element	In a given open state, the substrate may reach the active site and become entrapped by the capping of CBM46 onto CD and induced-fit conformational adjustments.	FIG
+113	115	CD	structure_element	In a given open state, the substrate may reach the active site and become entrapped by the capping of CBM46 onto CD and induced-fit conformational adjustments.	FIG
+60	63	apo	protein_state	After hydrolysis, the reaction product is released to yield apo-BlCel5B, which becomes ready for a new cycle.	FIG
+64	71	BlCel5B	protein	After hydrolysis, the reaction product is released to yield apo-BlCel5B, which becomes ready for a new cycle.	FIG
+12	19	BlCel5B	protein	Activity of BlCel5B constructs against tested substrates.	TABLE
+39	41	WT	protein_state	"Substrate (1%)	Relative Activity (%)	 	WT*	W479A	W481A	ΔCBM46	ΔIg-CBM46	 	β-glucan	100	79.1	63.6	nd	nd	 	CMC	25.5	12.2	2.4	nd	nd	 	Lichenan	52.4	41	28.6	nd	nd	 	Xyloglucan	45.2	41.2	30.8	nd	nd	 	Azo-Avicel	nd**	nd	nd	nd	nd	 	Arabinoxylan	nd	nd	nd	nd	nd	 	Galactomannan	nd	nd	nd	nd	nd	 	1,4-β-mannan	nd	nd	nd	nd	nd	 	"	TABLE
+43	48	W479A	mutant	"Substrate (1%)	Relative Activity (%)	 	WT*	W479A	W481A	ΔCBM46	ΔIg-CBM46	 	β-glucan	100	79.1	63.6	nd	nd	 	CMC	25.5	12.2	2.4	nd	nd	 	Lichenan	52.4	41	28.6	nd	nd	 	Xyloglucan	45.2	41.2	30.8	nd	nd	 	Azo-Avicel	nd**	nd	nd	nd	nd	 	Arabinoxylan	nd	nd	nd	nd	nd	 	Galactomannan	nd	nd	nd	nd	nd	 	1,4-β-mannan	nd	nd	nd	nd	nd	 	"	TABLE
+49	54	W481A	mutant	"Substrate (1%)	Relative Activity (%)	 	WT*	W479A	W481A	ΔCBM46	ΔIg-CBM46	 	β-glucan	100	79.1	63.6	nd	nd	 	CMC	25.5	12.2	2.4	nd	nd	 	Lichenan	52.4	41	28.6	nd	nd	 	Xyloglucan	45.2	41.2	30.8	nd	nd	 	Azo-Avicel	nd**	nd	nd	nd	nd	 	Arabinoxylan	nd	nd	nd	nd	nd	 	Galactomannan	nd	nd	nd	nd	nd	 	1,4-β-mannan	nd	nd	nd	nd	nd	 	"	TABLE
+55	61	ΔCBM46	mutant	"Substrate (1%)	Relative Activity (%)	 	WT*	W479A	W481A	ΔCBM46	ΔIg-CBM46	 	β-glucan	100	79.1	63.6	nd	nd	 	CMC	25.5	12.2	2.4	nd	nd	 	Lichenan	52.4	41	28.6	nd	nd	 	Xyloglucan	45.2	41.2	30.8	nd	nd	 	Azo-Avicel	nd**	nd	nd	nd	nd	 	Arabinoxylan	nd	nd	nd	nd	nd	 	Galactomannan	nd	nd	nd	nd	nd	 	1,4-β-mannan	nd	nd	nd	nd	nd	 	"	TABLE
+62	71	ΔIg-CBM46	mutant	"Substrate (1%)	Relative Activity (%)	 	WT*	W479A	W481A	ΔCBM46	ΔIg-CBM46	 	β-glucan	100	79.1	63.6	nd	nd	 	CMC	25.5	12.2	2.4	nd	nd	 	Lichenan	52.4	41	28.6	nd	nd	 	Xyloglucan	45.2	41.2	30.8	nd	nd	 	Azo-Avicel	nd**	nd	nd	nd	nd	 	Arabinoxylan	nd	nd	nd	nd	nd	 	Galactomannan	nd	nd	nd	nd	nd	 	1,4-β-mannan	nd	nd	nd	nd	nd	 	"	TABLE
+74	82	β-glucan	chemical	"Substrate (1%)	Relative Activity (%)	 	WT*	W479A	W481A	ΔCBM46	ΔIg-CBM46	 	β-glucan	100	79.1	63.6	nd	nd	 	CMC	25.5	12.2	2.4	nd	nd	 	Lichenan	52.4	41	28.6	nd	nd	 	Xyloglucan	45.2	41.2	30.8	nd	nd	 	Azo-Avicel	nd**	nd	nd	nd	nd	 	Arabinoxylan	nd	nd	nd	nd	nd	 	Galactomannan	nd	nd	nd	nd	nd	 	1,4-β-mannan	nd	nd	nd	nd	nd	 	"	TABLE
+105	108	CMC	chemical	"Substrate (1%)	Relative Activity (%)	 	WT*	W479A	W481A	ΔCBM46	ΔIg-CBM46	 	β-glucan	100	79.1	63.6	nd	nd	 	CMC	25.5	12.2	2.4	nd	nd	 	Lichenan	52.4	41	28.6	nd	nd	 	Xyloglucan	45.2	41.2	30.8	nd	nd	 	Azo-Avicel	nd**	nd	nd	nd	nd	 	Arabinoxylan	nd	nd	nd	nd	nd	 	Galactomannan	nd	nd	nd	nd	nd	 	1,4-β-mannan	nd	nd	nd	nd	nd	 	"	TABLE
+131	139	Lichenan	chemical	"Substrate (1%)	Relative Activity (%)	 	WT*	W479A	W481A	ΔCBM46	ΔIg-CBM46	 	β-glucan	100	79.1	63.6	nd	nd	 	CMC	25.5	12.2	2.4	nd	nd	 	Lichenan	52.4	41	28.6	nd	nd	 	Xyloglucan	45.2	41.2	30.8	nd	nd	 	Azo-Avicel	nd**	nd	nd	nd	nd	 	Arabinoxylan	nd	nd	nd	nd	nd	 	Galactomannan	nd	nd	nd	nd	nd	 	1,4-β-mannan	nd	nd	nd	nd	nd	 	"	TABLE
+161	171	Xyloglucan	chemical	"Substrate (1%)	Relative Activity (%)	 	WT*	W479A	W481A	ΔCBM46	ΔIg-CBM46	 	β-glucan	100	79.1	63.6	nd	nd	 	CMC	25.5	12.2	2.4	nd	nd	 	Lichenan	52.4	41	28.6	nd	nd	 	Xyloglucan	45.2	41.2	30.8	nd	nd	 	Azo-Avicel	nd**	nd	nd	nd	nd	 	Arabinoxylan	nd	nd	nd	nd	nd	 	Galactomannan	nd	nd	nd	nd	nd	 	1,4-β-mannan	nd	nd	nd	nd	nd	 	"	TABLE
+195	205	Azo-Avicel	chemical	"Substrate (1%)	Relative Activity (%)	 	WT*	W479A	W481A	ΔCBM46	ΔIg-CBM46	 	β-glucan	100	79.1	63.6	nd	nd	 	CMC	25.5	12.2	2.4	nd	nd	 	Lichenan	52.4	41	28.6	nd	nd	 	Xyloglucan	45.2	41.2	30.8	nd	nd	 	Azo-Avicel	nd**	nd	nd	nd	nd	 	Arabinoxylan	nd	nd	nd	nd	nd	 	Galactomannan	nd	nd	nd	nd	nd	 	1,4-β-mannan	nd	nd	nd	nd	nd	 	"	TABLE
+225	237	Arabinoxylan	chemical	"Substrate (1%)	Relative Activity (%)	 	WT*	W479A	W481A	ΔCBM46	ΔIg-CBM46	 	β-glucan	100	79.1	63.6	nd	nd	 	CMC	25.5	12.2	2.4	nd	nd	 	Lichenan	52.4	41	28.6	nd	nd	 	Xyloglucan	45.2	41.2	30.8	nd	nd	 	Azo-Avicel	nd**	nd	nd	nd	nd	 	Arabinoxylan	nd	nd	nd	nd	nd	 	Galactomannan	nd	nd	nd	nd	nd	 	1,4-β-mannan	nd	nd	nd	nd	nd	 	"	TABLE
+255	268	Galactomannan	chemical	"Substrate (1%)	Relative Activity (%)	 	WT*	W479A	W481A	ΔCBM46	ΔIg-CBM46	 	β-glucan	100	79.1	63.6	nd	nd	 	CMC	25.5	12.2	2.4	nd	nd	 	Lichenan	52.4	41	28.6	nd	nd	 	Xyloglucan	45.2	41.2	30.8	nd	nd	 	Azo-Avicel	nd**	nd	nd	nd	nd	 	Arabinoxylan	nd	nd	nd	nd	nd	 	Galactomannan	nd	nd	nd	nd	nd	 	1,4-β-mannan	nd	nd	nd	nd	nd	 	"	TABLE
+286	298	1,4-β-mannan	chemical	"Substrate (1%)	Relative Activity (%)	 	WT*	W479A	W481A	ΔCBM46	ΔIg-CBM46	 	β-glucan	100	79.1	63.6	nd	nd	 	CMC	25.5	12.2	2.4	nd	nd	 	Lichenan	52.4	41	28.6	nd	nd	 	Xyloglucan	45.2	41.2	30.8	nd	nd	 	Azo-Avicel	nd**	nd	nd	nd	nd	 	Arabinoxylan	nd	nd	nd	nd	nd	 	Galactomannan	nd	nd	nd	nd	nd	 	1,4-β-mannan	nd	nd	nd	nd	nd	 	"	TABLE
+1	3	WT	protein_state	*WT = wild type.	TABLE
+6	15	wild type	protein_state	*WT = wild type.	TABLE
diff --git a/annotation_CSV/PMC4980666.csv b/annotation_CSV/PMC4980666.csv
new file mode 100644
index 0000000000000000000000000000000000000000..a73b7d87a0317a9ed38d7b504b5154dfecb53743
--- /dev/null
+++ b/annotation_CSV/PMC4980666.csv
@@ -0,0 +1,670 @@
+anno_start	anno_end	anno_text	entity_type	sentence	section
+0	15	N-acylhydrazone	chemical	N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes	TITLE
+30	39	influenza	taxonomy_domain	N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes	TITLE
+40	45	virus	taxonomy_domain	N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes	TITLE
+46	48	PA	protein	N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes	TITLE
+49	61	endonuclease	protein_type	N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes	TITLE
+0	9	Influenza	taxonomy_domain	Influenza virus PA endonuclease has recently emerged as an attractive target for the development of novel antiviral therapeutics.	ABSTRACT
+10	15	virus	taxonomy_domain	Influenza virus PA endonuclease has recently emerged as an attractive target for the development of novel antiviral therapeutics.	ABSTRACT
+16	18	PA	protein	Influenza virus PA endonuclease has recently emerged as an attractive target for the development of novel antiviral therapeutics.	ABSTRACT
+19	31	endonuclease	protein_type	Influenza virus PA endonuclease has recently emerged as an attractive target for the development of novel antiviral therapeutics.	ABSTRACT
+46	50	Mg2+	chemical	This is an enzyme with divalent metal ion(s) (Mg2+ or Mn2+) in its catalytic site: chelation of these metal cofactors is an attractive strategy to inhibit enzymatic activity.	ABSTRACT
+54	58	Mn2+	chemical	This is an enzyme with divalent metal ion(s) (Mg2+ or Mn2+) in its catalytic site: chelation of these metal cofactors is an attractive strategy to inhibit enzymatic activity.	ABSTRACT
+67	81	catalytic site	site	This is an enzyme with divalent metal ion(s) (Mg2+ or Mn2+) in its catalytic site: chelation of these metal cofactors is an attractive strategy to inhibit enzymatic activity.	ABSTRACT
+83	92	chelation	bond_interaction	This is an enzyme with divalent metal ion(s) (Mg2+ or Mn2+) in its catalytic site: chelation of these metal cofactors is an attractive strategy to inhibit enzymatic activity.	ABSTRACT
+43	59	N-acylhydrazones	chemical	Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays.	ABSTRACT
+66	81	enzymatic assay	experimental_method	Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays.	ABSTRACT
+87	89	PA	protein	Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays.	ABSTRACT
+90	94	Nter	structure_element	Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays.	ABSTRACT
+95	107	endonuclease	protein_type	Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays.	ABSTRACT
+123	163	cell-based influenza vRNP reconstitution	experimental_method	Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays.	ABSTRACT
+168	186	virus yield assays	experimental_method	Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays.	ABSTRACT
+8	24	N-acylhydrazones	chemical	Several N-acylhydrazones were found to have promising anti-influenza activity in the low micromolar concentration range and good selectivity.	ABSTRACT
+59	68	influenza	taxonomy_domain	Several N-acylhydrazones were found to have promising anti-influenza activity in the low micromolar concentration range and good selectivity.	ABSTRACT
+0	29	Computational docking studies	experimental_method	Computational docking studies are carried on to investigate the key features that determine inhibition of the endonuclease enzyme by N-acylhydrazones.	ABSTRACT
+110	122	endonuclease	protein_type	Computational docking studies are carried on to investigate the key features that determine inhibition of the endonuclease enzyme by N-acylhydrazones.	ABSTRACT
+133	149	N-acylhydrazones	chemical	Computational docking studies are carried on to investigate the key features that determine inhibition of the endonuclease enzyme by N-acylhydrazones.	ABSTRACT
+31	48	crystal structure	evidence	Moreover, we here describe the crystal structure of PA-Nter in complex with one of the most active inhibitors, revealing its interactions within the protein’s active site.	ABSTRACT
+52	54	PA	protein	Moreover, we here describe the crystal structure of PA-Nter in complex with one of the most active inhibitors, revealing its interactions within the protein’s active site.	ABSTRACT
+55	59	Nter	structure_element	Moreover, we here describe the crystal structure of PA-Nter in complex with one of the most active inhibitors, revealing its interactions within the protein’s active site.	ABSTRACT
+60	75	in complex with	protein_state	Moreover, we here describe the crystal structure of PA-Nter in complex with one of the most active inhibitors, revealing its interactions within the protein’s active site.	ABSTRACT
+159	170	active site	site	Moreover, we here describe the crystal structure of PA-Nter in complex with one of the most active inhibitors, revealing its interactions within the protein’s active site.	ABSTRACT
+0	9	Influenza	taxonomy_domain	Influenza virus is an enveloped virus with a segmented negative-oriented single-stranded RNA genome, belonging to the Orthomyxoviridae.	INTRO
+10	15	virus	taxonomy_domain	Influenza virus is an enveloped virus with a segmented negative-oriented single-stranded RNA genome, belonging to the Orthomyxoviridae.	INTRO
+22	37	enveloped virus	taxonomy_domain	Influenza virus is an enveloped virus with a segmented negative-oriented single-stranded RNA genome, belonging to the Orthomyxoviridae.	INTRO
+55	92	negative-oriented single-stranded RNA	chemical	Influenza virus is an enveloped virus with a segmented negative-oriented single-stranded RNA genome, belonging to the Orthomyxoviridae.	INTRO
+118	134	Orthomyxoviridae	taxonomy_domain	Influenza virus is an enveloped virus with a segmented negative-oriented single-stranded RNA genome, belonging to the Orthomyxoviridae.	INTRO
+9	20	influenza A	taxonomy_domain	Seasonal influenza A and B viruses affect each year approximately 5–10% of the adult and 20–30% of the paediatric population, and there is a permanent risk of sudden influenza pandemics, such as the notorious ‘Spanish flu’ in 1918 and the swine-origin H1N1 pandemic in 2009.	INTRO
+25	26	B	taxonomy_domain	Seasonal influenza A and B viruses affect each year approximately 5–10% of the adult and 20–30% of the paediatric population, and there is a permanent risk of sudden influenza pandemics, such as the notorious ‘Spanish flu’ in 1918 and the swine-origin H1N1 pandemic in 2009.	INTRO
+27	34	viruses	taxonomy_domain	Seasonal influenza A and B viruses affect each year approximately 5–10% of the adult and 20–30% of the paediatric population, and there is a permanent risk of sudden influenza pandemics, such as the notorious ‘Spanish flu’ in 1918 and the swine-origin H1N1 pandemic in 2009.	INTRO
+166	175	influenza	taxonomy_domain	Seasonal influenza A and B viruses affect each year approximately 5–10% of the adult and 20–30% of the paediatric population, and there is a permanent risk of sudden influenza pandemics, such as the notorious ‘Spanish flu’ in 1918 and the swine-origin H1N1 pandemic in 2009.	INTRO
+252	256	H1N1	species	Seasonal influenza A and B viruses affect each year approximately 5–10% of the adult and 20–30% of the paediatric population, and there is a permanent risk of sudden influenza pandemics, such as the notorious ‘Spanish flu’ in 1918 and the swine-origin H1N1 pandemic in 2009.	INTRO
+20	29	influenza	taxonomy_domain	Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir).	INTRO
+30	35	virus	taxonomy_domain	Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir).	INTRO
+71	76	viral	taxonomy_domain	Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir).	INTRO
+77	91	M2 ion-channel	protein_type	Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir).	INTRO
+93	103	amantadine	chemical	Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir).	INTRO
+108	119	rimantadine	chemical	Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir).	INTRO
+131	136	viral	taxonomy_domain	Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir).	INTRO
+137	150	neuraminidase	protein_type	Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir).	INTRO
+152	161	zanamivir	chemical	Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir).	INTRO
+166	177	oseltamivir	chemical	Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir).	INTRO
+4	6	M2	protein_type	The M2 inhibitors have limited clinical utility due to their central nervous system side effects and widespread resistance, as in the case of the 2009 pandemic H1N1 virus; resistance is also a growing concern for oseltamivir.	INTRO
+160	164	H1N1	species	The M2 inhibitors have limited clinical utility due to their central nervous system side effects and widespread resistance, as in the case of the 2009 pandemic H1N1 virus; resistance is also a growing concern for oseltamivir.	INTRO
+165	170	virus	taxonomy_domain	The M2 inhibitors have limited clinical utility due to their central nervous system side effects and widespread resistance, as in the case of the 2009 pandemic H1N1 virus; resistance is also a growing concern for oseltamivir.	INTRO
+213	224	oseltamivir	chemical	The M2 inhibitors have limited clinical utility due to their central nervous system side effects and widespread resistance, as in the case of the 2009 pandemic H1N1 virus; resistance is also a growing concern for oseltamivir.	INTRO
+4	13	influenza	taxonomy_domain	The influenza virus polymerase complex is composed of three subunits: PB1, PB2 and PA.	INTRO
+14	19	virus	taxonomy_domain	The influenza virus polymerase complex is composed of three subunits: PB1, PB2 and PA.	INTRO
+20	30	polymerase	protein_type	The influenza virus polymerase complex is composed of three subunits: PB1, PB2 and PA.	INTRO
+70	73	PB1	protein	The influenza virus polymerase complex is composed of three subunits: PB1, PB2 and PA.	INTRO
+75	78	PB2	protein	The influenza virus polymerase complex is composed of three subunits: PB1, PB2 and PA.	INTRO
+83	85	PA	protein	The influenza virus polymerase complex is composed of three subunits: PB1, PB2 and PA.	INTRO
+4	6	PA	protein	The PA subunit performs the ‘cap-snatching’ endonuclease reaction, the PB2 subunit is responsible for initial binding of the capped RNAs, while the actual RNA synthesis is performed by the PB1 protein.	INTRO
+7	14	subunit	structure_element	The PA subunit performs the ‘cap-snatching’ endonuclease reaction, the PB2 subunit is responsible for initial binding of the capped RNAs, while the actual RNA synthesis is performed by the PB1 protein.	INTRO
+44	56	endonuclease	protein_type	The PA subunit performs the ‘cap-snatching’ endonuclease reaction, the PB2 subunit is responsible for initial binding of the capped RNAs, while the actual RNA synthesis is performed by the PB1 protein.	INTRO
+71	74	PB2	protein	The PA subunit performs the ‘cap-snatching’ endonuclease reaction, the PB2 subunit is responsible for initial binding of the capped RNAs, while the actual RNA synthesis is performed by the PB1 protein.	INTRO
+75	82	subunit	structure_element	The PA subunit performs the ‘cap-snatching’ endonuclease reaction, the PB2 subunit is responsible for initial binding of the capped RNAs, while the actual RNA synthesis is performed by the PB1 protein.	INTRO
+125	136	capped RNAs	chemical	The PA subunit performs the ‘cap-snatching’ endonuclease reaction, the PB2 subunit is responsible for initial binding of the capped RNAs, while the actual RNA synthesis is performed by the PB1 protein.	INTRO
+155	158	RNA	chemical	The PA subunit performs the ‘cap-snatching’ endonuclease reaction, the PB2 subunit is responsible for initial binding of the capped RNAs, while the actual RNA synthesis is performed by the PB1 protein.	INTRO
+189	192	PB1	protein	The PA subunit performs the ‘cap-snatching’ endonuclease reaction, the PB2 subunit is responsible for initial binding of the capped RNAs, while the actual RNA synthesis is performed by the PB1 protein.	INTRO
+30	35	viral	taxonomy_domain	Given its crucial role in the viral life cycle, the influenza virus polymerase is widely recognized as a superior target for antiviral drug development and, in particular, inhibition of the PA endonuclease has deserved much attention in recent years.	INTRO
+52	61	influenza	taxonomy_domain	Given its crucial role in the viral life cycle, the influenza virus polymerase is widely recognized as a superior target for antiviral drug development and, in particular, inhibition of the PA endonuclease has deserved much attention in recent years.	INTRO
+62	67	virus	taxonomy_domain	Given its crucial role in the viral life cycle, the influenza virus polymerase is widely recognized as a superior target for antiviral drug development and, in particular, inhibition of the PA endonuclease has deserved much attention in recent years.	INTRO
+68	78	polymerase	protein_type	Given its crucial role in the viral life cycle, the influenza virus polymerase is widely recognized as a superior target for antiviral drug development and, in particular, inhibition of the PA endonuclease has deserved much attention in recent years.	INTRO
+190	192	PA	protein	Given its crucial role in the viral life cycle, the influenza virus polymerase is widely recognized as a superior target for antiviral drug development and, in particular, inhibition of the PA endonuclease has deserved much attention in recent years.	INTRO
+193	205	endonuclease	protein_type	Given its crucial role in the viral life cycle, the influenza virus polymerase is widely recognized as a superior target for antiviral drug development and, in particular, inhibition of the PA endonuclease has deserved much attention in recent years.	INTRO
+4	16	endonuclease	protein_type	The endonuclease catalytic site resides in the N-terminal domain of PA (PA-Nter; residues 1~195).	INTRO
+17	31	catalytic site	site	The endonuclease catalytic site resides in the N-terminal domain of PA (PA-Nter; residues 1~195).	INTRO
+47	64	N-terminal domain	structure_element	The endonuclease catalytic site resides in the N-terminal domain of PA (PA-Nter; residues 1~195).	INTRO
+68	70	PA	protein	The endonuclease catalytic site resides in the N-terminal domain of PA (PA-Nter; residues 1~195).	INTRO
+72	74	PA	protein	The endonuclease catalytic site resides in the N-terminal domain of PA (PA-Nter; residues 1~195).	INTRO
+75	79	Nter	structure_element	The endonuclease catalytic site resides in the N-terminal domain of PA (PA-Nter; residues 1~195).	INTRO
+90	95	1~195	residue_range	The endonuclease catalytic site resides in the N-terminal domain of PA (PA-Nter; residues 1~195).	INTRO
+15	24	histidine	residue_name	It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions.	INTRO
+26	31	His41	residue_name_number	It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions.	INTRO
+56	74	strictly conserved	protein_state	It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions.	INTRO
+75	81	acidic	protein_state	It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions.	INTRO
+92	97	Glu80	residue_name_number	It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions.	INTRO
+99	105	Asp108	residue_name_number	It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions.	INTRO
+107	113	Glu119	residue_name_number	It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions.	INTRO
+122	132	coordinate	bond_interaction	It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions.	INTRO
+148	154	Ile120	residue_name_number	It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions.	INTRO
+175	184	manganese	chemical	It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions.	INTRO
+188	197	magnesium	chemical	It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions.	INTRO
+41	45	Mg2+	chemical	Since the intracellular concentration of Mg2+ is at least 1000-fold higher than that of Mn2+, magnesium may be more biologically relevant.	INTRO
+88	93	Mn2+,	chemical	Since the intracellular concentration of Mg2+ is at least 1000-fold higher than that of Mn2+, magnesium may be more biologically relevant.	INTRO
+94	103	magnesium	chemical	Since the intracellular concentration of Mg2+ is at least 1000-fold higher than that of Mn2+, magnesium may be more biologically relevant.	INTRO
+70	81	active site	site	A controversy about number and type of metal ions exists also for the active site of HIV-1 integrase.	INTRO
+85	90	HIV-1	species	A controversy about number and type of metal ions exists also for the active site of HIV-1 integrase.	INTRO
+91	100	integrase	protein_type	A controversy about number and type of metal ions exists also for the active site of HIV-1 integrase.	INTRO
+0	5	HIV-1	species	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+6	15	integrase	protein_type	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+113	118	metal	chemical	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+134	139	viral	taxonomy_domain	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+168	170	PA	protein	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+242	251	influenza	taxonomy_domain	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+252	264	endonuclease	protein_type	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+296	318	2,4-dioxobutanoic acid	chemical	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+332	341	flutimide	chemical	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+363	384	2-hydroxyphenyl amide	chemical	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+409	423	tetramic acids	chemical	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+425	449	5-hydroxypyrimidin-4-one	chemical	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+463	474	marchantins	chemical	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+479	488	green tea	taxonomy_domain	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+489	498	catechins	chemical	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+505	531	epigallocatechin-3-gallate	chemical	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+533	537	EGCG	chemical	HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).	INTRO
+102	104	PA	protein	In recent years, we focused our research on chemical scaffolds that are able to chelate metal ions of PA-Nter, resulting in inhibition of influenza virus replication.	INTRO
+105	109	Nter	structure_element	In recent years, we focused our research on chemical scaffolds that are able to chelate metal ions of PA-Nter, resulting in inhibition of influenza virus replication.	INTRO
+138	147	influenza	taxonomy_domain	In recent years, we focused our research on chemical scaffolds that are able to chelate metal ions of PA-Nter, resulting in inhibition of influenza virus replication.	INTRO
+148	153	virus	taxonomy_domain	In recent years, we focused our research on chemical scaffolds that are able to chelate metal ions of PA-Nter, resulting in inhibition of influenza virus replication.	INTRO
+0	16	N-acylhydrazones	chemical	N-acylhydrazones represent an appealing class of chelating ligands with a broad spectrum of biological activities, such as activity against HIV, hepatitis A, vaccinia and influenza virus.	INTRO
+80	88	spectrum	evidence	N-acylhydrazones represent an appealing class of chelating ligands with a broad spectrum of biological activities, such as activity against HIV, hepatitis A, vaccinia and influenza virus.	INTRO
+140	143	HIV	taxonomy_domain	N-acylhydrazones represent an appealing class of chelating ligands with a broad spectrum of biological activities, such as activity against HIV, hepatitis A, vaccinia and influenza virus.	INTRO
+145	156	hepatitis A	taxonomy_domain	N-acylhydrazones represent an appealing class of chelating ligands with a broad spectrum of biological activities, such as activity against HIV, hepatitis A, vaccinia and influenza virus.	INTRO
+158	166	vaccinia	taxonomy_domain	N-acylhydrazones represent an appealing class of chelating ligands with a broad spectrum of biological activities, such as activity against HIV, hepatitis A, vaccinia and influenza virus.	INTRO
+171	180	influenza	taxonomy_domain	N-acylhydrazones represent an appealing class of chelating ligands with a broad spectrum of biological activities, such as activity against HIV, hepatitis A, vaccinia and influenza virus.	INTRO
+181	186	virus	taxonomy_domain	N-acylhydrazones represent an appealing class of chelating ligands with a broad spectrum of biological activities, such as activity against HIV, hepatitis A, vaccinia and influenza virus.	INTRO
+70	86	N-acylhydrazones	chemical	In the present work, we report the biological activity of a series of N-acylhydrazones (Fig. 2), as determined in an enzymatic assay with PA-Nter endonuclease as well as in cell-based influenza viral ribonucleoprotein (vRNP) reconstitution and virus yield assays.	INTRO
+117	132	enzymatic assay	experimental_method	In the present work, we report the biological activity of a series of N-acylhydrazones (Fig. 2), as determined in an enzymatic assay with PA-Nter endonuclease as well as in cell-based influenza viral ribonucleoprotein (vRNP) reconstitution and virus yield assays.	INTRO
+138	140	PA	protein	In the present work, we report the biological activity of a series of N-acylhydrazones (Fig. 2), as determined in an enzymatic assay with PA-Nter endonuclease as well as in cell-based influenza viral ribonucleoprotein (vRNP) reconstitution and virus yield assays.	INTRO
+141	145	Nter	structure_element	In the present work, we report the biological activity of a series of N-acylhydrazones (Fig. 2), as determined in an enzymatic assay with PA-Nter endonuclease as well as in cell-based influenza viral ribonucleoprotein (vRNP) reconstitution and virus yield assays.	INTRO
+146	158	endonuclease	protein_type	In the present work, we report the biological activity of a series of N-acylhydrazones (Fig. 2), as determined in an enzymatic assay with PA-Nter endonuclease as well as in cell-based influenza viral ribonucleoprotein (vRNP) reconstitution and virus yield assays.	INTRO
+173	239	cell-based influenza viral ribonucleoprotein (vRNP) reconstitution	experimental_method	In the present work, we report the biological activity of a series of N-acylhydrazones (Fig. 2), as determined in an enzymatic assay with PA-Nter endonuclease as well as in cell-based influenza viral ribonucleoprotein (vRNP) reconstitution and virus yield assays.	INTRO
+244	262	virus yield assays	experimental_method	In the present work, we report the biological activity of a series of N-acylhydrazones (Fig. 2), as determined in an enzymatic assay with PA-Nter endonuclease as well as in cell-based influenza viral ribonucleoprotein (vRNP) reconstitution and virus yield assays.	INTRO
+8	24	N-acylhydrazones	chemical	Several N-acylhydrazones were found to have promising anti-influenza activity with 50% effective concentration values (EC50) in the range of 3–20 μM and good selectivity (Table 1 and Fig. 3).	INTRO
+59	68	influenza	taxonomy_domain	Several N-acylhydrazones were found to have promising anti-influenza activity with 50% effective concentration values (EC50) in the range of 3–20 μM and good selectivity (Table 1 and Fig. 3).	INTRO
+83	110	50% effective concentration	evidence	Several N-acylhydrazones were found to have promising anti-influenza activity with 50% effective concentration values (EC50) in the range of 3–20 μM and good selectivity (Table 1 and Fig. 3).	INTRO
+119	123	EC50	evidence	Several N-acylhydrazones were found to have promising anti-influenza activity with 50% effective concentration values (EC50) in the range of 3–20 μM and good selectivity (Table 1 and Fig. 3).	INTRO
+0	29	Computational docking studies	experimental_method	Computational docking studies of two candidate ligands in the PA-Nter active site gave information about the features that could determine inhibition of endonuclease activity.	INTRO
+62	64	PA	protein	Computational docking studies of two candidate ligands in the PA-Nter active site gave information about the features that could determine inhibition of endonuclease activity.	INTRO
+65	69	Nter	structure_element	Computational docking studies of two candidate ligands in the PA-Nter active site gave information about the features that could determine inhibition of endonuclease activity.	INTRO
+70	81	active site	site	Computational docking studies of two candidate ligands in the PA-Nter active site gave information about the features that could determine inhibition of endonuclease activity.	INTRO
+153	165	endonuclease	protein_type	Computational docking studies of two candidate ligands in the PA-Nter active site gave information about the features that could determine inhibition of endonuclease activity.	INTRO
+26	49	X-ray crystal structure	evidence	Moreover, we describe the X-ray crystal structure of PA-Nter in complex with one of the most active inhibitors.	INTRO
+53	55	PA	protein	Moreover, we describe the X-ray crystal structure of PA-Nter in complex with one of the most active inhibitors.	INTRO
+56	60	Nter	structure_element	Moreover, we describe the X-ray crystal structure of PA-Nter in complex with one of the most active inhibitors.	INTRO
+61	76	in complex with	protein_state	Moreover, we describe the X-ray crystal structure of PA-Nter in complex with one of the most active inhibitors.	INTRO
+0	16	N-acylhydrazones	chemical	N-acylhydrazones 1–27 (Fig. 2) were prepared in high yields by following literature methods (Fig. 2A); they were characterized by spectroscopic tools, mass spectrometry and elemental analysis.	RESULTS
+17	21	1–27	chemical	N-acylhydrazones 1–27 (Fig. 2) were prepared in high yields by following literature methods (Fig. 2A); they were characterized by spectroscopic tools, mass spectrometry and elemental analysis.	RESULTS
+151	168	mass spectrometry	experimental_method	N-acylhydrazones 1–27 (Fig. 2) were prepared in high yields by following literature methods (Fig. 2A); they were characterized by spectroscopic tools, mass spectrometry and elemental analysis.	RESULTS
+173	191	elemental analysis	experimental_method	N-acylhydrazones 1–27 (Fig. 2) were prepared in high yields by following literature methods (Fig. 2A); they were characterized by spectroscopic tools, mass spectrometry and elemental analysis.	RESULTS
+53	57	1–27	chemical	Even if isomerism around the C = N bond is possible, 1–27 are present in the E form in solution, as evidenced by the chemical shift values of the HC = N and NH protons in the 1H-NMR spectrum.	RESULTS
+175	181	1H-NMR	experimental_method	Even if isomerism around the C = N bond is possible, 1–27 are present in the E form in solution, as evidenced by the chemical shift values of the HC = N and NH protons in the 1H-NMR spectrum.	RESULTS
+182	190	spectrum	evidence	Even if isomerism around the C = N bond is possible, 1–27 are present in the E form in solution, as evidenced by the chemical shift values of the HC = N and NH protons in the 1H-NMR spectrum.	RESULTS
+52	53	3	chemical	Exceptions are represented by the alkyl-derivatives 3 and 4 (2:1 and 5:3 E:Z ratio, respectively).	RESULTS
+58	59	4	chemical	Exceptions are represented by the alkyl-derivatives 3 and 4 (2:1 and 5:3 E:Z ratio, respectively).	RESULTS
+74	88	acylhydrazones	chemical	If R’ (Fig. 2A) is a 2-hydroxy substituted phenyl ring, the corresponding acylhydrazones can coordinate one or, depending on denticity, two metal centers (modes A and B in Fig. 4).	RESULTS
+93	103	coordinate	bond_interaction	If R’ (Fig. 2A) is a 2-hydroxy substituted phenyl ring, the corresponding acylhydrazones can coordinate one or, depending on denticity, two metal centers (modes A and B in Fig. 4).	RESULTS
+14	57	N’-(2,3-dihydroxybenzylidene)-semicarbazide	chemical	Starting from N’-(2,3-dihydroxybenzylidene)-semicarbazide (1) and its methoxy-analogue (2), we modified the acylhydrazonic substituent R” (3–8, 18, 19, Fig. 2A).	RESULTS
+59	60	1	chemical	Starting from N’-(2,3-dihydroxybenzylidene)-semicarbazide (1) and its methoxy-analogue (2), we modified the acylhydrazonic substituent R” (3–8, 18, 19, Fig. 2A).	RESULTS
+88	89	2	chemical	Starting from N’-(2,3-dihydroxybenzylidene)-semicarbazide (1) and its methoxy-analogue (2), we modified the acylhydrazonic substituent R” (3–8, 18, 19, Fig. 2A).	RESULTS
+139	142	3–8	chemical	Starting from N’-(2,3-dihydroxybenzylidene)-semicarbazide (1) and its methoxy-analogue (2), we modified the acylhydrazonic substituent R” (3–8, 18, 19, Fig. 2A).	RESULTS
+144	146	18	chemical	Starting from N’-(2,3-dihydroxybenzylidene)-semicarbazide (1) and its methoxy-analogue (2), we modified the acylhydrazonic substituent R” (3–8, 18, 19, Fig. 2A).	RESULTS
+148	150	19	chemical	Starting from N’-(2,3-dihydroxybenzylidene)-semicarbazide (1) and its methoxy-analogue (2), we modified the acylhydrazonic substituent R” (3–8, 18, 19, Fig. 2A).	RESULTS
+3	5	18	chemical	In 18 and 19, also the gallic moiety can be involved in the chelation of the metal cofactors (mode C, Fig. 4).	RESULTS
+10	12	19	chemical	In 18 and 19, also the gallic moiety can be involved in the chelation of the metal cofactors (mode C, Fig. 4).	RESULTS
+23	29	gallic	chemical	In 18 and 19, also the gallic moiety can be involved in the chelation of the metal cofactors (mode C, Fig. 4).	RESULTS
+60	69	chelation	bond_interaction	In 18 and 19, also the gallic moiety can be involved in the chelation of the metal cofactors (mode C, Fig. 4).	RESULTS
+58	62	9–11	chemical	In order to investigate the role of hydroxyl substituents 9–11, 13–17, 20–23 and 27 were also synthesized.	RESULTS
+64	69	13–17	chemical	In order to investigate the role of hydroxyl substituents 9–11, 13–17, 20–23 and 27 were also synthesized.	RESULTS
+71	76	20–23	chemical	In order to investigate the role of hydroxyl substituents 9–11, 13–17, 20–23 and 27 were also synthesized.	RESULTS
+81	83	27	chemical	In order to investigate the role of hydroxyl substituents 9–11, 13–17, 20–23 and 27 were also synthesized.	RESULTS
+9	11	12	chemical	Compound 12 was synthesized in order to confirm the crucial influence of the gallic moiety.	RESULTS
+77	83	gallic	chemical	Compound 12 was synthesized in order to confirm the crucial influence of the gallic moiety.	RESULTS
+9	11	26	chemical	Finally, 26 was here considered, because it is an inhibitor of HIV RNase H, another enzyme with two magnesium ions in its active site.	RESULTS
+63	66	HIV	taxonomy_domain	Finally, 26 was here considered, because it is an inhibitor of HIV RNase H, another enzyme with two magnesium ions in its active site.	RESULTS
+67	74	RNase H	protein	Finally, 26 was here considered, because it is an inhibitor of HIV RNase H, another enzyme with two magnesium ions in its active site.	RESULTS
+100	109	magnesium	chemical	Finally, 26 was here considered, because it is an inhibitor of HIV RNase H, another enzyme with two magnesium ions in its active site.	RESULTS
+122	133	active site	site	Finally, 26 was here considered, because it is an inhibitor of HIV RNase H, another enzyme with two magnesium ions in its active site.	RESULTS
+37	53	N-acylhydrazones	chemical	Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+.	RESULTS
+74	83	chelation	bond_interaction	Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+.	RESULTS
+100	105	metal	chemical	Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+.	RESULTS
+125	134	influenza	taxonomy_domain	Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+.	RESULTS
+135	137	PA	protein	Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+.	RESULTS
+138	142	Nter	structure_element	Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+.	RESULTS
+143	154	active site	site	Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+.	RESULTS
+226	228	19	chemical	Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+.	RESULTS
+230	233	H2L	chemical	Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+.	RESULTS
+243	247	Mg2+	chemical	Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+.	RESULTS
+99	112	triethylamine	chemical	Different reaction conditions were used (1:1 and 1:2 metal to ligand ratio, up to 4 equivalents of triethylamine), but in any case the same chemical species Mg(HL)2∙4H2O was recovered and conveniently characterized.	RESULTS
+157	169	Mg(HL)2∙4H2O	chemical	Different reaction conditions were used (1:1 and 1:2 metal to ligand ratio, up to 4 equivalents of triethylamine), but in any case the same chemical species Mg(HL)2∙4H2O was recovered and conveniently characterized.	RESULTS
+37	44	d6-DMSO	chemical	The use of a coordinating solvent as d6-DMSO causes partial decoordination of the ligand, but the 1H-NMR spectrum in MeOD, instead, shows only the signals attributable to the complex.	RESULTS
+98	104	1H-NMR	experimental_method	The use of a coordinating solvent as d6-DMSO causes partial decoordination of the ligand, but the 1H-NMR spectrum in MeOD, instead, shows only the signals attributable to the complex.	RESULTS
+105	113	spectrum	evidence	The use of a coordinating solvent as d6-DMSO causes partial decoordination of the ligand, but the 1H-NMR spectrum in MeOD, instead, shows only the signals attributable to the complex.	RESULTS
+7	14	13C-NMR	experimental_method	In the 13C-NMR spectrum, the signal of the C = O quaternary carbon is practically unaffected by complexation, suggesting that the C = O group is weakly involved in the coordination to the metal ion.	RESULTS
+15	23	spectrum	evidence	In the 13C-NMR spectrum, the signal of the C = O quaternary carbon is practically unaffected by complexation, suggesting that the C = O group is weakly involved in the coordination to the metal ion.	RESULTS
+26	28	IR	experimental_method	This is confirmed, in the IR spectrum, by the shift of about 20 cm−1 of the C = O absorption, while a shift of 30–50 cm−1 is expected when the carbonylic oxygen is tightly bound to the metal ion.	RESULTS
+29	37	spectrum	evidence	This is confirmed, in the IR spectrum, by the shift of about 20 cm−1 of the C = O absorption, while a shift of 30–50 cm−1 is expected when the carbonylic oxygen is tightly bound to the metal ion.	RESULTS
+0	8	ESI-mass	experimental_method	ESI-mass spectra and elemental analysis confirmed the formula Mg(HL)2∙4H2O.	RESULTS
+9	16	spectra	evidence	ESI-mass spectra and elemental analysis confirmed the formula Mg(HL)2∙4H2O.	RESULTS
+21	39	elemental analysis	experimental_method	ESI-mass spectra and elemental analysis confirmed the formula Mg(HL)2∙4H2O.	RESULTS
+62	74	Mg(HL)2∙4H2O	chemical	ESI-mass spectra and elemental analysis confirmed the formula Mg(HL)2∙4H2O.	RESULTS
+28	43	N-acylhydrazone	chemical	The interaction between the N-acylhydrazone ligands and the magnesium cation was investigated also by means of UV-visible spectroscopy (UV-visible titrations of 23 and 19 with increasing amount of Mg(CH3COO)2 are shown in Figure S1).	RESULTS
+60	69	magnesium	chemical	The interaction between the N-acylhydrazone ligands and the magnesium cation was investigated also by means of UV-visible spectroscopy (UV-visible titrations of 23 and 19 with increasing amount of Mg(CH3COO)2 are shown in Figure S1).	RESULTS
+111	134	UV-visible spectroscopy	experimental_method	The interaction between the N-acylhydrazone ligands and the magnesium cation was investigated also by means of UV-visible spectroscopy (UV-visible titrations of 23 and 19 with increasing amount of Mg(CH3COO)2 are shown in Figure S1).	RESULTS
+136	157	UV-visible titrations	experimental_method	The interaction between the N-acylhydrazone ligands and the magnesium cation was investigated also by means of UV-visible spectroscopy (UV-visible titrations of 23 and 19 with increasing amount of Mg(CH3COO)2 are shown in Figure S1).	RESULTS
+161	163	23	chemical	The interaction between the N-acylhydrazone ligands and the magnesium cation was investigated also by means of UV-visible spectroscopy (UV-visible titrations of 23 and 19 with increasing amount of Mg(CH3COO)2 are shown in Figure S1).	RESULTS
+168	170	19	chemical	The interaction between the N-acylhydrazone ligands and the magnesium cation was investigated also by means of UV-visible spectroscopy (UV-visible titrations of 23 and 19 with increasing amount of Mg(CH3COO)2 are shown in Figure S1).	RESULTS
+176	193	increasing amount	experimental_method	The interaction between the N-acylhydrazone ligands and the magnesium cation was investigated also by means of UV-visible spectroscopy (UV-visible titrations of 23 and 19 with increasing amount of Mg(CH3COO)2 are shown in Figure S1).	RESULTS
+197	208	Mg(CH3COO)2	chemical	The interaction between the N-acylhydrazone ligands and the magnesium cation was investigated also by means of UV-visible spectroscopy (UV-visible titrations of 23 and 19 with increasing amount of Mg(CH3COO)2 are shown in Figure S1).	RESULTS
+4	12	spectrum	evidence	The spectrum of 19 includes a band at 313 nm assignable to n-π* transitions of the C = N and C = O groups.	RESULTS
+16	18	19	chemical	The spectrum of 19 includes a band at 313 nm assignable to n-π* transitions of the C = N and C = O groups.	RESULTS
+36	47	Mg(CH3COO)2	chemical	By adding increasing equivalents of Mg(CH3COO)2, the absorption around 400 nm increases, and a new band appears with a maximum at 397 nm.	RESULTS
+44	46	23	chemical	When the same experiment was performed with 23, a different behavior was observed.	RESULTS
+28	33	Mg2+,	chemical	Increasing concentration of Mg2+, in fact, caused a diminution in the maximum absorption, an isosbestic point is visible at about 345 nm, but a new band at 400 nm does not appear.	RESULTS
+8	10	19	chemical	Ligands 19 and 23 coordinate the Mg2+ ions in different ways: 19 chelates the metal ion by using the deprotonated salicyl oxygen and the iminic nitrogen, while for 23, the gallic moiety is supposed to be involved (Fig. 4A,B versus C), leading to different, less extensive, modifications of the UV spectrum.	RESULTS
+15	17	23	chemical	Ligands 19 and 23 coordinate the Mg2+ ions in different ways: 19 chelates the metal ion by using the deprotonated salicyl oxygen and the iminic nitrogen, while for 23, the gallic moiety is supposed to be involved (Fig. 4A,B versus C), leading to different, less extensive, modifications of the UV spectrum.	RESULTS
+18	28	coordinate	bond_interaction	Ligands 19 and 23 coordinate the Mg2+ ions in different ways: 19 chelates the metal ion by using the deprotonated salicyl oxygen and the iminic nitrogen, while for 23, the gallic moiety is supposed to be involved (Fig. 4A,B versus C), leading to different, less extensive, modifications of the UV spectrum.	RESULTS
+33	37	Mg2+	chemical	Ligands 19 and 23 coordinate the Mg2+ ions in different ways: 19 chelates the metal ion by using the deprotonated salicyl oxygen and the iminic nitrogen, while for 23, the gallic moiety is supposed to be involved (Fig. 4A,B versus C), leading to different, less extensive, modifications of the UV spectrum.	RESULTS
+62	64	19	chemical	Ligands 19 and 23 coordinate the Mg2+ ions in different ways: 19 chelates the metal ion by using the deprotonated salicyl oxygen and the iminic nitrogen, while for 23, the gallic moiety is supposed to be involved (Fig. 4A,B versus C), leading to different, less extensive, modifications of the UV spectrum.	RESULTS
+164	166	23	chemical	Ligands 19 and 23 coordinate the Mg2+ ions in different ways: 19 chelates the metal ion by using the deprotonated salicyl oxygen and the iminic nitrogen, while for 23, the gallic moiety is supposed to be involved (Fig. 4A,B versus C), leading to different, less extensive, modifications of the UV spectrum.	RESULTS
+294	296	UV	experimental_method	Ligands 19 and 23 coordinate the Mg2+ ions in different ways: 19 chelates the metal ion by using the deprotonated salicyl oxygen and the iminic nitrogen, while for 23, the gallic moiety is supposed to be involved (Fig. 4A,B versus C), leading to different, less extensive, modifications of the UV spectrum.	RESULTS
+297	305	spectrum	evidence	Ligands 19 and 23 coordinate the Mg2+ ions in different ways: 19 chelates the metal ion by using the deprotonated salicyl oxygen and the iminic nitrogen, while for 23, the gallic moiety is supposed to be involved (Fig. 4A,B versus C), leading to different, less extensive, modifications of the UV spectrum.	RESULTS
+18	20	PA	protein	Inhibition of the PA-Nter enzyme	RESULTS
+21	25	Nter	structure_element	Inhibition of the PA-Nter enzyme	RESULTS
+63	72	influenza	taxonomy_domain	All the compounds were tested for their ability to inhibit the influenza endonuclease in an enzymatic plasmid-based assay with recombinant PA-Nter, as well as in cell-based influenza methods (i.e. virus yield and vRNP reconstitution assays).	RESULTS
+73	85	endonuclease	protein_type	All the compounds were tested for their ability to inhibit the influenza endonuclease in an enzymatic plasmid-based assay with recombinant PA-Nter, as well as in cell-based influenza methods (i.e. virus yield and vRNP reconstitution assays).	RESULTS
+92	121	enzymatic plasmid-based assay	experimental_method	All the compounds were tested for their ability to inhibit the influenza endonuclease in an enzymatic plasmid-based assay with recombinant PA-Nter, as well as in cell-based influenza methods (i.e. virus yield and vRNP reconstitution assays).	RESULTS
+139	141	PA	protein	All the compounds were tested for their ability to inhibit the influenza endonuclease in an enzymatic plasmid-based assay with recombinant PA-Nter, as well as in cell-based influenza methods (i.e. virus yield and vRNP reconstitution assays).	RESULTS
+142	146	Nter	structure_element	All the compounds were tested for their ability to inhibit the influenza endonuclease in an enzymatic plasmid-based assay with recombinant PA-Nter, as well as in cell-based influenza methods (i.e. virus yield and vRNP reconstitution assays).	RESULTS
+162	190	cell-based influenza methods	experimental_method	All the compounds were tested for their ability to inhibit the influenza endonuclease in an enzymatic plasmid-based assay with recombinant PA-Nter, as well as in cell-based influenza methods (i.e. virus yield and vRNP reconstitution assays).	RESULTS
+197	239	virus yield and vRNP reconstitution assays	experimental_method	All the compounds were tested for their ability to inhibit the influenza endonuclease in an enzymatic plasmid-based assay with recombinant PA-Nter, as well as in cell-based influenza methods (i.e. virus yield and vRNP reconstitution assays).	RESULTS
+129	149	dose-response curves	evidence	The results are shown in Table 1 and summarized in Fig. 3 to visualize the structure-activity relationships; Figure S2 shows the dose-response curves for three representative compounds (i.e. 10, 13 and 23) in either the PA-enzyme or vRNP reconstitution assay.	RESULTS
+191	193	10	chemical	The results are shown in Table 1 and summarized in Fig. 3 to visualize the structure-activity relationships; Figure S2 shows the dose-response curves for three representative compounds (i.e. 10, 13 and 23) in either the PA-enzyme or vRNP reconstitution assay.	RESULTS
+195	197	13	chemical	The results are shown in Table 1 and summarized in Fig. 3 to visualize the structure-activity relationships; Figure S2 shows the dose-response curves for three representative compounds (i.e. 10, 13 and 23) in either the PA-enzyme or vRNP reconstitution assay.	RESULTS
+202	204	23	chemical	The results are shown in Table 1 and summarized in Fig. 3 to visualize the structure-activity relationships; Figure S2 shows the dose-response curves for three representative compounds (i.e. 10, 13 and 23) in either the PA-enzyme or vRNP reconstitution assay.	RESULTS
+220	258	PA-enzyme or vRNP reconstitution assay	experimental_method	The results are shown in Table 1 and summarized in Fig. 3 to visualize the structure-activity relationships; Figure S2 shows the dose-response curves for three representative compounds (i.e. 10, 13 and 23) in either the PA-enzyme or vRNP reconstitution assay.	RESULTS
+23	27	IC50	evidence	The moderate activity (IC50 = 24 μM) of N’-2,3-dihydroxybenzylidene semicarbazide (1) was completely lost when the NH2 moiety was replaced by a hydrophobic heptyl chain (3), but it is less affected when a phenyl or a 2-hydroxyphenyl is present (5 and 7, IC50 = 84 and 54 μM, respectively).	RESULTS
+40	81	N’-2,3-dihydroxybenzylidene semicarbazide	chemical	The moderate activity (IC50 = 24 μM) of N’-2,3-dihydroxybenzylidene semicarbazide (1) was completely lost when the NH2 moiety was replaced by a hydrophobic heptyl chain (3), but it is less affected when a phenyl or a 2-hydroxyphenyl is present (5 and 7, IC50 = 84 and 54 μM, respectively).	RESULTS
+83	84	1	chemical	The moderate activity (IC50 = 24 μM) of N’-2,3-dihydroxybenzylidene semicarbazide (1) was completely lost when the NH2 moiety was replaced by a hydrophobic heptyl chain (3), but it is less affected when a phenyl or a 2-hydroxyphenyl is present (5 and 7, IC50 = 84 and 54 μM, respectively).	RESULTS
+170	171	3	chemical	The moderate activity (IC50 = 24 μM) of N’-2,3-dihydroxybenzylidene semicarbazide (1) was completely lost when the NH2 moiety was replaced by a hydrophobic heptyl chain (3), but it is less affected when a phenyl or a 2-hydroxyphenyl is present (5 and 7, IC50 = 84 and 54 μM, respectively).	RESULTS
+245	246	5	chemical	The moderate activity (IC50 = 24 μM) of N’-2,3-dihydroxybenzylidene semicarbazide (1) was completely lost when the NH2 moiety was replaced by a hydrophobic heptyl chain (3), but it is less affected when a phenyl or a 2-hydroxyphenyl is present (5 and 7, IC50 = 84 and 54 μM, respectively).	RESULTS
+251	252	7	chemical	The moderate activity (IC50 = 24 μM) of N’-2,3-dihydroxybenzylidene semicarbazide (1) was completely lost when the NH2 moiety was replaced by a hydrophobic heptyl chain (3), but it is less affected when a phenyl or a 2-hydroxyphenyl is present (5 and 7, IC50 = 84 and 54 μM, respectively).	RESULTS
+254	258	IC50	evidence	The moderate activity (IC50 = 24 μM) of N’-2,3-dihydroxybenzylidene semicarbazide (1) was completely lost when the NH2 moiety was replaced by a hydrophobic heptyl chain (3), but it is less affected when a phenyl or a 2-hydroxyphenyl is present (5 and 7, IC50 = 84 and 54 μM, respectively).	RESULTS
+39	63	2,3-dihydroxybenzylidene	chemical	When the hydroxyl in position 3 on R1 (2,3-dihydroxybenzylidene) was replaced by a methoxy group (2-hydroxy-3-methoxybenzylidene), the activity disappeared (compounds 2, 4, 6 and 8).	RESULTS
+98	128	2-hydroxy-3-methoxybenzylidene	chemical	When the hydroxyl in position 3 on R1 (2,3-dihydroxybenzylidene) was replaced by a methoxy group (2-hydroxy-3-methoxybenzylidene), the activity disappeared (compounds 2, 4, 6 and 8).	RESULTS
+167	168	2	chemical	When the hydroxyl in position 3 on R1 (2,3-dihydroxybenzylidene) was replaced by a methoxy group (2-hydroxy-3-methoxybenzylidene), the activity disappeared (compounds 2, 4, 6 and 8).	RESULTS
+170	171	4	chemical	When the hydroxyl in position 3 on R1 (2,3-dihydroxybenzylidene) was replaced by a methoxy group (2-hydroxy-3-methoxybenzylidene), the activity disappeared (compounds 2, 4, 6 and 8).	RESULTS
+173	174	6	chemical	When the hydroxyl in position 3 on R1 (2,3-dihydroxybenzylidene) was replaced by a methoxy group (2-hydroxy-3-methoxybenzylidene), the activity disappeared (compounds 2, 4, 6 and 8).	RESULTS
+179	180	8	chemical	When the hydroxyl in position 3 on R1 (2,3-dihydroxybenzylidene) was replaced by a methoxy group (2-hydroxy-3-methoxybenzylidene), the activity disappeared (compounds 2, 4, 6 and 8).	RESULTS
+28	32	IC50	evidence	The activity is unaffected (IC50 values ranging from 45 to 75 μM) when going from two hydroxyls in R1 (7) to compounds with three hydroxyls (i.e. 9, 10 and 11).	RESULTS
+103	104	7	chemical	The activity is unaffected (IC50 values ranging from 45 to 75 μM) when going from two hydroxyls in R1 (7) to compounds with three hydroxyls (i.e. 9, 10 and 11).	RESULTS
+146	147	9	chemical	The activity is unaffected (IC50 values ranging from 45 to 75 μM) when going from two hydroxyls in R1 (7) to compounds with three hydroxyls (i.e. 9, 10 and 11).	RESULTS
+149	151	10	chemical	The activity is unaffected (IC50 values ranging from 45 to 75 μM) when going from two hydroxyls in R1 (7) to compounds with three hydroxyls (i.e. 9, 10 and 11).	RESULTS
+156	158	11	chemical	The activity is unaffected (IC50 values ranging from 45 to 75 μM) when going from two hydroxyls in R1 (7) to compounds with three hydroxyls (i.e. 9, 10 and 11).	RESULTS
+11	13	11	chemical	Similarly, 11 (R1 = 3,4,5-trihydroxyphenyl, R2 = 2-hydroxyphenyl) had comparable activity as 27 (R1 = 3,4,5-trihydroxyphenyl, R2 = NH2).	RESULTS
+93	95	27	chemical	Similarly, 11 (R1 = 3,4,5-trihydroxyphenyl, R2 = 2-hydroxyphenyl) had comparable activity as 27 (R1 = 3,4,5-trihydroxyphenyl, R2 = NH2).	RESULTS
+71	73	11	chemical	Within the series carrying a 2-hydroxyphenyl R2 group, the activity of 11 is particularly intriguing.	RESULTS
+0	2	11	chemical	11 does not have the possibility to chelate in a tridentate ONO fashion (mode A in Fig. 4), but it can coordinate two cations by means of its three OH groups in R1 (mode C, Fig. 4).	RESULTS
+103	113	coordinate	bond_interaction	11 does not have the possibility to chelate in a tridentate ONO fashion (mode A in Fig. 4), but it can coordinate two cations by means of its three OH groups in R1 (mode C, Fig. 4).	RESULTS
+53	70	crystal structure	evidence	Note that a similar chelating mode was observed in a crystal structure, solved by Cusack and coworkers, of PA-Nter endonuclease in complex with the inhibitor EGCG.	RESULTS
+107	109	PA	protein	Note that a similar chelating mode was observed in a crystal structure, solved by Cusack and coworkers, of PA-Nter endonuclease in complex with the inhibitor EGCG.	RESULTS
+110	114	Nter	structure_element	Note that a similar chelating mode was observed in a crystal structure, solved by Cusack and coworkers, of PA-Nter endonuclease in complex with the inhibitor EGCG.	RESULTS
+115	127	endonuclease	protein_type	Note that a similar chelating mode was observed in a crystal structure, solved by Cusack and coworkers, of PA-Nter endonuclease in complex with the inhibitor EGCG.	RESULTS
+128	143	in complex with	protein_state	Note that a similar chelating mode was observed in a crystal structure, solved by Cusack and coworkers, of PA-Nter endonuclease in complex with the inhibitor EGCG.	RESULTS
+158	162	EGCG	chemical	Note that a similar chelating mode was observed in a crystal structure, solved by Cusack and coworkers, of PA-Nter endonuclease in complex with the inhibitor EGCG.	RESULTS
+4	6	PA	protein	The PA-Nter inhibitory activity strongly depends on the number and position of hydroxyl substituents in R1 and R2: this is clearly highlighted by the data obtained with compounds 13–23, in which R2 is a 3,4,5-trihydroxyphenyl (gallic) group, the most active scaffold in our series.	RESULTS
+7	11	Nter	structure_element	The PA-Nter inhibitory activity strongly depends on the number and position of hydroxyl substituents in R1 and R2: this is clearly highlighted by the data obtained with compounds 13–23, in which R2 is a 3,4,5-trihydroxyphenyl (gallic) group, the most active scaffold in our series.	RESULTS
+179	184	13–23	chemical	The PA-Nter inhibitory activity strongly depends on the number and position of hydroxyl substituents in R1 and R2: this is clearly highlighted by the data obtained with compounds 13–23, in which R2 is a 3,4,5-trihydroxyphenyl (gallic) group, the most active scaffold in our series.	RESULTS
+69	71	13	chemical	The analogue carrying an unsubstituted aromatic ring as R1 (compound 13) had moderate activity (IC50 = 69 μM).	RESULTS
+96	100	IC50	evidence	The analogue carrying an unsubstituted aromatic ring as R1 (compound 13) had moderate activity (IC50 = 69 μM).	RESULTS
+52	54	14	chemical	When one OH was added at position 2 of the R1 ring (14), the activity was lost.	RESULTS
+83	85	15	chemical	Adding a second OH substituent at position 5 resulted in strong activity (compound 15, IC50 = 9 μM); medium activity for a 3-OH (18; IC50 = 83 μM), and marginal activity when the second OH is at position 4 (17, IC50 ≥ 370 μM).	RESULTS
+87	91	IC50	evidence	Adding a second OH substituent at position 5 resulted in strong activity (compound 15, IC50 = 9 μM); medium activity for a 3-OH (18; IC50 = 83 μM), and marginal activity when the second OH is at position 4 (17, IC50 ≥ 370 μM).	RESULTS
+129	131	18	chemical	Adding a second OH substituent at position 5 resulted in strong activity (compound 15, IC50 = 9 μM); medium activity for a 3-OH (18; IC50 = 83 μM), and marginal activity when the second OH is at position 4 (17, IC50 ≥ 370 μM).	RESULTS
+133	137	IC50	evidence	Adding a second OH substituent at position 5 resulted in strong activity (compound 15, IC50 = 9 μM); medium activity for a 3-OH (18; IC50 = 83 μM), and marginal activity when the second OH is at position 4 (17, IC50 ≥ 370 μM).	RESULTS
+207	209	17	chemical	Adding a second OH substituent at position 5 resulted in strong activity (compound 15, IC50 = 9 μM); medium activity for a 3-OH (18; IC50 = 83 μM), and marginal activity when the second OH is at position 4 (17, IC50 ≥ 370 μM).	RESULTS
+211	215	IC50	evidence	Adding a second OH substituent at position 5 resulted in strong activity (compound 15, IC50 = 9 μM); medium activity for a 3-OH (18; IC50 = 83 μM), and marginal activity when the second OH is at position 4 (17, IC50 ≥ 370 μM).	RESULTS
+35	37	19	chemical	The addition of a 3-methoxy group (19) abolished all inhibitory activity.	RESULTS
+107	112	14–19	chemical	This cannot be related to variations in the chelating features displayed by the R1 moiety, since compounds 14–19 all have, in theory, the capacity to chelate one metal ion through the ortho-OH and iminic nitrogen (mode A in Fig. 4).	RESULTS
+19	21	18	chemical	Moreover, compound 18 can, in principle, chelate the two M2+ ions in the active site according to mode B (Fig. 4), yet it (IC50 = 83 μM) has nine-fold lower activity than 15, that does not possess this two-metal chelating feature.	RESULTS
+57	60	M2+	chemical	Moreover, compound 18 can, in principle, chelate the two M2+ ions in the active site according to mode B (Fig. 4), yet it (IC50 = 83 μM) has nine-fold lower activity than 15, that does not possess this two-metal chelating feature.	RESULTS
+73	84	active site	site	Moreover, compound 18 can, in principle, chelate the two M2+ ions in the active site according to mode B (Fig. 4), yet it (IC50 = 83 μM) has nine-fold lower activity than 15, that does not possess this two-metal chelating feature.	RESULTS
+123	127	IC50	evidence	Moreover, compound 18 can, in principle, chelate the two M2+ ions in the active site according to mode B (Fig. 4), yet it (IC50 = 83 μM) has nine-fold lower activity than 15, that does not possess this two-metal chelating feature.	RESULTS
+171	173	15	chemical	Moreover, compound 18 can, in principle, chelate the two M2+ ions in the active site according to mode B (Fig. 4), yet it (IC50 = 83 μM) has nine-fold lower activity than 15, that does not possess this two-metal chelating feature.	RESULTS
+184	195	active site	site	Therefore, we hypothesized that the inhibitory activity of the series containing the gallic moiety is determined by: (i) the capacity of the moiety R2 to chelate two metal ions in the active site of the enzyme, according to mode C (Fig. 4); and (ii) the presence and position of one or more hydroxyl substituents in R1, which may possibly result in ligand-protein interactions (e.g. through hydrogen bonds).	RESULTS
+391	405	hydrogen bonds	bond_interaction	Therefore, we hypothesized that the inhibitory activity of the series containing the gallic moiety is determined by: (i) the capacity of the moiety R2 to chelate two metal ions in the active site of the enzyme, according to mode C (Fig. 4); and (ii) the presence and position of one or more hydroxyl substituents in R1, which may possibly result in ligand-protein interactions (e.g. through hydrogen bonds).	RESULTS
+33	63	molecular docking calculations	experimental_method	This assumption was supported by molecular docking calculations and X-ray analysis of inhibitor 23 in complex with PA-Nter (vide infra).	RESULTS
+68	82	X-ray analysis	experimental_method	This assumption was supported by molecular docking calculations and X-ray analysis of inhibitor 23 in complex with PA-Nter (vide infra).	RESULTS
+96	98	23	chemical	This assumption was supported by molecular docking calculations and X-ray analysis of inhibitor 23 in complex with PA-Nter (vide infra).	RESULTS
+99	114	in complex with	protein_state	This assumption was supported by molecular docking calculations and X-ray analysis of inhibitor 23 in complex with PA-Nter (vide infra).	RESULTS
+115	117	PA	protein	This assumption was supported by molecular docking calculations and X-ray analysis of inhibitor 23 in complex with PA-Nter (vide infra).	RESULTS
+118	122	Nter	structure_element	This assumption was supported by molecular docking calculations and X-ray analysis of inhibitor 23 in complex with PA-Nter (vide infra).	RESULTS
+34	36	15	chemical	Substitution of the 5-hydroxyl in 15 by a methoxy group (16) causes a dramatic drop in activity (IC50 = 9 and 454 μM for 15 and 16, respectively).	RESULTS
+57	59	16	chemical	Substitution of the 5-hydroxyl in 15 by a methoxy group (16) causes a dramatic drop in activity (IC50 = 9 and 454 μM for 15 and 16, respectively).	RESULTS
+97	101	IC50	evidence	Substitution of the 5-hydroxyl in 15 by a methoxy group (16) causes a dramatic drop in activity (IC50 = 9 and 454 μM for 15 and 16, respectively).	RESULTS
+121	123	15	chemical	Substitution of the 5-hydroxyl in 15 by a methoxy group (16) causes a dramatic drop in activity (IC50 = 9 and 454 μM for 15 and 16, respectively).	RESULTS
+128	130	16	chemical	Substitution of the 5-hydroxyl in 15 by a methoxy group (16) causes a dramatic drop in activity (IC50 = 9 and 454 μM for 15 and 16, respectively).	RESULTS
+81	83	20	chemical	In particular, all the compounds with a trihydroxylated phenyl group as R1 (i.e. 20, 21, 22 and 23) were able to inhibit PA-Nter quite potently.	RESULTS
+85	87	21	chemical	In particular, all the compounds with a trihydroxylated phenyl group as R1 (i.e. 20, 21, 22 and 23) were able to inhibit PA-Nter quite potently.	RESULTS
+89	91	22	chemical	In particular, all the compounds with a trihydroxylated phenyl group as R1 (i.e. 20, 21, 22 and 23) were able to inhibit PA-Nter quite potently.	RESULTS
+96	98	23	chemical	In particular, all the compounds with a trihydroxylated phenyl group as R1 (i.e. 20, 21, 22 and 23) were able to inhibit PA-Nter quite potently.	RESULTS
+121	123	PA	protein	In particular, all the compounds with a trihydroxylated phenyl group as R1 (i.e. 20, 21, 22 and 23) were able to inhibit PA-Nter quite potently.	RESULTS
+124	128	Nter	structure_element	In particular, all the compounds with a trihydroxylated phenyl group as R1 (i.e. 20, 21, 22 and 23) were able to inhibit PA-Nter quite potently.	RESULTS
+11	15	IC50	evidence	The lowest IC50 values were obtained for 21 and 23 (IC50 = 13 and 7 μM, respectively), which both have one of their three hydroxyl groups at position 5.	RESULTS
+41	43	21	chemical	The lowest IC50 values were obtained for 21 and 23 (IC50 = 13 and 7 μM, respectively), which both have one of their three hydroxyl groups at position 5.	RESULTS
+48	50	23	chemical	The lowest IC50 values were obtained for 21 and 23 (IC50 = 13 and 7 μM, respectively), which both have one of their three hydroxyl groups at position 5.	RESULTS
+52	56	IC50	evidence	The lowest IC50 values were obtained for 21 and 23 (IC50 = 13 and 7 μM, respectively), which both have one of their three hydroxyl groups at position 5.	RESULTS
+44	46	23	chemical	The most active compound in this series was 23, which lacks the hydroxyl group at position 2 of R1, further confirming that this function is undesirable or even detrimental for inhibitory activity against PA-Nter, as already noticed above for 14.	RESULTS
+205	207	PA	protein	The most active compound in this series was 23, which lacks the hydroxyl group at position 2 of R1, further confirming that this function is undesirable or even detrimental for inhibitory activity against PA-Nter, as already noticed above for 14.	RESULTS
+208	212	Nter	structure_element	The most active compound in this series was 23, which lacks the hydroxyl group at position 2 of R1, further confirming that this function is undesirable or even detrimental for inhibitory activity against PA-Nter, as already noticed above for 14.	RESULTS
+243	245	14	chemical	The most active compound in this series was 23, which lacks the hydroxyl group at position 2 of R1, further confirming that this function is undesirable or even detrimental for inhibitory activity against PA-Nter, as already noticed above for 14.	RESULTS
+64	73	chelation	bond_interaction	Consistent with a crucial role of the R2 gallic moiety in metal chelation, the strong activity of 15 was completely lost in its 3,4,5-trimethoxy analogue 12.	RESULTS
+98	100	15	chemical	Consistent with a crucial role of the R2 gallic moiety in metal chelation, the strong activity of 15 was completely lost in its 3,4,5-trimethoxy analogue 12.	RESULTS
+154	156	12	chemical	Consistent with a crucial role of the R2 gallic moiety in metal chelation, the strong activity of 15 was completely lost in its 3,4,5-trimethoxy analogue 12.	RESULTS
+83	87	IC50	evidence	On the other hand, the R2 gallic containing compounds displayed moderate activity (IC50 values around 40 μM) when R1 was absent (i.e. the 3,4,5-trihydroxybenzohydrazide 28, Fig. 2), or composed of an extended ring system (26) or a pyrrole ring (25).	RESULTS
+138	168	3,4,5-trihydroxybenzohydrazide	chemical	On the other hand, the R2 gallic containing compounds displayed moderate activity (IC50 values around 40 μM) when R1 was absent (i.e. the 3,4,5-trihydroxybenzohydrazide 28, Fig. 2), or composed of an extended ring system (26) or a pyrrole ring (25).	RESULTS
+169	171	28	chemical	On the other hand, the R2 gallic containing compounds displayed moderate activity (IC50 values around 40 μM) when R1 was absent (i.e. the 3,4,5-trihydroxybenzohydrazide 28, Fig. 2), or composed of an extended ring system (26) or a pyrrole ring (25).	RESULTS
+222	224	26	chemical	On the other hand, the R2 gallic containing compounds displayed moderate activity (IC50 values around 40 μM) when R1 was absent (i.e. the 3,4,5-trihydroxybenzohydrazide 28, Fig. 2), or composed of an extended ring system (26) or a pyrrole ring (25).	RESULTS
+245	247	25	chemical	On the other hand, the R2 gallic containing compounds displayed moderate activity (IC50 values around 40 μM) when R1 was absent (i.e. the 3,4,5-trihydroxybenzohydrazide 28, Fig. 2), or composed of an extended ring system (26) or a pyrrole ring (25).	RESULTS
+57	59	24	chemical	Still lower activity was seen with the pyridine analogue 24.	RESULTS
+102	104	PA	protein	Evidently, the 3,4,5-trihydroxybenzyl moiety at R2 is fundamental but not sufficient to ensure potent PA-Nter endonuclease inhibition, since the interactions of R1 with the amino acid side chains of the protein appear crucial in modulating activity.	RESULTS
+105	109	Nter	structure_element	Evidently, the 3,4,5-trihydroxybenzyl moiety at R2 is fundamental but not sufficient to ensure potent PA-Nter endonuclease inhibition, since the interactions of R1 with the amino acid side chains of the protein appear crucial in modulating activity.	RESULTS
+110	122	endonuclease	protein_type	Evidently, the 3,4,5-trihydroxybenzyl moiety at R2 is fundamental but not sufficient to ensure potent PA-Nter endonuclease inhibition, since the interactions of R1 with the amino acid side chains of the protein appear crucial in modulating activity.	RESULTS
+14	18	vRNP	complex_assembly	Inhibition of vRNP activity or virus replication in cells	RESULTS
+31	36	virus	taxonomy_domain	Inhibition of vRNP activity or virus replication in cells	RESULTS
+22	31	influenza	taxonomy_domain	To determine the anti-influenza virus activity of compounds 1–28 in cell culture, we performed an influenza vRNP reconstitution assay in human embryonic kidney 293 T (HEK293T) cells, then subjected the active compounds (i.e. EC50 < 100 μM) to a virus yield assay in influenza virus-infected Madin-Darby canine kidney (MDCK) cells (Table 1 and Fig. 3).	RESULTS
+32	37	virus	taxonomy_domain	To determine the anti-influenza virus activity of compounds 1–28 in cell culture, we performed an influenza vRNP reconstitution assay in human embryonic kidney 293 T (HEK293T) cells, then subjected the active compounds (i.e. EC50 < 100 μM) to a virus yield assay in influenza virus-infected Madin-Darby canine kidney (MDCK) cells (Table 1 and Fig. 3).	RESULTS
+60	64	1–28	chemical	To determine the anti-influenza virus activity of compounds 1–28 in cell culture, we performed an influenza vRNP reconstitution assay in human embryonic kidney 293 T (HEK293T) cells, then subjected the active compounds (i.e. EC50 < 100 μM) to a virus yield assay in influenza virus-infected Madin-Darby canine kidney (MDCK) cells (Table 1 and Fig. 3).	RESULTS
+98	133	influenza vRNP reconstitution assay	experimental_method	To determine the anti-influenza virus activity of compounds 1–28 in cell culture, we performed an influenza vRNP reconstitution assay in human embryonic kidney 293 T (HEK293T) cells, then subjected the active compounds (i.e. EC50 < 100 μM) to a virus yield assay in influenza virus-infected Madin-Darby canine kidney (MDCK) cells (Table 1 and Fig. 3).	RESULTS
+137	142	human	species	To determine the anti-influenza virus activity of compounds 1–28 in cell culture, we performed an influenza vRNP reconstitution assay in human embryonic kidney 293 T (HEK293T) cells, then subjected the active compounds (i.e. EC50 < 100 μM) to a virus yield assay in influenza virus-infected Madin-Darby canine kidney (MDCK) cells (Table 1 and Fig. 3).	RESULTS
+225	229	EC50	evidence	To determine the anti-influenza virus activity of compounds 1–28 in cell culture, we performed an influenza vRNP reconstitution assay in human embryonic kidney 293 T (HEK293T) cells, then subjected the active compounds (i.e. EC50 < 100 μM) to a virus yield assay in influenza virus-infected Madin-Darby canine kidney (MDCK) cells (Table 1 and Fig. 3).	RESULTS
+245	262	virus yield assay	experimental_method	To determine the anti-influenza virus activity of compounds 1–28 in cell culture, we performed an influenza vRNP reconstitution assay in human embryonic kidney 293 T (HEK293T) cells, then subjected the active compounds (i.e. EC50 < 100 μM) to a virus yield assay in influenza virus-infected Madin-Darby canine kidney (MDCK) cells (Table 1 and Fig. 3).	RESULTS
+266	275	influenza	taxonomy_domain	To determine the anti-influenza virus activity of compounds 1–28 in cell culture, we performed an influenza vRNP reconstitution assay in human embryonic kidney 293 T (HEK293T) cells, then subjected the active compounds (i.e. EC50 < 100 μM) to a virus yield assay in influenza virus-infected Madin-Darby canine kidney (MDCK) cells (Table 1 and Fig. 3).	RESULTS
+276	281	virus	taxonomy_domain	To determine the anti-influenza virus activity of compounds 1–28 in cell culture, we performed an influenza vRNP reconstitution assay in human embryonic kidney 293 T (HEK293T) cells, then subjected the active compounds (i.e. EC50 < 100 μM) to a virus yield assay in influenza virus-infected Madin-Darby canine kidney (MDCK) cells (Table 1 and Fig. 3).	RESULTS
+9	24	N-acylhydrazone	chemical	For some N-acylhydrazone compounds, we observed quite potent and selective activity in the vRNP reconstitution assay.	RESULTS
+91	116	vRNP reconstitution assay	experimental_method	For some N-acylhydrazone compounds, we observed quite potent and selective activity in the vRNP reconstitution assay.	RESULTS
+45	50	viral	taxonomy_domain	This indicates that they are able to inhibit viral RNA synthesis and suggests that they could be classified as original PA inhibitors.	RESULTS
+51	54	RNA	chemical	This indicates that they are able to inhibit viral RNA synthesis and suggests that they could be classified as original PA inhibitors.	RESULTS
+120	122	PA	protein	This indicates that they are able to inhibit viral RNA synthesis and suggests that they could be classified as original PA inhibitors.	RESULTS
+11	15	EC50	evidence	Values for EC50 (vRNP) or EC90 (virus yield) in the range of 0.4–18 μM were obtained for compounds 15 and 20–23, which all carry a 3,4,5-trihydroxyphenyl as R2, and possess either two (15) or three (20–23) hydroxyl substituents in the R1 moiety.	RESULTS
+17	21	vRNP	complex_assembly	Values for EC50 (vRNP) or EC90 (virus yield) in the range of 0.4–18 μM were obtained for compounds 15 and 20–23, which all carry a 3,4,5-trihydroxyphenyl as R2, and possess either two (15) or three (20–23) hydroxyl substituents in the R1 moiety.	RESULTS
+26	30	EC90	evidence	Values for EC50 (vRNP) or EC90 (virus yield) in the range of 0.4–18 μM were obtained for compounds 15 and 20–23, which all carry a 3,4,5-trihydroxyphenyl as R2, and possess either two (15) or three (20–23) hydroxyl substituents in the R1 moiety.	RESULTS
+32	37	virus	taxonomy_domain	Values for EC50 (vRNP) or EC90 (virus yield) in the range of 0.4–18 μM were obtained for compounds 15 and 20–23, which all carry a 3,4,5-trihydroxyphenyl as R2, and possess either two (15) or three (20–23) hydroxyl substituents in the R1 moiety.	RESULTS
+99	101	15	chemical	Values for EC50 (vRNP) or EC90 (virus yield) in the range of 0.4–18 μM were obtained for compounds 15 and 20–23, which all carry a 3,4,5-trihydroxyphenyl as R2, and possess either two (15) or three (20–23) hydroxyl substituents in the R1 moiety.	RESULTS
+106	111	20–23	chemical	Values for EC50 (vRNP) or EC90 (virus yield) in the range of 0.4–18 μM were obtained for compounds 15 and 20–23, which all carry a 3,4,5-trihydroxyphenyl as R2, and possess either two (15) or three (20–23) hydroxyl substituents in the R1 moiety.	RESULTS
+185	187	15	chemical	Values for EC50 (vRNP) or EC90 (virus yield) in the range of 0.4–18 μM were obtained for compounds 15 and 20–23, which all carry a 3,4,5-trihydroxyphenyl as R2, and possess either two (15) or three (20–23) hydroxyl substituents in the R1 moiety.	RESULTS
+199	201	20	chemical	Values for EC50 (vRNP) or EC90 (virus yield) in the range of 0.4–18 μM were obtained for compounds 15 and 20–23, which all carry a 3,4,5-trihydroxyphenyl as R2, and possess either two (15) or three (20–23) hydroxyl substituents in the R1 moiety.	RESULTS
+202	204	23	chemical	Values for EC50 (vRNP) or EC90 (virus yield) in the range of 0.4–18 μM were obtained for compounds 15 and 20–23, which all carry a 3,4,5-trihydroxyphenyl as R2, and possess either two (15) or three (20–23) hydroxyl substituents in the R1 moiety.	RESULTS
+10	34	enzymatic PA-Nter assays	experimental_method	As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.	RESULTS
+88	90	21	chemical	As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.	RESULTS
+92	94	22	chemical	As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.	RESULTS
+99	101	23	chemical	As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.	RESULTS
+186	187	9	chemical	As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.	RESULTS
+189	191	10	chemical	As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.	RESULTS
+196	198	11	chemical	As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.	RESULTS
+201	203	10	chemical	As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.	RESULTS
+208	210	22	chemical	As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.	RESULTS
+239	243	EC50	evidence	As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.	RESULTS
+251	276	vRNP reconstitution assay	experimental_method	As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.	RESULTS
+4	13	hydrazide	chemical	The hydrazide 28 displayed weak (virus yield) to moderate (vRNP reconstitution) activity, albeit less than the most active molecules in the 3,4,5-trihydroxyphenyl series (i.e. 18 and 21–23).	RESULTS
+14	16	28	chemical	The hydrazide 28 displayed weak (virus yield) to moderate (vRNP reconstitution) activity, albeit less than the most active molecules in the 3,4,5-trihydroxyphenyl series (i.e. 18 and 21–23).	RESULTS
+33	38	virus	taxonomy_domain	The hydrazide 28 displayed weak (virus yield) to moderate (vRNP reconstitution) activity, albeit less than the most active molecules in the 3,4,5-trihydroxyphenyl series (i.e. 18 and 21–23).	RESULTS
+59	78	vRNP reconstitution	experimental_method	The hydrazide 28 displayed weak (virus yield) to moderate (vRNP reconstitution) activity, albeit less than the most active molecules in the 3,4,5-trihydroxyphenyl series (i.e. 18 and 21–23).	RESULTS
+176	178	18	chemical	The hydrazide 28 displayed weak (virus yield) to moderate (vRNP reconstitution) activity, albeit less than the most active molecules in the 3,4,5-trihydroxyphenyl series (i.e. 18 and 21–23).	RESULTS
+183	188	21–23	chemical	The hydrazide 28 displayed weak (virus yield) to moderate (vRNP reconstitution) activity, albeit less than the most active molecules in the 3,4,5-trihydroxyphenyl series (i.e. 18 and 21–23).	RESULTS
+121	123	28	chemical	Even if there are no data indicating that the compounds reported in the paper are subject to hydrolysis, the activity of 28 could raise the concern that for some N-acylhydrazones the antiviral activity in cell culture may be related to their intracellular hydrolysis.	RESULTS
+162	178	N-acylhydrazones	chemical	Even if there are no data indicating that the compounds reported in the paper are subject to hydrolysis, the activity of 28 could raise the concern that for some N-acylhydrazones the antiviral activity in cell culture may be related to their intracellular hydrolysis.	RESULTS
+86	90	EC50	evidence	However, this is unlikely, since the antiviral potency showed large differences (i.e. EC50 values between 0.42 and 29 μM) for compounds with the same R2 but different R1 groups, meaning that R1 does play a role in modulating the antiviral effect.	RESULTS
+32	56	2,3-dihydroxybenzylidene	chemical	Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8.	RESULTS
+63	64	3	chemical	Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8.	RESULTS
+66	67	5	chemical	Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8.	RESULTS
+72	73	7	chemical	Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8.	RESULTS
+78	108	2-hydroxy-3-methoxybenzylidene	chemical	Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8.	RESULTS
+122	123	4	chemical	Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8.	RESULTS
+125	126	6	chemical	Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8.	RESULTS
+131	132	8	chemical	Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8.	RESULTS
+177	187	vRNP assay	experimental_method	Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8.	RESULTS
+194	198	CC50	evidence	Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8.	RESULTS
+224	241	selectivity index	evidence	Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8.	RESULTS
+252	256	CC50	evidence	Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8.	RESULTS
+260	264	EC50	evidence	Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8.	RESULTS
+27	29	18	chemical	Two notable exceptions are 18 and 19 (containing a 2,3-dihydroxybenzylidene or 2-hydroxy-3-methoxybenzylidene R1, respectively) which were not cytotoxic at 200 μM and displayed favorable antiviral selectivity.	RESULTS
+34	36	19	chemical	Two notable exceptions are 18 and 19 (containing a 2,3-dihydroxybenzylidene or 2-hydroxy-3-methoxybenzylidene R1, respectively) which were not cytotoxic at 200 μM and displayed favorable antiviral selectivity.	RESULTS
+51	75	2,3-dihydroxybenzylidene	chemical	Two notable exceptions are 18 and 19 (containing a 2,3-dihydroxybenzylidene or 2-hydroxy-3-methoxybenzylidene R1, respectively) which were not cytotoxic at 200 μM and displayed favorable antiviral selectivity.	RESULTS
+79	109	2-hydroxy-3-methoxybenzylidene	chemical	Two notable exceptions are 18 and 19 (containing a 2,3-dihydroxybenzylidene or 2-hydroxy-3-methoxybenzylidene R1, respectively) which were not cytotoxic at 200 μM and displayed favorable antiviral selectivity.	RESULTS
+5	20	N-acylhydrazone	chemical	Some N-acylhydrazone compounds were devoid of activity in the enzymatic assay, yet showed good to moderate efficacy in cell culture (e.g. 14 and 19, having EC50 values of 2.2 and 7.1 μM, respectively).	RESULTS
+62	77	enzymatic assay	experimental_method	Some N-acylhydrazone compounds were devoid of activity in the enzymatic assay, yet showed good to moderate efficacy in cell culture (e.g. 14 and 19, having EC50 values of 2.2 and 7.1 μM, respectively).	RESULTS
+138	140	14	chemical	Some N-acylhydrazone compounds were devoid of activity in the enzymatic assay, yet showed good to moderate efficacy in cell culture (e.g. 14 and 19, having EC50 values of 2.2 and 7.1 μM, respectively).	RESULTS
+145	147	19	chemical	Some N-acylhydrazone compounds were devoid of activity in the enzymatic assay, yet showed good to moderate efficacy in cell culture (e.g. 14 and 19, having EC50 values of 2.2 and 7.1 μM, respectively).	RESULTS
+156	160	EC50	evidence	Some N-acylhydrazone compounds were devoid of activity in the enzymatic assay, yet showed good to moderate efficacy in cell culture (e.g. 14 and 19, having EC50 values of 2.2 and 7.1 μM, respectively).	RESULTS
+39	40	9	chemical	For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay.	RESULTS
+42	44	11	chemical	For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay.	RESULTS
+46	48	13	chemical	For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay.	RESULTS
+50	55	15–21	chemical	For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay.	RESULTS
+57	59	23	chemical	For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay.	RESULTS
+61	63	24	chemical	For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay.	RESULTS
+68	70	26	chemical	For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay.	RESULTS
+112	129	cell-based assays	experimental_method	For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay.	RESULTS
+141	145	EC50	evidence	For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay.	RESULTS
+169	179	vRNP assay	experimental_method	For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay.	RESULTS
+219	223	EC90	evidence	For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay.	RESULTS
+238	255	virus yield assay	experimental_method	For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay.	RESULTS
+58	59	7	chemical	On the other hand, this difference was 8-fold or more for 7, 10, 14, 22, 25 and 28.	RESULTS
+61	63	10	chemical	On the other hand, this difference was 8-fold or more for 7, 10, 14, 22, 25 and 28.	RESULTS
+65	67	14	chemical	On the other hand, this difference was 8-fold or more for 7, 10, 14, 22, 25 and 28.	RESULTS
+69	71	22	chemical	On the other hand, this difference was 8-fold or more for 7, 10, 14, 22, 25 and 28.	RESULTS
+73	75	25	chemical	On the other hand, this difference was 8-fold or more for 7, 10, 14, 22, 25 and 28.	RESULTS
+80	82	28	chemical	On the other hand, this difference was 8-fold or more for 7, 10, 14, 22, 25 and 28.	RESULTS
+5	20	N-acylhydrazone	chemical	Some N-acylhydrazone compounds showed good to moderate efficacy in the vRNP assay (e.g. 14 and 19, having EC50 values of 2.3 and 5.7 μM, respectively), yet were devoid of activity in the enzymatic assay.	RESULTS
+71	81	vRNP assay	experimental_method	Some N-acylhydrazone compounds showed good to moderate efficacy in the vRNP assay (e.g. 14 and 19, having EC50 values of 2.3 and 5.7 μM, respectively), yet were devoid of activity in the enzymatic assay.	RESULTS
+88	90	14	chemical	Some N-acylhydrazone compounds showed good to moderate efficacy in the vRNP assay (e.g. 14 and 19, having EC50 values of 2.3 and 5.7 μM, respectively), yet were devoid of activity in the enzymatic assay.	RESULTS
+95	97	19	chemical	Some N-acylhydrazone compounds showed good to moderate efficacy in the vRNP assay (e.g. 14 and 19, having EC50 values of 2.3 and 5.7 μM, respectively), yet were devoid of activity in the enzymatic assay.	RESULTS
+106	110	EC50	evidence	Some N-acylhydrazone compounds showed good to moderate efficacy in the vRNP assay (e.g. 14 and 19, having EC50 values of 2.3 and 5.7 μM, respectively), yet were devoid of activity in the enzymatic assay.	RESULTS
+187	202	enzymatic assay	experimental_method	Some N-acylhydrazone compounds showed good to moderate efficacy in the vRNP assay (e.g. 14 and 19, having EC50 values of 2.3 and 5.7 μM, respectively), yet were devoid of activity in the enzymatic assay.	RESULTS
+52	57	viral	taxonomy_domain	This observation suggests that they may inhibit the viral polymerase in an endonuclease-independent manner.	RESULTS
+58	68	polymerase	protein_type	This observation suggests that they may inhibit the viral polymerase in an endonuclease-independent manner.	RESULTS
+75	87	endonuclease	protein_type	This observation suggests that they may inhibit the viral polymerase in an endonuclease-independent manner.	RESULTS
+61	77	N-acylhydrazones	chemical	To achieve a clear insight into the antiviral profile of the N-acylhydrazones, specific mechanistic experiments are currently ongoing in our laboratory, in which we are analyzing in full depth their effects on virus entry, polymerase-dependent RNA synthesis or the late stage (maturation and release) of the virus replication cycle.	RESULTS
+210	215	virus	taxonomy_domain	To achieve a clear insight into the antiviral profile of the N-acylhydrazones, specific mechanistic experiments are currently ongoing in our laboratory, in which we are analyzing in full depth their effects on virus entry, polymerase-dependent RNA synthesis or the late stage (maturation and release) of the virus replication cycle.	RESULTS
+223	233	polymerase	protein_type	To achieve a clear insight into the antiviral profile of the N-acylhydrazones, specific mechanistic experiments are currently ongoing in our laboratory, in which we are analyzing in full depth their effects on virus entry, polymerase-dependent RNA synthesis or the late stage (maturation and release) of the virus replication cycle.	RESULTS
+244	247	RNA	chemical	To achieve a clear insight into the antiviral profile of the N-acylhydrazones, specific mechanistic experiments are currently ongoing in our laboratory, in which we are analyzing in full depth their effects on virus entry, polymerase-dependent RNA synthesis or the late stage (maturation and release) of the virus replication cycle.	RESULTS
+308	313	virus	taxonomy_domain	To achieve a clear insight into the antiviral profile of the N-acylhydrazones, specific mechanistic experiments are currently ongoing in our laboratory, in which we are analyzing in full depth their effects on virus entry, polymerase-dependent RNA synthesis or the late stage (maturation and release) of the virus replication cycle.	RESULTS
+0	15	Docking studies	experimental_method	Docking studies	RESULTS
+76	95	docking simulations	experimental_method	In order to explore the possible binding mode of the synthesized compounds, docking simulations by GOLD program were performed by using the structural coordinates (PDB code 4AWM) for the PA-Nter endonuclease in complex with EGCG.	RESULTS
+99	111	GOLD program	experimental_method	In order to explore the possible binding mode of the synthesized compounds, docking simulations by GOLD program were performed by using the structural coordinates (PDB code 4AWM) for the PA-Nter endonuclease in complex with EGCG.	RESULTS
+187	189	PA	protein	In order to explore the possible binding mode of the synthesized compounds, docking simulations by GOLD program were performed by using the structural coordinates (PDB code 4AWM) for the PA-Nter endonuclease in complex with EGCG.	RESULTS
+190	194	Nter	structure_element	In order to explore the possible binding mode of the synthesized compounds, docking simulations by GOLD program were performed by using the structural coordinates (PDB code 4AWM) for the PA-Nter endonuclease in complex with EGCG.	RESULTS
+195	207	endonuclease	protein_type	In order to explore the possible binding mode of the synthesized compounds, docking simulations by GOLD program were performed by using the structural coordinates (PDB code 4AWM) for the PA-Nter endonuclease in complex with EGCG.	RESULTS
+208	223	in complex with	protein_state	In order to explore the possible binding mode of the synthesized compounds, docking simulations by GOLD program were performed by using the structural coordinates (PDB code 4AWM) for the PA-Nter endonuclease in complex with EGCG.	RESULTS
+224	228	EGCG	chemical	In order to explore the possible binding mode of the synthesized compounds, docking simulations by GOLD program were performed by using the structural coordinates (PDB code 4AWM) for the PA-Nter endonuclease in complex with EGCG.	RESULTS
+128	140	superimposed	experimental_method	Considering that the position of the side-chains of some residues changes depending on which pocket the ligand is occupying, we superimposed some X-ray structures of complexes between PA-Nter endonuclease and known active ligands.	RESULTS
+146	162	X-ray structures	evidence	Considering that the position of the side-chains of some residues changes depending on which pocket the ligand is occupying, we superimposed some X-ray structures of complexes between PA-Nter endonuclease and known active ligands.	RESULTS
+184	186	PA	protein	Considering that the position of the side-chains of some residues changes depending on which pocket the ligand is occupying, we superimposed some X-ray structures of complexes between PA-Nter endonuclease and known active ligands.	RESULTS
+187	191	Nter	structure_element	Considering that the position of the side-chains of some residues changes depending on which pocket the ligand is occupying, we superimposed some X-ray structures of complexes between PA-Nter endonuclease and known active ligands.	RESULTS
+192	204	endonuclease	protein_type	Considering that the position of the side-chains of some residues changes depending on which pocket the ligand is occupying, we superimposed some X-ray structures of complexes between PA-Nter endonuclease and known active ligands.	RESULTS
+50	55	Tyr24	residue_name_number	It was observed that the side-chain of amino acid Tyr24 shows greater movement than the other residues and for this reason we considered it as a flexible residue during the docking procedure.	RESULTS
+145	153	flexible	protein_state	It was observed that the side-chain of amino acid Tyr24 shows greater movement than the other residues and for this reason we considered it as a flexible residue during the docking procedure.	RESULTS
+173	190	docking procedure	experimental_method	It was observed that the side-chain of amino acid Tyr24 shows greater movement than the other residues and for this reason we considered it as a flexible residue during the docking procedure.	RESULTS
+7	32	test docking calculations	experimental_method	First, test docking calculations, using EGCG, L-742,001 and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one (Fig. 1), were carried out to compare experimental and predicted binding modes and validate docking procedure.	RESULTS
+40	44	EGCG	chemical	First, test docking calculations, using EGCG, L-742,001 and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one (Fig. 1), were carried out to compare experimental and predicted binding modes and validate docking procedure.	RESULTS
+46	55	L-742,001	chemical	First, test docking calculations, using EGCG, L-742,001 and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one (Fig. 1), were carried out to compare experimental and predicted binding modes and validate docking procedure.	RESULTS
+60	119	2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one	chemical	First, test docking calculations, using EGCG, L-742,001 and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one (Fig. 1), were carried out to compare experimental and predicted binding modes and validate docking procedure.	RESULTS
+212	229	docking procedure	experimental_method	First, test docking calculations, using EGCG, L-742,001 and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one (Fig. 1), were carried out to compare experimental and predicted binding modes and validate docking procedure.	RESULTS
+74	78	rmsd	evidence	Their best docking poses agreed well with the experimental binding modes (rmsd values of 0.8, 1.2 and 0.7, respectively).	RESULTS
+6	13	docking	experimental_method	Next, docking of several N-acylhydrazones was performed and this generated a number of possible binding conformations, highlighting that the active site cavity of the PA endonuclease is quite spacious, as already demonstrated by crystallographic studies, and confirming the ability of this scaffold to chelate the two M2+ ions in different ways (Mode A-C in Fig. 4).	RESULTS
+25	41	N-acylhydrazones	chemical	Next, docking of several N-acylhydrazones was performed and this generated a number of possible binding conformations, highlighting that the active site cavity of the PA endonuclease is quite spacious, as already demonstrated by crystallographic studies, and confirming the ability of this scaffold to chelate the two M2+ ions in different ways (Mode A-C in Fig. 4).	RESULTS
+141	159	active site cavity	site	Next, docking of several N-acylhydrazones was performed and this generated a number of possible binding conformations, highlighting that the active site cavity of the PA endonuclease is quite spacious, as already demonstrated by crystallographic studies, and confirming the ability of this scaffold to chelate the two M2+ ions in different ways (Mode A-C in Fig. 4).	RESULTS
+167	169	PA	protein	Next, docking of several N-acylhydrazones was performed and this generated a number of possible binding conformations, highlighting that the active site cavity of the PA endonuclease is quite spacious, as already demonstrated by crystallographic studies, and confirming the ability of this scaffold to chelate the two M2+ ions in different ways (Mode A-C in Fig. 4).	RESULTS
+170	182	endonuclease	protein_type	Next, docking of several N-acylhydrazones was performed and this generated a number of possible binding conformations, highlighting that the active site cavity of the PA endonuclease is quite spacious, as already demonstrated by crystallographic studies, and confirming the ability of this scaffold to chelate the two M2+ ions in different ways (Mode A-C in Fig. 4).	RESULTS
+229	253	crystallographic studies	experimental_method	Next, docking of several N-acylhydrazones was performed and this generated a number of possible binding conformations, highlighting that the active site cavity of the PA endonuclease is quite spacious, as already demonstrated by crystallographic studies, and confirming the ability of this scaffold to chelate the two M2+ ions in different ways (Mode A-C in Fig. 4).	RESULTS
+318	321	M2+	chemical	Next, docking of several N-acylhydrazones was performed and this generated a number of possible binding conformations, highlighting that the active site cavity of the PA endonuclease is quite spacious, as already demonstrated by crystallographic studies, and confirming the ability of this scaffold to chelate the two M2+ ions in different ways (Mode A-C in Fig. 4).	RESULTS
+59	78	GOLD cluster docked	experimental_method	Figure 5 displays the first (panel A) and second (panel B) GOLD cluster docked solutions for compound 23.	RESULTS
+102	104	23	chemical	Figure 5 displays the first (panel A) and second (panel B) GOLD cluster docked solutions for compound 23.	RESULTS
+18	28	structures	evidence	These two complex structures represent the largest clusters with similar fitness values (59.20 and 58.65, respectively).	RESULTS
+15	17	23	chemical	In both cases, 23 appears able to coordinate the two M2+ ions in the active site through the three contiguous OH groups (Fig. 5).	RESULTS
+34	44	coordinate	bond_interaction	In both cases, 23 appears able to coordinate the two M2+ ions in the active site through the three contiguous OH groups (Fig. 5).	RESULTS
+53	56	M2+	chemical	In both cases, 23 appears able to coordinate the two M2+ ions in the active site through the three contiguous OH groups (Fig. 5).	RESULTS
+69	80	active site	site	In both cases, 23 appears able to coordinate the two M2+ ions in the active site through the three contiguous OH groups (Fig. 5).	RESULTS
+13	15	23	chemical	In addition, 23 was predicted to form two hydrogen bonding interactions, i.e. with the catalytic Lys134 on the one side and Glu26 on the other side.	RESULTS
+42	71	hydrogen bonding interactions	bond_interaction	In addition, 23 was predicted to form two hydrogen bonding interactions, i.e. with the catalytic Lys134 on the one side and Glu26 on the other side.	RESULTS
+87	96	catalytic	protein_state	In addition, 23 was predicted to form two hydrogen bonding interactions, i.e. with the catalytic Lys134 on the one side and Glu26 on the other side.	RESULTS
+97	103	Lys134	residue_name_number	In addition, 23 was predicted to form two hydrogen bonding interactions, i.e. with the catalytic Lys134 on the one side and Glu26 on the other side.	RESULTS
+124	129	Glu26	residue_name_number	In addition, 23 was predicted to form two hydrogen bonding interactions, i.e. with the catalytic Lys134 on the one side and Glu26 on the other side.	RESULTS
+51	53	23	chemical	Furthermore, in these two different binding modes, 23 forms π–π interactions with the aromatic ring of Tyr24, in a fashion similar to that described for other endonuclease inhibitors, i.e. EGCG and L-742,001.	RESULTS
+60	76	π–π interactions	bond_interaction	Furthermore, in these two different binding modes, 23 forms π–π interactions with the aromatic ring of Tyr24, in a fashion similar to that described for other endonuclease inhibitors, i.e. EGCG and L-742,001.	RESULTS
+103	108	Tyr24	residue_name_number	Furthermore, in these two different binding modes, 23 forms π–π interactions with the aromatic ring of Tyr24, in a fashion similar to that described for other endonuclease inhibitors, i.e. EGCG and L-742,001.	RESULTS
+159	171	endonuclease	protein_type	Furthermore, in these two different binding modes, 23 forms π–π interactions with the aromatic ring of Tyr24, in a fashion similar to that described for other endonuclease inhibitors, i.e. EGCG and L-742,001.	RESULTS
+189	193	EGCG	chemical	Furthermore, in these two different binding modes, 23 forms π–π interactions with the aromatic ring of Tyr24, in a fashion similar to that described for other endonuclease inhibitors, i.e. EGCG and L-742,001.	RESULTS
+198	207	L-742,001	chemical	Furthermore, in these two different binding modes, 23 forms π–π interactions with the aromatic ring of Tyr24, in a fashion similar to that described for other endonuclease inhibitors, i.e. EGCG and L-742,001.	RESULTS
+42	44	15	chemical	The best docked conformation for compound 15 (Fig. 6, fitness value 68.56), which has an activity slightly lower than 23, reveals a different role for the gallic moiety.	RESULTS
+54	67	fitness value	evidence	The best docked conformation for compound 15 (Fig. 6, fitness value 68.56), which has an activity slightly lower than 23, reveals a different role for the gallic moiety.	RESULTS
+29	58	hydrogen bonding interactions	bond_interaction	The ligand seems to form two hydrogen bonding interactions with Tyr130 as well as a cation–π interaction with Lys134.	RESULTS
+64	70	Tyr130	residue_name_number	The ligand seems to form two hydrogen bonding interactions with Tyr130 as well as a cation–π interaction with Lys134.	RESULTS
+84	104	cation–π interaction	bond_interaction	The ligand seems to form two hydrogen bonding interactions with Tyr130 as well as a cation–π interaction with Lys134.	RESULTS
+110	116	Lys134	residue_name_number	The ligand seems to form two hydrogen bonding interactions with Tyr130 as well as a cation–π interaction with Lys134.	RESULTS
+0	6	Tyr130	residue_name_number	Tyr130 lies in a pocket that also contains Arg124, a residue that was proposed to have a crucial role in binding of the RNA substrate.	RESULTS
+17	23	pocket	site	Tyr130 lies in a pocket that also contains Arg124, a residue that was proposed to have a crucial role in binding of the RNA substrate.	RESULTS
+43	49	Arg124	residue_name_number	Tyr130 lies in a pocket that also contains Arg124, a residue that was proposed to have a crucial role in binding of the RNA substrate.	RESULTS
+120	123	RNA	chemical	Tyr130 lies in a pocket that also contains Arg124, a residue that was proposed to have a crucial role in binding of the RNA substrate.	RESULTS
+9	11	15	chemical	Compound 15 appears further stabilized by hydrogen bonding interactions between two hydroxyl groups and Arg82 and Asp108.	RESULTS
+42	71	hydrogen bonding interactions	bond_interaction	Compound 15 appears further stabilized by hydrogen bonding interactions between two hydroxyl groups and Arg82 and Asp108.	RESULTS
+104	109	Arg82	residue_name_number	Compound 15 appears further stabilized by hydrogen bonding interactions between two hydroxyl groups and Arg82 and Asp108.	RESULTS
+114	120	Asp108	residue_name_number	Compound 15 appears further stabilized by hydrogen bonding interactions between two hydroxyl groups and Arg82 and Asp108.	RESULTS
+14	23	chelation	bond_interaction	In this case, chelation of the two M2+ ions is carried out by involving the imine group (mode A in Fig. 4).	RESULTS
+35	38	M2+	chemical	In this case, chelation of the two M2+ ions is carried out by involving the imine group (mode A in Fig. 4).	RESULTS
+44	46	23	chemical	It is important to highlight that compounds 23 and 15, although in different ways, both are able to chelate the metal cofactors and to establish interactions with highly conserved aminoacids (Tyr24, Glu26, Arg124, Tyr130 and Lys134) that are very important for both endonuclease activity and transcription in vitro.	RESULTS
+51	53	15	chemical	It is important to highlight that compounds 23 and 15, although in different ways, both are able to chelate the metal cofactors and to establish interactions with highly conserved aminoacids (Tyr24, Glu26, Arg124, Tyr130 and Lys134) that are very important for both endonuclease activity and transcription in vitro.	RESULTS
+163	179	highly conserved	protein_state	It is important to highlight that compounds 23 and 15, although in different ways, both are able to chelate the metal cofactors and to establish interactions with highly conserved aminoacids (Tyr24, Glu26, Arg124, Tyr130 and Lys134) that are very important for both endonuclease activity and transcription in vitro.	RESULTS
+192	197	Tyr24	residue_name_number	It is important to highlight that compounds 23 and 15, although in different ways, both are able to chelate the metal cofactors and to establish interactions with highly conserved aminoacids (Tyr24, Glu26, Arg124, Tyr130 and Lys134) that are very important for both endonuclease activity and transcription in vitro.	RESULTS
+199	204	Glu26	residue_name_number	It is important to highlight that compounds 23 and 15, although in different ways, both are able to chelate the metal cofactors and to establish interactions with highly conserved aminoacids (Tyr24, Glu26, Arg124, Tyr130 and Lys134) that are very important for both endonuclease activity and transcription in vitro.	RESULTS
+206	212	Arg124	residue_name_number	It is important to highlight that compounds 23 and 15, although in different ways, both are able to chelate the metal cofactors and to establish interactions with highly conserved aminoacids (Tyr24, Glu26, Arg124, Tyr130 and Lys134) that are very important for both endonuclease activity and transcription in vitro.	RESULTS
+214	220	Tyr130	residue_name_number	It is important to highlight that compounds 23 and 15, although in different ways, both are able to chelate the metal cofactors and to establish interactions with highly conserved aminoacids (Tyr24, Glu26, Arg124, Tyr130 and Lys134) that are very important for both endonuclease activity and transcription in vitro.	RESULTS
+225	231	Lys134	residue_name_number	It is important to highlight that compounds 23 and 15, although in different ways, both are able to chelate the metal cofactors and to establish interactions with highly conserved aminoacids (Tyr24, Glu26, Arg124, Tyr130 and Lys134) that are very important for both endonuclease activity and transcription in vitro.	RESULTS
+266	278	endonuclease	protein_type	It is important to highlight that compounds 23 and 15, although in different ways, both are able to chelate the metal cofactors and to establish interactions with highly conserved aminoacids (Tyr24, Glu26, Arg124, Tyr130 and Lys134) that are very important for both endonuclease activity and transcription in vitro.	RESULTS
+91	93	15	chemical	The crucial role of such interactions is underlined by the differences in activity between 15 (IC50 = 9.0 μM) and 19 (>500 μM): their coordinating features are similar, since both coordinate to the divalent metal ion through the phenolic oxygen, the iminic nitrogen and the carbonylic oxygen (mode A in Fig. 4), but the biological activity could be related to their different ability to engage interactions with the protein environment.	RESULTS
+95	99	IC50	evidence	The crucial role of such interactions is underlined by the differences in activity between 15 (IC50 = 9.0 μM) and 19 (>500 μM): their coordinating features are similar, since both coordinate to the divalent metal ion through the phenolic oxygen, the iminic nitrogen and the carbonylic oxygen (mode A in Fig. 4), but the biological activity could be related to their different ability to engage interactions with the protein environment.	RESULTS
+114	116	19	chemical	The crucial role of such interactions is underlined by the differences in activity between 15 (IC50 = 9.0 μM) and 19 (>500 μM): their coordinating features are similar, since both coordinate to the divalent metal ion through the phenolic oxygen, the iminic nitrogen and the carbonylic oxygen (mode A in Fig. 4), but the biological activity could be related to their different ability to engage interactions with the protein environment.	RESULTS
+180	190	coordinate	bond_interaction	The crucial role of such interactions is underlined by the differences in activity between 15 (IC50 = 9.0 μM) and 19 (>500 μM): their coordinating features are similar, since both coordinate to the divalent metal ion through the phenolic oxygen, the iminic nitrogen and the carbonylic oxygen (mode A in Fig. 4), but the biological activity could be related to their different ability to engage interactions with the protein environment.	RESULTS
+0	24	Crystallographic Studies	experimental_method	Crystallographic Studies	RESULTS
+22	36	co-crystallize	experimental_method	Attempts were made to co-crystallize PA-Nter with 15, 20, 21 and 23 in one to four molar excess.	RESULTS
+37	39	PA	protein	Attempts were made to co-crystallize PA-Nter with 15, 20, 21 and 23 in one to four molar excess.	RESULTS
+40	44	Nter	structure_element	Attempts were made to co-crystallize PA-Nter with 15, 20, 21 and 23 in one to four molar excess.	RESULTS
+50	52	15	chemical	Attempts were made to co-crystallize PA-Nter with 15, 20, 21 and 23 in one to four molar excess.	RESULTS
+54	56	20	chemical	Attempts were made to co-crystallize PA-Nter with 15, 20, 21 and 23 in one to four molar excess.	RESULTS
+58	60	21	chemical	Attempts were made to co-crystallize PA-Nter with 15, 20, 21 and 23 in one to four molar excess.	RESULTS
+65	67	23	chemical	Attempts were made to co-crystallize PA-Nter with 15, 20, 21 and 23 in one to four molar excess.	RESULTS
+6	14	crystals	evidence	While crystals appeared and diffracted well, upon data processing, no or very little electron density for the inhibitors was observed.	RESULTS
+85	101	electron density	evidence	While crystals appeared and diffracted well, upon data processing, no or very little electron density for the inhibitors was observed.	RESULTS
+17	20	apo	protein_state	Attempts to soak apo crystals in crystallization solution containing 5 mM inhibitor overnight also did not result in substantial electron density for the inhibitor.	RESULTS
+21	29	crystals	evidence	Attempts to soak apo crystals in crystallization solution containing 5 mM inhibitor overnight also did not result in substantial electron density for the inhibitor.	RESULTS
+129	145	electron density	evidence	Attempts to soak apo crystals in crystallization solution containing 5 mM inhibitor overnight also did not result in substantial electron density for the inhibitor.	RESULTS
+101	104	apo	protein_state	As a last resort, dry powder of the inhibitor was sprinkled over the crystallization drop containing apo crystals and left over night.	RESULTS
+105	113	crystals	evidence	As a last resort, dry powder of the inhibitor was sprinkled over the crystallization drop containing apo crystals and left over night.	RESULTS
+44	46	23	chemical	This experiment was successful for compound 23, the crystals diffracted to 2.15 Å and diffraction data were collected (PDB ID 5EGA).	RESULTS
+52	60	crystals	evidence	This experiment was successful for compound 23, the crystals diffracted to 2.15 Å and diffraction data were collected (PDB ID 5EGA).	RESULTS
+12	21	structure	evidence	The refined structure shows unambiguous electron density for the inhibitor (Table S1 and Fig. 7).	RESULTS
+40	56	electron density	evidence	The refined structure shows unambiguous electron density for the inhibitor (Table S1 and Fig. 7).	RESULTS
+4	21	complex structure	evidence	The complex structure confirms one of the two binding modes predicted by the docking simulations (Fig. 5, panel B).	RESULTS
+77	96	docking simulations	experimental_method	The complex structure confirms one of the two binding modes predicted by the docking simulations (Fig. 5, panel B).	RESULTS
+32	41	manganese	chemical	The galloyl moiety chelates the manganese ions, while the trihydroxyphenyl group stacks against the Tyr24 side chain.	RESULTS
+100	105	Tyr24	residue_name_number	The galloyl moiety chelates the manganese ions, while the trihydroxyphenyl group stacks against the Tyr24 side chain.	RESULTS
+84	98	hydrogen bonds	bond_interaction	It is interesting to note that two of these hydroxyl groups are in position to form hydrogen bonds with the side chain of Glu26 and Lys34 (Fig. 7).	RESULTS
+122	127	Glu26	residue_name_number	It is interesting to note that two of these hydroxyl groups are in position to form hydrogen bonds with the side chain of Glu26 and Lys34 (Fig. 7).	RESULTS
+132	137	Lys34	residue_name_number	It is interesting to note that two of these hydroxyl groups are in position to form hydrogen bonds with the side chain of Glu26 and Lys34 (Fig. 7).	RESULTS
+147	152	Glu26	residue_name_number	These interactions suggest that other functional groups, e.g. halogens, could be used in place of the hydroxyl groups for better interactions with Glu26 and Lys34 side chains, and the inhibitory potency of these compounds could be further improved.	RESULTS
+157	162	Lys34	residue_name_number	These interactions suggest that other functional groups, e.g. halogens, could be used in place of the hydroxyl groups for better interactions with Glu26 and Lys34 side chains, and the inhibitory potency of these compounds could be further improved.	RESULTS
+51	60	influenza	taxonomy_domain	The development of new agents for the treatment of influenza infection that exert their action by inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is a strategy that recently is gaining a lot of interest.	CONCL
+116	128	endonuclease	protein_type	The development of new agents for the treatment of influenza infection that exert their action by inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is a strategy that recently is gaining a lot of interest.	CONCL
+141	150	influenza	taxonomy_domain	The development of new agents for the treatment of influenza infection that exert their action by inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is a strategy that recently is gaining a lot of interest.	CONCL
+151	179	RNA-dependent RNA polymerase	protein_type	The development of new agents for the treatment of influenza infection that exert their action by inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is a strategy that recently is gaining a lot of interest.	CONCL
+35	50	N-acylhydrazone	chemical	The results here presented add the N-acylhydrazone scaffold to the library of the chelating molecules with potent antiviral activity (EC90 < 5 μM, virus yield assay in influenza virus-infected MDCK cells).	CONCL
+134	138	EC90	evidence	The results here presented add the N-acylhydrazone scaffold to the library of the chelating molecules with potent antiviral activity (EC90 < 5 μM, virus yield assay in influenza virus-infected MDCK cells).	CONCL
+147	164	virus yield assay	experimental_method	The results here presented add the N-acylhydrazone scaffold to the library of the chelating molecules with potent antiviral activity (EC90 < 5 μM, virus yield assay in influenza virus-infected MDCK cells).	CONCL
+168	177	influenza	taxonomy_domain	The results here presented add the N-acylhydrazone scaffold to the library of the chelating molecules with potent antiviral activity (EC90 < 5 μM, virus yield assay in influenza virus-infected MDCK cells).	CONCL
+178	183	virus	taxonomy_domain	The results here presented add the N-acylhydrazone scaffold to the library of the chelating molecules with potent antiviral activity (EC90 < 5 μM, virus yield assay in influenza virus-infected MDCK cells).	CONCL
+4	13	structure	evidence	The structure of the N-acylhydrazone 23 co-crystallized with PA-Nter is important not only because confirms that the polyhydroxypheyl group efficiently coordinates two metal ions in the active site of the enzyme, but also because highlights the importance of the (flexible) inhibitor backbone in order to engage effective interactions with crucial aminoacids of the protein.	CONCL
+21	36	N-acylhydrazone	chemical	The structure of the N-acylhydrazone 23 co-crystallized with PA-Nter is important not only because confirms that the polyhydroxypheyl group efficiently coordinates two metal ions in the active site of the enzyme, but also because highlights the importance of the (flexible) inhibitor backbone in order to engage effective interactions with crucial aminoacids of the protein.	CONCL
+37	39	23	chemical	The structure of the N-acylhydrazone 23 co-crystallized with PA-Nter is important not only because confirms that the polyhydroxypheyl group efficiently coordinates two metal ions in the active site of the enzyme, but also because highlights the importance of the (flexible) inhibitor backbone in order to engage effective interactions with crucial aminoacids of the protein.	CONCL
+40	55	co-crystallized	experimental_method	The structure of the N-acylhydrazone 23 co-crystallized with PA-Nter is important not only because confirms that the polyhydroxypheyl group efficiently coordinates two metal ions in the active site of the enzyme, but also because highlights the importance of the (flexible) inhibitor backbone in order to engage effective interactions with crucial aminoacids of the protein.	CONCL
+61	63	PA	protein	The structure of the N-acylhydrazone 23 co-crystallized with PA-Nter is important not only because confirms that the polyhydroxypheyl group efficiently coordinates two metal ions in the active site of the enzyme, but also because highlights the importance of the (flexible) inhibitor backbone in order to engage effective interactions with crucial aminoacids of the protein.	CONCL
+64	68	Nter	structure_element	The structure of the N-acylhydrazone 23 co-crystallized with PA-Nter is important not only because confirms that the polyhydroxypheyl group efficiently coordinates two metal ions in the active site of the enzyme, but also because highlights the importance of the (flexible) inhibitor backbone in order to engage effective interactions with crucial aminoacids of the protein.	CONCL
+152	163	coordinates	bond_interaction	The structure of the N-acylhydrazone 23 co-crystallized with PA-Nter is important not only because confirms that the polyhydroxypheyl group efficiently coordinates two metal ions in the active site of the enzyme, but also because highlights the importance of the (flexible) inhibitor backbone in order to engage effective interactions with crucial aminoacids of the protein.	CONCL
+168	173	metal	chemical	The structure of the N-acylhydrazone 23 co-crystallized with PA-Nter is important not only because confirms that the polyhydroxypheyl group efficiently coordinates two metal ions in the active site of the enzyme, but also because highlights the importance of the (flexible) inhibitor backbone in order to engage effective interactions with crucial aminoacids of the protein.	CONCL
+186	197	active site	site	The structure of the N-acylhydrazone 23 co-crystallized with PA-Nter is important not only because confirms that the polyhydroxypheyl group efficiently coordinates two metal ions in the active site of the enzyme, but also because highlights the importance of the (flexible) inhibitor backbone in order to engage effective interactions with crucial aminoacids of the protein.	CONCL
+18	30	endonuclease	protein_type	Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase could represent another example, after carbonic anhydrase, histone deacetylase, and HIV-1 integrase, of metal binding as a successful strategy in drug design.	CONCL
+43	52	influenza	taxonomy_domain	Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase could represent another example, after carbonic anhydrase, histone deacetylase, and HIV-1 integrase, of metal binding as a successful strategy in drug design.	CONCL
+53	81	RNA-dependent RNA polymerase	protein_type	Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase could represent another example, after carbonic anhydrase, histone deacetylase, and HIV-1 integrase, of metal binding as a successful strategy in drug design.	CONCL
+121	139	carbonic anhydrase	protein_type	Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase could represent another example, after carbonic anhydrase, histone deacetylase, and HIV-1 integrase, of metal binding as a successful strategy in drug design.	CONCL
+141	160	histone deacetylase	protein_type	Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase could represent another example, after carbonic anhydrase, histone deacetylase, and HIV-1 integrase, of metal binding as a successful strategy in drug design.	CONCL
+166	171	HIV-1	species	Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase could represent another example, after carbonic anhydrase, histone deacetylase, and HIV-1 integrase, of metal binding as a successful strategy in drug design.	CONCL
+172	181	integrase	protein_type	Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase could represent another example, after carbonic anhydrase, histone deacetylase, and HIV-1 integrase, of metal binding as a successful strategy in drug design.	CONCL
+186	191	metal	chemical	Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase could represent another example, after carbonic anhydrase, histone deacetylase, and HIV-1 integrase, of metal binding as a successful strategy in drug design.	CONCL
+15	20	water	chemical	The ligand and water molecules were discarded and the hydrogens were added to the protein by Discovery Studio 2.5.	METHODS
+233	244	presence of	protein_state	One microgram of recombinant PA-Nter (residues 1–217 from the PA protein of influenza virus strain A/X-31) was incubated with 1 μg (16.7 nM) of single-stranded circular DNA plasmid M13mp18 (Bayou Biolabs, Metairie, Louisiana) in the presence of the test compounds and at a final volume of 25 μL. The assay buffer contained 50 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM β-mercaptoethanol and 1 mM MnCl2.	METHODS
+42	53	presence of	protein_state	After incubation at 37 °C for 24 h in the presence of serial dilutions of the test compounds, the ONE-Glo luciferase assay system (Promega, Madison, WI) was used to determine luciferase activity.	METHODS
+53	57	EC99	evidence	The compound concentration values causing a 2-log10 (EC99) and a 1-log10 (EC90) reduction in viral RNA (vRNA) copy number at 24 h p.i., as compared to the virus control receiving no compound, were calculated by interpolation from data of at least three experiments.	METHODS
+74	78	EC90	evidence	The compound concentration values causing a 2-log10 (EC99) and a 1-log10 (EC90) reduction in viral RNA (vRNA) copy number at 24 h p.i., as compared to the virus control receiving no compound, were calculated by interpolation from data of at least three experiments.	METHODS
+17	25	PANΔLoop	mutant	A PAN construct (PANΔLoop) with a loop (residues 51–72) deleted and replaced with GGS from A/California/04/2009 H1N1 strain was used for the crystallographic studies.	METHODS
+21	29	PANΔLoop	mutant	The apo structure of PANΔLoop (PDB ID: 5DES) was used as starting model for molecular replacement.	METHODS
+52	61	influenza	taxonomy_domain	Chemical structures of some prototype inhibitors of influenza virus endonuclease.	FIG
+62	67	virus	taxonomy_domain	Chemical structures of some prototype inhibitors of influenza virus endonuclease.	FIG
+68	80	endonuclease	protein_type	Chemical structures of some prototype inhibitors of influenza virus endonuclease.	FIG
+22	38	enzymatic assays	experimental_method	Inhibitor activity in enzymatic assays (IC50, μM) as reported in: aref., bref., cref., dref..	FIG
+40	44	IC50	evidence	Inhibitor activity in enzymatic assays (IC50, μM) as reported in: aref., bref., cref., dref..	FIG
+22	38	N-acylhydrazones	chemical	General synthesis for N-acylhydrazones 1–27 and hydrazides 28 and 29 (A).	FIG
+39	43	1–27	chemical	General synthesis for N-acylhydrazones 1–27 and hydrazides 28 and 29 (A).	FIG
+48	58	hydrazides	chemical	General synthesis for N-acylhydrazones 1–27 and hydrazides 28 and 29 (A).	FIG
+59	61	28	chemical	General synthesis for N-acylhydrazones 1–27 and hydrazides 28 and 29 (A).	FIG
+66	68	29	chemical	General synthesis for N-acylhydrazones 1–27 and hydrazides 28 and 29 (A).	FIG
+33	37	1–27	chemical	Chemical structures of compounds 1–27 (B).	FIG
+62	66	1–27	chemical	Overview of the structure-activity relationship for compounds 1–27.	FIG
+48	64	N-acylhydrazones	chemical	Scheme of possible binding modes of the studied N-acylhydrazones.	FIG
+25	44	GOLD cluster docked	experimental_method	First (A) and second (B) GOLD cluster docked solutions of compound 23 (orange and cyan, respectively) in complex with PA endonuclease.	FIG
+67	69	23	chemical	First (A) and second (B) GOLD cluster docked solutions of compound 23 (orange and cyan, respectively) in complex with PA endonuclease.	FIG
+102	117	in complex with	protein_state	First (A) and second (B) GOLD cluster docked solutions of compound 23 (orange and cyan, respectively) in complex with PA endonuclease.	FIG
+118	120	PA	protein	First (A) and second (B) GOLD cluster docked solutions of compound 23 (orange and cyan, respectively) in complex with PA endonuclease.	FIG
+121	133	endonuclease	protein_type	First (A) and second (B) GOLD cluster docked solutions of compound 23 (orange and cyan, respectively) in complex with PA endonuclease.	FIG
+20	26	pocket	site	Key residues of the pocket are presented using PyMOL [ http://www.pymol.org] and LIGPLUS [Laskowski, R. A.; Swindells, M. B. Journal of chemical information and modeling 2011, 51, 2778].	FIG
+81	88	LIGPLUS	experimental_method	Key residues of the pocket are presented using PyMOL [ http://www.pymol.org] and LIGPLUS [Laskowski, R. A.; Swindells, M. B. Journal of chemical information and modeling 2011, 51, 2778].	FIG
+20	26	pocket	site	Key residues of the pocket are presented using PyMOL [ http://www.pymol.org] and LIGPLUS [Laskowski, R. A.; Swindells, M. B. Journal of chemical information and modeling 2011, 51, 2778].	FIG
+81	88	LIGPLUS	experimental_method	Key residues of the pocket are presented using PyMOL [ http://www.pymol.org] and LIGPLUS [Laskowski, R. A.; Swindells, M. B. Journal of chemical information and modeling 2011, 51, 2778].	FIG
+0	14	Hydrogen bonds	bond_interaction	Hydrogen bonds are illustrated by dotted lines, while the divalent metal ions are shown as purple spheres.	FIG
+71	90	GOLD cluster docked	experimental_method	Schematic drawings of the interactions of the first (C) and second (D) GOLD cluster docked solutions generated using LIGPLUS.	FIG
+117	124	LIGPLUS	experimental_method	Schematic drawings of the interactions of the first (C) and second (D) GOLD cluster docked solutions generated using LIGPLUS.	FIG
+17	31	hydrogen bonds	bond_interaction	Dashed lines are hydrogen bonds and ‘eyelashes’ show residues involved in hydrophobic interactions.	FIG
+74	98	hydrophobic interactions	bond_interaction	Dashed lines are hydrogen bonds and ‘eyelashes’ show residues involved in hydrophobic interactions.	FIG
+17	31	hydrogen bonds	bond_interaction	Dashed lines are hydrogen bonds and ‘eyelashes’ show residues involved in hydrophobic interactions.	FIG
+74	98	hydrophobic interactions	bond_interaction	Dashed lines are hydrogen bonds and ‘eyelashes’ show residues involved in hydrophobic interactions.	FIG
+29	31	15	chemical	(A) Binding mode of compound 15 (orange) in complex with PA endonuclease.	FIG
+41	56	in complex with	protein_state	(A) Binding mode of compound 15 (orange) in complex with PA endonuclease.	FIG
+57	59	PA	protein	(A) Binding mode of compound 15 (orange) in complex with PA endonuclease.	FIG
+60	72	endonuclease	protein_type	(A) Binding mode of compound 15 (orange) in complex with PA endonuclease.	FIG
+0	14	Hydrogen bonds	bond_interaction	Hydrogen bonds are illustrated by dotted lines while the divalent metal ions are shown as purple spheres.	FIG
+54	56	15	chemical	(B) Schematic drawing of the interactions of compound 15 generated using LIGPLUS.	FIG
+73	80	LIGPLUS	experimental_method	(B) Schematic drawing of the interactions of compound 15 generated using LIGPLUS.	FIG
+0	17	Crystal structure	evidence	Crystal structure of PANΔLoop in complex with compound 23.	FIG
+21	29	PANΔLoop	mutant	Crystal structure of PANΔLoop in complex with compound 23.	FIG
+30	45	in complex with	protein_state	Crystal structure of PANΔLoop in complex with compound 23.	FIG
+55	57	23	chemical	Crystal structure of PANΔLoop in complex with compound 23.	FIG
+0	11	Active site	site	Active site residues are shown in sticks with green carbons, manganese atoms are shown as purple spheres and water molecules as red spheres.	FIG
+61	70	manganese	chemical	Active site residues are shown in sticks with green carbons, manganese atoms are shown as purple spheres and water molecules as red spheres.	FIG
+109	114	water	chemical	Active site residues are shown in sticks with green carbons, manganese atoms are shown as purple spheres and water molecules as red spheres.	FIG
+9	11	23	chemical	Compound 23 is shown in sticks with yellow carbons.	FIG
+0	27	2Fo-Fc electron density map	evidence	2Fo-Fc electron density map contoured at 1σ is shown as blue mesh.	FIG
+0	14	Hydrogen bonds	bond_interaction	Hydrogen bonds and metal coordination are shown with dotted lines.	FIG
+19	37	metal coordination	bond_interaction	Hydrogen bonds and metal coordination are shown with dotted lines.	FIG
+4	10	H-bond	bond_interaction	The H-bond distances from the side chain carboxyl group of Glu26 to p-OH and m-OH of the trihydroxyphenyl group of the inhibitor are 2.7 Å and 3.0 Å, respectively.	FIG
+59	64	Glu26	residue_name_number	The H-bond distances from the side chain carboxyl group of Glu26 to p-OH and m-OH of the trihydroxyphenyl group of the inhibitor are 2.7 Å and 3.0 Å, respectively.	FIG
+4	10	H-bond	bond_interaction	The H-bond distance from the side chain of Lys34 to p-OH of the trihydroxyphenyl group is 3.6 Å. The H-bond distance to the water molecule from m-OH of the galloyl moiety is 3.0 Å, which in turn is H-bonded to the side chain of Tyr130 with a distance of 2.7 Å. Crystal structure has been deposited in the RCSB Protein Data Bank with PDB ID: 5EGA.	FIG
+43	48	Lys34	residue_name_number	The H-bond distance from the side chain of Lys34 to p-OH of the trihydroxyphenyl group is 3.6 Å. The H-bond distance to the water molecule from m-OH of the galloyl moiety is 3.0 Å, which in turn is H-bonded to the side chain of Tyr130 with a distance of 2.7 Å. Crystal structure has been deposited in the RCSB Protein Data Bank with PDB ID: 5EGA.	FIG
+101	107	H-bond	bond_interaction	The H-bond distance from the side chain of Lys34 to p-OH of the trihydroxyphenyl group is 3.6 Å. The H-bond distance to the water molecule from m-OH of the galloyl moiety is 3.0 Å, which in turn is H-bonded to the side chain of Tyr130 with a distance of 2.7 Å. Crystal structure has been deposited in the RCSB Protein Data Bank with PDB ID: 5EGA.	FIG
+124	129	water	chemical	The H-bond distance from the side chain of Lys34 to p-OH of the trihydroxyphenyl group is 3.6 Å. The H-bond distance to the water molecule from m-OH of the galloyl moiety is 3.0 Å, which in turn is H-bonded to the side chain of Tyr130 with a distance of 2.7 Å. Crystal structure has been deposited in the RCSB Protein Data Bank with PDB ID: 5EGA.	FIG
+198	206	H-bonded	bond_interaction	The H-bond distance from the side chain of Lys34 to p-OH of the trihydroxyphenyl group is 3.6 Å. The H-bond distance to the water molecule from m-OH of the galloyl moiety is 3.0 Å, which in turn is H-bonded to the side chain of Tyr130 with a distance of 2.7 Å. Crystal structure has been deposited in the RCSB Protein Data Bank with PDB ID: 5EGA.	FIG
+228	234	Tyr130	residue_name_number	The H-bond distance from the side chain of Lys34 to p-OH of the trihydroxyphenyl group is 3.6 Å. The H-bond distance to the water molecule from m-OH of the galloyl moiety is 3.0 Å, which in turn is H-bonded to the side chain of Tyr130 with a distance of 2.7 Å. Crystal structure has been deposited in the RCSB Protein Data Bank with PDB ID: 5EGA.	FIG
+261	278	Crystal structure	evidence	The H-bond distance from the side chain of Lys34 to p-OH of the trihydroxyphenyl group is 3.6 Å. The H-bond distance to the water molecule from m-OH of the galloyl moiety is 3.0 Å, which in turn is H-bonded to the side chain of Tyr130 with a distance of 2.7 Å. Crystal structure has been deposited in the RCSB Protein Data Bank with PDB ID: 5EGA.	FIG
+27	43	N-acylhydrazones	chemical	Inhibitory activity of the N-acylhydrazones 1–27 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays.	TABLE
+44	48	1–27	chemical	Inhibitory activity of the N-acylhydrazones 1–27 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays.	TABLE
+53	62	hydrazide	chemical	Inhibitory activity of the N-acylhydrazones 1–27 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays.	TABLE
+63	65	28	chemical	Inhibitory activity of the N-acylhydrazones 1–27 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays.	TABLE
+73	88	enzymatic assay	experimental_method	Inhibitory activity of the N-acylhydrazones 1–27 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays.	TABLE
+94	103	influenza	taxonomy_domain	Inhibitory activity of the N-acylhydrazones 1–27 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays.	TABLE
+104	109	virus	taxonomy_domain	Inhibitory activity of the N-acylhydrazones 1–27 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays.	TABLE
+110	112	PA	protein	Inhibitory activity of the N-acylhydrazones 1–27 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays.	TABLE
+113	117	Nter	structure_element	Inhibitory activity of the N-acylhydrazones 1–27 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays.	TABLE
+118	130	endonuclease	protein_type	Inhibitory activity of the N-acylhydrazones 1–27 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays.	TABLE
+138	169	cellular influenza virus assays	experimental_method	Inhibitory activity of the N-acylhydrazones 1–27 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays.	TABLE
+0	21	"Compound	Enzyme assay"	experimental_method	"Compound	Enzyme assay with PA-Ntera	Virus yield assay in influenza virus-infected MDCK cellsb	vRNP reconstitution assay in HEK293T cellsc	 	Antiviral activity	Cytotoxicity	SId	Activity	Cytotoxicity	 	IC50	EC99	EC90	CC50	EC50	CC50	 	(1)	24	NDf	ND	ND	 	107	>200	 	(2)	>500	ND	ND	ND	 	>100	>200	 	(3)	>500	ND	ND	>200	 	5.9	48	 	(4)	>500	ND	ND	>200	 	6.3	33	 	(5)	67	>25	>25	≥146	 	2.6	10	 	(6)	>500	>50	>50	>200	 	15	14	 	(7)	54	172	100	>200	>2.0	3.2	8.9	 	(8)	>500	>12.5	>12.5	>200	 	1.9	15	 	(9)	34	16	5.3	>200	>38	5.5	>200	 	(10)	68	14	8.5	111	>13	0.40	132	 	(11)	45	30	12	>200	>17	5.6	>200	 	(12)	>500	>12.5	>12.5	>200	 	20	39	 	(13)	69	71	34	>200	>5.9	6.3	>200	 	(14)	>500	63	37	>200	>5.4	2.3	>200	 	(15)	8.9	18	7.5	≥172	≥23	14	>200	 	(16)	454	67	28	>200	>7.1	5.2	>200	 	(17)	482	21	8.1	>200	>25	7.1	>200	 	(18)	83	6.2	2.2	>200	>91	3.3	>200	 	(19)	>500	53	26	>200	>7.7	5.7	>200	 	(20)	18	35	11	>200	>18	2.2	>200	 	(21)	13	8.3	3.6	>200	>56	2.5	>200	 	(22)	75	7.4	3.4	>200	>59	0.42	>200	 	(23)	8.7	11	3.5	>200	>57	3.1	>200	 	(24)	131	58	26	>200	>7.7	25	>200	 	(25)	40	132	70	>200	>2.9	4.1	>200	 	(26)	30	36	13	>200	>15	5.5	>200	 	(27)	36	ND	ND	ND	 	21	>200	 	(28)	40	158	85	>200	>2.4	7.2	>200	 	DPBAe	5.3	ND	ND	ND	 	ND	ND	 	Ribavirin	ND	13	8.5	>200	>24	9.4	>200	 	"	TABLE
+27	29	PA	protein	"Compound	Enzyme assay with PA-Ntera	Virus yield assay in influenza virus-infected MDCK cellsb	vRNP reconstitution assay in HEK293T cellsc	 	Antiviral activity	Cytotoxicity	SId	Activity	Cytotoxicity	 	IC50	EC99	EC90	CC50	EC50	CC50	 	(1)	24	NDf	ND	ND	 	107	>200	 	(2)	>500	ND	ND	ND	 	>100	>200	 	(3)	>500	ND	ND	>200	 	5.9	48	 	(4)	>500	ND	ND	>200	 	6.3	33	 	(5)	67	>25	>25	≥146	 	2.6	10	 	(6)	>500	>50	>50	>200	 	15	14	 	(7)	54	172	100	>200	>2.0	3.2	8.9	 	(8)	>500	>12.5	>12.5	>200	 	1.9	15	 	(9)	34	16	5.3	>200	>38	5.5	>200	 	(10)	68	14	8.5	111	>13	0.40	132	 	(11)	45	30	12	>200	>17	5.6	>200	 	(12)	>500	>12.5	>12.5	>200	 	20	39	 	(13)	69	71	34	>200	>5.9	6.3	>200	 	(14)	>500	63	37	>200	>5.4	2.3	>200	 	(15)	8.9	18	7.5	≥172	≥23	14	>200	 	(16)	454	67	28	>200	>7.1	5.2	>200	 	(17)	482	21	8.1	>200	>25	7.1	>200	 	(18)	83	6.2	2.2	>200	>91	3.3	>200	 	(19)	>500	53	26	>200	>7.7	5.7	>200	 	(20)	18	35	11	>200	>18	2.2	>200	 	(21)	13	8.3	3.6	>200	>56	2.5	>200	 	(22)	75	7.4	3.4	>200	>59	0.42	>200	 	(23)	8.7	11	3.5	>200	>57	3.1	>200	 	(24)	131	58	26	>200	>7.7	25	>200	 	(25)	40	132	70	>200	>2.9	4.1	>200	 	(26)	30	36	13	>200	>15	5.5	>200	 	(27)	36	ND	ND	ND	 	21	>200	 	(28)	40	158	85	>200	>2.4	7.2	>200	 	DPBAe	5.3	ND	ND	ND	 	ND	ND	 	Ribavirin	ND	13	8.5	>200	>24	9.4	>200	 	"	TABLE
+36	53	Virus yield assay	experimental_method	"Compound	Enzyme assay with PA-Ntera	Virus yield assay in influenza virus-infected MDCK cellsb	vRNP reconstitution assay in HEK293T cellsc	 	Antiviral activity	Cytotoxicity	SId	Activity	Cytotoxicity	 	IC50	EC99	EC90	CC50	EC50	CC50	 	(1)	24	NDf	ND	ND	 	107	>200	 	(2)	>500	ND	ND	ND	 	>100	>200	 	(3)	>500	ND	ND	>200	 	5.9	48	 	(4)	>500	ND	ND	>200	 	6.3	33	 	(5)	67	>25	>25	≥146	 	2.6	10	 	(6)	>500	>50	>50	>200	 	15	14	 	(7)	54	172	100	>200	>2.0	3.2	8.9	 	(8)	>500	>12.5	>12.5	>200	 	1.9	15	 	(9)	34	16	5.3	>200	>38	5.5	>200	 	(10)	68	14	8.5	111	>13	0.40	132	 	(11)	45	30	12	>200	>17	5.6	>200	 	(12)	>500	>12.5	>12.5	>200	 	20	39	 	(13)	69	71	34	>200	>5.9	6.3	>200	 	(14)	>500	63	37	>200	>5.4	2.3	>200	 	(15)	8.9	18	7.5	≥172	≥23	14	>200	 	(16)	454	67	28	>200	>7.1	5.2	>200	 	(17)	482	21	8.1	>200	>25	7.1	>200	 	(18)	83	6.2	2.2	>200	>91	3.3	>200	 	(19)	>500	53	26	>200	>7.7	5.7	>200	 	(20)	18	35	11	>200	>18	2.2	>200	 	(21)	13	8.3	3.6	>200	>56	2.5	>200	 	(22)	75	7.4	3.4	>200	>59	0.42	>200	 	(23)	8.7	11	3.5	>200	>57	3.1	>200	 	(24)	131	58	26	>200	>7.7	25	>200	 	(25)	40	132	70	>200	>2.9	4.1	>200	 	(26)	30	36	13	>200	>15	5.5	>200	 	(27)	36	ND	ND	ND	 	21	>200	 	(28)	40	158	85	>200	>2.4	7.2	>200	 	DPBAe	5.3	ND	ND	ND	 	ND	ND	 	Ribavirin	ND	13	8.5	>200	>24	9.4	>200	 	"	TABLE
+57	66	influenza	taxonomy_domain	"Compound	Enzyme assay with PA-Ntera	Virus yield assay in influenza virus-infected MDCK cellsb	vRNP reconstitution assay in HEK293T cellsc	 	Antiviral activity	Cytotoxicity	SId	Activity	Cytotoxicity	 	IC50	EC99	EC90	CC50	EC50	CC50	 	(1)	24	NDf	ND	ND	 	107	>200	 	(2)	>500	ND	ND	ND	 	>100	>200	 	(3)	>500	ND	ND	>200	 	5.9	48	 	(4)	>500	ND	ND	>200	 	6.3	33	 	(5)	67	>25	>25	≥146	 	2.6	10	 	(6)	>500	>50	>50	>200	 	15	14	 	(7)	54	172	100	>200	>2.0	3.2	8.9	 	(8)	>500	>12.5	>12.5	>200	 	1.9	15	 	(9)	34	16	5.3	>200	>38	5.5	>200	 	(10)	68	14	8.5	111	>13	0.40	132	 	(11)	45	30	12	>200	>17	5.6	>200	 	(12)	>500	>12.5	>12.5	>200	 	20	39	 	(13)	69	71	34	>200	>5.9	6.3	>200	 	(14)	>500	63	37	>200	>5.4	2.3	>200	 	(15)	8.9	18	7.5	≥172	≥23	14	>200	 	(16)	454	67	28	>200	>7.1	5.2	>200	 	(17)	482	21	8.1	>200	>25	7.1	>200	 	(18)	83	6.2	2.2	>200	>91	3.3	>200	 	(19)	>500	53	26	>200	>7.7	5.7	>200	 	(20)	18	35	11	>200	>18	2.2	>200	 	(21)	13	8.3	3.6	>200	>56	2.5	>200	 	(22)	75	7.4	3.4	>200	>59	0.42	>200	 	(23)	8.7	11	3.5	>200	>57	3.1	>200	 	(24)	131	58	26	>200	>7.7	25	>200	 	(25)	40	132	70	>200	>2.9	4.1	>200	 	(26)	30	36	13	>200	>15	5.5	>200	 	(27)	36	ND	ND	ND	 	21	>200	 	(28)	40	158	85	>200	>2.4	7.2	>200	 	DPBAe	5.3	ND	ND	ND	 	ND	ND	 	Ribavirin	ND	13	8.5	>200	>24	9.4	>200	 	"	TABLE
+67	72	virus	taxonomy_domain	"Compound	Enzyme assay with PA-Ntera	Virus yield assay in influenza virus-infected MDCK cellsb	vRNP reconstitution assay in HEK293T cellsc	 	Antiviral activity	Cytotoxicity	SId	Activity	Cytotoxicity	 	IC50	EC99	EC90	CC50	EC50	CC50	 	(1)	24	NDf	ND	ND	 	107	>200	 	(2)	>500	ND	ND	ND	 	>100	>200	 	(3)	>500	ND	ND	>200	 	5.9	48	 	(4)	>500	ND	ND	>200	 	6.3	33	 	(5)	67	>25	>25	≥146	 	2.6	10	 	(6)	>500	>50	>50	>200	 	15	14	 	(7)	54	172	100	>200	>2.0	3.2	8.9	 	(8)	>500	>12.5	>12.5	>200	 	1.9	15	 	(9)	34	16	5.3	>200	>38	5.5	>200	 	(10)	68	14	8.5	111	>13	0.40	132	 	(11)	45	30	12	>200	>17	5.6	>200	 	(12)	>500	>12.5	>12.5	>200	 	20	39	 	(13)	69	71	34	>200	>5.9	6.3	>200	 	(14)	>500	63	37	>200	>5.4	2.3	>200	 	(15)	8.9	18	7.5	≥172	≥23	14	>200	 	(16)	454	67	28	>200	>7.1	5.2	>200	 	(17)	482	21	8.1	>200	>25	7.1	>200	 	(18)	83	6.2	2.2	>200	>91	3.3	>200	 	(19)	>500	53	26	>200	>7.7	5.7	>200	 	(20)	18	35	11	>200	>18	2.2	>200	 	(21)	13	8.3	3.6	>200	>56	2.5	>200	 	(22)	75	7.4	3.4	>200	>59	0.42	>200	 	(23)	8.7	11	3.5	>200	>57	3.1	>200	 	(24)	131	58	26	>200	>7.7	25	>200	 	(25)	40	132	70	>200	>2.9	4.1	>200	 	(26)	30	36	13	>200	>15	5.5	>200	 	(27)	36	ND	ND	ND	 	21	>200	 	(28)	40	158	85	>200	>2.4	7.2	>200	 	DPBAe	5.3	ND	ND	ND	 	ND	ND	 	Ribavirin	ND	13	8.5	>200	>24	9.4	>200	 	"	TABLE
+94	119	vRNP reconstitution assay	experimental_method	"Compound	Enzyme assay with PA-Ntera	Virus yield assay in influenza virus-infected MDCK cellsb	vRNP reconstitution assay in HEK293T cellsc	 	Antiviral activity	Cytotoxicity	SId	Activity	Cytotoxicity	 	IC50	EC99	EC90	CC50	EC50	CC50	 	(1)	24	NDf	ND	ND	 	107	>200	 	(2)	>500	ND	ND	ND	 	>100	>200	 	(3)	>500	ND	ND	>200	 	5.9	48	 	(4)	>500	ND	ND	>200	 	6.3	33	 	(5)	67	>25	>25	≥146	 	2.6	10	 	(6)	>500	>50	>50	>200	 	15	14	 	(7)	54	172	100	>200	>2.0	3.2	8.9	 	(8)	>500	>12.5	>12.5	>200	 	1.9	15	 	(9)	34	16	5.3	>200	>38	5.5	>200	 	(10)	68	14	8.5	111	>13	0.40	132	 	(11)	45	30	12	>200	>17	5.6	>200	 	(12)	>500	>12.5	>12.5	>200	 	20	39	 	(13)	69	71	34	>200	>5.9	6.3	>200	 	(14)	>500	63	37	>200	>5.4	2.3	>200	 	(15)	8.9	18	7.5	≥172	≥23	14	>200	 	(16)	454	67	28	>200	>7.1	5.2	>200	 	(17)	482	21	8.1	>200	>25	7.1	>200	 	(18)	83	6.2	2.2	>200	>91	3.3	>200	 	(19)	>500	53	26	>200	>7.7	5.7	>200	 	(20)	18	35	11	>200	>18	2.2	>200	 	(21)	13	8.3	3.6	>200	>56	2.5	>200	 	(22)	75	7.4	3.4	>200	>59	0.42	>200	 	(23)	8.7	11	3.5	>200	>57	3.1	>200	 	(24)	131	58	26	>200	>7.7	25	>200	 	(25)	40	132	70	>200	>2.9	4.1	>200	 	(26)	30	36	13	>200	>15	5.5	>200	 	(27)	36	ND	ND	ND	 	21	>200	 	(28)	40	158	85	>200	>2.4	7.2	>200	 	DPBAe	5.3	ND	ND	ND	 	ND	ND	 	Ribavirin	ND	13	8.5	>200	>24	9.4	>200	 	"	TABLE
+200	204	IC50	evidence	"Compound	Enzyme assay with PA-Ntera	Virus yield assay in influenza virus-infected MDCK cellsb	vRNP reconstitution assay in HEK293T cellsc	 	Antiviral activity	Cytotoxicity	SId	Activity	Cytotoxicity	 	IC50	EC99	EC90	CC50	EC50	CC50	 	(1)	24	NDf	ND	ND	 	107	>200	 	(2)	>500	ND	ND	ND	 	>100	>200	 	(3)	>500	ND	ND	>200	 	5.9	48	 	(4)	>500	ND	ND	>200	 	6.3	33	 	(5)	67	>25	>25	≥146	 	2.6	10	 	(6)	>500	>50	>50	>200	 	15	14	 	(7)	54	172	100	>200	>2.0	3.2	8.9	 	(8)	>500	>12.5	>12.5	>200	 	1.9	15	 	(9)	34	16	5.3	>200	>38	5.5	>200	 	(10)	68	14	8.5	111	>13	0.40	132	 	(11)	45	30	12	>200	>17	5.6	>200	 	(12)	>500	>12.5	>12.5	>200	 	20	39	 	(13)	69	71	34	>200	>5.9	6.3	>200	 	(14)	>500	63	37	>200	>5.4	2.3	>200	 	(15)	8.9	18	7.5	≥172	≥23	14	>200	 	(16)	454	67	28	>200	>7.1	5.2	>200	 	(17)	482	21	8.1	>200	>25	7.1	>200	 	(18)	83	6.2	2.2	>200	>91	3.3	>200	 	(19)	>500	53	26	>200	>7.7	5.7	>200	 	(20)	18	35	11	>200	>18	2.2	>200	 	(21)	13	8.3	3.6	>200	>56	2.5	>200	 	(22)	75	7.4	3.4	>200	>59	0.42	>200	 	(23)	8.7	11	3.5	>200	>57	3.1	>200	 	(24)	131	58	26	>200	>7.7	25	>200	 	(25)	40	132	70	>200	>2.9	4.1	>200	 	(26)	30	36	13	>200	>15	5.5	>200	 	(27)	36	ND	ND	ND	 	21	>200	 	(28)	40	158	85	>200	>2.4	7.2	>200	 	DPBAe	5.3	ND	ND	ND	 	ND	ND	 	Ribavirin	ND	13	8.5	>200	>24	9.4	>200	 	"	TABLE
+205	209	EC99	evidence	"Compound	Enzyme assay with PA-Ntera	Virus yield assay in influenza virus-infected MDCK cellsb	vRNP reconstitution assay in HEK293T cellsc	 	Antiviral activity	Cytotoxicity	SId	Activity	Cytotoxicity	 	IC50	EC99	EC90	CC50	EC50	CC50	 	(1)	24	NDf	ND	ND	 	107	>200	 	(2)	>500	ND	ND	ND	 	>100	>200	 	(3)	>500	ND	ND	>200	 	5.9	48	 	(4)	>500	ND	ND	>200	 	6.3	33	 	(5)	67	>25	>25	≥146	 	2.6	10	 	(6)	>500	>50	>50	>200	 	15	14	 	(7)	54	172	100	>200	>2.0	3.2	8.9	 	(8)	>500	>12.5	>12.5	>200	 	1.9	15	 	(9)	34	16	5.3	>200	>38	5.5	>200	 	(10)	68	14	8.5	111	>13	0.40	132	 	(11)	45	30	12	>200	>17	5.6	>200	 	(12)	>500	>12.5	>12.5	>200	 	20	39	 	(13)	69	71	34	>200	>5.9	6.3	>200	 	(14)	>500	63	37	>200	>5.4	2.3	>200	 	(15)	8.9	18	7.5	≥172	≥23	14	>200	 	(16)	454	67	28	>200	>7.1	5.2	>200	 	(17)	482	21	8.1	>200	>25	7.1	>200	 	(18)	83	6.2	2.2	>200	>91	3.3	>200	 	(19)	>500	53	26	>200	>7.7	5.7	>200	 	(20)	18	35	11	>200	>18	2.2	>200	 	(21)	13	8.3	3.6	>200	>56	2.5	>200	 	(22)	75	7.4	3.4	>200	>59	0.42	>200	 	(23)	8.7	11	3.5	>200	>57	3.1	>200	 	(24)	131	58	26	>200	>7.7	25	>200	 	(25)	40	132	70	>200	>2.9	4.1	>200	 	(26)	30	36	13	>200	>15	5.5	>200	 	(27)	36	ND	ND	ND	 	21	>200	 	(28)	40	158	85	>200	>2.4	7.2	>200	 	DPBAe	5.3	ND	ND	ND	 	ND	ND	 	Ribavirin	ND	13	8.5	>200	>24	9.4	>200	 	"	TABLE
+210	214	EC90	evidence	"Compound	Enzyme assay with PA-Ntera	Virus yield assay in influenza virus-infected MDCK cellsb	vRNP reconstitution assay in HEK293T cellsc	 	Antiviral activity	Cytotoxicity	SId	Activity	Cytotoxicity	 	IC50	EC99	EC90	CC50	EC50	CC50	 	(1)	24	NDf	ND	ND	 	107	>200	 	(2)	>500	ND	ND	ND	 	>100	>200	 	(3)	>500	ND	ND	>200	 	5.9	48	 	(4)	>500	ND	ND	>200	 	6.3	33	 	(5)	67	>25	>25	≥146	 	2.6	10	 	(6)	>500	>50	>50	>200	 	15	14	 	(7)	54	172	100	>200	>2.0	3.2	8.9	 	(8)	>500	>12.5	>12.5	>200	 	1.9	15	 	(9)	34	16	5.3	>200	>38	5.5	>200	 	(10)	68	14	8.5	111	>13	0.40	132	 	(11)	45	30	12	>200	>17	5.6	>200	 	(12)	>500	>12.5	>12.5	>200	 	20	39	 	(13)	69	71	34	>200	>5.9	6.3	>200	 	(14)	>500	63	37	>200	>5.4	2.3	>200	 	(15)	8.9	18	7.5	≥172	≥23	14	>200	 	(16)	454	67	28	>200	>7.1	5.2	>200	 	(17)	482	21	8.1	>200	>25	7.1	>200	 	(18)	83	6.2	2.2	>200	>91	3.3	>200	 	(19)	>500	53	26	>200	>7.7	5.7	>200	 	(20)	18	35	11	>200	>18	2.2	>200	 	(21)	13	8.3	3.6	>200	>56	2.5	>200	 	(22)	75	7.4	3.4	>200	>59	0.42	>200	 	(23)	8.7	11	3.5	>200	>57	3.1	>200	 	(24)	131	58	26	>200	>7.7	25	>200	 	(25)	40	132	70	>200	>2.9	4.1	>200	 	(26)	30	36	13	>200	>15	5.5	>200	 	(27)	36	ND	ND	ND	 	21	>200	 	(28)	40	158	85	>200	>2.4	7.2	>200	 	DPBAe	5.3	ND	ND	ND	 	ND	ND	 	Ribavirin	ND	13	8.5	>200	>24	9.4	>200	 	"	TABLE
+215	219	CC50	evidence	"Compound	Enzyme assay with PA-Ntera	Virus yield assay in influenza virus-infected MDCK cellsb	vRNP reconstitution assay in HEK293T cellsc	 	Antiviral activity	Cytotoxicity	SId	Activity	Cytotoxicity	 	IC50	EC99	EC90	CC50	EC50	CC50	 	(1)	24	NDf	ND	ND	 	107	>200	 	(2)	>500	ND	ND	ND	 	>100	>200	 	(3)	>500	ND	ND	>200	 	5.9	48	 	(4)	>500	ND	ND	>200	 	6.3	33	 	(5)	67	>25	>25	≥146	 	2.6	10	 	(6)	>500	>50	>50	>200	 	15	14	 	(7)	54	172	100	>200	>2.0	3.2	8.9	 	(8)	>500	>12.5	>12.5	>200	 	1.9	15	 	(9)	34	16	5.3	>200	>38	5.5	>200	 	(10)	68	14	8.5	111	>13	0.40	132	 	(11)	45	30	12	>200	>17	5.6	>200	 	(12)	>500	>12.5	>12.5	>200	 	20	39	 	(13)	69	71	34	>200	>5.9	6.3	>200	 	(14)	>500	63	37	>200	>5.4	2.3	>200	 	(15)	8.9	18	7.5	≥172	≥23	14	>200	 	(16)	454	67	28	>200	>7.1	5.2	>200	 	(17)	482	21	8.1	>200	>25	7.1	>200	 	(18)	83	6.2	2.2	>200	>91	3.3	>200	 	(19)	>500	53	26	>200	>7.7	5.7	>200	 	(20)	18	35	11	>200	>18	2.2	>200	 	(21)	13	8.3	3.6	>200	>56	2.5	>200	 	(22)	75	7.4	3.4	>200	>59	0.42	>200	 	(23)	8.7	11	3.5	>200	>57	3.1	>200	 	(24)	131	58	26	>200	>7.7	25	>200	 	(25)	40	132	70	>200	>2.9	4.1	>200	 	(26)	30	36	13	>200	>15	5.5	>200	 	(27)	36	ND	ND	ND	 	21	>200	 	(28)	40	158	85	>200	>2.4	7.2	>200	 	DPBAe	5.3	ND	ND	ND	 	ND	ND	 	Ribavirin	ND	13	8.5	>200	>24	9.4	>200	 	"	TABLE
+220	224	EC50	evidence	"Compound	Enzyme assay with PA-Ntera	Virus yield assay in influenza virus-infected MDCK cellsb	vRNP reconstitution assay in HEK293T cellsc	 	Antiviral activity	Cytotoxicity	SId	Activity	Cytotoxicity	 	IC50	EC99	EC90	CC50	EC50	CC50	 	(1)	24	NDf	ND	ND	 	107	>200	 	(2)	>500	ND	ND	ND	 	>100	>200	 	(3)	>500	ND	ND	>200	 	5.9	48	 	(4)	>500	ND	ND	>200	 	6.3	33	 	(5)	67	>25	>25	≥146	 	2.6	10	 	(6)	>500	>50	>50	>200	 	15	14	 	(7)	54	172	100	>200	>2.0	3.2	8.9	 	(8)	>500	>12.5	>12.5	>200	 	1.9	15	 	(9)	34	16	5.3	>200	>38	5.5	>200	 	(10)	68	14	8.5	111	>13	0.40	132	 	(11)	45	30	12	>200	>17	5.6	>200	 	(12)	>500	>12.5	>12.5	>200	 	20	39	 	(13)	69	71	34	>200	>5.9	6.3	>200	 	(14)	>500	63	37	>200	>5.4	2.3	>200	 	(15)	8.9	18	7.5	≥172	≥23	14	>200	 	(16)	454	67	28	>200	>7.1	5.2	>200	 	(17)	482	21	8.1	>200	>25	7.1	>200	 	(18)	83	6.2	2.2	>200	>91	3.3	>200	 	(19)	>500	53	26	>200	>7.7	5.7	>200	 	(20)	18	35	11	>200	>18	2.2	>200	 	(21)	13	8.3	3.6	>200	>56	2.5	>200	 	(22)	75	7.4	3.4	>200	>59	0.42	>200	 	(23)	8.7	11	3.5	>200	>57	3.1	>200	 	(24)	131	58	26	>200	>7.7	25	>200	 	(25)	40	132	70	>200	>2.9	4.1	>200	 	(26)	30	36	13	>200	>15	5.5	>200	 	(27)	36	ND	ND	ND	 	21	>200	 	(28)	40	158	85	>200	>2.4	7.2	>200	 	DPBAe	5.3	ND	ND	ND	 	ND	ND	 	Ribavirin	ND	13	8.5	>200	>24	9.4	>200	 	"	TABLE
+225	229	CC50	evidence	"Compound	Enzyme assay with PA-Ntera	Virus yield assay in influenza virus-infected MDCK cellsb	vRNP reconstitution assay in HEK293T cellsc	 	Antiviral activity	Cytotoxicity	SId	Activity	Cytotoxicity	 	IC50	EC99	EC90	CC50	EC50	CC50	 	(1)	24	NDf	ND	ND	 	107	>200	 	(2)	>500	ND	ND	ND	 	>100	>200	 	(3)	>500	ND	ND	>200	 	5.9	48	 	(4)	>500	ND	ND	>200	 	6.3	33	 	(5)	67	>25	>25	≥146	 	2.6	10	 	(6)	>500	>50	>50	>200	 	15	14	 	(7)	54	172	100	>200	>2.0	3.2	8.9	 	(8)	>500	>12.5	>12.5	>200	 	1.9	15	 	(9)	34	16	5.3	>200	>38	5.5	>200	 	(10)	68	14	8.5	111	>13	0.40	132	 	(11)	45	30	12	>200	>17	5.6	>200	 	(12)	>500	>12.5	>12.5	>200	 	20	39	 	(13)	69	71	34	>200	>5.9	6.3	>200	 	(14)	>500	63	37	>200	>5.4	2.3	>200	 	(15)	8.9	18	7.5	≥172	≥23	14	>200	 	(16)	454	67	28	>200	>7.1	5.2	>200	 	(17)	482	21	8.1	>200	>25	7.1	>200	 	(18)	83	6.2	2.2	>200	>91	3.3	>200	 	(19)	>500	53	26	>200	>7.7	5.7	>200	 	(20)	18	35	11	>200	>18	2.2	>200	 	(21)	13	8.3	3.6	>200	>56	2.5	>200	 	(22)	75	7.4	3.4	>200	>59	0.42	>200	 	(23)	8.7	11	3.5	>200	>57	3.1	>200	 	(24)	131	58	26	>200	>7.7	25	>200	 	(25)	40	132	70	>200	>2.9	4.1	>200	 	(26)	30	36	13	>200	>15	5.5	>200	 	(27)	36	ND	ND	ND	 	21	>200	 	(28)	40	158	85	>200	>2.4	7.2	>200	 	DPBAe	5.3	ND	ND	ND	 	ND	ND	 	Ribavirin	ND	13	8.5	>200	>24	9.4	>200	 	"	TABLE
+13	15	PA	protein	aRecombinant PA-Nter was incubated with the ssDNA plasmid substrate, a Mn2+-containing buffer and test compounds.	TABLE
+16	20	Nter	structure_element	aRecombinant PA-Nter was incubated with the ssDNA plasmid substrate, a Mn2+-containing buffer and test compounds.	TABLE
+25	34	incubated	experimental_method	aRecombinant PA-Nter was incubated with the ssDNA plasmid substrate, a Mn2+-containing buffer and test compounds.	TABLE
+44	49	ssDNA	chemical	aRecombinant PA-Nter was incubated with the ssDNA plasmid substrate, a Mn2+-containing buffer and test compounds.	TABLE
+71	75	Mn2+	chemical	aRecombinant PA-Nter was incubated with the ssDNA plasmid substrate, a Mn2+-containing buffer and test compounds.	TABLE
+4	8	IC50	evidence	The IC50 represents the compound concentration (in μM) required to obtain 50% inhibition of cleavage, calculated by nonlinear least-squares regression analysis (using GraphPad Prism software) of the results from 2–4 independent experiments.	TABLE
+116	159	nonlinear least-squares regression analysis	experimental_method	The IC50 represents the compound concentration (in μM) required to obtain 50% inhibition of cleavage, calculated by nonlinear least-squares regression analysis (using GraphPad Prism software) of the results from 2–4 independent experiments.	TABLE
+31	42	influenza A	taxonomy_domain	bMDCK cells were infected with influenza A virus (strain A/PR/8/34) and incubated with the compounds during 24 h. The virus yield in the supernatant was assessed by real-time qPCR.	TABLE
+43	48	virus	taxonomy_domain	bMDCK cells were infected with influenza A virus (strain A/PR/8/34) and incubated with the compounds during 24 h. The virus yield in the supernatant was assessed by real-time qPCR.	TABLE
+118	123	virus	taxonomy_domain	bMDCK cells were infected with influenza A virus (strain A/PR/8/34) and incubated with the compounds during 24 h. The virus yield in the supernatant was assessed by real-time qPCR.	TABLE
+165	179	real-time qPCR	experimental_method	bMDCK cells were infected with influenza A virus (strain A/PR/8/34) and incubated with the compounds during 24 h. The virus yield in the supernatant was assessed by real-time qPCR.	TABLE
+4	8	EC99	evidence	The EC99 and EC90 values represent the compound concentrations (in μM) producing a 2-log10 or 1-log10 reduction in virus titer, respectively, determined in 2–3 independent experiments.	TABLE
+13	17	EC90	evidence	The EC99 and EC90 values represent the compound concentrations (in μM) producing a 2-log10 or 1-log10 reduction in virus titer, respectively, determined in 2–3 independent experiments.	TABLE
+115	120	virus	taxonomy_domain	The EC99 and EC90 values represent the compound concentrations (in μM) producing a 2-log10 or 1-log10 reduction in virus titer, respectively, determined in 2–3 independent experiments.	TABLE
+74	78	CC50	evidence	The cytotoxicity, assessed in uninfected MDCK cells, was expressed as the CC50 value (50% cytotoxic concentration, determined with the MTS cell viability assay, in μM).	TABLE
+135	159	MTS cell viability assay	experimental_method	The cytotoxicity, assessed in uninfected MDCK cells, was expressed as the CC50 value (50% cytotoxic concentration, determined with the MTS cell viability assay, in μM).	TABLE
+20	34	co-transfected	experimental_method	cHEK293T cells were co-transfected with the four vRNP-reconstituting plasmids and the luciferase reporter plasmid in the presence of the test compounds.	TABLE
+49	53	vRNP	complex_assembly	cHEK293T cells were co-transfected with the four vRNP-reconstituting plasmids and the luciferase reporter plasmid in the presence of the test compounds.	TABLE
+121	132	presence of	protein_state	cHEK293T cells were co-transfected with the four vRNP-reconstituting plasmids and the luciferase reporter plasmid in the presence of the test compounds.	TABLE
+4	8	EC50	evidence	The EC50 represents the compound concentration (in μM) producing 50% reduction in vRNP-driven firefly reporter signal, estimated at 24 h after transfection.	TABLE
+82	86	vRNP	complex_assembly	The EC50 represents the compound concentration (in μM) producing 50% reduction in vRNP-driven firefly reporter signal, estimated at 24 h after transfection.	TABLE
+4	8	EC50	evidence	The EC50 value was derived from data from 2–4 independent experiments, by nonlinear least-squares regression analysis (using GraphPad Prism software).	TABLE
+74	117	nonlinear least-squares regression analysis	experimental_method	The EC50 value was derived from data from 2–4 independent experiments, by nonlinear least-squares regression analysis (using GraphPad Prism software).	TABLE
+4	8	CC50	evidence	The CC50 (in μM), i.e. the 50% cytotoxic concentration, was determined in untransfected HEK293T cells by MTS cell viability assay.	TABLE
+105	129	MTS cell viability assay	experimental_method	The CC50 (in μM), i.e. the 50% cytotoxic concentration, was determined in untransfected HEK293T cells by MTS cell viability assay.	TABLE
+0	3	dSI	evidence	dSI, selectivity index, defined as the ratio between the CC50 and EC90.	TABLE
+5	22	selectivity index	evidence	dSI, selectivity index, defined as the ratio between the CC50 and EC90.	TABLE
+57	61	CC50	evidence	dSI, selectivity index, defined as the ratio between the CC50 and EC90.	TABLE
+66	70	EC90	evidence	dSI, selectivity index, defined as the ratio between the CC50 and EC90.	TABLE
+0	5	eDPBA	chemical	eDPBA, 2,4-dioxo-4-phenylbutanoic acid.	TABLE
+7	38	2,4-dioxo-4-phenylbutanoic acid	chemical	eDPBA, 2,4-dioxo-4-phenylbutanoic acid.	TABLE
diff --git a/annotation_CSV/PMC4981400.csv b/annotation_CSV/PMC4981400.csv
new file mode 100644
index 0000000000000000000000000000000000000000..ba0ef07a39621e330afd153cafd2e62428308a6b
--- /dev/null
+++ b/annotation_CSV/PMC4981400.csv
@@ -0,0 +1,571 @@
+anno_start	anno_end	anno_text	entity_type	sentence	section
+0	17	Crystal Structure	evidence	Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA	TITLE
+25	29	SPOC	structure_element	Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA	TITLE
+44	55	Arabidopsis	taxonomy_domain	Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA	TITLE
+56	75	Flowering Regulator	protein_type	Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA	TITLE
+76	79	FPA	protein	Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA	TITLE
+4	15	Arabidopsis	taxonomy_domain	The Arabidopsis protein FPA controls flowering time by regulating the alternative 3′-end processing of the FLOWERING LOCUS (FLC) antisense RNA.	ABSTRACT
+24	27	FPA	protein	The Arabidopsis protein FPA controls flowering time by regulating the alternative 3′-end processing of the FLOWERING LOCUS (FLC) antisense RNA.	ABSTRACT
+107	122	FLOWERING LOCUS	gene	The Arabidopsis protein FPA controls flowering time by regulating the alternative 3′-end processing of the FLOWERING LOCUS (FLC) antisense RNA.	ABSTRACT
+124	127	FLC	gene	The Arabidopsis protein FPA controls flowering time by regulating the alternative 3′-end processing of the FLOWERING LOCUS (FLC) antisense RNA.	ABSTRACT
+129	142	antisense RNA	chemical	The Arabidopsis protein FPA controls flowering time by regulating the alternative 3′-end processing of the FLOWERING LOCUS (FLC) antisense RNA.	ABSTRACT
+0	3	FPA	protein	FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain.	ABSTRACT
+19	29	split ends	protein_type	FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain.	ABSTRACT
+31	35	SPEN	protein_type	FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain.	ABSTRACT
+82	104	RNA recognition motifs	structure_element	FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain.	ABSTRACT
+106	110	RRMs	structure_element	FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain.	ABSTRACT
+118	154	SPEN paralog and ortholog C-terminal	structure_element	FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain.	ABSTRACT
+156	160	SPOC	structure_element	FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain.	ABSTRACT
+4	8	SPOC	structure_element	The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower.	ABSTRACT
+19	35	highly conserved	protein_state	The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower.	ABSTRACT
+42	45	FPA	protein	The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower.	ABSTRACT
+58	64	plants	taxonomy_domain	The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower.	ABSTRACT
+112	116	SPEN	protein_type	The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower.	ABSTRACT
+23	40	crystal structure	evidence	We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution.	ABSTRACT
+44	64	Arabidopsis thaliana	species	We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution.	ABSTRACT
+65	68	FPA	protein	We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution.	ABSTRACT
+69	73	SPOC	structure_element	We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution.	ABSTRACT
+12	21	structure	evidence	The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them.	ABSTRACT
+48	52	SPOC	structure_element	The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them.	ABSTRACT
+63	68	human	species	The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them.	ABSTRACT
+69	108	SMRT/HDAC1 Associated Repressor Protein	protein	The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them.	ABSTRACT
+110	115	SHARP	protein	The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them.	ABSTRACT
+0	32	Structural and sequence analyses	experimental_method	Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs.	ABSTRACT
+44	57	surface patch	site	Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs.	ABSTRACT
+66	75	conserved	protein_state	Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs.	ABSTRACT
+82	87	plant	taxonomy_domain	Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs.	ABSTRACT
+88	91	FPA	protein	Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs.	ABSTRACT
+0	9	Mutations	experimental_method	Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.	ABSTRACT
+34	47	surface patch	site	Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.	ABSTRACT
+64	67	FPA	protein	Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.	ABSTRACT
+106	110	SPOC	structure_element	Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.	ABSTRACT
+150	153	FPA	protein	Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.	ABSTRACT
+168	171	RNA	chemical	Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.	ABSTRACT
+213	216	FPA	protein	Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.	ABSTRACT
+217	221	SPOC	structure_element	Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.	ABSTRACT
+319	324	SHARP	protein	Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.	ABSTRACT
+325	329	SPOC	structure_element	Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.	ABSTRACT
+0	10	Eukaryotic	taxonomy_domain	Eukaryotic messenger RNAs (mRNAs) are made as precursors through transcription by RNA polymerase II (Pol II), and these primary transcripts undergo extensive processing, including 3′-end cleavage and polyadenylation.	INTRO
+11	25	messenger RNAs	chemical	Eukaryotic messenger RNAs (mRNAs) are made as precursors through transcription by RNA polymerase II (Pol II), and these primary transcripts undergo extensive processing, including 3′-end cleavage and polyadenylation.	INTRO
+27	32	mRNAs	chemical	Eukaryotic messenger RNAs (mRNAs) are made as precursors through transcription by RNA polymerase II (Pol II), and these primary transcripts undergo extensive processing, including 3′-end cleavage and polyadenylation.	INTRO
+82	99	RNA polymerase II	complex_assembly	Eukaryotic messenger RNAs (mRNAs) are made as precursors through transcription by RNA polymerase II (Pol II), and these primary transcripts undergo extensive processing, including 3′-end cleavage and polyadenylation.	INTRO
+101	107	Pol II	complex_assembly	Eukaryotic messenger RNAs (mRNAs) are made as precursors through transcription by RNA polymerase II (Pol II), and these primary transcripts undergo extensive processing, including 3′-end cleavage and polyadenylation.	INTRO
+103	113	eukaryotes	taxonomy_domain	In addition, alternative 3′-end cleavage and polyadenylation is an essential and ubiquitous process in eukaryotes.	INTRO
+14	24	split ends	protein_type	Recently, the split ends (SPEN) family of proteins was identified as RNA binding proteins that regulate alternative 3′-end cleavage and polyadenylation.	INTRO
+26	30	SPEN	protein_type	Recently, the split ends (SPEN) family of proteins was identified as RNA binding proteins that regulate alternative 3′-end cleavage and polyadenylation.	INTRO
+69	89	RNA binding proteins	protein_type	Recently, the split ends (SPEN) family of proteins was identified as RNA binding proteins that regulate alternative 3′-end cleavage and polyadenylation.	INTRO
+48	70	RNA recognition motifs	structure_element	They are characterized by possessing N-terminal RNA recognition motifs (RRMs) and a conserved SPEN paralog and ortholog C-terminal (SPOC) domain (Fig 1A).	INTRO
+72	76	RRMs	structure_element	They are characterized by possessing N-terminal RNA recognition motifs (RRMs) and a conserved SPEN paralog and ortholog C-terminal (SPOC) domain (Fig 1A).	INTRO
+84	93	conserved	protein_state	They are characterized by possessing N-terminal RNA recognition motifs (RRMs) and a conserved SPEN paralog and ortholog C-terminal (SPOC) domain (Fig 1A).	INTRO
+94	130	SPEN paralog and ortholog C-terminal	structure_element	They are characterized by possessing N-terminal RNA recognition motifs (RRMs) and a conserved SPEN paralog and ortholog C-terminal (SPOC) domain (Fig 1A).	INTRO
+132	136	SPOC	structure_element	They are characterized by possessing N-terminal RNA recognition motifs (RRMs) and a conserved SPEN paralog and ortholog C-terminal (SPOC) domain (Fig 1A).	INTRO
+4	8	SPOC	structure_element	The SPOC domain is believed to mediate protein-protein interactions and has diverse functions among SPEN family proteins, but the molecular mechanism of these functions is not well understood.	INTRO
+100	104	SPEN	protein_type	The SPOC domain is believed to mediate protein-protein interactions and has diverse functions among SPEN family proteins, but the molecular mechanism of these functions is not well understood.	INTRO
+0	21	Sequence conservation	evidence	Sequence conservation of SPOC domains.	FIG
+25	29	SPOC	structure_element	Sequence conservation of SPOC domains.	FIG
+23	34	A. thaliana	species	Domain organization of A. thaliana FPA. (B).	FIG
+35	38	FPA	protein	Domain organization of A. thaliana FPA. (B).	FIG
+0	18	Sequence alignment	experimental_method	Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP.	FIG
+26	30	SPOC	structure_element	Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP.	FIG
+42	62	Arabidopsis thaliana	species	Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP.	FIG
+63	66	FPA	protein	Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP.	FIG
+68	73	human	species	Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP.	FIG
+74	79	RBM15	protein	Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP.	FIG
+81	91	Drosophila	taxonomy_domain	Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP.	FIG
+92	96	SPEN	protein_type	Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP.	FIG
+98	103	mouse	taxonomy_domain	Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP.	FIG
+104	108	MINT	protein	Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP.	FIG
+114	119	human	species	Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP.	FIG
+120	125	SHARP	protein	Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP.	FIG
+12	27	surface patch 1	site	Residues in surface patch 1 are indicated with the orange dots, and those in surface patch 2 with the green dots.	FIG
+77	92	surface patch 2	site	Residues in surface patch 1 are indicated with the orange dots, and those in surface patch 2 with the green dots.	FIG
+40	49	structure	evidence	The secondary structure elements in the structure of FPA SPOC are labeled.	FIG
+53	56	FPA	protein	The secondary structure elements in the structure of FPA SPOC are labeled.	FIG
+57	61	SPOC	structure_element	The secondary structure elements in the structure of FPA SPOC are labeled.	FIG
+18	36	strictly conserved	protein_state	Residues that are strictly conserved among the five proteins are shown in white with a red background, and those that are mostly conserved in red.	FIG
+122	138	mostly conserved	protein_state	Residues that are strictly conserved among the five proteins are shown in white with a red background, and those that are mostly conserved in red.	FIG
+0	3	FPA	protein	FPA, a SPEN family protein in Arabidopsis thaliana and other plants, was found to regulate the 3′-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene.	INTRO
+7	11	SPEN	protein_type	FPA, a SPEN family protein in Arabidopsis thaliana and other plants, was found to regulate the 3′-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene.	INTRO
+30	50	Arabidopsis thaliana	species	FPA, a SPEN family protein in Arabidopsis thaliana and other plants, was found to regulate the 3′-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene.	INTRO
+61	67	plants	taxonomy_domain	FPA, a SPEN family protein in Arabidopsis thaliana and other plants, was found to regulate the 3′-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene.	INTRO
+150	164	antisense RNAs	chemical	FPA, a SPEN family protein in Arabidopsis thaliana and other plants, was found to regulate the 3′-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene.	INTRO
+168	183	FLOWERING LOCUS	gene	FPA, a SPEN family protein in Arabidopsis thaliana and other plants, was found to regulate the 3′-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene.	INTRO
+185	188	FLC	gene	FPA, a SPEN family protein in Arabidopsis thaliana and other plants, was found to regulate the 3′-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene.	INTRO
+0	3	FPA	protein	FPA promotes the 3′-end processing of class I FLC antisense RNAs, which includes the proximal polyadenylation site.	INTRO
+46	49	FLC	gene	FPA promotes the 3′-end processing of class I FLC antisense RNAs, which includes the proximal polyadenylation site.	INTRO
+50	64	antisense RNAs	chemical	FPA promotes the 3′-end processing of class I FLC antisense RNAs, which includes the proximal polyadenylation site.	INTRO
+94	114	polyadenylation site	site	FPA promotes the 3′-end processing of class I FLC antisense RNAs, which includes the proximal polyadenylation site.	INTRO
+24	43	histone demethylase	protein_type	This is associated with histone demethylase activity and down-regulation of FLC transcription.	INTRO
+76	79	FLC	gene	This is associated with histone demethylase activity and down-regulation of FLC transcription.	INTRO
+11	15	SPOC	structure_element	Although a SPOC domain is found in all the SPEN family proteins, its sequence conservation is rather low.	INTRO
+43	47	SPEN	protein_type	Although a SPOC domain is found in all the SPEN family proteins, its sequence conservation is rather low.	INTRO
+47	51	SPOC	structure_element	For example, the sequence identity between the SPOC domains of A. thaliana FPA and human SMRT/HDAC1 Associated Repressor Protein (SHARP) is only 19% (Fig 1B).	INTRO
+63	74	A. thaliana	species	For example, the sequence identity between the SPOC domains of A. thaliana FPA and human SMRT/HDAC1 Associated Repressor Protein (SHARP) is only 19% (Fig 1B).	INTRO
+75	78	FPA	protein	For example, the sequence identity between the SPOC domains of A. thaliana FPA and human SMRT/HDAC1 Associated Repressor Protein (SHARP) is only 19% (Fig 1B).	INTRO
+83	88	human	species	For example, the sequence identity between the SPOC domains of A. thaliana FPA and human SMRT/HDAC1 Associated Repressor Protein (SHARP) is only 19% (Fig 1B).	INTRO
+89	128	SMRT/HDAC1 Associated Repressor Protein	protein	For example, the sequence identity between the SPOC domains of A. thaliana FPA and human SMRT/HDAC1 Associated Repressor Protein (SHARP) is only 19% (Fig 1B).	INTRO
+130	135	SHARP	protein	For example, the sequence identity between the SPOC domains of A. thaliana FPA and human SMRT/HDAC1 Associated Repressor Protein (SHARP) is only 19% (Fig 1B).	INTRO
+15	20	SHARP	protein	Currently, the SHARP SPOC domain is the only one with structural information.	INTRO
+21	25	SPOC	structure_element	Currently, the SHARP SPOC domain is the only one with structural information.	INTRO
+126	129	FPA	protein	As a first step toward understanding the molecular basis for the regulation of alternative 3′-end processing and flowering by FPA, we have determined the crystal structure of the SPOC domain of A. thaliana FPA at 2.7 Å resolution.	INTRO
+154	171	crystal structure	evidence	As a first step toward understanding the molecular basis for the regulation of alternative 3′-end processing and flowering by FPA, we have determined the crystal structure of the SPOC domain of A. thaliana FPA at 2.7 Å resolution.	INTRO
+179	183	SPOC	structure_element	As a first step toward understanding the molecular basis for the regulation of alternative 3′-end processing and flowering by FPA, we have determined the crystal structure of the SPOC domain of A. thaliana FPA at 2.7 Å resolution.	INTRO
+194	205	A. thaliana	species	As a first step toward understanding the molecular basis for the regulation of alternative 3′-end processing and flowering by FPA, we have determined the crystal structure of the SPOC domain of A. thaliana FPA at 2.7 Å resolution.	INTRO
+206	209	FPA	protein	As a first step toward understanding the molecular basis for the regulation of alternative 3′-end processing and flowering by FPA, we have determined the crystal structure of the SPOC domain of A. thaliana FPA at 2.7 Å resolution.	INTRO
+12	21	structure	evidence	The overall structure is similar to that of the SHARP SPOC domain, although there are also substantial conformational differences between them.	INTRO
+48	53	SHARP	protein	The overall structure is similar to that of the SHARP SPOC domain, although there are also substantial conformational differences between them.	INTRO
+54	58	SPOC	structure_element	The overall structure is similar to that of the SHARP SPOC domain, although there are also substantial conformational differences between them.	INTRO
+4	13	structure	evidence	The structure reveals a surface patch that is conserved among FPA homologs.	INTRO
+24	37	surface patch	site	The structure reveals a surface patch that is conserved among FPA homologs.	INTRO
+46	55	conserved	protein_state	The structure reveals a surface patch that is conserved among FPA homologs.	INTRO
+62	65	FPA	protein	The structure reveals a surface patch that is conserved among FPA homologs.	INTRO
+0	9	Structure	evidence	Structure of FPA SPOC domain	RESULTS
+13	16	FPA	protein	Structure of FPA SPOC domain	RESULTS
+17	21	SPOC	structure_element	Structure of FPA SPOC domain	RESULTS
+4	21	crystal structure	evidence	The crystal structure of the SPOC domain of A. thaliana FPA has been determined at 2.7 Å resolution using the selenomethionyl single-wavelength anomalous dispersion method.	RESULTS
+29	33	SPOC	structure_element	The crystal structure of the SPOC domain of A. thaliana FPA has been determined at 2.7 Å resolution using the selenomethionyl single-wavelength anomalous dispersion method.	RESULTS
+44	55	A. thaliana	species	The crystal structure of the SPOC domain of A. thaliana FPA has been determined at 2.7 Å resolution using the selenomethionyl single-wavelength anomalous dispersion method.	RESULTS
+56	59	FPA	protein	The crystal structure of the SPOC domain of A. thaliana FPA has been determined at 2.7 Å resolution using the selenomethionyl single-wavelength anomalous dispersion method.	RESULTS
+110	171	selenomethionyl single-wavelength anomalous dispersion method	experimental_method	The crystal structure of the SPOC domain of A. thaliana FPA has been determined at 2.7 Å resolution using the selenomethionyl single-wavelength anomalous dispersion method.	RESULTS
+44	51	433–565	residue_range	The expression construct contained residues 433–565 of FPA, but only residues 439–460 and 465–565 are ordered in the crystal.	RESULTS
+55	58	FPA	protein	The expression construct contained residues 433–565 of FPA, but only residues 439–460 and 465–565 are ordered in the crystal.	RESULTS
+78	85	439–460	residue_range	The expression construct contained residues 433–565 of FPA, but only residues 439–460 and 465–565 are ordered in the crystal.	RESULTS
+90	97	465–565	residue_range	The expression construct contained residues 433–565 of FPA, but only residues 439–460 and 465–565 are ordered in the crystal.	RESULTS
+117	124	crystal	evidence	The expression construct contained residues 433–565 of FPA, but only residues 439–460 and 465–565 are ordered in the crystal.	RESULTS
+4	16	atomic model	evidence	The atomic model has good agreement with the X-ray diffraction data and the expected bond lengths, bond angles and other geometric parameters (Table 1).	RESULTS
+45	67	X-ray diffraction data	evidence	The atomic model has good agreement with the X-ray diffraction data and the expected bond lengths, bond angles and other geometric parameters (Table 1).	RESULTS
+59	76	Ramachandran plot	evidence	All the residues are located in the favored regions of the Ramachandran plot (data not shown).	RESULTS
+4	13	structure	evidence	The structure has been deposited in the Protein Data Bank, with accession code 5KXF.	RESULTS
+159	167	R factor	evidence	"Resolution range (Å)1	50–2.7 (2.8–2.7)	 	Number of observations	78,008	 	Rmerge (%)	10.5 (45.3)	 	I/σI	24.1 (6.3)	 	Redundancy		 	Completeness (%)	100 (100)	 	R factor (%)	19.2 (25.0)	 	Free R factor (%)	25.4 (35.4)	 	Rms deviation in bond lengths (Å)	0.017	 	Rms deviation in bond angles (°)	1.9	 	"	TABLE
+186	199	Free R factor	evidence	"Resolution range (Å)1	50–2.7 (2.8–2.7)	 	Number of observations	78,008	 	Rmerge (%)	10.5 (45.3)	 	I/σI	24.1 (6.3)	 	Redundancy		 	Completeness (%)	100 (100)	 	R factor (%)	19.2 (25.0)	 	Free R factor (%)	25.4 (35.4)	 	Rms deviation in bond lengths (Å)	0.017	 	Rms deviation in bond angles (°)	1.9	 	"	TABLE
+4	21	crystal structure	evidence	The crystal structure of the FPA SPOC domain contains a seven-stranded, mostly anti-parallel β-barrel (β1-β7) and three helices (αA-αC) (Fig 2A).	RESULTS
+29	32	FPA	protein	The crystal structure of the FPA SPOC domain contains a seven-stranded, mostly anti-parallel β-barrel (β1-β7) and three helices (αA-αC) (Fig 2A).	RESULTS
+33	37	SPOC	structure_element	The crystal structure of the FPA SPOC domain contains a seven-stranded, mostly anti-parallel β-barrel (β1-β7) and three helices (αA-αC) (Fig 2A).	RESULTS
+56	101	seven-stranded, mostly anti-parallel β-barrel	structure_element	The crystal structure of the FPA SPOC domain contains a seven-stranded, mostly anti-parallel β-barrel (β1-β7) and three helices (αA-αC) (Fig 2A).	RESULTS
+103	108	β1-β7	structure_element	The crystal structure of the FPA SPOC domain contains a seven-stranded, mostly anti-parallel β-barrel (β1-β7) and three helices (αA-αC) (Fig 2A).	RESULTS
+120	127	helices	structure_element	The crystal structure of the FPA SPOC domain contains a seven-stranded, mostly anti-parallel β-barrel (β1-β7) and three helices (αA-αC) (Fig 2A).	RESULTS
+129	134	αA-αC	structure_element	The crystal structure of the FPA SPOC domain contains a seven-stranded, mostly anti-parallel β-barrel (β1-β7) and three helices (αA-αC) (Fig 2A).	RESULTS
+28	35	strands	structure_element	Only two of the neighboring strands, β1 and β3, are parallel to each other.	RESULTS
+37	39	β1	structure_element	Only two of the neighboring strands, β1 and β3, are parallel to each other.	RESULTS
+44	46	β3	structure_element	Only two of the neighboring strands, β1 and β3, are parallel to each other.	RESULTS
+0	5	Helix	structure_element	Helix αB covers one end of the barrel, while helices αA and αC are located next to each other at one side of the barrel (Fig 2B).	RESULTS
+6	8	αB	structure_element	Helix αB covers one end of the barrel, while helices αA and αC are located next to each other at one side of the barrel (Fig 2B).	RESULTS
+31	37	barrel	structure_element	Helix αB covers one end of the barrel, while helices αA and αC are located next to each other at one side of the barrel (Fig 2B).	RESULTS
+45	52	helices	structure_element	Helix αB covers one end of the barrel, while helices αA and αC are located next to each other at one side of the barrel (Fig 2B).	RESULTS
+53	55	αA	structure_element	Helix αB covers one end of the barrel, while helices αA and αC are located next to each other at one side of the barrel (Fig 2B).	RESULTS
+60	62	αC	structure_element	Helix αB covers one end of the barrel, while helices αA and αC are located next to each other at one side of the barrel (Fig 2B).	RESULTS
+113	119	barrel	structure_element	Helix αB covers one end of the barrel, while helices αA and αC are located next to each other at one side of the barrel (Fig 2B).	RESULTS
+21	29	β-barrel	structure_element	The other end of the β-barrel is covered by the loop connecting strands β2 and β3, which contains the disordered 461–464 segment.	RESULTS
+48	52	loop	structure_element	The other end of the β-barrel is covered by the loop connecting strands β2 and β3, which contains the disordered 461–464 segment.	RESULTS
+64	71	strands	structure_element	The other end of the β-barrel is covered by the loop connecting strands β2 and β3, which contains the disordered 461–464 segment.	RESULTS
+72	74	β2	structure_element	The other end of the β-barrel is covered by the loop connecting strands β2 and β3, which contains the disordered 461–464 segment.	RESULTS
+79	81	β3	structure_element	The other end of the β-barrel is covered by the loop connecting strands β2 and β3, which contains the disordered 461–464 segment.	RESULTS
+102	112	disordered	protein_state	The other end of the β-barrel is covered by the loop connecting strands β2 and β3, which contains the disordered 461–464 segment.	RESULTS
+113	120	461–464	residue_range	The other end of the β-barrel is covered by the loop connecting strands β2 and β3, which contains the disordered 461–464 segment.	RESULTS
+18	24	barrel	structure_element	The center of the barrel is filled with hydrophobic side chains and is not accessible to the solvent.	RESULTS
+0	17	Crystal structure	evidence	Crystal structure of the SPOC domain of A. thaliana FPA.	FIG
+25	29	SPOC	structure_element	Crystal structure of the SPOC domain of A. thaliana FPA.	FIG
+40	51	A. thaliana	species	Crystal structure of the SPOC domain of A. thaliana FPA.	FIG
+52	55	FPA	protein	Crystal structure of the SPOC domain of A. thaliana FPA.	FIG
+25	34	structure	evidence	Schematic drawing of the structure of FPA SPOC domain, colored from blue at the N terminus to red at the C terminus.	FIG
+38	41	FPA	protein	Schematic drawing of the structure of FPA SPOC domain, colored from blue at the N terminus to red at the C terminus.	FIG
+42	46	SPOC	structure_element	Schematic drawing of the structure of FPA SPOC domain, colored from blue at the N terminus to red at the C terminus.	FIG
+33	41	β-barrel	structure_element	The view is from the side of the β-barrel.	FIG
+4	14	disordered	protein_state	The disordered segment (residues 460–465) is indicated with the dotted line.	FIG
+33	40	460–465	residue_range	The disordered segment (residues 460–465) is indicated with the dotted line.	FIG
+0	9	Structure	evidence	Structure of the FPA SPOC domain, viewed from the end of the β-barrel, after 90° rotation around the horizontal axis from panel A. All structure figures were produced with PyMOL (www.pymol.org).	FIG
+17	20	FPA	protein	Structure of the FPA SPOC domain, viewed from the end of the β-barrel, after 90° rotation around the horizontal axis from panel A. All structure figures were produced with PyMOL (www.pymol.org).	FIG
+21	25	SPOC	structure_element	Structure of the FPA SPOC domain, viewed from the end of the β-barrel, after 90° rotation around the horizontal axis from panel A. All structure figures were produced with PyMOL (www.pymol.org).	FIG
+61	69	β-barrel	structure_element	Structure of the FPA SPOC domain, viewed from the end of the β-barrel, after 90° rotation around the horizontal axis from panel A. All structure figures were produced with PyMOL (www.pymol.org).	FIG
+0	34	Comparisons to structural homologs	experimental_method	Comparisons to structural homologs of the SPOC domain	RESULTS
+42	46	SPOC	structure_element	Comparisons to structural homologs of the SPOC domain	RESULTS
+37	40	FPA	protein	Only five structural homologs of the FPA SPOC domain were found in the Protein Data Bank with the DaliLite server, suggesting that the SPOC domain structure is relatively unique.	RESULTS
+41	45	SPOC	structure_element	Only five structural homologs of the FPA SPOC domain were found in the Protein Data Bank with the DaliLite server, suggesting that the SPOC domain structure is relatively unique.	RESULTS
+98	113	DaliLite server	experimental_method	Only five structural homologs of the FPA SPOC domain were found in the Protein Data Bank with the DaliLite server, suggesting that the SPOC domain structure is relatively unique.	RESULTS
+135	139	SPOC	structure_element	Only five structural homologs of the FPA SPOC domain were found in the Protein Data Bank with the DaliLite server, suggesting that the SPOC domain structure is relatively unique.	RESULTS
+147	156	structure	evidence	Only five structural homologs of the FPA SPOC domain were found in the Protein Data Bank with the DaliLite server, suggesting that the SPOC domain structure is relatively unique.	RESULTS
+19	23	SPOC	structure_element	The top hit is the SPOC domain of human SHARP (Fig 3A), with a Z score of 12.3.	RESULTS
+34	39	human	species	The top hit is the SPOC domain of human SHARP (Fig 3A), with a Z score of 12.3.	RESULTS
+40	45	SHARP	protein	The top hit is the SPOC domain of human SHARP (Fig 3A), with a Z score of 12.3.	RESULTS
+63	70	Z score	evidence	The top hit is the SPOC domain of human SHARP (Fig 3A), with a Z score of 12.3.	RESULTS
+47	55	β-barrel	structure_element	The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D).	RESULTS
+79	83	Ku70	protein	The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D).	RESULTS
+88	92	Ku80	protein	The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D).	RESULTS
+94	101	Z score	evidence	The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D).	RESULTS
+134	154	chromodomain protein	protein_type	The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D).	RESULTS
+155	159	Chp1	protein	The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D).	RESULTS
+161	168	Z score	evidence	The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D).	RESULTS
+193	221	activator interacting domain	structure_element	The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D).	RESULTS
+223	227	ACID	structure_element	The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D).	RESULTS
+236	241	Med25	protein	The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D).	RESULTS
+275	282	Z score	evidence	The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D).	RESULTS
+34	41	Z score	evidence	The next structural homolog has a Z score of 3.0.	RESULTS
+27	30	FPA	protein	Structural homologs of the FPA SPOC domain.	FIG
+31	35	SPOC	structure_element	Structural homologs of the FPA SPOC domain.	FIG
+0	7	Overlay	experimental_method	Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray).	FIG
+15	25	structures	evidence	Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray).	FIG
+33	36	FPA	protein	Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray).	FIG
+37	41	SPOC	structure_element	Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray).	FIG
+64	69	SHARP	protein	Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray).	FIG
+70	74	SPOC	structure_element	Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray).	FIG
+24	45	doubly-phosphorylated	protein_state	The bound position of a doubly-phosphorylated peptide from SMRT is shown in magenta.	FIG
+46	53	peptide	chemical	The bound position of a doubly-phosphorylated peptide from SMRT is shown in magenta.	FIG
+59	63	SMRT	protein	The bound position of a doubly-phosphorylated peptide from SMRT is shown in magenta.	FIG
+0	7	Overlay	experimental_method	Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 β-barrel domain (gray).	FIG
+15	25	structures	evidence	Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 β-barrel domain (gray).	FIG
+33	36	FPA	protein	Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 β-barrel domain (gray).	FIG
+37	41	SPOC	structure_element	Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 β-barrel domain (gray).	FIG
+64	68	Ku70	protein	Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 β-barrel domain (gray).	FIG
+69	77	β-barrel	structure_element	Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 β-barrel domain (gray).	FIG
+0	4	Ku80	protein	Ku80 contains a homologous domain (green), which forms a hetero-dimer with that in Ku70.	FIG
+57	69	hetero-dimer	oligomeric_state	Ku80 contains a homologous domain (green), which forms a hetero-dimer with that in Ku70.	FIG
+83	87	Ku70	protein	Ku80 contains a homologous domain (green), which forms a hetero-dimer with that in Ku70.	FIG
+71	76	dsDNA	chemical	The two domains, and inserted segments on them, mediate the binding of dsDNA (orange).	FIG
+67	75	β-barrel	structure_element	The red rectangle highlights the region of contact between the two β-barrel domains.	FIG
+0	7	Overlay	experimental_method	Overlay of the structures of the FPA SPOC domain (cyan) and the homologous domain in Chp1 (gray).	FIG
+15	25	structures	evidence	Overlay of the structures of the FPA SPOC domain (cyan) and the homologous domain in Chp1 (gray).	FIG
+33	36	FPA	protein	Overlay of the structures of the FPA SPOC domain (cyan) and the homologous domain in Chp1 (gray).	FIG
+37	41	SPOC	structure_element	Overlay of the structures of the FPA SPOC domain (cyan) and the homologous domain in Chp1 (gray).	FIG
+85	89	Chp1	protein	Overlay of the structures of the FPA SPOC domain (cyan) and the homologous domain in Chp1 (gray).	FIG
+23	27	Chp1	protein	The binding partner of Chp1, Tas3, is shown in green.	FIG
+29	33	Tas3	protein	The binding partner of Chp1, Tas3, is shown in green.	FIG
+57	69	binding site	site	The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (D).	FIG
+77	82	SMART	protein	The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (D).	FIG
+83	97	phosphopeptide	ptm	The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (D).	FIG
+101	106	SHARP	protein	The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (D).	FIG
+107	111	SPOC	structure_element	The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (D).	FIG
+128	132	loop	structure_element	The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (D).	FIG
+136	140	Tas3	protein	The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (D).	FIG
+0	7	Overlay	experimental_method	Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray).	FIG
+15	25	structures	evidence	Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray).	FIG
+33	36	FPA	protein	Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray).	FIG
+37	41	SPOC	structure_element	Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray).	FIG
+64	69	Med25	protein	Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray).	FIG
+70	74	ACID	structure_element	Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray).	FIG
+0	5	SHARP	protein	SHARP is a transcriptional co-repressor in the nuclear receptor and Notch/RBP-Jκ signaling pathways.	RESULTS
+11	39	transcriptional co-repressor	protein_type	SHARP is a transcriptional co-repressor in the nuclear receptor and Notch/RBP-Jκ signaling pathways.	RESULTS
+47	63	nuclear receptor	protein_type	SHARP is a transcriptional co-repressor in the nuclear receptor and Notch/RBP-Jκ signaling pathways.	RESULTS
+68	73	Notch	protein	SHARP is a transcriptional co-repressor in the nuclear receptor and Notch/RBP-Jκ signaling pathways.	RESULTS
+74	80	RBP-Jκ	protein	SHARP is a transcriptional co-repressor in the nuclear receptor and Notch/RBP-Jκ signaling pathways.	RESULTS
+4	8	SPOC	structure_element	The SPOC domain of SHARP interacts directly with silencing mediator for retinoid and thyroid receptor (SMRT), nuclear receptor co-repressor (N-CoR), HDAC, and other components to represses transcription.	RESULTS
+19	24	SHARP	protein	The SPOC domain of SHARP interacts directly with silencing mediator for retinoid and thyroid receptor (SMRT), nuclear receptor co-repressor (N-CoR), HDAC, and other components to represses transcription.	RESULTS
+49	101	silencing mediator for retinoid and thyroid receptor	protein	The SPOC domain of SHARP interacts directly with silencing mediator for retinoid and thyroid receptor (SMRT), nuclear receptor co-repressor (N-CoR), HDAC, and other components to represses transcription.	RESULTS
+103	107	SMRT	protein	The SPOC domain of SHARP interacts directly with silencing mediator for retinoid and thyroid receptor (SMRT), nuclear receptor co-repressor (N-CoR), HDAC, and other components to represses transcription.	RESULTS
+110	139	nuclear receptor co-repressor	protein_type	The SPOC domain of SHARP interacts directly with silencing mediator for retinoid and thyroid receptor (SMRT), nuclear receptor co-repressor (N-CoR), HDAC, and other components to represses transcription.	RESULTS
+141	146	N-CoR	protein_type	The SPOC domain of SHARP interacts directly with silencing mediator for retinoid and thyroid receptor (SMRT), nuclear receptor co-repressor (N-CoR), HDAC, and other components to represses transcription.	RESULTS
+149	153	HDAC	protein	The SPOC domain of SHARP interacts directly with silencing mediator for retinoid and thyroid receptor (SMRT), nuclear receptor co-repressor (N-CoR), HDAC, and other components to represses transcription.	RESULTS
+18	27	structure	evidence	While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the β-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A).	RESULTS
+35	38	FPA	protein	While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the β-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A).	RESULTS
+39	43	SPOC	structure_element	While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the β-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A).	RESULTS
+77	82	SHARP	protein	While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the β-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A).	RESULTS
+83	87	SPOC	structure_element	While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the β-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A).	RESULTS
+155	164	β-strands	structure_element	While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the β-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A).	RESULTS
+173	180	helices	structure_element	While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the β-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A).	RESULTS
+198	203	loops	structure_element	While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the β-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A).	RESULTS
+17	22	SHARP	protein	In addition, the SHARP SPOC domain has three extra helices.	RESULTS
+23	27	SPOC	structure_element	In addition, the SHARP SPOC domain has three extra helices.	RESULTS
+51	58	helices	structure_element	In addition, the SHARP SPOC domain has three extra helices.	RESULTS
+40	48	β-barrel	structure_element	One of them covers the other end of the β-barrel, and the other two shield an additional surface of the side of the β-barrel from solvent.	RESULTS
+116	124	β-barrel	structure_element	One of them covers the other end of the β-barrel, and the other two shield an additional surface of the side of the β-barrel from solvent.	RESULTS
+2	23	doubly-phosphorylated	protein_state	A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands β1 and β3 (Fig 3A).	RESULTS
+24	31	peptide	chemical	A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands β1 and β3 (Fig 3A).	RESULTS
+37	41	SMRT	protein	A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands β1 and β3 (Fig 3A).	RESULTS
+45	53	bound to	protein_state	A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands β1 and β3 (Fig 3A).	RESULTS
+70	76	barrel	structure_element	A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands β1 and β3 (Fig 3A).	RESULTS
+83	90	strands	structure_element	A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands β1 and β3 (Fig 3A).	RESULTS
+91	93	β1	structure_element	A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands β1 and β3 (Fig 3A).	RESULTS
+98	100	β3	structure_element	A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands β1 and β3 (Fig 3A).	RESULTS
+54	57	FPA	protein	Such a binding mode probably would not be possible in FPA, as the peptide would clash with the β1-β2 loop.	RESULTS
+66	73	peptide	chemical	Such a binding mode probably would not be possible in FPA, as the peptide would clash with the β1-β2 loop.	RESULTS
+95	105	β1-β2 loop	structure_element	Such a binding mode probably would not be possible in FPA, as the peptide would clash with the β1-β2 loop.	RESULTS
+4	13	Ku70-Ku80	complex_assembly	The Ku70-Ku80 hetero-dimer is involved in DNA double-strand break repair and the β-barrel domain contributes to DNA binding.	RESULTS
+14	26	hetero-dimer	oligomeric_state	The Ku70-Ku80 hetero-dimer is involved in DNA double-strand break repair and the β-barrel domain contributes to DNA binding.	RESULTS
+81	89	β-barrel	structure_element	The Ku70-Ku80 hetero-dimer is involved in DNA double-strand break repair and the β-barrel domain contributes to DNA binding.	RESULTS
+112	115	DNA	chemical	The Ku70-Ku80 hetero-dimer is involved in DNA double-strand break repair and the β-barrel domain contributes to DNA binding.	RESULTS
+13	21	β-barrel	structure_element	In fact, the β-barrel domains of Ku70 and Ku80 form a hetero-dimer, primarily through interactions between the loops connecting the third and fourth strands of the barrel (Fig 3B).	RESULTS
+33	37	Ku70	protein	In fact, the β-barrel domains of Ku70 and Ku80 form a hetero-dimer, primarily through interactions between the loops connecting the third and fourth strands of the barrel (Fig 3B).	RESULTS
+42	46	Ku80	protein	In fact, the β-barrel domains of Ku70 and Ku80 form a hetero-dimer, primarily through interactions between the loops connecting the third and fourth strands of the barrel (Fig 3B).	RESULTS
+54	66	hetero-dimer	oligomeric_state	In fact, the β-barrel domains of Ku70 and Ku80 form a hetero-dimer, primarily through interactions between the loops connecting the third and fourth strands of the barrel (Fig 3B).	RESULTS
+111	116	loops	structure_element	In fact, the β-barrel domains of Ku70 and Ku80 form a hetero-dimer, primarily through interactions between the loops connecting the third and fourth strands of the barrel (Fig 3B).	RESULTS
+132	156	third and fourth strands	structure_element	In fact, the β-barrel domains of Ku70 and Ku80 form a hetero-dimer, primarily through interactions between the loops connecting the third and fourth strands of the barrel (Fig 3B).	RESULTS
+164	170	barrel	structure_element	In fact, the β-barrel domains of Ku70 and Ku80 form a hetero-dimer, primarily through interactions between the loops connecting the third and fourth strands of the barrel (Fig 3B).	RESULTS
+25	34	β-barrels	structure_element	The open ends of the two β-barrels face the DNA binding sites, and contact the phosphodiester backbone of the dsDNA.	RESULTS
+44	61	DNA binding sites	site	The open ends of the two β-barrels face the DNA binding sites, and contact the phosphodiester backbone of the dsDNA.	RESULTS
+110	115	dsDNA	chemical	The open ends of the two β-barrels face the DNA binding sites, and contact the phosphodiester backbone of the dsDNA.	RESULTS
+15	26	long insert	structure_element	In addition, a long insert connecting strands β2 and β3 in the two domains form an arch-like structure, encircling the dsDNA.	RESULTS
+38	45	strands	structure_element	In addition, a long insert connecting strands β2 and β3 in the two domains form an arch-like structure, encircling the dsDNA.	RESULTS
+46	48	β2	structure_element	In addition, a long insert connecting strands β2 and β3 in the two domains form an arch-like structure, encircling the dsDNA.	RESULTS
+53	55	β3	structure_element	In addition, a long insert connecting strands β2 and β3 in the two domains form an arch-like structure, encircling the dsDNA.	RESULTS
+83	102	arch-like structure	structure_element	In addition, a long insert connecting strands β2 and β3 in the two domains form an arch-like structure, encircling the dsDNA.	RESULTS
+119	124	dsDNA	chemical	In addition, a long insert connecting strands β2 and β3 in the two domains form an arch-like structure, encircling the dsDNA.	RESULTS
+0	4	Chp1	protein	Chp1 is a subunit of the RNA-induced initiation of transcriptional gene silencing (RITS) complex.	RESULTS
+25	81	RNA-induced initiation of transcriptional gene silencing	complex_assembly	Chp1 is a subunit of the RNA-induced initiation of transcriptional gene silencing (RITS) complex.	RESULTS
+83	87	RITS	complex_assembly	Chp1 is a subunit of the RNA-induced initiation of transcriptional gene silencing (RITS) complex.	RESULTS
+15	19	Chp1	protein	The partner of Chp1, Tas3, is bound between the barrel domain and the second domain of Chp1, and the linker between the two domains is also crucial for this interaction (Fig 3C).	RESULTS
+21	25	Tas3	protein	The partner of Chp1, Tas3, is bound between the barrel domain and the second domain of Chp1, and the linker between the two domains is also crucial for this interaction (Fig 3C).	RESULTS
+48	61	barrel domain	structure_element	The partner of Chp1, Tas3, is bound between the barrel domain and the second domain of Chp1, and the linker between the two domains is also crucial for this interaction (Fig 3C).	RESULTS
+70	83	second domain	structure_element	The partner of Chp1, Tas3, is bound between the barrel domain and the second domain of Chp1, and the linker between the two domains is also crucial for this interaction (Fig 3C).	RESULTS
+87	91	Chp1	protein	The partner of Chp1, Tas3, is bound between the barrel domain and the second domain of Chp1, and the linker between the two domains is also crucial for this interaction (Fig 3C).	RESULTS
+101	107	linker	structure_element	The partner of Chp1, Tas3, is bound between the barrel domain and the second domain of Chp1, and the linker between the two domains is also crucial for this interaction (Fig 3C).	RESULTS
+33	41	β-barrel	structure_element	It is probably unlikely that the β-barrel itself is sufficient to bind Tas3.	RESULTS
+71	75	Tas3	protein	It is probably unlikely that the β-barrel itself is sufficient to bind Tas3.	RESULTS
+17	21	loop	structure_element	Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).	RESULTS
+25	29	Tas3	protein	Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).	RESULTS
+39	45	strand	structure_element	Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).	RESULTS
+46	48	β3	structure_element	Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).	RESULTS
+56	69	barrel domain	structure_element	Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).	RESULTS
+143	147	SMRT	protein	Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).	RESULTS
+148	155	peptide	chemical	Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).	RESULTS
+156	171	in complex with	protein_state	Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).	RESULTS
+172	177	SHARP	protein	Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).	RESULTS
+178	182	SPOC	structure_element	Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).	RESULTS
+0	8	Mediator	protein_type	Mediator is a coactivator complex that promotes transcription by Pol II.	RESULTS
+65	71	Pol II	complex_assembly	Mediator is a coactivator complex that promotes transcription by Pol II.	RESULTS
+4	9	Med25	protein	The Med25 subunit ACID is the target of the potent activator VP16 of the herpes simplex virus.	RESULTS
+18	22	ACID	structure_element	The Med25 subunit ACID is the target of the potent activator VP16 of the herpes simplex virus.	RESULTS
+61	65	VP16	protein	The Med25 subunit ACID is the target of the potent activator VP16 of the herpes simplex virus.	RESULTS
+73	93	herpes simplex virus	species	The Med25 subunit ACID is the target of the potent activator VP16 of the herpes simplex virus.	RESULTS
+4	13	structure	evidence	The structure of ACID contains a helix at the C-terminus as well as an extended β1-β2 loop.	RESULTS
+17	21	ACID	structure_element	The structure of ACID contains a helix at the C-terminus as well as an extended β1-β2 loop.	RESULTS
+33	38	helix	structure_element	The structure of ACID contains a helix at the C-terminus as well as an extended β1-β2 loop.	RESULTS
+80	90	β1-β2 loop	structure_element	The structure of ACID contains a helix at the C-terminus as well as an extended β1-β2 loop.	RESULTS
+17	29	binding site	site	Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands β1 and β3, that is used by the SHARP and Tas3 SPOC domains for binding their partners.	RESULTS
+34	38	VP16	protein	Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands β1 and β3, that is used by the SHARP and Tas3 SPOC domains for binding their partners.	RESULTS
+75	88	surface patch	site	Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands β1 and β3, that is used by the SHARP and Tas3 SPOC domains for binding their partners.	RESULTS
+95	102	strands	structure_element	Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands β1 and β3, that is used by the SHARP and Tas3 SPOC domains for binding their partners.	RESULTS
+103	105	β1	structure_element	Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands β1 and β3, that is used by the SHARP and Tas3 SPOC domains for binding their partners.	RESULTS
+110	112	β3	structure_element	Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands β1 and β3, that is used by the SHARP and Tas3 SPOC domains for binding their partners.	RESULTS
+134	139	SHARP	protein	Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands β1 and β3, that is used by the SHARP and Tas3 SPOC domains for binding their partners.	RESULTS
+144	148	Tas3	protein	Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands β1 and β3, that is used by the SHARP and Tas3 SPOC domains for binding their partners.	RESULTS
+149	153	SPOC	structure_element	Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands β1 and β3, that is used by the SHARP and Tas3 SPOC domains for binding their partners.	RESULTS
+2	11	conserved	protein_state	A conserved surface patch in the FPA SPOC domain	RESULTS
+12	25	surface patch	site	A conserved surface patch in the FPA SPOC domain	RESULTS
+33	36	FPA	protein	A conserved surface patch in the FPA SPOC domain	RESULTS
+37	41	SPOC	structure_element	A conserved surface patch in the FPA SPOC domain	RESULTS
+19	23	SPOC	structure_element	An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A).	RESULTS
+49	62	surface patch	site	An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A).	RESULTS
+68	75	strands	structure_element	An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A).	RESULTS
+76	78	β1	structure_element	An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A).	RESULTS
+80	82	β3	structure_element	An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A).	RESULTS
+84	86	β5	structure_element	An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A).	RESULTS
+91	93	β6	structure_element	An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A).	RESULTS
+102	111	conserved	protein_state	An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A).	RESULTS
+118	123	plant	taxonomy_domain	An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A).	RESULTS
+124	127	FPA	protein	An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A).	RESULTS
+5	18	surface patch	site	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+42	53	sub-patches	site	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+69	75	Lys447	residue_name_number	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+80	86	strand	structure_element	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+87	89	β1	structure_element	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+92	98	Arg477	residue_name_number	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+100	102	β3	structure_element	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+105	111	Tyr515	residue_name_number	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+113	115	αB	structure_element	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+121	127	Arg521	residue_name_number	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+129	131	β5	structure_element	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+140	149	sub-patch	site	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+164	170	His486	residue_name_number	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+172	174	αA	structure_element	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+177	183	Thr478	residue_name_number	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+185	187	β3	structure_element	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+190	196	Val524	residue_name_number	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+198	200	β5	structure_element	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+206	212	Phe534	residue_name_number	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+214	216	β6	structure_element	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+231	240	sub-patch	site	This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B).	RESULTS
+4	23	first surface patch	site	The first surface patch is electropositive in nature (Fig 4C), and residues Arg477 and Tyr515 are also conserved in the SHARP SPOC domain (Fig 1B).	RESULTS
+27	42	electropositive	protein_state	The first surface patch is electropositive in nature (Fig 4C), and residues Arg477 and Tyr515 are also conserved in the SHARP SPOC domain (Fig 1B).	RESULTS
+76	82	Arg477	residue_name_number	The first surface patch is electropositive in nature (Fig 4C), and residues Arg477 and Tyr515 are also conserved in the SHARP SPOC domain (Fig 1B).	RESULTS
+87	93	Tyr515	residue_name_number	The first surface patch is electropositive in nature (Fig 4C), and residues Arg477 and Tyr515 are also conserved in the SHARP SPOC domain (Fig 1B).	RESULTS
+103	112	conserved	protein_state	The first surface patch is electropositive in nature (Fig 4C), and residues Arg477 and Tyr515 are also conserved in the SHARP SPOC domain (Fig 1B).	RESULTS
+120	125	SHARP	protein	The first surface patch is electropositive in nature (Fig 4C), and residues Arg477 and Tyr515 are also conserved in the SHARP SPOC domain (Fig 1B).	RESULTS
+126	130	SPOC	structure_element	The first surface patch is electropositive in nature (Fig 4C), and residues Arg477 and Tyr515 are also conserved in the SHARP SPOC domain (Fig 1B).	RESULTS
+20	34	phosphorylated	protein_state	In fact, one of the phosphorylated residues of the SMRT peptide interacts with this surface patch (Fig 3A), suggesting that the FPA SPOC domain might also interact with a phosphorylated segment here.	RESULTS
+51	55	SMRT	protein	In fact, one of the phosphorylated residues of the SMRT peptide interacts with this surface patch (Fig 3A), suggesting that the FPA SPOC domain might also interact with a phosphorylated segment here.	RESULTS
+56	63	peptide	chemical	In fact, one of the phosphorylated residues of the SMRT peptide interacts with this surface patch (Fig 3A), suggesting that the FPA SPOC domain might also interact with a phosphorylated segment here.	RESULTS
+84	97	surface patch	site	In fact, one of the phosphorylated residues of the SMRT peptide interacts with this surface patch (Fig 3A), suggesting that the FPA SPOC domain might also interact with a phosphorylated segment here.	RESULTS
+128	131	FPA	protein	In fact, one of the phosphorylated residues of the SMRT peptide interacts with this surface patch (Fig 3A), suggesting that the FPA SPOC domain might also interact with a phosphorylated segment here.	RESULTS
+132	136	SPOC	structure_element	In fact, one of the phosphorylated residues of the SMRT peptide interacts with this surface patch (Fig 3A), suggesting that the FPA SPOC domain might also interact with a phosphorylated segment here.	RESULTS
+171	185	phosphorylated	protein_state	In fact, one of the phosphorylated residues of the SMRT peptide interacts with this surface patch (Fig 3A), suggesting that the FPA SPOC domain might also interact with a phosphorylated segment here.	RESULTS
+19	39	second surface patch	site	In comparison, the second surface patch is more hydrophobic in nature (Fig 4C).	RESULTS
+48	59	hydrophobic	protein_state	In comparison, the second surface patch is more hydrophobic in nature (Fig 4C).	RESULTS
+2	11	conserved	protein_state	A conserved surface patch of FPA SPOC domain.	FIG
+12	25	surface patch	site	A conserved surface patch of FPA SPOC domain.	FIG
+29	32	FPA	protein	A conserved surface patch of FPA SPOC domain.	FIG
+33	37	SPOC	structure_element	A conserved surface patch of FPA SPOC domain.	FIG
+38	41	FPA	protein	Two views of the molecular surface of FPA SPOC domain colored based on sequence conservation among plant FPA homologs.	FIG
+42	46	SPOC	structure_element	Two views of the molecular surface of FPA SPOC domain colored based on sequence conservation among plant FPA homologs.	FIG
+99	104	plant	taxonomy_domain	Two views of the molecular surface of FPA SPOC domain colored based on sequence conservation among plant FPA homologs.	FIG
+105	108	FPA	protein	Two views of the molecular surface of FPA SPOC domain colored based on sequence conservation among plant FPA homologs.	FIG
+16	25	conserved	protein_state	Residues in the conserved surface patch of FPA SPOC domain.	FIG
+26	39	surface patch	site	Residues in the conserved surface patch of FPA SPOC domain.	FIG
+43	46	FPA	protein	Residues in the conserved surface patch of FPA SPOC domain.	FIG
+47	51	SPOC	structure_element	Residues in the conserved surface patch of FPA SPOC domain.	FIG
+81	96	first sub-patch	site	The side chains of the residues are shown in stick models, colored orange in the first sub-patch and green in the second. (C).	FIG
+21	24	FPA	protein	Molecular surface of FPA SPOC domain colored based on electrostatic potential.	FIG
+25	29	SPOC	structure_element	Molecular surface of FPA SPOC domain colored based on electrostatic potential.	FIG
+62	65	FPA	protein	Testing the requirement of specific conserved amino acids for FPA functions	RESULTS
+45	54	conserved	protein_state	We next examined the potential impact of the conserved surface patch on FPA function in vivo.	RESULTS
+55	68	surface patch	site	We next examined the potential impact of the conserved surface patch on FPA function in vivo.	RESULTS
+72	75	FPA	protein	We next examined the potential impact of the conserved surface patch on FPA function in vivo.	RESULTS
+3	10	mutated	experimental_method	We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain (Fig 1B) and were found to be functionally important.	RESULTS
+25	31	Arg477	residue_name_number	We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain (Fig 1B) and were found to be functionally important.	RESULTS
+36	42	Tyr515	residue_name_number	We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain (Fig 1B) and were found to be functionally important.	RESULTS
+51	64	surface patch	site	We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain (Fig 1B) and were found to be functionally important.	RESULTS
+81	90	conserved	protein_state	We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain (Fig 1B) and were found to be functionally important.	RESULTS
+98	103	SHARP	protein	We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain (Fig 1B) and were found to be functionally important.	RESULTS
+104	108	SPOC	structure_element	We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain (Fig 1B) and were found to be functionally important.	RESULTS
+4	13	mutations	experimental_method	The mutations were introduced into a transgene designed to express FPA from its native control elements (promoter, introns and 3′ UTR).	RESULTS
+19	29	introduced	experimental_method	The mutations were introduced into a transgene designed to express FPA from its native control elements (promoter, introns and 3′ UTR).	RESULTS
+67	70	FPA	protein	The mutations were introduced into a transgene designed to express FPA from its native control elements (promoter, introns and 3′ UTR).	RESULTS
+35	53	stably transformed	experimental_method	The resulting transgenes were then stably transformed into an fpa-8 mutant background so that the impact of the mutations on FPA function could be assessed.	RESULTS
+62	67	fpa-8	gene	The resulting transgenes were then stably transformed into an fpa-8 mutant background so that the impact of the mutations on FPA function could be assessed.	RESULTS
+68	74	mutant	protein_state	The resulting transgenes were then stably transformed into an fpa-8 mutant background so that the impact of the mutations on FPA function could be assessed.	RESULTS
+112	121	mutations	experimental_method	The resulting transgenes were then stably transformed into an fpa-8 mutant background so that the impact of the mutations on FPA function could be assessed.	RESULTS
+125	128	FPA	protein	The resulting transgenes were then stably transformed into an fpa-8 mutant background so that the impact of the mutations on FPA function could be assessed.	RESULTS
+35	56	expression constructs	experimental_method	Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig).	RESULTS
+62	67	fpa-8	gene	Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig).	RESULTS
+88	97	wild-type	protein_state	Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig).	RESULTS
+98	101	FPA	protein	Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig).	RESULTS
+119	122	FPA	protein	Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig).	RESULTS
+131	148	expression levels	evidence	Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig).	RESULTS
+157	166	wild-type	protein_state	Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig).	RESULTS
+222	225	FPA	protein	Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig).	RESULTS
+241	244	RNA	chemical	Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig).	RESULTS
+278	281	FPA	protein	Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig).	RESULTS
+282	290	pre-mRNA	chemical	Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig).	RESULTS
+57	62	R477A	mutant	We examined independent transgenic lines expressing each R477A and Y515A mutation.	RESULTS
+67	72	Y515A	mutant	We examined independent transgenic lines expressing each R477A and Y515A mutation.	RESULTS
+73	81	mutation	experimental_method	We examined independent transgenic lines expressing each R477A and Y515A mutation.	RESULTS
+53	56	FPA	protein	In each case, we confirmed that detectable levels of FPA protein expression were restored close to wild-type levels in protein blot analyses using antibodies that specifically recognize FPA (S2 Fig).	RESULTS
+99	108	wild-type	protein_state	In each case, we confirmed that detectable levels of FPA protein expression were restored close to wild-type levels in protein blot analyses using antibodies that specifically recognize FPA (S2 Fig).	RESULTS
+119	131	protein blot	experimental_method	In each case, we confirmed that detectable levels of FPA protein expression were restored close to wild-type levels in protein blot analyses using antibodies that specifically recognize FPA (S2 Fig).	RESULTS
+186	189	FPA	protein	In each case, we confirmed that detectable levels of FPA protein expression were restored close to wild-type levels in protein blot analyses using antibodies that specifically recognize FPA (S2 Fig).	RESULTS
+35	48	surface patch	site	We then examined the impact of the surface patch mutations on FPA’s function in controlling RNA 3′-end formation by determining whether the mutant proteins functioned in FPA autoregulation and the repression of FLC expression.	RESULTS
+49	58	mutations	experimental_method	We then examined the impact of the surface patch mutations on FPA’s function in controlling RNA 3′-end formation by determining whether the mutant proteins functioned in FPA autoregulation and the repression of FLC expression.	RESULTS
+62	65	FPA	protein	We then examined the impact of the surface patch mutations on FPA’s function in controlling RNA 3′-end formation by determining whether the mutant proteins functioned in FPA autoregulation and the repression of FLC expression.	RESULTS
+140	146	mutant	protein_state	We then examined the impact of the surface patch mutations on FPA’s function in controlling RNA 3′-end formation by determining whether the mutant proteins functioned in FPA autoregulation and the repression of FLC expression.	RESULTS
+170	173	FPA	protein	We then examined the impact of the surface patch mutations on FPA’s function in controlling RNA 3′-end formation by determining whether the mutant proteins functioned in FPA autoregulation and the repression of FLC expression.	RESULTS
+211	214	FLC	gene	We then examined the impact of the surface patch mutations on FPA’s function in controlling RNA 3′-end formation by determining whether the mutant proteins functioned in FPA autoregulation and the repression of FLC expression.	RESULTS
+0	3	FPA	protein	FPA autoregulates its expression by promoting cleavage and polyadenylation within intron 1 of its own pre-mRNA, resulting in a truncated transcript that does not encode functional protein.	RESULTS
+102	110	pre-mRNA	chemical	FPA autoregulates its expression by promoting cleavage and polyadenylation within intron 1 of its own pre-mRNA, resulting in a truncated transcript that does not encode functional protein.	RESULTS
+8	29	RNA gel blot analyses	experimental_method	We used RNA gel blot analyses to reveal that in each of three independent transgenic lines for each single mutant, rescue of proximally polyadenylated FPA pre-mRNA can be detected (Fig 5A and 5B).	RESULTS
+107	113	mutant	protein_state	We used RNA gel blot analyses to reveal that in each of three independent transgenic lines for each single mutant, rescue of proximally polyadenylated FPA pre-mRNA can be detected (Fig 5A and 5B).	RESULTS
+151	154	FPA	protein	We used RNA gel blot analyses to reveal that in each of three independent transgenic lines for each single mutant, rescue of proximally polyadenylated FPA pre-mRNA can be detected (Fig 5A and 5B).	RESULTS
+155	163	pre-mRNA	chemical	We used RNA gel blot analyses to reveal that in each of three independent transgenic lines for each single mutant, rescue of proximally polyadenylated FPA pre-mRNA can be detected (Fig 5A and 5B).	RESULTS
+79	82	FPA	protein	We therefore conclude that neither of these mutations disrupted the ability of FPA to promote RNA 3′-end formation in its own transcript.	RESULTS
+21	24	FPA	protein	Impact of individual FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA.	FIG
+25	29	SPOC	structure_element	Impact of individual FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA.	FIG
+37	46	mutations	experimental_method	Impact of individual FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA.	FIG
+81	84	FPA	protein	Impact of individual FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA.	FIG
+85	93	pre-mRNA	chemical	Impact of individual FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA.	FIG
+0	12	RNA gel blot	experimental_method	RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A (A), or FPA::FPA Y515A (B) using poly(A)+ purified mRNAs.	FIG
+25	27	WT	protein_state	RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A (A), or FPA::FPA Y515A (B) using poly(A)+ purified mRNAs.	FIG
+28	39	A. thaliana	species	RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A (A), or FPA::FPA Y515A (B) using poly(A)+ purified mRNAs.	FIG
+67	73	plants	taxonomy_domain	RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A (A), or FPA::FPA Y515A (B) using poly(A)+ purified mRNAs.	FIG
+74	79	fpa-8	gene	RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A (A), or FPA::FPA Y515A (B) using poly(A)+ purified mRNAs.	FIG
+84	89	fpa-8	gene	RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A (A), or FPA::FPA Y515A (B) using poly(A)+ purified mRNAs.	FIG
+90	97	mutants	protein_state	RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A (A), or FPA::FPA Y515A (B) using poly(A)+ purified mRNAs.	FIG
+116	119	FPA	protein	RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A (A), or FPA::FPA Y515A (B) using poly(A)+ purified mRNAs.	FIG
+121	130	FPA R477A	mutant	RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A (A), or FPA::FPA Y515A (B) using poly(A)+ purified mRNAs.	FIG
+139	142	FPA	protein	RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A (A), or FPA::FPA Y515A (B) using poly(A)+ purified mRNAs.	FIG
+144	153	FPA Y515A	mutant	RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A (A), or FPA::FPA Y515A (B) using poly(A)+ purified mRNAs.	FIG
+182	187	mRNAs	chemical	RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A (A), or FPA::FPA Y515A (B) using poly(A)+ purified mRNAs.	FIG
+45	48	FPA	protein	A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs.	FIG
+49	53	mRNA	chemical	A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs.	FIG
+73	76	FPA	protein	A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs.	FIG
+86	91	mRNAs	chemical	A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs.	FIG
+45	48	FPA	protein	A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs.	FIG
+49	53	mRNA	chemical	A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs.	FIG
+73	76	FPA	protein	A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs.	FIG
+86	91	mRNAs	chemical	A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs.	FIG
+39	42	FPA	protein	Proximally and distally polyadenylated FPA transcripts are marked with arrows.	FIG
+103	106	FPA	protein	The ratio of distal:proximal polyadenylated forms is given under each lane. (C,D) Impact of individual FPA SPOC domain mutations on FLC transcript levels.	FIG
+107	111	SPOC	structure_element	The ratio of distal:proximal polyadenylated forms is given under each lane. (C,D) Impact of individual FPA SPOC domain mutations on FLC transcript levels.	FIG
+119	128	mutations	experimental_method	The ratio of distal:proximal polyadenylated forms is given under each lane. (C,D) Impact of individual FPA SPOC domain mutations on FLC transcript levels.	FIG
+132	135	FLC	gene	The ratio of distal:proximal polyadenylated forms is given under each lane. (C,D) Impact of individual FPA SPOC domain mutations on FLC transcript levels.	FIG
+0	7	qRT-PCR	experimental_method	qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A (C), FPA::FPA Y515A (D) plants.	FIG
+42	45	RNA	chemical	qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A (C), FPA::FPA Y515A (D) plants.	FIG
+67	72	fpa-8	gene	qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A (C), FPA::FPA Y515A (D) plants.	FIG
+79	82	FPA	protein	qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A (C), FPA::FPA Y515A (D) plants.	FIG
+83	86	YFP	experimental_method	qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A (C), FPA::FPA Y515A (D) plants.	FIG
+91	94	FPA	protein	qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A (C), FPA::FPA Y515A (D) plants.	FIG
+96	105	FPA R477A	mutant	qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A (C), FPA::FPA Y515A (D) plants.	FIG
+111	114	FPA	protein	qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A (C), FPA::FPA Y515A (D) plants.	FIG
+116	125	FPA Y515A	mutant	qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A (C), FPA::FPA Y515A (D) plants.	FIG
+130	136	plants	taxonomy_domain	qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A (C), FPA::FPA Y515A (D) plants.	FIG
+0	10	Histograms	evidence	Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.	FIG
+54	57	PCR	experimental_method	Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.	FIG
+0	10	Histograms	evidence	Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.	FIG
+54	57	PCR	experimental_method	Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.	FIG
+78	81	FPA	protein	We next examined whether the corresponding mutations disrupted the ability of FPA to control FLC expression.	RESULTS
+93	96	FLC	gene	We next examined whether the corresponding mutations disrupted the ability of FPA to control FLC expression.	RESULTS
+8	15	RT-qPCR	experimental_method	We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).	RESULTS
+45	48	FLC	gene	We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).	RESULTS
+49	53	mRNA	chemical	We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).	RESULTS
+119	126	mutated	protein_state	We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).	RESULTS
+127	130	FPA	protein	We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).	RESULTS
+163	166	FLC	gene	We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).	RESULTS
+179	184	fpa-8	gene	We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).	RESULTS
+185	192	mutants	protein_state	We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).	RESULTS
+215	224	wild-type	protein_state	We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).	RESULTS
+253	256	FPA	protein	We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).	RESULTS
+257	261	SPOC	structure_element	We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).	RESULTS
+262	271	conserved	protein_state	We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).	RESULTS
+272	277	patch	site	We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).	RESULTS
+278	284	mutant	protein_state	We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).	RESULTS
+11	24	surface patch	site	Since each surface patch mutation appeared to be insufficient to disrupt FPA functions on its own, we combined both mutations into the same transgene.	RESULTS
+25	33	mutation	experimental_method	Since each surface patch mutation appeared to be insufficient to disrupt FPA functions on its own, we combined both mutations into the same transgene.	RESULTS
+73	76	FPA	protein	Since each surface patch mutation appeared to be insufficient to disrupt FPA functions on its own, we combined both mutations into the same transgene.	RESULTS
+33	42	wild-type	protein_state	We could again confirm that near wild-type levels of FPA protein were expressed from three independent transgenic lines expressing the FPA R477A;Y515A doubly mutated protein in an fpa-8 mutant background (S3 Fig).	RESULTS
+53	56	FPA	protein	We could again confirm that near wild-type levels of FPA protein were expressed from three independent transgenic lines expressing the FPA R477A;Y515A doubly mutated protein in an fpa-8 mutant background (S3 Fig).	RESULTS
+135	150	FPA R477A;Y515A	mutant	We could again confirm that near wild-type levels of FPA protein were expressed from three independent transgenic lines expressing the FPA R477A;Y515A doubly mutated protein in an fpa-8 mutant background (S3 Fig).	RESULTS
+151	165	doubly mutated	protein_state	We could again confirm that near wild-type levels of FPA protein were expressed from three independent transgenic lines expressing the FPA R477A;Y515A doubly mutated protein in an fpa-8 mutant background (S3 Fig).	RESULTS
+180	185	fpa-8	gene	We could again confirm that near wild-type levels of FPA protein were expressed from three independent transgenic lines expressing the FPA R477A;Y515A doubly mutated protein in an fpa-8 mutant background (S3 Fig).	RESULTS
+186	192	mutant	protein_state	We could again confirm that near wild-type levels of FPA protein were expressed from three independent transgenic lines expressing the FPA R477A;Y515A doubly mutated protein in an fpa-8 mutant background (S3 Fig).	RESULTS
+14	29	FPA R477A;Y515A	mutant	We found that FPA R477A;Y515A protein functioned like wild-type FPA to restore FPA pre-mRNA proximal polyadenylation (Fig 6A) and FLC expression to wild-type levels (Fig 6B).	RESULTS
+54	63	wild-type	protein_state	We found that FPA R477A;Y515A protein functioned like wild-type FPA to restore FPA pre-mRNA proximal polyadenylation (Fig 6A) and FLC expression to wild-type levels (Fig 6B).	RESULTS
+64	67	FPA	protein	We found that FPA R477A;Y515A protein functioned like wild-type FPA to restore FPA pre-mRNA proximal polyadenylation (Fig 6A) and FLC expression to wild-type levels (Fig 6B).	RESULTS
+79	82	FPA	protein	We found that FPA R477A;Y515A protein functioned like wild-type FPA to restore FPA pre-mRNA proximal polyadenylation (Fig 6A) and FLC expression to wild-type levels (Fig 6B).	RESULTS
+83	91	pre-mRNA	chemical	We found that FPA R477A;Y515A protein functioned like wild-type FPA to restore FPA pre-mRNA proximal polyadenylation (Fig 6A) and FLC expression to wild-type levels (Fig 6B).	RESULTS
+130	133	FLC	gene	We found that FPA R477A;Y515A protein functioned like wild-type FPA to restore FPA pre-mRNA proximal polyadenylation (Fig 6A) and FLC expression to wild-type levels (Fig 6B).	RESULTS
+148	157	wild-type	protein_state	We found that FPA R477A;Y515A protein functioned like wild-type FPA to restore FPA pre-mRNA proximal polyadenylation (Fig 6A) and FLC expression to wild-type levels (Fig 6B).	RESULTS
+17	20	FPA	protein	Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and FLC expression.	FIG
+21	25	SPOC	structure_element	Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and FLC expression.	FIG
+33	42	mutations	experimental_method	Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and FLC expression.	FIG
+77	80	FPA	protein	Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and FLC expression.	FIG
+81	89	pre-mRNA	chemical	Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and FLC expression.	FIG
+94	97	FLC	gene	Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and FLC expression.	FIG
+4	16	RNA gel blot	experimental_method	(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs.	FIG
+29	31	WT	protein_state	(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs.	FIG
+32	43	A. thaliana	species	(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs.	FIG
+71	77	plants	taxonomy_domain	(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs.	FIG
+78	83	fpa-8	gene	(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs.	FIG
+88	93	fpa-8	gene	(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs.	FIG
+94	101	mutants	protein_state	(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs.	FIG
+113	116	FPA	protein	(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs.	FIG
+118	133	FPA R477A;Y515A	mutant	(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs.	FIG
+158	163	mRNAs	chemical	(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs.	FIG
+65	68	FPA	protein	Black arrows indicate the proximally and distally polyadenylated FPA mRNAs.	FIG
+69	74	mRNAs	chemical	Black arrows indicate the proximally and distally polyadenylated FPA mRNAs.	FIG
+0	7	qRT-PCR	experimental_method	qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, and FPA::FPA R477A;Y515A plants.	FIG
+42	45	RNA	chemical	qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, and FPA::FPA R477A;Y515A plants.	FIG
+67	72	fpa-8	gene	qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, and FPA::FPA R477A;Y515A plants.	FIG
+78	81	FPA	protein	qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, and FPA::FPA R477A;Y515A plants.	FIG
+83	98	FPA R477A;Y515A	mutant	qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, and FPA::FPA R477A;Y515A plants.	FIG
+99	105	plants	taxonomy_domain	qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, and FPA::FPA R477A;Y515A plants.	FIG
+46	50	SPOC	structure_element	Together our findings suggest that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation, or that this combination of mutations is not sufficient to critically disrupt the function of the FPA SPOC domain.	RESULTS
+90	93	FPA	protein	Together our findings suggest that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation, or that this combination of mutations is not sufficient to critically disrupt the function of the FPA SPOC domain.	RESULTS
+108	111	RNA	chemical	Together our findings suggest that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation, or that this combination of mutations is not sufficient to critically disrupt the function of the FPA SPOC domain.	RESULTS
+158	167	mutations	experimental_method	Together our findings suggest that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation, or that this combination of mutations is not sufficient to critically disrupt the function of the FPA SPOC domain.	RESULTS
+228	231	FPA	protein	Together our findings suggest that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation, or that this combination of mutations is not sufficient to critically disrupt the function of the FPA SPOC domain.	RESULTS
+232	236	SPOC	structure_element	Together our findings suggest that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation, or that this combination of mutations is not sufficient to critically disrupt the function of the FPA SPOC domain.	RESULTS
+24	33	mutations	experimental_method	Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain.	RESULTS
+41	46	SHARP	protein	Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain.	RESULTS
+47	51	SPOC	structure_element	Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain.	RESULTS
+89	105	unphosphorylated	protein_state	Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain.	RESULTS
+106	110	SMRT	protein	Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain.	RESULTS
+111	119	peptides	chemical	Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain.	RESULTS
+202	205	FPA	protein	Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain.	RESULTS
+206	210	SPOC	structure_element	Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain.	RESULTS
+273	277	SPOC	structure_element	Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain.	RESULTS
diff --git a/annotation_CSV/PMC4993997.csv b/annotation_CSV/PMC4993997.csv
new file mode 100644
index 0000000000000000000000000000000000000000..77d0d9b05ce4ee3b3a86fb83c03aeaedc012ac6c
--- /dev/null
+++ b/annotation_CSV/PMC4993997.csv
@@ -0,0 +1,1052 @@
+anno_start	anno_end	anno_text	entity_type	sentence	section
+26	31	human	species	Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase	TITLE
+32	37	Naa60	protein	Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase	TITLE
+39	43	NatF	complex_assembly	Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase	TITLE
+78	95	acetyltransferase	protein_type	Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase	TITLE
+0	22	N-terminal acetylation	ptm	N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains.	ABSTRACT
+24	38	Nt-acetylation	ptm	N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains.	ABSTRACT
+56	85	N-terminal acetyltransferases	protein_type	N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains.	ABSTRACT
+87	91	NATs	protein_type	N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains.	ABSTRACT
+145	152	peptide	chemical	N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains.	ABSTRACT
+0	5	Naa60	protein	Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes.	ABSTRACT
+18	22	NatF	complex_assembly	Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes.	ABSTRACT
+49	52	NAT	protein_type	Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes.	ABSTRACT
+67	91	multicellular eukaryotes	taxonomy_domain	Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes.	ABSTRACT
+80	94	Nt-acetylation	ptm	This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine.	ABSTRACT
+142	169	lysine Nε-acetyltransferase	protein_type	This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine.	ABSTRACT
+171	174	KAT	protein_type	This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine.	ABSTRACT
+201	212	acetylation	ptm	This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine.	ABSTRACT
+216	222	lysine	residue_name	This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine.	ABSTRACT
+20	38	crystal structures	evidence	Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA).	ABSTRACT
+42	47	human	species	Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA).	ABSTRACT
+48	53	Naa60	protein	Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA).	ABSTRACT
+55	61	hNaa60	protein	Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA).	ABSTRACT
+63	78	in complex with	protein_state	Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA).	ABSTRACT
+79	96	Acetyl-Coenzyme A	chemical	Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA).	ABSTRACT
+98	104	Ac-CoA	chemical	Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA).	ABSTRACT
+109	119	Coenzyme A	chemical	Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA).	ABSTRACT
+121	124	CoA	chemical	Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA).	ABSTRACT
+4	10	hNaa60	protein	The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures.	ABSTRACT
+31	48	amphipathic helix	structure_element	The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures.	ABSTRACT
+63	74	GNAT domain	structure_element	The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures.	ABSTRACT
+120	126	hNaa60	protein	The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures.	ABSTRACT
+136	149	β7-β8 hairpin	structure_element	The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures.	ABSTRACT
+189	195	hNaa60	protein	The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures.	ABSTRACT
+196	201	1-242	residue_range	The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures.	ABSTRACT
+207	220	hNaa60(1-199)	mutant	The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures.	ABSTRACT
+221	239	crystal structures	evidence	The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures.	ABSTRACT
+44	50	Phe 34	residue_name_number	Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions.	ABSTRACT
+85	93	coenzyme	chemical	Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions.	ABSTRACT
+10	55	structural comparison and biochemical studies	experimental_method	Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.	ABSTRACT
+71	77	Tyr 97	residue_name_number	Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.	ABSTRACT
+82	89	His 138	residue_name_number	Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.	ABSTRACT
+141	154	non-conserved	protein_state	Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.	ABSTRACT
+155	170	β3-β4 long loop	structure_element	Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.	ABSTRACT
+205	211	hNaa60	protein	Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.	ABSTRACT
+0	11	Acetylation	ptm	Acetylation is one of the most ubiquitous modifications that plays a vital role in many biological processes, such as transcriptional regulation, protein-protein interaction, enzyme activity, protein stability, antibiotic resistance, biological rhythm and so on.	INTRO
+8	19	acetylation	ptm	Protein acetylation can be grouped into lysine Nε-acetylation and peptide N-terminal acetylation (Nt-acetylation).	INTRO
+40	61	lysine Nε-acetylation	ptm	Protein acetylation can be grouped into lysine Nε-acetylation and peptide N-terminal acetylation (Nt-acetylation).	INTRO
+66	73	peptide	chemical	Protein acetylation can be grouped into lysine Nε-acetylation and peptide N-terminal acetylation (Nt-acetylation).	INTRO
+74	96	N-terminal acetylation	ptm	Protein acetylation can be grouped into lysine Nε-acetylation and peptide N-terminal acetylation (Nt-acetylation).	INTRO
+98	112	Nt-acetylation	ptm	Protein acetylation can be grouped into lysine Nε-acetylation and peptide N-terminal acetylation (Nt-acetylation).	INTRO
+11	25	Nε-acetylation	ptm	Generally, Nε-acetylation refers to the transfer of an acetyl group from an acetyl coenzyme A (Ac-CoA) to the ε-amino group of lysine.	INTRO
+55	61	acetyl	chemical	Generally, Nε-acetylation refers to the transfer of an acetyl group from an acetyl coenzyme A (Ac-CoA) to the ε-amino group of lysine.	INTRO
+76	93	acetyl coenzyme A	chemical	Generally, Nε-acetylation refers to the transfer of an acetyl group from an acetyl coenzyme A (Ac-CoA) to the ε-amino group of lysine.	INTRO
+95	101	Ac-CoA	chemical	Generally, Nε-acetylation refers to the transfer of an acetyl group from an acetyl coenzyme A (Ac-CoA) to the ε-amino group of lysine.	INTRO
+127	133	lysine	residue_name	Generally, Nε-acetylation refers to the transfer of an acetyl group from an acetyl coenzyme A (Ac-CoA) to the ε-amino group of lysine.	INTRO
+42	67	lysine acetyltransferases	protein_type	This kind of modification is catalyzed by lysine acetyltransferases (KATs), some of which are named histone acetyltransferases (HATs) because early studies focused mostly on the post-transcriptional acetylation of histones.	INTRO
+69	73	KATs	protein_type	This kind of modification is catalyzed by lysine acetyltransferases (KATs), some of which are named histone acetyltransferases (HATs) because early studies focused mostly on the post-transcriptional acetylation of histones.	INTRO
+100	126	histone acetyltransferases	protein_type	This kind of modification is catalyzed by lysine acetyltransferases (KATs), some of which are named histone acetyltransferases (HATs) because early studies focused mostly on the post-transcriptional acetylation of histones.	INTRO
+128	132	HATs	protein_type	This kind of modification is catalyzed by lysine acetyltransferases (KATs), some of which are named histone acetyltransferases (HATs) because early studies focused mostly on the post-transcriptional acetylation of histones.	INTRO
+199	210	acetylation	ptm	This kind of modification is catalyzed by lysine acetyltransferases (KATs), some of which are named histone acetyltransferases (HATs) because early studies focused mostly on the post-transcriptional acetylation of histones.	INTRO
+214	222	histones	protein_type	This kind of modification is catalyzed by lysine acetyltransferases (KATs), some of which are named histone acetyltransferases (HATs) because early studies focused mostly on the post-transcriptional acetylation of histones.	INTRO
+61	75	Nε-acetylation	ptm	Despite the prominent accomplishments in the field regarding Nε-acetylation by KATs for over 50 years, the significance of the more evolutionarily conserved Nt-acetylation is still inconclusive.	INTRO
+79	83	KATs	protein_type	Despite the prominent accomplishments in the field regarding Nε-acetylation by KATs for over 50 years, the significance of the more evolutionarily conserved Nt-acetylation is still inconclusive.	INTRO
+157	171	Nt-acetylation	ptm	Despite the prominent accomplishments in the field regarding Nε-acetylation by KATs for over 50 years, the significance of the more evolutionarily conserved Nt-acetylation is still inconclusive.	INTRO
+0	14	Nt-acetylation	ptm	Nt-acetylation is an abundant and evolutionarily conserved modification occurring in bacteria, archaea and eukaryotes.	INTRO
+85	93	bacteria	taxonomy_domain	Nt-acetylation is an abundant and evolutionarily conserved modification occurring in bacteria, archaea and eukaryotes.	INTRO
+95	102	archaea	taxonomy_domain	Nt-acetylation is an abundant and evolutionarily conserved modification occurring in bacteria, archaea and eukaryotes.	INTRO
+107	117	eukaryotes	taxonomy_domain	Nt-acetylation is an abundant and evolutionarily conserved modification occurring in bacteria, archaea and eukaryotes.	INTRO
+45	50	human	species	It is estimated that about 80–90% of soluble human proteins and 50–70% of yeast proteins are subjected to Nt-acetylation, where an acetyl moiety is transferred from Ac-CoA to the α-amino group of the first residue.	INTRO
+74	79	yeast	taxonomy_domain	It is estimated that about 80–90% of soluble human proteins and 50–70% of yeast proteins are subjected to Nt-acetylation, where an acetyl moiety is transferred from Ac-CoA to the α-amino group of the first residue.	INTRO
+106	120	Nt-acetylation	ptm	It is estimated that about 80–90% of soluble human proteins and 50–70% of yeast proteins are subjected to Nt-acetylation, where an acetyl moiety is transferred from Ac-CoA to the α-amino group of the first residue.	INTRO
+131	137	acetyl	chemical	It is estimated that about 80–90% of soluble human proteins and 50–70% of yeast proteins are subjected to Nt-acetylation, where an acetyl moiety is transferred from Ac-CoA to the α-amino group of the first residue.	INTRO
+165	171	Ac-CoA	chemical	It is estimated that about 80–90% of soluble human proteins and 50–70% of yeast proteins are subjected to Nt-acetylation, where an acetyl moiety is transferred from Ac-CoA to the α-amino group of the first residue.	INTRO
+34	48	Nt-acetylation	ptm	Recently Nt-acetylome expands the Nt-acetylation to transmembrane proteins.	INTRO
+7	21	Nε-acetylation	ptm	Unlike Nε-acetylation that can be eliminated by deacetylases, Nt-acetylation is considered irreversible since no corresponding deacetylase is found to date.	INTRO
+48	60	deacetylases	protein_type	Unlike Nε-acetylation that can be eliminated by deacetylases, Nt-acetylation is considered irreversible since no corresponding deacetylase is found to date.	INTRO
+62	76	Nt-acetylation	ptm	Unlike Nε-acetylation that can be eliminated by deacetylases, Nt-acetylation is considered irreversible since no corresponding deacetylase is found to date.	INTRO
+91	103	irreversible	protein_state	Unlike Nε-acetylation that can be eliminated by deacetylases, Nt-acetylation is considered irreversible since no corresponding deacetylase is found to date.	INTRO
+127	138	deacetylase	protein_type	Unlike Nε-acetylation that can be eliminated by deacetylases, Nt-acetylation is considered irreversible since no corresponding deacetylase is found to date.	INTRO
+9	23	Nt-acetylation	ptm	Although Nt-acetylation has been regarded as a co-translational modification traditionally, there is evidence that post-translational Nt-acetylation exists.	INTRO
+134	148	Nt-acetylation	ptm	Although Nt-acetylation has been regarded as a co-translational modification traditionally, there is evidence that post-translational Nt-acetylation exists.	INTRO
+110	124	Nt-acetylation	ptm	During the past decades, a large number of Nt-acetylome researches have shed light on the functional roles of Nt-acetylation, including protein degradation, subcellular localization, protein-protein interaction, protein-membrane interaction, plant development, stress-response and protein stability.	INTRO
+242	247	plant	taxonomy_domain	During the past decades, a large number of Nt-acetylome researches have shed light on the functional roles of Nt-acetylation, including protein degradation, subcellular localization, protein-protein interaction, protein-membrane interaction, plant development, stress-response and protein stability.	INTRO
+4	18	Nt-acetylation	ptm	The Nt-acetylation is carried out by N-terminal acetyltransferases (NATs) that belong to the GNAT superfamily.	INTRO
+37	66	N-terminal acetyltransferases	protein_type	The Nt-acetylation is carried out by N-terminal acetyltransferases (NATs) that belong to the GNAT superfamily.	INTRO
+68	72	NATs	protein_type	The Nt-acetylation is carried out by N-terminal acetyltransferases (NATs) that belong to the GNAT superfamily.	INTRO
+93	109	GNAT superfamily	protein_type	The Nt-acetylation is carried out by N-terminal acetyltransferases (NATs) that belong to the GNAT superfamily.	INTRO
+13	17	NATs	protein_type	To date, six NATs (NatA/B/C/D/E/F) have been identified in eukaryotes.	INTRO
+19	23	NatA	complex_assembly	To date, six NATs (NatA/B/C/D/E/F) have been identified in eukaryotes.	INTRO
+24	25	B	complex_assembly	To date, six NATs (NatA/B/C/D/E/F) have been identified in eukaryotes.	INTRO
+26	27	C	complex_assembly	To date, six NATs (NatA/B/C/D/E/F) have been identified in eukaryotes.	INTRO
+28	29	D	complex_assembly	To date, six NATs (NatA/B/C/D/E/F) have been identified in eukaryotes.	INTRO
+30	31	E	complex_assembly	To date, six NATs (NatA/B/C/D/E/F) have been identified in eukaryotes.	INTRO
+32	33	F	complex_assembly	To date, six NATs (NatA/B/C/D/E/F) have been identified in eukaryotes.	INTRO
+59	69	eukaryotes	taxonomy_domain	To date, six NATs (NatA/B/C/D/E/F) have been identified in eukaryotes.	INTRO
+20	34	Nt-acetylation	ptm	About 40 percent of Nt-acetylation of soluble proteins in cells is catalyzed by NatA complex which is composed of the catalytic subunit Naa10p and the auxiliary subunit Naa15p.	INTRO
+80	84	NatA	complex_assembly	About 40 percent of Nt-acetylation of soluble proteins in cells is catalyzed by NatA complex which is composed of the catalytic subunit Naa10p and the auxiliary subunit Naa15p.	INTRO
+136	142	Naa10p	protein	About 40 percent of Nt-acetylation of soluble proteins in cells is catalyzed by NatA complex which is composed of the catalytic subunit Naa10p and the auxiliary subunit Naa15p.	INTRO
+169	175	Naa15p	protein	About 40 percent of Nt-acetylation of soluble proteins in cells is catalyzed by NatA complex which is composed of the catalytic subunit Naa10p and the auxiliary subunit Naa15p.	INTRO
+0	4	NatE	complex_assembly	NatE was found to physically interact with the NatA complex without any observation of impact on NatA-activity.	INTRO
+47	51	NatA	complex_assembly	NatE was found to physically interact with the NatA complex without any observation of impact on NatA-activity.	INTRO
+97	101	NatA	complex_assembly	NatE was found to physically interact with the NatA complex without any observation of impact on NatA-activity.	INTRO
+34	38	NATs	protein_type	Two other multimeric complexes of NATs are NatB and NatC which contain the catalytic subunits Naa20 and Naa30 and the auxiliary subunits Naa25 and Naa35/Naa38, respectively.	INTRO
+43	47	NatB	complex_assembly	Two other multimeric complexes of NATs are NatB and NatC which contain the catalytic subunits Naa20 and Naa30 and the auxiliary subunits Naa25 and Naa35/Naa38, respectively.	INTRO
+52	56	NatC	complex_assembly	Two other multimeric complexes of NATs are NatB and NatC which contain the catalytic subunits Naa20 and Naa30 and the auxiliary subunits Naa25 and Naa35/Naa38, respectively.	INTRO
+94	99	Naa20	protein	Two other multimeric complexes of NATs are NatB and NatC which contain the catalytic subunits Naa20 and Naa30 and the auxiliary subunits Naa25 and Naa35/Naa38, respectively.	INTRO
+104	109	Naa30	protein	Two other multimeric complexes of NATs are NatB and NatC which contain the catalytic subunits Naa20 and Naa30 and the auxiliary subunits Naa25 and Naa35/Naa38, respectively.	INTRO
+137	142	Naa25	protein	Two other multimeric complexes of NATs are NatB and NatC which contain the catalytic subunits Naa20 and Naa30 and the auxiliary subunits Naa25 and Naa35/Naa38, respectively.	INTRO
+147	152	Naa35	protein	Two other multimeric complexes of NATs are NatB and NatC which contain the catalytic subunits Naa20 and Naa30 and the auxiliary subunits Naa25 and Naa35/Naa38, respectively.	INTRO
+153	158	Naa38	protein	Two other multimeric complexes of NATs are NatB and NatC which contain the catalytic subunits Naa20 and Naa30 and the auxiliary subunits Naa25 and Naa35/Naa38, respectively.	INTRO
+41	46	Naa40	protein	Furthermore, only the catalytic subunits Naa40 and Naa60 were found for NatD and NatF, respectively.	INTRO
+51	56	Naa60	protein	Furthermore, only the catalytic subunits Naa40 and Naa60 were found for NatD and NatF, respectively.	INTRO
+72	76	NatD	complex_assembly	Furthermore, only the catalytic subunits Naa40 and Naa60 were found for NatD and NatF, respectively.	INTRO
+81	85	NatF	complex_assembly	Furthermore, only the catalytic subunits Naa40 and Naa60 were found for NatD and NatF, respectively.	INTRO
+8	22	Nt-acetylation	ptm	Besides Nt-acetylation, accumulating reports have proposed Nε-acetylation carried out by NATs.	INTRO
+59	73	Nε-acetylation	ptm	Besides Nt-acetylation, accumulating reports have proposed Nε-acetylation carried out by NATs.	INTRO
+89	93	NATs	protein_type	Besides Nt-acetylation, accumulating reports have proposed Nε-acetylation carried out by NATs.	INTRO
+53	67	Nt-acetylation	ptm	There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family.	INTRO
+76	81	yeast	taxonomy_domain	There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family.	INTRO
+86	91	human	species	There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family.	INTRO
+147	151	NatF	complex_assembly	There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family.	INTRO
+166	194	N-terminal acetyltransferase	protein_type	There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family.	INTRO
+223	227	NatF	complex_assembly	There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family.	INTRO
+240	245	NAA60	protein	There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family.	INTRO
+264	306	Histone acetyltransferase type B protein 4	protein	There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family.	INTRO
+308	312	HAT4	protein	There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family.	INTRO
+315	320	Naa60	protein	There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family.	INTRO
+324	346	N-acetyltransferase 15	protein	There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family.	INTRO
+348	353	NAT15	protein	There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family.	INTRO
+386	389	NAT	protein_type	There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family.	INTRO
+13	17	NATs	protein_type	Unlike other NATs that are highly conserved among lower and higher eukaryotes, NatF only exists in higher eukaryotes.	INTRO
+27	43	highly conserved	protein_state	Unlike other NATs that are highly conserved among lower and higher eukaryotes, NatF only exists in higher eukaryotes.	INTRO
+50	55	lower	taxonomy_domain	Unlike other NATs that are highly conserved among lower and higher eukaryotes, NatF only exists in higher eukaryotes.	INTRO
+60	77	higher eukaryotes	taxonomy_domain	Unlike other NATs that are highly conserved among lower and higher eukaryotes, NatF only exists in higher eukaryotes.	INTRO
+79	83	NatF	complex_assembly	Unlike other NATs that are highly conserved among lower and higher eukaryotes, NatF only exists in higher eukaryotes.	INTRO
+99	116	higher eukaryotes	taxonomy_domain	Unlike other NATs that are highly conserved among lower and higher eukaryotes, NatF only exists in higher eukaryotes.	INTRO
+37	41	NatF	complex_assembly	Subsequent researches indicated that NatF displays its catalytic ability with both Nt-acetylation and lysine Nε-acetylation.	INTRO
+83	97	Nt-acetylation	ptm	Subsequent researches indicated that NatF displays its catalytic ability with both Nt-acetylation and lysine Nε-acetylation.	INTRO
+102	123	lysine Nε-acetylation	ptm	Subsequent researches indicated that NatF displays its catalytic ability with both Nt-acetylation and lysine Nε-acetylation.	INTRO
+6	34	N-terminal acetyltransferase	protein_type	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+36	40	NatF	complex_assembly	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+67	78	acetylation	ptm	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+187	195	Met-Lys-	structure_element	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+197	205	Met-Val-	structure_element	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+207	215	Met-Ala-	structure_element	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+220	228	Met-Met-	structure_element	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+293	297	NatC	complex_assembly	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+302	306	NatE	complex_assembly	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+327	331	NatF	complex_assembly	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+342	366	lysine acetyltransferase	protein_type	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+390	408	lysine acetylation	ptm	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+417	424	histone	protein_type	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+425	427	H4	protein_type	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+439	441	H4	protein_type	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+441	444	K20	residue_name_number	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+446	448	H4	protein_type	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+448	451	K79	residue_name_number	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+456	458	H4	protein_type	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+458	461	K91	residue_name_number	As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.	INTRO
+29	33	NatF	complex_assembly	Another important feature of NatF is that this protein is anchored on the Golgi apparatus through its C-terminal membrane-integrating region and takes part in the maintaining of Golgi integrity.	INTRO
+113	140	membrane-integrating region	structure_element	Another important feature of NatF is that this protein is anchored on the Golgi apparatus through its C-terminal membrane-integrating region and takes part in the maintaining of Golgi integrity.	INTRO
+81	85	NatF	complex_assembly	With its unique intracellular organellar localization and substrate selectivity, NatF appears to provide more evolutionary information among the NAT family members.	INTRO
+145	148	NAT	protein_type	With its unique intracellular organellar localization and substrate selectivity, NatF appears to provide more evolutionary information among the NAT family members.	INTRO
+27	31	NatF	complex_assembly	It was recently found that NatF facilitates nucleosomes assembly and that NAA60 knockdown in MCF7-cell inhibits cell proliferation, sensitizes cells to DNA damage and induces cell apoptosis.	INTRO
+44	55	nucleosomes	complex_assembly	It was recently found that NatF facilitates nucleosomes assembly and that NAA60 knockdown in MCF7-cell inhibits cell proliferation, sensitizes cells to DNA damage and induces cell apoptosis.	INTRO
+74	79	NAA60	protein	It was recently found that NatF facilitates nucleosomes assembly and that NAA60 knockdown in MCF7-cell inhibits cell proliferation, sensitizes cells to DNA damage and induces cell apoptosis.	INTRO
+3	13	Drosophila	taxonomy_domain	In Drosophila cells, NAA60 knockdown induces chromosomal segregation defects during anaphase including lagging chromosomes and chromosomal bridges.	INTRO
+21	26	NAA60	protein	In Drosophila cells, NAA60 knockdown induces chromosomal segregation defects during anaphase including lagging chromosomes and chromosomal bridges.	INTRO
+66	70	NatF	complex_assembly	Much recent attention has also been focused on the requirement of NatF for regulation of organellar structure.	INTRO
+15	20	NAA60	protein	In HeLa cells, NAA60 knockdown causes Golgi apparatus fragmentation which can be rescued by overexpression Naa60.	INTRO
+92	106	overexpression	experimental_method	In HeLa cells, NAA60 knockdown causes Golgi apparatus fragmentation which can be rescued by overexpression Naa60.	INTRO
+107	112	Naa60	protein	In HeLa cells, NAA60 knockdown causes Golgi apparatus fragmentation which can be rescued by overexpression Naa60.	INTRO
+79	83	NATs	protein_type	The systematic investigation of publicly available microarray data showed that NATs share distinct tissue-specific expression patterns in Drosophila and NatF shows a higher expression level in central nervous system of Drosophila.	INTRO
+138	148	Drosophila	taxonomy_domain	The systematic investigation of publicly available microarray data showed that NATs share distinct tissue-specific expression patterns in Drosophila and NatF shows a higher expression level in central nervous system of Drosophila.	INTRO
+153	157	NatF	complex_assembly	The systematic investigation of publicly available microarray data showed that NATs share distinct tissue-specific expression patterns in Drosophila and NatF shows a higher expression level in central nervous system of Drosophila.	INTRO
+219	229	Drosophila	taxonomy_domain	The systematic investigation of publicly available microarray data showed that NATs share distinct tissue-specific expression patterns in Drosophila and NatF shows a higher expression level in central nervous system of Drosophila.	INTRO
+18	24	solved	experimental_method	In this study, we solved the structures of human Naa60 (NatF) in complex with coenzyme.	INTRO
+29	39	structures	evidence	In this study, we solved the structures of human Naa60 (NatF) in complex with coenzyme.	INTRO
+43	48	human	species	In this study, we solved the structures of human Naa60 (NatF) in complex with coenzyme.	INTRO
+49	54	Naa60	protein	In this study, we solved the structures of human Naa60 (NatF) in complex with coenzyme.	INTRO
+56	60	NatF	complex_assembly	In this study, we solved the structures of human Naa60 (NatF) in complex with coenzyme.	INTRO
+62	77	in complex with	protein_state	In this study, we solved the structures of human Naa60 (NatF) in complex with coenzyme.	INTRO
+78	86	coenzyme	chemical	In this study, we solved the structures of human Naa60 (NatF) in complex with coenzyme.	INTRO
+4	10	hNaa60	protein	The hNaa60 protein contains a unique amphipathic α-helix (α5) following its GNAT domain that might account for the Golgi localization of this protein.	INTRO
+37	56	amphipathic α-helix	structure_element	The hNaa60 protein contains a unique amphipathic α-helix (α5) following its GNAT domain that might account for the Golgi localization of this protein.	INTRO
+58	60	α5	structure_element	The hNaa60 protein contains a unique amphipathic α-helix (α5) following its GNAT domain that might account for the Golgi localization of this protein.	INTRO
+76	87	GNAT domain	structure_element	The hNaa60 protein contains a unique amphipathic α-helix (α5) following its GNAT domain that might account for the Golgi localization of this protein.	INTRO
+0	18	Crystal structures	evidence	Crystal structures showed that the β7-β8 hairpin rotated about 50 degrees upon removing the C-terminal region of the protein and this movement substantially changed the geometry of the substrate-binding pocket.	INTRO
+35	48	β7-β8 hairpin	structure_element	Crystal structures showed that the β7-β8 hairpin rotated about 50 degrees upon removing the C-terminal region of the protein and this movement substantially changed the geometry of the substrate-binding pocket.	INTRO
+92	109	C-terminal region	structure_element	Crystal structures showed that the β7-β8 hairpin rotated about 50 degrees upon removing the C-terminal region of the protein and this movement substantially changed the geometry of the substrate-binding pocket.	INTRO
+185	209	substrate-binding pocket	site	Crystal structures showed that the β7-β8 hairpin rotated about 50 degrees upon removing the C-terminal region of the protein and this movement substantially changed the geometry of the substrate-binding pocket.	INTRO
+25	31	Phe 34	residue_name_number	Remarkably, we find that Phe 34 may participate in the proper positioning of the coenzyme for the transfer reaction to occur.	INTRO
+81	89	coenzyme	chemical	Remarkably, we find that Phe 34 may participate in the proper positioning of the coenzyme for the transfer reaction to occur.	INTRO
+8	28	structure comparison	experimental_method	Further structure comparison and biochemical studies also identified other key structural elements essential for the enzyme activity of Naa60.	INTRO
+33	52	biochemical studies	experimental_method	Further structure comparison and biochemical studies also identified other key structural elements essential for the enzyme activity of Naa60.	INTRO
+136	141	Naa60	protein	Further structure comparison and biochemical studies also identified other key structural elements essential for the enzyme activity of Naa60.	INTRO
+8	17	structure	evidence	Overall structure of hNaa60	RESULTS
+21	27	hNaa60	protein	Overall structure of hNaa60	RESULTS
+88	94	hNaa60	protein	In the effort to prepare the protein for structural studies, we tried a large number of hNaa60 constructs but all failed due to heavy precipitation or aggregation.	RESULTS
+0	18	Sequence alignment	experimental_method	Sequence alignment of Naa60 from different species revealed a Glu-Glu-Arg (EER) versus Val-Val-Pro (VVP) sequence difference near the N-terminus of the protein in Xenopus Laevis versus Homo sapiens (Fig. 1A).	RESULTS
+22	27	Naa60	protein	Sequence alignment of Naa60 from different species revealed a Glu-Glu-Arg (EER) versus Val-Val-Pro (VVP) sequence difference near the N-terminus of the protein in Xenopus Laevis versus Homo sapiens (Fig. 1A).	RESULTS
+62	73	Glu-Glu-Arg	structure_element	Sequence alignment of Naa60 from different species revealed a Glu-Glu-Arg (EER) versus Val-Val-Pro (VVP) sequence difference near the N-terminus of the protein in Xenopus Laevis versus Homo sapiens (Fig. 1A).	RESULTS
+75	78	EER	structure_element	Sequence alignment of Naa60 from different species revealed a Glu-Glu-Arg (EER) versus Val-Val-Pro (VVP) sequence difference near the N-terminus of the protein in Xenopus Laevis versus Homo sapiens (Fig. 1A).	RESULTS
+87	98	Val-Val-Pro	structure_element	Sequence alignment of Naa60 from different species revealed a Glu-Glu-Arg (EER) versus Val-Val-Pro (VVP) sequence difference near the N-terminus of the protein in Xenopus Laevis versus Homo sapiens (Fig. 1A).	RESULTS
+100	103	VVP	structure_element	Sequence alignment of Naa60 from different species revealed a Glu-Glu-Arg (EER) versus Val-Val-Pro (VVP) sequence difference near the N-terminus of the protein in Xenopus Laevis versus Homo sapiens (Fig. 1A).	RESULTS
+163	177	Xenopus Laevis	species	Sequence alignment of Naa60 from different species revealed a Glu-Glu-Arg (EER) versus Val-Val-Pro (VVP) sequence difference near the N-terminus of the protein in Xenopus Laevis versus Homo sapiens (Fig. 1A).	RESULTS
+185	197	Homo sapiens	species	Sequence alignment of Naa60 from different species revealed a Glu-Glu-Arg (EER) versus Val-Val-Pro (VVP) sequence difference near the N-terminus of the protein in Xenopus Laevis versus Homo sapiens (Fig. 1A).	RESULTS
+169	176	mutated	experimental_method	Considering that terminal residues may lack higher-order structure and hydrophobic residues in this region may expose to solvent and hence cause protein aggregation, we mutated residues 4–6 from VVP to EER for the purpose of improving solubility of this protein.	RESULTS
+186	189	4–6	residue_range	Considering that terminal residues may lack higher-order structure and hydrophobic residues in this region may expose to solvent and hence cause protein aggregation, we mutated residues 4–6 from VVP to EER for the purpose of improving solubility of this protein.	RESULTS
+195	205	VVP to EER	mutant	Considering that terminal residues may lack higher-order structure and hydrophobic residues in this region may expose to solvent and hence cause protein aggregation, we mutated residues 4–6 from VVP to EER for the purpose of improving solubility of this protein.	RESULTS
+80	86	hNaa60	protein	According to previous studies, this N-terminal region should not interfere with hNaa60’s Golgi localization.	RESULTS
+14	20	hNaa60	protein	We tried many hNaa60 constructs with the three-residues mutation but only the truncated variant 1-199 and the full-length protein behaved well.	RESULTS
+56	64	mutation	experimental_method	We tried many hNaa60 constructs with the three-residues mutation but only the truncated variant 1-199 and the full-length protein behaved well.	RESULTS
+78	87	truncated	protein_state	We tried many hNaa60 constructs with the three-residues mutation but only the truncated variant 1-199 and the full-length protein behaved well.	RESULTS
+96	101	1-199	residue_range	We tried many hNaa60 constructs with the three-residues mutation but only the truncated variant 1-199 and the full-length protein behaved well.	RESULTS
+110	121	full-length	protein_state	We tried many hNaa60 constructs with the three-residues mutation but only the truncated variant 1-199 and the full-length protein behaved well.	RESULTS
+16	23	crystal	evidence	We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C).	RESULTS
+31	40	truncated	protein_state	We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C).	RESULTS
+49	54	1-199	residue_range	We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C).	RESULTS
+55	70	in complex with	protein_state	We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C).	RESULTS
+71	74	CoA	chemical	We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C).	RESULTS
+120	127	crystal	evidence	We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C).	RESULTS
+135	146	full-length	protein_state	We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C).	RESULTS
+174	179	1-242	residue_range	We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C).	RESULTS
+181	196	in complex with	protein_state	We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C).	RESULTS
+197	203	Ac-CoA	chemical	We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C).	RESULTS
+34	41	mutants	protein_state	Hereafter, all deletions or point mutants of hNaa60 we describe here are with the EER mutation.	RESULTS
+45	51	hNaa60	protein	Hereafter, all deletions or point mutants of hNaa60 we describe here are with the EER mutation.	RESULTS
+82	85	EER	structure_element	Hereafter, all deletions or point mutants of hNaa60 we describe here are with the EER mutation.	RESULTS
+86	94	mutation	experimental_method	Hereafter, all deletions or point mutants of hNaa60 we describe here are with the EER mutation.	RESULTS
+4	22	crystal structures	evidence	The crystal structures of hNaa60(1-242)/Ac-CoA and hNaa60(1-199)/CoA were determined by molecular replacement and refined to 1.38 Å and 1.60 Å resolution, respectively (Table 1).	RESULTS
+26	46	hNaa60(1-242)/Ac-CoA	complex_assembly	The crystal structures of hNaa60(1-242)/Ac-CoA and hNaa60(1-199)/CoA were determined by molecular replacement and refined to 1.38 Å and 1.60 Å resolution, respectively (Table 1).	RESULTS
+51	68	hNaa60(1-199)/CoA	complex_assembly	The crystal structures of hNaa60(1-242)/Ac-CoA and hNaa60(1-199)/CoA were determined by molecular replacement and refined to 1.38 Å and 1.60 Å resolution, respectively (Table 1).	RESULTS
+88	109	molecular replacement	experimental_method	The crystal structures of hNaa60(1-242)/Ac-CoA and hNaa60(1-199)/CoA were determined by molecular replacement and refined to 1.38 Å and 1.60 Å resolution, respectively (Table 1).	RESULTS
+4	25	electron density maps	evidence	The electron density maps were of sufficient quality to trace residues 1-211 of hNaa60(1-242) and residues 5-199 of hNaa60(1-199).	RESULTS
+71	76	1-211	residue_range	The electron density maps were of sufficient quality to trace residues 1-211 of hNaa60(1-242) and residues 5-199 of hNaa60(1-199).	RESULTS
+80	86	hNaa60	protein	The electron density maps were of sufficient quality to trace residues 1-211 of hNaa60(1-242) and residues 5-199 of hNaa60(1-199).	RESULTS
+87	92	1-242	residue_range	The electron density maps were of sufficient quality to trace residues 1-211 of hNaa60(1-242) and residues 5-199 of hNaa60(1-199).	RESULTS
+107	112	5-199	residue_range	The electron density maps were of sufficient quality to trace residues 1-211 of hNaa60(1-242) and residues 5-199 of hNaa60(1-199).	RESULTS
+116	129	hNaa60(1-199)	mutant	The electron density maps were of sufficient quality to trace residues 1-211 of hNaa60(1-242) and residues 5-199 of hNaa60(1-199).	RESULTS
+4	13	structure	evidence	The structure of hNaa60 protein contains a central domain exhibiting a classic GCN5-related N-acetyltransferase (GNAT) folding, along with the extended N- and C-terminal regions (Fig. 1B,C).	RESULTS
+17	23	hNaa60	protein	The structure of hNaa60 protein contains a central domain exhibiting a classic GCN5-related N-acetyltransferase (GNAT) folding, along with the extended N- and C-terminal regions (Fig. 1B,C).	RESULTS
+43	57	central domain	structure_element	The structure of hNaa60 protein contains a central domain exhibiting a classic GCN5-related N-acetyltransferase (GNAT) folding, along with the extended N- and C-terminal regions (Fig. 1B,C).	RESULTS
+79	111	GCN5-related N-acetyltransferase	protein_type	The structure of hNaa60 protein contains a central domain exhibiting a classic GCN5-related N-acetyltransferase (GNAT) folding, along with the extended N- and C-terminal regions (Fig. 1B,C).	RESULTS
+113	117	GNAT	protein_type	The structure of hNaa60 protein contains a central domain exhibiting a classic GCN5-related N-acetyltransferase (GNAT) folding, along with the extended N- and C-terminal regions (Fig. 1B,C).	RESULTS
+143	151	extended	protein_state	The structure of hNaa60 protein contains a central domain exhibiting a classic GCN5-related N-acetyltransferase (GNAT) folding, along with the extended N- and C-terminal regions (Fig. 1B,C).	RESULTS
+152	177	N- and C-terminal regions	structure_element	The structure of hNaa60 protein contains a central domain exhibiting a classic GCN5-related N-acetyltransferase (GNAT) folding, along with the extended N- and C-terminal regions (Fig. 1B,C).	RESULTS
+4	18	central domain	structure_element	The central domain includes nine β strands (β1-β9) and four α-helixes (α1-α4) and is highly similar to the known hNaa50p and other reported NATs (Fig. 1D).	RESULTS
+33	42	β strands	structure_element	The central domain includes nine β strands (β1-β9) and four α-helixes (α1-α4) and is highly similar to the known hNaa50p and other reported NATs (Fig. 1D).	RESULTS
+44	49	β1-β9	structure_element	The central domain includes nine β strands (β1-β9) and four α-helixes (α1-α4) and is highly similar to the known hNaa50p and other reported NATs (Fig. 1D).	RESULTS
+60	69	α-helixes	structure_element	The central domain includes nine β strands (β1-β9) and four α-helixes (α1-α4) and is highly similar to the known hNaa50p and other reported NATs (Fig. 1D).	RESULTS
+71	76	α1-α4	structure_element	The central domain includes nine β strands (β1-β9) and four α-helixes (α1-α4) and is highly similar to the known hNaa50p and other reported NATs (Fig. 1D).	RESULTS
+85	99	highly similar	protein_state	The central domain includes nine β strands (β1-β9) and four α-helixes (α1-α4) and is highly similar to the known hNaa50p and other reported NATs (Fig. 1D).	RESULTS
+113	120	hNaa50p	protein	The central domain includes nine β strands (β1-β9) and four α-helixes (α1-α4) and is highly similar to the known hNaa50p and other reported NATs (Fig. 1D).	RESULTS
+140	144	NATs	protein_type	The central domain includes nine β strands (β1-β9) and four α-helixes (α1-α4) and is highly similar to the known hNaa50p and other reported NATs (Fig. 1D).	RESULTS
+12	18	hNaa60	protein	However, in hNaa60, there is an extra 20-residue loop between β3 and β4 that forms a small subdomain with well-defined 3D structure (Fig. 1B–D).	RESULTS
+32	53	extra 20-residue loop	structure_element	However, in hNaa60, there is an extra 20-residue loop between β3 and β4 that forms a small subdomain with well-defined 3D structure (Fig. 1B–D).	RESULTS
+62	64	β3	structure_element	However, in hNaa60, there is an extra 20-residue loop between β3 and β4 that forms a small subdomain with well-defined 3D structure (Fig. 1B–D).	RESULTS
+69	71	β4	structure_element	However, in hNaa60, there is an extra 20-residue loop between β3 and β4 that forms a small subdomain with well-defined 3D structure (Fig. 1B–D).	RESULTS
+85	100	small subdomain	structure_element	However, in hNaa60, there is an extra 20-residue loop between β3 and β4 that forms a small subdomain with well-defined 3D structure (Fig. 1B–D).	RESULTS
+17	30	β7-β8 strands	structure_element	Furthermore, the β7-β8 strands form an approximately antiparallel β-hairpin structure remarkably different from that in hNaa50p (Fig. 1D).	RESULTS
+39	85	approximately antiparallel β-hairpin structure	structure_element	Furthermore, the β7-β8 strands form an approximately antiparallel β-hairpin structure remarkably different from that in hNaa50p (Fig. 1D).	RESULTS
+120	127	hNaa50p	protein	Furthermore, the β7-β8 strands form an approximately antiparallel β-hairpin structure remarkably different from that in hNaa50p (Fig. 1D).	RESULTS
+4	29	N- and C-terminal regions	structure_element	The N- and C-terminal regions form helical structures (α0 and α5) stretching out from the central GCN5-domain (Fig. 1C).	RESULTS
+35	53	helical structures	structure_element	The N- and C-terminal regions form helical structures (α0 and α5) stretching out from the central GCN5-domain (Fig. 1C).	RESULTS
+55	57	α0	structure_element	The N- and C-terminal regions form helical structures (α0 and α5) stretching out from the central GCN5-domain (Fig. 1C).	RESULTS
+62	64	α5	structure_element	The N- and C-terminal regions form helical structures (α0 and α5) stretching out from the central GCN5-domain (Fig. 1C).	RESULTS
+98	109	GCN5-domain	structure_element	The N- and C-terminal regions form helical structures (α0 and α5) stretching out from the central GCN5-domain (Fig. 1C).	RESULTS
+55	61	hNaa60	protein	Interestingly, we found that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) (Figure S1), indicating that residues 200–242 may have some auto-inhibitory effect on the activity of the enzyme.	RESULTS
+62	67	1-242	residue_range	Interestingly, we found that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) (Figure S1), indicating that residues 200–242 may have some auto-inhibitory effect on the activity of the enzyme.	RESULTS
+96	109	hNaa60(1-199)	mutant	Interestingly, we found that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) (Figure S1), indicating that residues 200–242 may have some auto-inhibitory effect on the activity of the enzyme.	RESULTS
+148	155	200–242	residue_range	Interestingly, we found that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) (Figure S1), indicating that residues 200–242 may have some auto-inhibitory effect on the activity of the enzyme.	RESULTS
+50	56	hNaa60	protein	However, since this region was not visible in the hNaa60(1-242) crystal structure, we do not yet understand how this happens.	RESULTS
+57	62	1-242	residue_range	However, since this region was not visible in the hNaa60(1-242) crystal structure, we do not yet understand how this happens.	RESULTS
+64	81	crystal structure	evidence	However, since this region was not visible in the hNaa60(1-242) crystal structure, we do not yet understand how this happens.	RESULTS
+34	40	hNaa60	protein	Another possibility is that since hNaa60 is localized on Golgi apparatus, the observed low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies.	RESULTS
+107	118	full-length	protein_state	Another possibility is that since hNaa60 is localized on Golgi apparatus, the observed low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies.	RESULTS
+119	125	hNaa60	protein	Another possibility is that since hNaa60 is localized on Golgi apparatus, the observed low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies.	RESULTS
+48	55	mutants	protein_state	For the convenience of studying the kinetics of mutants, the mutagenesis studies described hereafter were all based on hNaa60 (1-199).	RESULTS
+61	80	mutagenesis studies	experimental_method	For the convenience of studying the kinetics of mutants, the mutagenesis studies described hereafter were all based on hNaa60 (1-199).	RESULTS
+119	133	hNaa60 (1-199)	mutant	For the convenience of studying the kinetics of mutants, the mutagenesis studies described hereafter were all based on hNaa60 (1-199).	RESULTS
+3	22	amphipathic α-helix	structure_element	An amphipathic α-helix in the C-terminal region may contribute to Golgi localization of hNaa60	RESULTS
+30	47	C-terminal region	structure_element	An amphipathic α-helix in the C-terminal region may contribute to Golgi localization of hNaa60	RESULTS
+88	94	hNaa60	protein	An amphipathic α-helix in the C-terminal region may contribute to Golgi localization of hNaa60	RESULTS
+13	19	hNaa60	protein	There is one hNaa60 molecule in the asymmetric unit in the hNaa60(1-242)/Ac-CoA structure.	RESULTS
+59	79	hNaa60(1-242)/Ac-CoA	complex_assembly	There is one hNaa60 molecule in the asymmetric unit in the hNaa60(1-242)/Ac-CoA structure.	RESULTS
+80	89	structure	evidence	There is one hNaa60 molecule in the asymmetric unit in the hNaa60(1-242)/Ac-CoA structure.	RESULTS
+4	21	C-terminal region	structure_element	The C-terminal region extended from the GCN5-domain forms an amphipathic helix (α5) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between α5-helix and a hydrophobic groove between the N-terminal β1 and β3 strands of the neighbor molecule (Fig. 2A).	RESULTS
+40	51	GCN5-domain	structure_element	The C-terminal region extended from the GCN5-domain forms an amphipathic helix (α5) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between α5-helix and a hydrophobic groove between the N-terminal β1 and β3 strands of the neighbor molecule (Fig. 2A).	RESULTS
+61	78	amphipathic helix	structure_element	The C-terminal region extended from the GCN5-domain forms an amphipathic helix (α5) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between α5-helix and a hydrophobic groove between the N-terminal β1 and β3 strands of the neighbor molecule (Fig. 2A).	RESULTS
+80	82	α5	structure_element	The C-terminal region extended from the GCN5-domain forms an amphipathic helix (α5) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between α5-helix and a hydrophobic groove between the N-terminal β1 and β3 strands of the neighbor molecule (Fig. 2A).	RESULTS
+152	176	hydrophobic interactions	bond_interaction	The C-terminal region extended from the GCN5-domain forms an amphipathic helix (α5) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between α5-helix and a hydrophobic groove between the N-terminal β1 and β3 strands of the neighbor molecule (Fig. 2A).	RESULTS
+185	193	α5-helix	structure_element	The C-terminal region extended from the GCN5-domain forms an amphipathic helix (α5) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between α5-helix and a hydrophobic groove between the N-terminal β1 and β3 strands of the neighbor molecule (Fig. 2A).	RESULTS
+200	218	hydrophobic groove	site	The C-terminal region extended from the GCN5-domain forms an amphipathic helix (α5) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between α5-helix and a hydrophobic groove between the N-terminal β1 and β3 strands of the neighbor molecule (Fig. 2A).	RESULTS
+242	244	β1	structure_element	The C-terminal region extended from the GCN5-domain forms an amphipathic helix (α5) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between α5-helix and a hydrophobic groove between the N-terminal β1 and β3 strands of the neighbor molecule (Fig. 2A).	RESULTS
+249	259	β3 strands	structure_element	The C-terminal region extended from the GCN5-domain forms an amphipathic helix (α5) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between α5-helix and a hydrophobic groove between the N-terminal β1 and β3 strands of the neighbor molecule (Fig. 2A).	RESULTS
+4	24	C-terminal extension	structure_element	The C-terminal extension following α5-helix forms a β-turn that wraps around and interacts with the neighbor protein molecule through hydrophobic interactions, too.	RESULTS
+35	43	α5-helix	structure_element	The C-terminal extension following α5-helix forms a β-turn that wraps around and interacts with the neighbor protein molecule through hydrophobic interactions, too.	RESULTS
+52	58	β-turn	structure_element	The C-terminal extension following α5-helix forms a β-turn that wraps around and interacts with the neighbor protein molecule through hydrophobic interactions, too.	RESULTS
+134	158	hydrophobic interactions	bond_interaction	The C-terminal extension following α5-helix forms a β-turn that wraps around and interacts with the neighbor protein molecule through hydrophobic interactions, too.	RESULTS
+7	24	hNaa60(1-199)/CoA	complex_assembly	In the hNaa60(1-199)/CoA structure, a part of the α5-helix is deleted due to truncation of the C-terminal region (Fig. 1B).	RESULTS
+25	34	structure	evidence	In the hNaa60(1-199)/CoA structure, a part of the α5-helix is deleted due to truncation of the C-terminal region (Fig. 1B).	RESULTS
+50	58	α5-helix	structure_element	In the hNaa60(1-199)/CoA structure, a part of the α5-helix is deleted due to truncation of the C-terminal region (Fig. 1B).	RESULTS
+95	112	C-terminal region	structure_element	In the hNaa60(1-199)/CoA structure, a part of the α5-helix is deleted due to truncation of the C-terminal region (Fig. 1B).	RESULTS
+41	49	α5-helix	structure_element	Interestingly, the remaining residues in α5-helix still form an amphipathic helix although the hydrophobic interaction with the N-terminal hydrophobic groove of a neighbor molecule is abolished and the helix is largely exposed in solvent due to different crystal packing (Fig. 2B).	RESULTS
+64	81	amphipathic helix	structure_element	Interestingly, the remaining residues in α5-helix still form an amphipathic helix although the hydrophobic interaction with the N-terminal hydrophobic groove of a neighbor molecule is abolished and the helix is largely exposed in solvent due to different crystal packing (Fig. 2B).	RESULTS
+95	118	hydrophobic interaction	bond_interaction	Interestingly, the remaining residues in α5-helix still form an amphipathic helix although the hydrophobic interaction with the N-terminal hydrophobic groove of a neighbor molecule is abolished and the helix is largely exposed in solvent due to different crystal packing (Fig. 2B).	RESULTS
+139	157	hydrophobic groove	site	Interestingly, the remaining residues in α5-helix still form an amphipathic helix although the hydrophobic interaction with the N-terminal hydrophobic groove of a neighbor molecule is abolished and the helix is largely exposed in solvent due to different crystal packing (Fig. 2B).	RESULTS
+202	207	helix	structure_element	Interestingly, the remaining residues in α5-helix still form an amphipathic helix although the hydrophobic interaction with the N-terminal hydrophobic groove of a neighbor molecule is abolished and the helix is largely exposed in solvent due to different crystal packing (Fig. 2B).	RESULTS
+255	270	crystal packing	evidence	Interestingly, the remaining residues in α5-helix still form an amphipathic helix although the hydrophobic interaction with the N-terminal hydrophobic groove of a neighbor molecule is abolished and the helix is largely exposed in solvent due to different crystal packing (Fig. 2B).	RESULTS
+39	46	182–216	residue_range	A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.	RESULTS
+85	91	hNaa60	protein	A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.	RESULTS
+119	128	structure	evidence	A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.	RESULTS
+134	149	solvent-exposed	protein_state	A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.	RESULTS
+150	167	amphipathic helix	structure_element	A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.	RESULTS
+169	171	α5	structure_element	A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.	RESULTS
+192	199	190-202	residue_range	A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.	RESULTS
+259	266	Ile 190	residue_name_number	A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.	RESULTS
+268	275	Leu 191	residue_name_number	A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.	RESULTS
+277	284	Ile 194	residue_name_number	A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.	RESULTS
+286	293	Leu 197	residue_name_number	A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.	RESULTS
+298	305	Leu 201	residue_name_number	A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.	RESULTS
+398	404	hNaa60	protein	A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.	RESULTS
+579	587	KalSec14	protein	A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.	RESULTS
+589	593	Atg3	protein	A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.	RESULTS
+595	601	PB1-F2	protein	A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.	RESULTS
+4	17	β7-β8 hairpin	structure_element	The β7-β8 hairpin showed alternative conformations in the hNaa60 crystal structures	RESULTS
+58	64	hNaa60	protein	The β7-β8 hairpin showed alternative conformations in the hNaa60 crystal structures	RESULTS
+65	83	crystal structures	evidence	The β7-β8 hairpin showed alternative conformations in the hNaa60 crystal structures	RESULTS
+0	13	Superposition	experimental_method	Superposition of hNaa60(1-242)/Ac-CoA, hNaa60(1-199)/CoA and hNaa50/CoA/peptide (PDB 3TFY) revealed considerable difference in the β7-β8 hairpin region despite the overall stability and similarity of the GNAT domain (Fig. 1D).	RESULTS
+17	37	hNaa60(1-242)/Ac-CoA	complex_assembly	Superposition of hNaa60(1-242)/Ac-CoA, hNaa60(1-199)/CoA and hNaa50/CoA/peptide (PDB 3TFY) revealed considerable difference in the β7-β8 hairpin region despite the overall stability and similarity of the GNAT domain (Fig. 1D).	RESULTS
+39	56	hNaa60(1-199)/CoA	complex_assembly	Superposition of hNaa60(1-242)/Ac-CoA, hNaa60(1-199)/CoA and hNaa50/CoA/peptide (PDB 3TFY) revealed considerable difference in the β7-β8 hairpin region despite the overall stability and similarity of the GNAT domain (Fig. 1D).	RESULTS
+61	79	hNaa50/CoA/peptide	complex_assembly	Superposition of hNaa60(1-242)/Ac-CoA, hNaa60(1-199)/CoA and hNaa50/CoA/peptide (PDB 3TFY) revealed considerable difference in the β7-β8 hairpin region despite the overall stability and similarity of the GNAT domain (Fig. 1D).	RESULTS
+131	144	β7-β8 hairpin	structure_element	Superposition of hNaa60(1-242)/Ac-CoA, hNaa60(1-199)/CoA and hNaa50/CoA/peptide (PDB 3TFY) revealed considerable difference in the β7-β8 hairpin region despite the overall stability and similarity of the GNAT domain (Fig. 1D).	RESULTS
+204	215	GNAT domain	structure_element	Superposition of hNaa60(1-242)/Ac-CoA, hNaa60(1-199)/CoA and hNaa50/CoA/peptide (PDB 3TFY) revealed considerable difference in the β7-β8 hairpin region despite the overall stability and similarity of the GNAT domain (Fig. 1D).	RESULTS
+3	9	hNaa60	protein	In hNaa60(1-242), the β7-β8 hairpin is located in close proximity to the α1-α2 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (β6-β7 loop).	RESULTS
+10	15	1-242	residue_range	In hNaa60(1-242), the β7-β8 hairpin is located in close proximity to the α1-α2 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (β6-β7 loop).	RESULTS
+22	35	β7-β8 hairpin	structure_element	In hNaa60(1-242), the β7-β8 hairpin is located in close proximity to the α1-α2 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (β6-β7 loop).	RESULTS
+73	83	α1-α2 loop	structure_element	In hNaa60(1-242), the β7-β8 hairpin is located in close proximity to the α1-α2 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (β6-β7 loop).	RESULTS
+109	131	substrate binding site	site	In hNaa60(1-242), the β7-β8 hairpin is located in close proximity to the α1-α2 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (β6-β7 loop).	RESULTS
+145	151	hNaa50	protein	In hNaa60(1-242), the β7-β8 hairpin is located in close proximity to the α1-α2 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (β6-β7 loop).	RESULTS
+185	193	flexible	protein_state	In hNaa60(1-242), the β7-β8 hairpin is located in close proximity to the α1-α2 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (β6-β7 loop).	RESULTS
+194	198	loop	structure_element	In hNaa60(1-242), the β7-β8 hairpin is located in close proximity to the α1-α2 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (β6-β7 loop).	RESULTS
+213	223	β6-β7 loop	structure_element	In hNaa60(1-242), the β7-β8 hairpin is located in close proximity to the α1-α2 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (β6-β7 loop).	RESULTS
+5	13	removing	experimental_method	Upon removing the C-terminal region of hNaa60, we observed that hNaa60 (1-199) molecules pack in a different way involving the β7-β8 hairpin in the crystal, leading to about 50 degree rotation of the hairpin which moves away from the α1-α2 loop (Figs 1D and 2C).	RESULTS
+18	35	C-terminal region	structure_element	Upon removing the C-terminal region of hNaa60, we observed that hNaa60 (1-199) molecules pack in a different way involving the β7-β8 hairpin in the crystal, leading to about 50 degree rotation of the hairpin which moves away from the α1-α2 loop (Figs 1D and 2C).	RESULTS
+39	45	hNaa60	protein	Upon removing the C-terminal region of hNaa60, we observed that hNaa60 (1-199) molecules pack in a different way involving the β7-β8 hairpin in the crystal, leading to about 50 degree rotation of the hairpin which moves away from the α1-α2 loop (Figs 1D and 2C).	RESULTS
+64	78	hNaa60 (1-199)	mutant	Upon removing the C-terminal region of hNaa60, we observed that hNaa60 (1-199) molecules pack in a different way involving the β7-β8 hairpin in the crystal, leading to about 50 degree rotation of the hairpin which moves away from the α1-α2 loop (Figs 1D and 2C).	RESULTS
+127	140	β7-β8 hairpin	structure_element	Upon removing the C-terminal region of hNaa60, we observed that hNaa60 (1-199) molecules pack in a different way involving the β7-β8 hairpin in the crystal, leading to about 50 degree rotation of the hairpin which moves away from the α1-α2 loop (Figs 1D and 2C).	RESULTS
+148	155	crystal	evidence	Upon removing the C-terminal region of hNaa60, we observed that hNaa60 (1-199) molecules pack in a different way involving the β7-β8 hairpin in the crystal, leading to about 50 degree rotation of the hairpin which moves away from the α1-α2 loop (Figs 1D and 2C).	RESULTS
+200	207	hairpin	structure_element	Upon removing the C-terminal region of hNaa60, we observed that hNaa60 (1-199) molecules pack in a different way involving the β7-β8 hairpin in the crystal, leading to about 50 degree rotation of the hairpin which moves away from the α1-α2 loop (Figs 1D and 2C).	RESULTS
+234	244	α1-α2 loop	structure_element	Upon removing the C-terminal region of hNaa60, we observed that hNaa60 (1-199) molecules pack in a different way involving the β7-β8 hairpin in the crystal, leading to about 50 degree rotation of the hairpin which moves away from the α1-α2 loop (Figs 1D and 2C).	RESULTS
+69	91	substrate binding site	site	This conformational change substantially altered the geometry of the substrate binding site, which could potentially change the way in which the substrate accesses the active site of the enzyme.	RESULTS
+168	179	active site	site	This conformational change substantially altered the geometry of the substrate binding site, which could potentially change the way in which the substrate accesses the active site of the enzyme.	RESULTS
+3	9	hNaa60	protein	In hNaa60(1-242), the β7-β8 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only.	RESULTS
+10	15	1-242	residue_range	In hNaa60(1-242), the β7-β8 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only.	RESULTS
+22	35	β7-β8 hairpin	structure_element	In hNaa60(1-242), the β7-β8 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only.	RESULTS
+47	58	active site	site	In hNaa60(1-242), the β7-β8 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only.	RESULTS
+96	102	hNaa50	protein	In hNaa60(1-242), the β7-β8 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only.	RESULTS
+168	179	active site	site	In hNaa60(1-242), the β7-β8 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only.	RESULTS
+231	237	tunnel	site	In hNaa60(1-242), the β7-β8 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only.	RESULTS
+244	250	Ac-CoA	chemical	In hNaa60(1-242), the β7-β8 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only.	RESULTS
+251	254	CoA	chemical	In hNaa60(1-242), the β7-β8 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only.	RESULTS
+306	309	NAT	protein_type	In hNaa60(1-242), the β7-β8 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only.	RESULTS
+0	3	KAT	protein_type	KAT activity of hNaa60 toward histone H4 has been noted in previous study, and our enzyme kinetic data also indicated that hNaa60 can acetylate H3-H4 tetramer in vitro (Figure S3).	RESULTS
+16	22	hNaa60	protein	KAT activity of hNaa60 toward histone H4 has been noted in previous study, and our enzyme kinetic data also indicated that hNaa60 can acetylate H3-H4 tetramer in vitro (Figure S3).	RESULTS
+30	37	histone	protein_type	KAT activity of hNaa60 toward histone H4 has been noted in previous study, and our enzyme kinetic data also indicated that hNaa60 can acetylate H3-H4 tetramer in vitro (Figure S3).	RESULTS
+38	40	H4	protein_type	KAT activity of hNaa60 toward histone H4 has been noted in previous study, and our enzyme kinetic data also indicated that hNaa60 can acetylate H3-H4 tetramer in vitro (Figure S3).	RESULTS
+83	102	enzyme kinetic data	evidence	KAT activity of hNaa60 toward histone H4 has been noted in previous study, and our enzyme kinetic data also indicated that hNaa60 can acetylate H3-H4 tetramer in vitro (Figure S3).	RESULTS
+123	129	hNaa60	protein	KAT activity of hNaa60 toward histone H4 has been noted in previous study, and our enzyme kinetic data also indicated that hNaa60 can acetylate H3-H4 tetramer in vitro (Figure S3).	RESULTS
+144	149	H3-H4	complex_assembly	KAT activity of hNaa60 toward histone H4 has been noted in previous study, and our enzyme kinetic data also indicated that hNaa60 can acetylate H3-H4 tetramer in vitro (Figure S3).	RESULTS
+150	158	tetramer	oligomeric_state	KAT activity of hNaa60 toward histone H4 has been noted in previous study, and our enzyme kinetic data also indicated that hNaa60 can acetylate H3-H4 tetramer in vitro (Figure S3).	RESULTS
+29	40	acetylation	ptm	Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4).	RESULTS
+51	58	histone	protein_type	Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4).	RESULTS
+59	64	H3-H4	complex_assembly	Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4).	RESULTS
+65	73	tetramer	oligomeric_state	Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4).	RESULTS
+80	97	mass spectrometry	experimental_method	Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4).	RESULTS
+125	131	lysine	residue_name	Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4).	RESULTS
+187	198	acetylation	ptm	Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4).	RESULTS
+217	228	acetylation	ptm	Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4).	RESULTS
+257	270	hNaa60(1-199)	mutant	Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4).	RESULTS
+18	64	liquid chromatography-tandem mass spectrometry	experimental_method	We also conducted liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis on a synthetic peptide (NH2-MKGKEEKEGGAR-COOH) after treatment with hNaa60(1-199), and the data confirmed that both the N-terminal α-amine and lysine side-chain ε-amine were robustly acetylated after the treatment (Table S1).	RESULTS
+66	74	LC/MS/MS	experimental_method	We also conducted liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis on a synthetic peptide (NH2-MKGKEEKEGGAR-COOH) after treatment with hNaa60(1-199), and the data confirmed that both the N-terminal α-amine and lysine side-chain ε-amine were robustly acetylated after the treatment (Table S1).	RESULTS
+100	107	peptide	chemical	We also conducted liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis on a synthetic peptide (NH2-MKGKEEKEGGAR-COOH) after treatment with hNaa60(1-199), and the data confirmed that both the N-terminal α-amine and lysine side-chain ε-amine were robustly acetylated after the treatment (Table S1).	RESULTS
+109	130	NH2-MKGKEEKEGGAR-COOH	chemical	We also conducted liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis on a synthetic peptide (NH2-MKGKEEKEGGAR-COOH) after treatment with hNaa60(1-199), and the data confirmed that both the N-terminal α-amine and lysine side-chain ε-amine were robustly acetylated after the treatment (Table S1).	RESULTS
+153	166	hNaa60(1-199)	mutant	We also conducted liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis on a synthetic peptide (NH2-MKGKEEKEGGAR-COOH) after treatment with hNaa60(1-199), and the data confirmed that both the N-terminal α-amine and lysine side-chain ε-amine were robustly acetylated after the treatment (Table S1).	RESULTS
+228	234	lysine	residue_name	We also conducted liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis on a synthetic peptide (NH2-MKGKEEKEGGAR-COOH) after treatment with hNaa60(1-199), and the data confirmed that both the N-terminal α-amine and lysine side-chain ε-amine were robustly acetylated after the treatment (Table S1).	RESULTS
+268	278	acetylated	protein_state	We also conducted liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis on a synthetic peptide (NH2-MKGKEEKEGGAR-COOH) after treatment with hNaa60(1-199), and the data confirmed that both the N-terminal α-amine and lysine side-chain ε-amine were robustly acetylated after the treatment (Table S1).	RESULTS
+7	31	structural investigation	experimental_method	Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site.	RESULTS
+41	45	NATs	protein_type	Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site.	RESULTS
+64	74	β6-β7 loop	structure_element	Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site.	RESULTS
+97	110	β7-β8 hairpin	structure_element	Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site.	RESULTS
+114	120	hNaa60	protein	Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site.	RESULTS
+130	140	α1-α2 loop	structure_element	Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site.	RESULTS
+154	176	substrate-binding site	site	Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site.	RESULTS
+180	184	NATs	protein_type	Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site.	RESULTS
+198	204	lysine	residue_name	Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site.	RESULTS
+223	226	KAT	protein_type	Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site.	RESULTS
+262	273	active site	site	Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site.	RESULTS
+8	21	superposition	experimental_method	Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D).	RESULTS
+25	31	hNaa60	protein	Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D).	RESULTS
+32	37	1-242	residue_range	Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D).	RESULTS
+39	48	structure	evidence	Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D).	RESULTS
+60	65	Hat1p	protein	Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D).	RESULTS
+77	80	KAT	protein_type	Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D).	RESULTS
+82	97	in complex with	protein_state	Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D).	RESULTS
+100	107	histone	protein_type	Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D).	RESULTS
+108	110	H4	protein_type	Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D).	RESULTS
+111	118	peptide	chemical	Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D).	RESULTS
+164	166	H4	protein_type	Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D).	RESULTS
+167	174	peptide	chemical	Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D).	RESULTS
+178	181	KAT	protein_type	Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D).	RESULTS
+202	215	β7-β8 hairpin	structure_element	Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D).	RESULTS
+219	225	hNaa60	protein	Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D).	RESULTS
+226	231	1-242	residue_range	Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D).	RESULTS
+22	35	hNaa60(1-199)	mutant	Interestingly, in the hNaa60(1-199) crystal structure, the displaced β7-β8 hairpin opened a second way for the substrate to access the active center that would readily accommodate the binding of the H4 peptide (Fig. 2E), thus implied a potential explanation for KAT activity of this enzyme from a structural biological view.	RESULTS
+36	53	crystal structure	evidence	Interestingly, in the hNaa60(1-199) crystal structure, the displaced β7-β8 hairpin opened a second way for the substrate to access the active center that would readily accommodate the binding of the H4 peptide (Fig. 2E), thus implied a potential explanation for KAT activity of this enzyme from a structural biological view.	RESULTS
+69	82	β7-β8 hairpin	structure_element	Interestingly, in the hNaa60(1-199) crystal structure, the displaced β7-β8 hairpin opened a second way for the substrate to access the active center that would readily accommodate the binding of the H4 peptide (Fig. 2E), thus implied a potential explanation for KAT activity of this enzyme from a structural biological view.	RESULTS
+135	148	active center	site	Interestingly, in the hNaa60(1-199) crystal structure, the displaced β7-β8 hairpin opened a second way for the substrate to access the active center that would readily accommodate the binding of the H4 peptide (Fig. 2E), thus implied a potential explanation for KAT activity of this enzyme from a structural biological view.	RESULTS
+199	201	H4	protein_type	Interestingly, in the hNaa60(1-199) crystal structure, the displaced β7-β8 hairpin opened a second way for the substrate to access the active center that would readily accommodate the binding of the H4 peptide (Fig. 2E), thus implied a potential explanation for KAT activity of this enzyme from a structural biological view.	RESULTS
+202	209	peptide	chemical	Interestingly, in the hNaa60(1-199) crystal structure, the displaced β7-β8 hairpin opened a second way for the substrate to access the active center that would readily accommodate the binding of the H4 peptide (Fig. 2E), thus implied a potential explanation for KAT activity of this enzyme from a structural biological view.	RESULTS
+262	265	KAT	protein_type	Interestingly, in the hNaa60(1-199) crystal structure, the displaced β7-β8 hairpin opened a second way for the substrate to access the active center that would readily accommodate the binding of the H4 peptide (Fig. 2E), thus implied a potential explanation for KAT activity of this enzyme from a structural biological view.	RESULTS
+15	21	hNaa60	protein	However, since hNaa60(1-242) and hNaa60(1-199) were crystallized in different crystal forms, the observed conformational change of the β7-β8 hairpin may simply be an artifact related to the different crystal packing.	RESULTS
+22	27	1-242	residue_range	However, since hNaa60(1-242) and hNaa60(1-199) were crystallized in different crystal forms, the observed conformational change of the β7-β8 hairpin may simply be an artifact related to the different crystal packing.	RESULTS
+33	39	hNaa60	protein	However, since hNaa60(1-242) and hNaa60(1-199) were crystallized in different crystal forms, the observed conformational change of the β7-β8 hairpin may simply be an artifact related to the different crystal packing.	RESULTS
+52	64	crystallized	experimental_method	However, since hNaa60(1-242) and hNaa60(1-199) were crystallized in different crystal forms, the observed conformational change of the β7-β8 hairpin may simply be an artifact related to the different crystal packing.	RESULTS
+78	91	crystal forms	evidence	However, since hNaa60(1-242) and hNaa60(1-199) were crystallized in different crystal forms, the observed conformational change of the β7-β8 hairpin may simply be an artifact related to the different crystal packing.	RESULTS
+135	148	β7-β8 hairpin	structure_element	However, since hNaa60(1-242) and hNaa60(1-199) were crystallized in different crystal forms, the observed conformational change of the β7-β8 hairpin may simply be an artifact related to the different crystal packing.	RESULTS
+200	215	crystal packing	evidence	However, since hNaa60(1-242) and hNaa60(1-199) were crystallized in different crystal forms, the observed conformational change of the β7-β8 hairpin may simply be an artifact related to the different crystal packing.	RESULTS
+12	15	KAT	protein_type	Whether the KAT substrates bind to the β7-β8 hairpin displaced conformation of the enzyme needs to be verified by further structural and functional studies.	RESULTS
+39	52	β7-β8 hairpin	structure_element	Whether the KAT substrates bind to the β7-β8 hairpin displaced conformation of the enzyme needs to be verified by further structural and functional studies.	RESULTS
+122	155	structural and functional studies	experimental_method	Whether the KAT substrates bind to the β7-β8 hairpin displaced conformation of the enzyme needs to be verified by further structural and functional studies.	RESULTS
+0	6	Phe 34	residue_name_number	Phe 34 facilitates proper positioning of the cofactor for acetyl-transfer	RESULTS
+58	64	acetyl	chemical	Phe 34 facilitates proper positioning of the cofactor for acetyl-transfer	RESULTS
+4	20	electron density	evidence	The electron density of Phe 34 side-chain is well defined in the hNaa60(1-242)/Ac-CoA structure, but becomes invisible in the hNaa60(1-199)/CoA structure, indicating displacement of the Phe 34 side-chain in the latter (Fig. 3A,B).	RESULTS
+24	30	Phe 34	residue_name_number	The electron density of Phe 34 side-chain is well defined in the hNaa60(1-242)/Ac-CoA structure, but becomes invisible in the hNaa60(1-199)/CoA structure, indicating displacement of the Phe 34 side-chain in the latter (Fig. 3A,B).	RESULTS
+65	85	hNaa60(1-242)/Ac-CoA	complex_assembly	The electron density of Phe 34 side-chain is well defined in the hNaa60(1-242)/Ac-CoA structure, but becomes invisible in the hNaa60(1-199)/CoA structure, indicating displacement of the Phe 34 side-chain in the latter (Fig. 3A,B).	RESULTS
+86	95	structure	evidence	The electron density of Phe 34 side-chain is well defined in the hNaa60(1-242)/Ac-CoA structure, but becomes invisible in the hNaa60(1-199)/CoA structure, indicating displacement of the Phe 34 side-chain in the latter (Fig. 3A,B).	RESULTS
+126	143	hNaa60(1-199)/CoA	complex_assembly	The electron density of Phe 34 side-chain is well defined in the hNaa60(1-242)/Ac-CoA structure, but becomes invisible in the hNaa60(1-199)/CoA structure, indicating displacement of the Phe 34 side-chain in the latter (Fig. 3A,B).	RESULTS
+144	153	structure	evidence	The electron density of Phe 34 side-chain is well defined in the hNaa60(1-242)/Ac-CoA structure, but becomes invisible in the hNaa60(1-199)/CoA structure, indicating displacement of the Phe 34 side-chain in the latter (Fig. 3A,B).	RESULTS
+186	192	Phe 34	residue_name_number	The electron density of Phe 34 side-chain is well defined in the hNaa60(1-242)/Ac-CoA structure, but becomes invisible in the hNaa60(1-199)/CoA structure, indicating displacement of the Phe 34 side-chain in the latter (Fig. 3A,B).	RESULTS
+18	26	malonate	chemical	A solvent-derived malonate molecule is found beside Phe 34 and the ethanethioate moiety of Ac-CoA in the high-resolution hNaa60(1-242)/Ac-CoA structure (Fig. 3A).	RESULTS
+52	58	Phe 34	residue_name_number	A solvent-derived malonate molecule is found beside Phe 34 and the ethanethioate moiety of Ac-CoA in the high-resolution hNaa60(1-242)/Ac-CoA structure (Fig. 3A).	RESULTS
+67	80	ethanethioate	chemical	A solvent-derived malonate molecule is found beside Phe 34 and the ethanethioate moiety of Ac-CoA in the high-resolution hNaa60(1-242)/Ac-CoA structure (Fig. 3A).	RESULTS
+91	97	Ac-CoA	chemical	A solvent-derived malonate molecule is found beside Phe 34 and the ethanethioate moiety of Ac-CoA in the high-resolution hNaa60(1-242)/Ac-CoA structure (Fig. 3A).	RESULTS
+121	141	hNaa60(1-242)/Ac-CoA	complex_assembly	A solvent-derived malonate molecule is found beside Phe 34 and the ethanethioate moiety of Ac-CoA in the high-resolution hNaa60(1-242)/Ac-CoA structure (Fig. 3A).	RESULTS
+142	151	structure	evidence	A solvent-derived malonate molecule is found beside Phe 34 and the ethanethioate moiety of Ac-CoA in the high-resolution hNaa60(1-242)/Ac-CoA structure (Fig. 3A).	RESULTS
+0	13	Superposition	experimental_method	Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A).	RESULTS
+22	31	structure	evidence	Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A).	RESULTS
+43	62	hNaa50p/CoA/peptide	complex_assembly	Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A).	RESULTS
+78	86	malonate	chemical	Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A).	RESULTS
+128	138	methionine	residue_name	Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A).	RESULTS
+156	163	peptide	chemical	Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A).	RESULTS
+176	182	Phe 34	residue_name_number	Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A).	RESULTS
+186	192	hNaa60	protein	Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A).	RESULTS
+210	216	Phe 27	residue_name_number	Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A).	RESULTS
+220	226	hNaa50	protein	Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A).	RESULTS
+22	31	structure	evidence	Interestingly, in the structure of hNaa60(1-199)/CoA, the terminal thiol of CoA adopts alternative conformations.	RESULTS
+35	52	hNaa60(1-199)/CoA	complex_assembly	Interestingly, in the structure of hNaa60(1-199)/CoA, the terminal thiol of CoA adopts alternative conformations.	RESULTS
+76	79	CoA	chemical	Interestingly, in the structure of hNaa60(1-199)/CoA, the terminal thiol of CoA adopts alternative conformations.	RESULTS
+33	38	amine	chemical	One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the α1-α2 loop and away from the substrate amine (Fig. 3B).	RESULTS
+60	72	superimposed	experimental_method	One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the α1-α2 loop and away from the substrate amine (Fig. 3B).	RESULTS
+73	91	hNaa50/CoA/peptide	complex_assembly	One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the α1-α2 loop and away from the substrate amine (Fig. 3B).	RESULTS
+92	101	structure	evidence	One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the α1-α2 loop and away from the substrate amine (Fig. 3B).	RESULTS
+128	141	ethanethioate	chemical	One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the α1-α2 loop and away from the substrate amine (Fig. 3B).	RESULTS
+145	151	Ac-CoA	chemical	One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the α1-α2 loop and away from the substrate amine (Fig. 3B).	RESULTS
+159	168	structure	evidence	One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the α1-α2 loop and away from the substrate amine (Fig. 3B).	RESULTS
+172	192	hNaa60(1-242)/Ac-CoA	complex_assembly	One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the α1-α2 loop and away from the substrate amine (Fig. 3B).	RESULTS
+223	233	α1-α2 loop	structure_element	One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the α1-α2 loop and away from the substrate amine (Fig. 3B).	RESULTS
+37	53	electron density	evidence	To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).	RESULTS
+130	146	electron density	evidence	To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).	RESULTS
+178	184	Phe 34	residue_name_number	To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).	RESULTS
+189	195	solved	experimental_method	To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).	RESULTS
+200	217	crystal structure	evidence	To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).	RESULTS
+221	243	hNaa60(1-199) F34A/CoA	complex_assembly	To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).	RESULTS
+249	258	structure	evidence	To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).	RESULTS
+267	273	mutant	protein_state	To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).	RESULTS
+295	312	hNaa60(1-199)/CoA	complex_assembly	To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).	RESULTS
+347	363	electron density	evidence	To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).	RESULTS
+0	6	Phe 27	residue_name_number	Phe 27 in hNaa50p (equivalent to Phe 34 in hNaa60) has been implicated to facilitate the binding of N-terminal methionine of the substrate peptide through hydrophobic interaction.	RESULTS
+10	17	hNaa50p	protein	Phe 27 in hNaa50p (equivalent to Phe 34 in hNaa60) has been implicated to facilitate the binding of N-terminal methionine of the substrate peptide through hydrophobic interaction.	RESULTS
+33	39	Phe 34	residue_name_number	Phe 27 in hNaa50p (equivalent to Phe 34 in hNaa60) has been implicated to facilitate the binding of N-terminal methionine of the substrate peptide through hydrophobic interaction.	RESULTS
+43	49	hNaa60	protein	Phe 27 in hNaa50p (equivalent to Phe 34 in hNaa60) has been implicated to facilitate the binding of N-terminal methionine of the substrate peptide through hydrophobic interaction.	RESULTS
+111	121	methionine	residue_name	Phe 27 in hNaa50p (equivalent to Phe 34 in hNaa60) has been implicated to facilitate the binding of N-terminal methionine of the substrate peptide through hydrophobic interaction.	RESULTS
+139	146	peptide	chemical	Phe 27 in hNaa50p (equivalent to Phe 34 in hNaa60) has been implicated to facilitate the binding of N-terminal methionine of the substrate peptide through hydrophobic interaction.	RESULTS
+155	178	hydrophobic interaction	bond_interaction	Phe 27 in hNaa50p (equivalent to Phe 34 in hNaa60) has been implicated to facilitate the binding of N-terminal methionine of the substrate peptide through hydrophobic interaction.	RESULTS
+16	29	hNaa60/Ac-CoA	complex_assembly	However, in the hNaa60/Ac-CoA structure, a hydrophilic malonate molecule is found at the same location where the N-terminal methionine should bind as is indicated by the superposition (Fig. 3A), suggesting that Phe 34 may accommodate binding of hydrophilic substrate, too.	RESULTS
+30	39	structure	evidence	However, in the hNaa60/Ac-CoA structure, a hydrophilic malonate molecule is found at the same location where the N-terminal methionine should bind as is indicated by the superposition (Fig. 3A), suggesting that Phe 34 may accommodate binding of hydrophilic substrate, too.	RESULTS
+55	63	malonate	chemical	However, in the hNaa60/Ac-CoA structure, a hydrophilic malonate molecule is found at the same location where the N-terminal methionine should bind as is indicated by the superposition (Fig. 3A), suggesting that Phe 34 may accommodate binding of hydrophilic substrate, too.	RESULTS
+124	134	methionine	residue_name	However, in the hNaa60/Ac-CoA structure, a hydrophilic malonate molecule is found at the same location where the N-terminal methionine should bind as is indicated by the superposition (Fig. 3A), suggesting that Phe 34 may accommodate binding of hydrophilic substrate, too.	RESULTS
+170	183	superposition	experimental_method	However, in the hNaa60/Ac-CoA structure, a hydrophilic malonate molecule is found at the same location where the N-terminal methionine should bind as is indicated by the superposition (Fig. 3A), suggesting that Phe 34 may accommodate binding of hydrophilic substrate, too.	RESULTS
+211	217	Phe 34	residue_name_number	However, in the hNaa60/Ac-CoA structure, a hydrophilic malonate molecule is found at the same location where the N-terminal methionine should bind as is indicated by the superposition (Fig. 3A), suggesting that Phe 34 may accommodate binding of hydrophilic substrate, too.	RESULTS
+25	31	Phe 34	residue_name_number	Moreover, orientation of Phe 34 side-chain seems to be co-related to positioning of the terminus of the co-enzyme and important for placing it at a location in close proximity to the substrate amine.	RESULTS
+23	29	Phe 34	residue_name_number	We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme.	RESULTS
+97	100	Met	residue_name	We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme.	RESULTS
+113	119	mutate	experimental_method	We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme.	RESULTS
+128	131	Phe	residue_name	We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme.	RESULTS
+135	138	Ala	residue_name	We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme.	RESULTS
+205	211	Phe 34	residue_name_number	We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme.	RESULTS
+257	270	ethanethioate	chemical	We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme.	RESULTS
+281	287	Ac-CoA	chemical	We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme.	RESULTS
+293	301	mutation	experimental_method	We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme.	RESULTS
+12	31	enzyme kinetic data	evidence	Indeed, our enzyme kinetic data showed that hNaa60(1-199) F34A mutant showed no detectable activity (Fig. 5A).	RESULTS
+44	57	hNaa60(1-199)	mutant	Indeed, our enzyme kinetic data showed that hNaa60(1-199) F34A mutant showed no detectable activity (Fig. 5A).	RESULTS
+58	62	F34A	mutant	Indeed, our enzyme kinetic data showed that hNaa60(1-199) F34A mutant showed no detectable activity (Fig. 5A).	RESULTS
+63	69	mutant	protein_state	Indeed, our enzyme kinetic data showed that hNaa60(1-199) F34A mutant showed no detectable activity (Fig. 5A).	RESULTS
+109	115	mutant	protein_state	In order to rule out the possibility that the observed loss of activity may be related to bad folding of the mutant protein, we studied the circular dichroism (CD) spectrum of the protein (Fig. 5B) and determined its crystal structure (Fig. 3C).	RESULTS
+140	158	circular dichroism	experimental_method	In order to rule out the possibility that the observed loss of activity may be related to bad folding of the mutant protein, we studied the circular dichroism (CD) spectrum of the protein (Fig. 5B) and determined its crystal structure (Fig. 3C).	RESULTS
+160	162	CD	experimental_method	In order to rule out the possibility that the observed loss of activity may be related to bad folding of the mutant protein, we studied the circular dichroism (CD) spectrum of the protein (Fig. 5B) and determined its crystal structure (Fig. 3C).	RESULTS
+164	172	spectrum	evidence	In order to rule out the possibility that the observed loss of activity may be related to bad folding of the mutant protein, we studied the circular dichroism (CD) spectrum of the protein (Fig. 5B) and determined its crystal structure (Fig. 3C).	RESULTS
+217	234	crystal structure	evidence	In order to rule out the possibility that the observed loss of activity may be related to bad folding of the mutant protein, we studied the circular dichroism (CD) spectrum of the protein (Fig. 5B) and determined its crystal structure (Fig. 3C).	RESULTS
+29	33	F34A	mutant	Both studies proved that the F34A mutant protein is well-folded.	RESULTS
+34	40	mutant	protein_state	Both studies proved that the F34A mutant protein is well-folded.	RESULTS
+52	63	well-folded	protein_state	Both studies proved that the F34A mutant protein is well-folded.	RESULTS
+50	60	α1-α2 loop	structure_element	Many studies have addressed the crucial effect of α1-α2 loop on catalysis, showing that some residues located in this area are involved in the binding of substrates.	RESULTS
+16	22	Phe 34	residue_name_number	We propose that Phe 34 may play a dual role both in interacting with the peptide substrate (recognition) and in positioning of the ethanethioate moiety of Ac-CoA to the right location to facilitate acetyl-transfer.	RESULTS
+73	80	peptide	chemical	We propose that Phe 34 may play a dual role both in interacting with the peptide substrate (recognition) and in positioning of the ethanethioate moiety of Ac-CoA to the right location to facilitate acetyl-transfer.	RESULTS
+131	144	ethanethioate	chemical	We propose that Phe 34 may play a dual role both in interacting with the peptide substrate (recognition) and in positioning of the ethanethioate moiety of Ac-CoA to the right location to facilitate acetyl-transfer.	RESULTS
+155	161	Ac-CoA	chemical	We propose that Phe 34 may play a dual role both in interacting with the peptide substrate (recognition) and in positioning of the ethanethioate moiety of Ac-CoA to the right location to facilitate acetyl-transfer.	RESULTS
+198	204	acetyl	chemical	We propose that Phe 34 may play a dual role both in interacting with the peptide substrate (recognition) and in positioning of the ethanethioate moiety of Ac-CoA to the right location to facilitate acetyl-transfer.	RESULTS
+21	27	hNaa60	protein	Structural basis for hNaa60 substrate binding	RESULTS
+70	76	hNaa60	protein	Several studies have demonstrated that the substrate specificities of hNaa60 and hNaa50 are highly overlapped.	RESULTS
+81	87	hNaa50	protein	Several studies have demonstrated that the substrate specificities of hNaa60 and hNaa50 are highly overlapped.	RESULTS
+4	13	structure	evidence	The structure of hNaa50p/CoA/peptide provides detailed information about the position of substrate N-terminal residues in the active site of hNaa50.	RESULTS
+17	36	hNaa50p/CoA/peptide	complex_assembly	The structure of hNaa50p/CoA/peptide provides detailed information about the position of substrate N-terminal residues in the active site of hNaa50.	RESULTS
+126	137	active site	site	The structure of hNaa50p/CoA/peptide provides detailed information about the position of substrate N-terminal residues in the active site of hNaa50.	RESULTS
+141	147	hNaa50	protein	The structure of hNaa50p/CoA/peptide provides detailed information about the position of substrate N-terminal residues in the active site of hNaa50.	RESULTS
+14	25	active site	site	Comparing the active site of hNaa60(1-242)/Ac-CoA with hNaa50p/CoA/peptide revealed that key catalytic and substrate binding residues are highly conserved in both proteins (Fig. 4A).	RESULTS
+29	49	hNaa60(1-242)/Ac-CoA	complex_assembly	Comparing the active site of hNaa60(1-242)/Ac-CoA with hNaa50p/CoA/peptide revealed that key catalytic and substrate binding residues are highly conserved in both proteins (Fig. 4A).	RESULTS
+55	74	hNaa50p/CoA/peptide	complex_assembly	Comparing the active site of hNaa60(1-242)/Ac-CoA with hNaa50p/CoA/peptide revealed that key catalytic and substrate binding residues are highly conserved in both proteins (Fig. 4A).	RESULTS
+93	133	catalytic and substrate binding residues	site	Comparing the active site of hNaa60(1-242)/Ac-CoA with hNaa50p/CoA/peptide revealed that key catalytic and substrate binding residues are highly conserved in both proteins (Fig. 4A).	RESULTS
+138	154	highly conserved	protein_state	Comparing the active site of hNaa60(1-242)/Ac-CoA with hNaa50p/CoA/peptide revealed that key catalytic and substrate binding residues are highly conserved in both proteins (Fig. 4A).	RESULTS
+27	34	hNaa50p	protein	With respect to catalysis, hNaa50p has been shown to employ residues Tyr 73 and His 112 to abstract proton from the α-amino group from the substrate’s first residue through a well-ordered water.	RESULTS
+69	75	Tyr 73	residue_name_number	With respect to catalysis, hNaa50p has been shown to employ residues Tyr 73 and His 112 to abstract proton from the α-amino group from the substrate’s first residue through a well-ordered water.	RESULTS
+80	87	His 112	residue_name_number	With respect to catalysis, hNaa50p has been shown to employ residues Tyr 73 and His 112 to abstract proton from the α-amino group from the substrate’s first residue through a well-ordered water.	RESULTS
+175	187	well-ordered	protein_state	With respect to catalysis, hNaa50p has been shown to employ residues Tyr 73 and His 112 to abstract proton from the α-amino group from the substrate’s first residue through a well-ordered water.	RESULTS
+188	193	water	chemical	With respect to catalysis, hNaa50p has been shown to employ residues Tyr 73 and His 112 to abstract proton from the α-amino group from the substrate’s first residue through a well-ordered water.	RESULTS
+2	14	well-ordered	protein_state	A well-ordered water was also found between Tyr 97 and His 138 in hNaa60 (1-199)/CoA and hNaa60 (1-242)/Ac-CoA (Fig. 4B).	RESULTS
+15	20	water	chemical	A well-ordered water was also found between Tyr 97 and His 138 in hNaa60 (1-199)/CoA and hNaa60 (1-242)/Ac-CoA (Fig. 4B).	RESULTS
+44	50	Tyr 97	residue_name_number	A well-ordered water was also found between Tyr 97 and His 138 in hNaa60 (1-199)/CoA and hNaa60 (1-242)/Ac-CoA (Fig. 4B).	RESULTS
+55	62	His 138	residue_name_number	A well-ordered water was also found between Tyr 97 and His 138 in hNaa60 (1-199)/CoA and hNaa60 (1-242)/Ac-CoA (Fig. 4B).	RESULTS
+66	84	hNaa60 (1-199)/CoA	complex_assembly	A well-ordered water was also found between Tyr 97 and His 138 in hNaa60 (1-199)/CoA and hNaa60 (1-242)/Ac-CoA (Fig. 4B).	RESULTS
+89	110	hNaa60 (1-242)/Ac-CoA	complex_assembly	A well-ordered water was also found between Tyr 97 and His 138 in hNaa60 (1-199)/CoA and hNaa60 (1-242)/Ac-CoA (Fig. 4B).	RESULTS
+29	35	Tyr 97	residue_name_number	To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B).	RESULTS
+40	47	His 138	residue_name_number	To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B).	RESULTS
+51	57	hNaa60	protein	To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B).	RESULTS
+72	79	mutated	experimental_method	To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B).	RESULTS
+98	105	alanine	residue_name	To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B).	RESULTS
+110	123	phenylalanine	residue_name	To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B).	RESULTS
+168	175	mutants	protein_state	To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B).	RESULTS
+188	202	kinetic assays	experimental_method	To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B).	RESULTS
+207	218	well-folded	protein_state	To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B).	RESULTS
+222	224	CD	experimental_method	To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B).	RESULTS
+225	232	spectra	evidence	To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B).	RESULTS
+45	53	SDS-PAGE	experimental_method	Purity of all proteins were also analyzed by SDS-PAGE (Figure S5).	RESULTS
+24	31	mutants	protein_state	As show in Fig. 5A, the mutants Y97A, Y97F, H138A and H138F abolished the activity of hNaa60.	RESULTS
+32	36	Y97A	mutant	As show in Fig. 5A, the mutants Y97A, Y97F, H138A and H138F abolished the activity of hNaa60.	RESULTS
+38	42	Y97F	mutant	As show in Fig. 5A, the mutants Y97A, Y97F, H138A and H138F abolished the activity of hNaa60.	RESULTS
+44	49	H138A	mutant	As show in Fig. 5A, the mutants Y97A, Y97F, H138A and H138F abolished the activity of hNaa60.	RESULTS
+54	59	H138F	mutant	As show in Fig. 5A, the mutants Y97A, Y97F, H138A and H138F abolished the activity of hNaa60.	RESULTS
+60	82	abolished the activity	protein_state	As show in Fig. 5A, the mutants Y97A, Y97F, H138A and H138F abolished the activity of hNaa60.	RESULTS
+86	92	hNaa60	protein	As show in Fig. 5A, the mutants Y97A, Y97F, H138A and H138F abolished the activity of hNaa60.	RESULTS
+16	22	mutate	experimental_method	In contrast, to mutate the nearby solvent exposed residue Glu 37 to Ala (E37A) has little impact on the activity of hNaa60 (Figs 4B and 5A).	RESULTS
+34	49	solvent exposed	protein_state	In contrast, to mutate the nearby solvent exposed residue Glu 37 to Ala (E37A) has little impact on the activity of hNaa60 (Figs 4B and 5A).	RESULTS
+58	64	Glu 37	residue_name_number	In contrast, to mutate the nearby solvent exposed residue Glu 37 to Ala (E37A) has little impact on the activity of hNaa60 (Figs 4B and 5A).	RESULTS
+68	71	Ala	residue_name	In contrast, to mutate the nearby solvent exposed residue Glu 37 to Ala (E37A) has little impact on the activity of hNaa60 (Figs 4B and 5A).	RESULTS
+73	77	E37A	mutant	In contrast, to mutate the nearby solvent exposed residue Glu 37 to Ala (E37A) has little impact on the activity of hNaa60 (Figs 4B and 5A).	RESULTS
+116	122	hNaa60	protein	In contrast, to mutate the nearby solvent exposed residue Glu 37 to Ala (E37A) has little impact on the activity of hNaa60 (Figs 4B and 5A).	RESULTS
+19	52	structural and functional studies	experimental_method	In conclusion, the structural and functional studies indicate that hNaa60 applies the same two base mechanism through Tyr 97, His 138 and a well-ordered water as was described for hNaa50.	RESULTS
+67	73	hNaa60	protein	In conclusion, the structural and functional studies indicate that hNaa60 applies the same two base mechanism through Tyr 97, His 138 and a well-ordered water as was described for hNaa50.	RESULTS
+118	124	Tyr 97	residue_name_number	In conclusion, the structural and functional studies indicate that hNaa60 applies the same two base mechanism through Tyr 97, His 138 and a well-ordered water as was described for hNaa50.	RESULTS
+126	133	His 138	residue_name_number	In conclusion, the structural and functional studies indicate that hNaa60 applies the same two base mechanism through Tyr 97, His 138 and a well-ordered water as was described for hNaa50.	RESULTS
+140	152	well-ordered	protein_state	In conclusion, the structural and functional studies indicate that hNaa60 applies the same two base mechanism through Tyr 97, His 138 and a well-ordered water as was described for hNaa50.	RESULTS
+153	158	water	chemical	In conclusion, the structural and functional studies indicate that hNaa60 applies the same two base mechanism through Tyr 97, His 138 and a well-ordered water as was described for hNaa50.	RESULTS
+180	186	hNaa50	protein	In conclusion, the structural and functional studies indicate that hNaa60 applies the same two base mechanism through Tyr 97, His 138 and a well-ordered water as was described for hNaa50.	RESULTS
+4	12	malonate	chemical	The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A).	RESULTS
+38	58	hNaa60(1-242)/Ac-CoA	complex_assembly	The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A).	RESULTS
+59	76	crystal structure	evidence	The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A).	RESULTS
+132	138	hNaa60	protein	The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A).	RESULTS
+166	177	active site	site	The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A).	RESULTS
+206	209	Met	residue_name	The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A).	RESULTS
+227	234	peptide	chemical	The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A).	RESULTS
+242	255	superposition	experimental_method	The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A).	RESULTS
+265	284	hNaa50p/CoA/peptide	complex_assembly	The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A).	RESULTS
+285	294	structure	evidence	The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A).	RESULTS
+9	15	Tyr 38	residue_name_number	Residues Tyr 38, Asn 143 and Tyr 165 are located around the malonate and interact with it through direct hydrogen bonds or water bridge (Fig. 4C).	RESULTS
+17	24	Asn 143	residue_name_number	Residues Tyr 38, Asn 143 and Tyr 165 are located around the malonate and interact with it through direct hydrogen bonds or water bridge (Fig. 4C).	RESULTS
+29	36	Tyr 165	residue_name_number	Residues Tyr 38, Asn 143 and Tyr 165 are located around the malonate and interact with it through direct hydrogen bonds or water bridge (Fig. 4C).	RESULTS
+60	68	malonate	chemical	Residues Tyr 38, Asn 143 and Tyr 165 are located around the malonate and interact with it through direct hydrogen bonds or water bridge (Fig. 4C).	RESULTS
+105	119	hydrogen bonds	bond_interaction	Residues Tyr 38, Asn 143 and Tyr 165 are located around the malonate and interact with it through direct hydrogen bonds or water bridge (Fig. 4C).	RESULTS
+123	135	water bridge	bond_interaction	Residues Tyr 38, Asn 143 and Tyr 165 are located around the malonate and interact with it through direct hydrogen bonds or water bridge (Fig. 4C).	RESULTS
+9	17	malonate	chemical	Although malonate is negatively charged, which is different from that of lysine ε-amine or peptide N-terminal amine, similar hydrophilic interactions may take place when substrate amine presents in the same position, since Tyr 38, Asn 143 and Tyr 165 are not positively or negatively charged.	RESULTS
+73	79	lysine	residue_name	Although malonate is negatively charged, which is different from that of lysine ε-amine or peptide N-terminal amine, similar hydrophilic interactions may take place when substrate amine presents in the same position, since Tyr 38, Asn 143 and Tyr 165 are not positively or negatively charged.	RESULTS
+91	98	peptide	chemical	Although malonate is negatively charged, which is different from that of lysine ε-amine or peptide N-terminal amine, similar hydrophilic interactions may take place when substrate amine presents in the same position, since Tyr 38, Asn 143 and Tyr 165 are not positively or negatively charged.	RESULTS
+125	149	hydrophilic interactions	bond_interaction	Although malonate is negatively charged, which is different from that of lysine ε-amine or peptide N-terminal amine, similar hydrophilic interactions may take place when substrate amine presents in the same position, since Tyr 38, Asn 143 and Tyr 165 are not positively or negatively charged.	RESULTS
+223	229	Tyr 38	residue_name_number	Although malonate is negatively charged, which is different from that of lysine ε-amine or peptide N-terminal amine, similar hydrophilic interactions may take place when substrate amine presents in the same position, since Tyr 38, Asn 143 and Tyr 165 are not positively or negatively charged.	RESULTS
+231	238	Asn 143	residue_name_number	Although malonate is negatively charged, which is different from that of lysine ε-amine or peptide N-terminal amine, similar hydrophilic interactions may take place when substrate amine presents in the same position, since Tyr 38, Asn 143 and Tyr 165 are not positively or negatively charged.	RESULTS
+243	250	Tyr 165	residue_name_number	Although malonate is negatively charged, which is different from that of lysine ε-amine or peptide N-terminal amine, similar hydrophilic interactions may take place when substrate amine presents in the same position, since Tyr 38, Asn 143 and Tyr 165 are not positively or negatively charged.	RESULTS
+57	61	Y38A	mutant	In agreement with this hypothesis, it was found that the Y38A, N143A and Y165A mutants all showed remarkably reduced activities as compared to WT, implying that these residues may be critical for substrate binding (Figs 4C and 5A).	RESULTS
+63	68	N143A	mutant	In agreement with this hypothesis, it was found that the Y38A, N143A and Y165A mutants all showed remarkably reduced activities as compared to WT, implying that these residues may be critical for substrate binding (Figs 4C and 5A).	RESULTS
+73	78	Y165A	mutant	In agreement with this hypothesis, it was found that the Y38A, N143A and Y165A mutants all showed remarkably reduced activities as compared to WT, implying that these residues may be critical for substrate binding (Figs 4C and 5A).	RESULTS
+79	86	mutants	protein_state	In agreement with this hypothesis, it was found that the Y38A, N143A and Y165A mutants all showed remarkably reduced activities as compared to WT, implying that these residues may be critical for substrate binding (Figs 4C and 5A).	RESULTS
+143	145	WT	protein_state	In agreement with this hypothesis, it was found that the Y38A, N143A and Y165A mutants all showed remarkably reduced activities as compared to WT, implying that these residues may be critical for substrate binding (Figs 4C and 5A).	RESULTS
+4	14	β3-β4 loop	structure_element	The β3-β4 loop participates in the regulation of hNaa60-activity	RESULTS
+49	55	hNaa60	protein	The β3-β4 loop participates in the regulation of hNaa60-activity	RESULTS
+17	19	β3	structure_element	Residues between β3 and β4 of hNaa60 form a unique 20-residue long loop (residues 73–92) that is a short turn in many other NAT members (Fig. 1D).	RESULTS
+24	26	β4	structure_element	Residues between β3 and β4 of hNaa60 form a unique 20-residue long loop (residues 73–92) that is a short turn in many other NAT members (Fig. 1D).	RESULTS
+30	36	hNaa60	protein	Residues between β3 and β4 of hNaa60 form a unique 20-residue long loop (residues 73–92) that is a short turn in many other NAT members (Fig. 1D).	RESULTS
+51	71	20-residue long loop	structure_element	Residues between β3 and β4 of hNaa60 form a unique 20-residue long loop (residues 73–92) that is a short turn in many other NAT members (Fig. 1D).	RESULTS
+82	87	73–92	residue_range	Residues between β3 and β4 of hNaa60 form a unique 20-residue long loop (residues 73–92) that is a short turn in many other NAT members (Fig. 1D).	RESULTS
+99	109	short turn	structure_element	Residues between β3 and β4 of hNaa60 form a unique 20-residue long loop (residues 73–92) that is a short turn in many other NAT members (Fig. 1D).	RESULTS
+124	127	NAT	protein_type	Residues between β3 and β4 of hNaa60 form a unique 20-residue long loop (residues 73–92) that is a short turn in many other NAT members (Fig. 1D).	RESULTS
+30	46	auto-acetylation	ptm	Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain.	RESULTS
+50	56	hNaa60	protein	Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain.	RESULTS
+56	59	K79	residue_name_number	Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain.	RESULTS
+92	98	hNaa60	protein	Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain.	RESULTS
+142	148	Lys 79	residue_name_number	Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain.	RESULTS
+152	162	acetylated	protein_state	Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain.	RESULTS
+170	188	crystal structures	evidence	Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain.	RESULTS
+216	232	electron density	evidence	Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain.	RESULTS
+236	242	Lys 79	residue_name_number	Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain.	RESULTS
+18	35	mass spectrometry	experimental_method	We therefore used mass spectrometry to analyze if Lys 79 was acetylated in our bacterially purified proteins, and observed no modification on this residue (Figure S6).	RESULTS
+50	56	Lys 79	residue_name_number	We therefore used mass spectrometry to analyze if Lys 79 was acetylated in our bacterially purified proteins, and observed no modification on this residue (Figure S6).	RESULTS
+61	71	acetylated	protein_state	We therefore used mass spectrometry to analyze if Lys 79 was acetylated in our bacterially purified proteins, and observed no modification on this residue (Figure S6).	RESULTS
+24	30	hNaa60	protein	To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively.	RESULTS
+30	33	K79	residue_name_number	To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively.	RESULTS
+34	50	auto-acetylation	ptm	To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively.	RESULTS
+79	83	K79R	mutant	To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively.	RESULTS
+88	92	K79Q	mutant	To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively.	RESULTS
+93	100	mutants	protein_state	To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively.	RESULTS
+117	130	un-acetylated	protein_state	To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively.	RESULTS
+135	145	acetylated	protein_state	To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively.	RESULTS
+154	160	Lys 79	residue_name_number	To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively.	RESULTS
+20	24	K79R	mutant	Interestingly, both K79R and K79Q mutants led to an increase in the catalytic activity of hNaa60, while K79A mutant led to modest decrease of the activity (Fig. 5A).	RESULTS
+29	33	K79Q	mutant	Interestingly, both K79R and K79Q mutants led to an increase in the catalytic activity of hNaa60, while K79A mutant led to modest decrease of the activity (Fig. 5A).	RESULTS
+34	41	mutants	protein_state	Interestingly, both K79R and K79Q mutants led to an increase in the catalytic activity of hNaa60, while K79A mutant led to modest decrease of the activity (Fig. 5A).	RESULTS
+90	96	hNaa60	protein	Interestingly, both K79R and K79Q mutants led to an increase in the catalytic activity of hNaa60, while K79A mutant led to modest decrease of the activity (Fig. 5A).	RESULTS
+104	108	K79A	mutant	Interestingly, both K79R and K79Q mutants led to an increase in the catalytic activity of hNaa60, while K79A mutant led to modest decrease of the activity (Fig. 5A).	RESULTS
+109	115	mutant	protein_state	Interestingly, both K79R and K79Q mutants led to an increase in the catalytic activity of hNaa60, while K79A mutant led to modest decrease of the activity (Fig. 5A).	RESULTS
+29	40	acetylation	ptm	These data indicate that the acetylation of Lys 79 is not required for optimal catalytic activity of hNaa60 in vitro.	RESULTS
+44	50	Lys 79	residue_name_number	These data indicate that the acetylation of Lys 79 is not required for optimal catalytic activity of hNaa60 in vitro.	RESULTS
+101	107	hNaa60	protein	These data indicate that the acetylation of Lys 79 is not required for optimal catalytic activity of hNaa60 in vitro.	RESULTS
+21	31	β3-β4 loop	structure_element	It is noted that the β3-β4 loop of hNaa60 acts like a door leaf to partly cover the substrate-binding pathway.	RESULTS
+35	41	hNaa60	protein	It is noted that the β3-β4 loop of hNaa60 acts like a door leaf to partly cover the substrate-binding pathway.	RESULTS
+84	109	substrate-binding pathway	site	It is noted that the β3-β4 loop of hNaa60 acts like a door leaf to partly cover the substrate-binding pathway.	RESULTS
+30	40	β3-β4 loop	structure_element	We hence hypothesize that the β3-β4 loop may interfere with the access of the peptide substrates and that the solvent-exposing Lys 79 may play a potential role to remove the door leaf when it hovers in solvent (Fig. 4D).	RESULTS
+78	85	peptide	chemical	We hence hypothesize that the β3-β4 loop may interfere with the access of the peptide substrates and that the solvent-exposing Lys 79 may play a potential role to remove the door leaf when it hovers in solvent (Fig. 4D).	RESULTS
+110	126	solvent-exposing	protein_state	We hence hypothesize that the β3-β4 loop may interfere with the access of the peptide substrates and that the solvent-exposing Lys 79 may play a potential role to remove the door leaf when it hovers in solvent (Fig. 4D).	RESULTS
+127	133	Lys 79	residue_name_number	We hence hypothesize that the β3-β4 loop may interfere with the access of the peptide substrates and that the solvent-exposing Lys 79 may play a potential role to remove the door leaf when it hovers in solvent (Fig. 4D).	RESULTS
+16	22	Glu 80	residue_name_number	Acidic residues Glu 80, Asp 81 and Asp 83 interact with His 138, His 159 and His 158 to maintain the conformation of the β3-β4 loop, thus contribute to control the substrate binding (Fig. 4D).	RESULTS
+24	30	Asp 81	residue_name_number	Acidic residues Glu 80, Asp 81 and Asp 83 interact with His 138, His 159 and His 158 to maintain the conformation of the β3-β4 loop, thus contribute to control the substrate binding (Fig. 4D).	RESULTS
+35	41	Asp 83	residue_name_number	Acidic residues Glu 80, Asp 81 and Asp 83 interact with His 138, His 159 and His 158 to maintain the conformation of the β3-β4 loop, thus contribute to control the substrate binding (Fig. 4D).	RESULTS
+56	63	His 138	residue_name_number	Acidic residues Glu 80, Asp 81 and Asp 83 interact with His 138, His 159 and His 158 to maintain the conformation of the β3-β4 loop, thus contribute to control the substrate binding (Fig. 4D).	RESULTS
+65	72	His 159	residue_name_number	Acidic residues Glu 80, Asp 81 and Asp 83 interact with His 138, His 159 and His 158 to maintain the conformation of the β3-β4 loop, thus contribute to control the substrate binding (Fig. 4D).	RESULTS
+77	84	His 158	residue_name_number	Acidic residues Glu 80, Asp 81 and Asp 83 interact with His 138, His 159 and His 158 to maintain the conformation of the β3-β4 loop, thus contribute to control the substrate binding (Fig. 4D).	RESULTS
+121	131	β3-β4 loop	structure_element	Acidic residues Glu 80, Asp 81 and Asp 83 interact with His 138, His 159 and His 158 to maintain the conformation of the β3-β4 loop, thus contribute to control the substrate binding (Fig. 4D).	RESULTS
+30	37	mutated	experimental_method	To verify this hypothesis, we mutated Glu 80, Asp 81 and Asp 83 to Ala respectively.	RESULTS
+38	44	Glu 80	residue_name_number	To verify this hypothesis, we mutated Glu 80, Asp 81 and Asp 83 to Ala respectively.	RESULTS
+46	52	Asp 81	residue_name_number	To verify this hypothesis, we mutated Glu 80, Asp 81 and Asp 83 to Ala respectively.	RESULTS
+57	63	Asp 83	residue_name_number	To verify this hypothesis, we mutated Glu 80, Asp 81 and Asp 83 to Ala respectively.	RESULTS
+67	70	Ala	residue_name	To verify this hypothesis, we mutated Glu 80, Asp 81 and Asp 83 to Ala respectively.	RESULTS
+29	33	E80A	mutant	In line with our hypothesis, E80A, D81A and D83A mutants exhibit at least 2-fold increase in hNaa60-activity (Fig. 5A).	RESULTS
+35	39	D81A	mutant	In line with our hypothesis, E80A, D81A and D83A mutants exhibit at least 2-fold increase in hNaa60-activity (Fig. 5A).	RESULTS
+44	48	D83A	mutant	In line with our hypothesis, E80A, D81A and D83A mutants exhibit at least 2-fold increase in hNaa60-activity (Fig. 5A).	RESULTS
+49	56	mutants	protein_state	In line with our hypothesis, E80A, D81A and D83A mutants exhibit at least 2-fold increase in hNaa60-activity (Fig. 5A).	RESULTS
+93	99	hNaa60	protein	In line with our hypothesis, E80A, D81A and D83A mutants exhibit at least 2-fold increase in hNaa60-activity (Fig. 5A).	RESULTS
+19	28	structure	evidence	Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.	RESULTS
+45	48	NAT	protein_type	Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.	RESULTS
+54	69	S. solfataricus	species	Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.	RESULTS
+86	111	10-residue long extension	structure_element	Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.	RESULTS
+120	122	β3	structure_element	Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.	RESULTS
+127	129	β4	structure_element	Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.	RESULTS
+139	172	structure and biochemical studies	experimental_method	Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.	RESULTS
+189	198	extension	structure_element	Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.	RESULTS
+202	207	SsNat	protein	Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.	RESULTS
+254	265	active site	site	Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.	RESULTS
+281	286	SsNat	protein	Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.	RESULTS
+0	14	Nt-acetylation	ptm	Nt-acetylation, which is carried out by the NAT family acetyltransferases, is an ancient and essential modification of proteins.	DISCUSS
+44	73	NAT family acetyltransferases	protein_type	Nt-acetylation, which is carried out by the NAT family acetyltransferases, is an ancient and essential modification of proteins.	DISCUSS
+14	18	NATs	protein_type	Although many NATs are highly conserved from lower to higher eukaryotes and the substrate bias of them appears to be partially overlapped, there is a significant increase in the overall level of N-terminal acetylation from lower to higher eukaryotes.	DISCUSS
+23	39	highly conserved	protein_state	Although many NATs are highly conserved from lower to higher eukaryotes and the substrate bias of them appears to be partially overlapped, there is a significant increase in the overall level of N-terminal acetylation from lower to higher eukaryotes.	DISCUSS
+45	50	lower	taxonomy_domain	Although many NATs are highly conserved from lower to higher eukaryotes and the substrate bias of them appears to be partially overlapped, there is a significant increase in the overall level of N-terminal acetylation from lower to higher eukaryotes.	DISCUSS
+54	71	higher eukaryotes	taxonomy_domain	Although many NATs are highly conserved from lower to higher eukaryotes and the substrate bias of them appears to be partially overlapped, there is a significant increase in the overall level of N-terminal acetylation from lower to higher eukaryotes.	DISCUSS
+195	217	N-terminal acetylation	ptm	Although many NATs are highly conserved from lower to higher eukaryotes and the substrate bias of them appears to be partially overlapped, there is a significant increase in the overall level of N-terminal acetylation from lower to higher eukaryotes.	DISCUSS
+223	228	lower	taxonomy_domain	Although many NATs are highly conserved from lower to higher eukaryotes and the substrate bias of them appears to be partially overlapped, there is a significant increase in the overall level of N-terminal acetylation from lower to higher eukaryotes.	DISCUSS
+232	249	higher eukaryotes	taxonomy_domain	Although many NATs are highly conserved from lower to higher eukaryotes and the substrate bias of them appears to be partially overlapped, there is a significant increase in the overall level of N-terminal acetylation from lower to higher eukaryotes.	DISCUSS
+50	55	Naa60	protein	In this study we provide structural insights into Naa60 found only in multicellular eukaryotes.	DISCUSS
+70	94	multicellular eukaryotes	taxonomy_domain	In this study we provide structural insights into Naa60 found only in multicellular eukaryotes.	DISCUSS
+18	24	hNaa60	protein	The N-terminus of hNaa60 harbors three hydrophobic residues (VVP) that makes it very difficult to express and purify the protein.	DISCUSS
+61	64	VVP	structure_element	The N-terminus of hNaa60 harbors three hydrophobic residues (VVP) that makes it very difficult to express and purify the protein.	DISCUSS
+27	36	replacing	experimental_method	This problem was solved by replacing residues 4–6 from VVP to EER that are found in Naa60 from Xenopus Laevis.	DISCUSS
+46	49	4–6	residue_range	This problem was solved by replacing residues 4–6 from VVP to EER that are found in Naa60 from Xenopus Laevis.	DISCUSS
+55	58	VVP	structure_element	This problem was solved by replacing residues 4–6 from VVP to EER that are found in Naa60 from Xenopus Laevis.	DISCUSS
+62	65	EER	structure_element	This problem was solved by replacing residues 4–6 from VVP to EER that are found in Naa60 from Xenopus Laevis.	DISCUSS
+84	89	Naa60	protein	This problem was solved by replacing residues 4–6 from VVP to EER that are found in Naa60 from Xenopus Laevis.	DISCUSS
+95	109	Xenopus Laevis	species	This problem was solved by replacing residues 4–6 from VVP to EER that are found in Naa60 from Xenopus Laevis.	DISCUSS
+6	11	Naa60	protein	Since Naa60 from human and from Xenopus Laevis are highly homologous (Fig. 1A), we speculate that these two proteins should have the same biological function.	DISCUSS
+17	22	human	species	Since Naa60 from human and from Xenopus Laevis are highly homologous (Fig. 1A), we speculate that these two proteins should have the same biological function.	DISCUSS
+32	46	Xenopus Laevis	species	Since Naa60 from human and from Xenopus Laevis are highly homologous (Fig. 1A), we speculate that these two proteins should have the same biological function.	DISCUSS
+51	68	highly homologous	protein_state	Since Naa60 from human and from Xenopus Laevis are highly homologous (Fig. 1A), we speculate that these two proteins should have the same biological function.	DISCUSS
+33	43	VVP to EER	mutant	Therefore it is deduced that the VVP to EER replacement on the N-terminus of hNaa60 may not interfere with its function.	DISCUSS
+44	55	replacement	experimental_method	Therefore it is deduced that the VVP to EER replacement on the N-terminus of hNaa60 may not interfere with its function.	DISCUSS
+77	83	hNaa60	protein	Therefore it is deduced that the VVP to EER replacement on the N-terminus of hNaa60 may not interfere with its function.	DISCUSS
+16	22	hNaa60	protein	However, in the hNaa60(1-242) structure the N-terminus adopts an α-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing.	DISCUSS
+23	28	1-242	residue_range	However, in the hNaa60(1-242) structure the N-terminus adopts an α-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing.	DISCUSS
+30	39	structure	evidence	However, in the hNaa60(1-242) structure the N-terminus adopts an α-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing.	DISCUSS
+65	84	α-helical structure	structure_element	However, in the hNaa60(1-242) structure the N-terminus adopts an α-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing.	DISCUSS
+126	127	6	residue_number	However, in the hNaa60(1-242) structure the N-terminus adopts an α-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing.	DISCUSS
+131	138	proline	residue_name	However, in the hNaa60(1-242) structure the N-terminus adopts an α-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing.	DISCUSS
+161	174	hNaa60(1-199)	mutant	However, in the hNaa60(1-242) structure the N-terminus adopts an α-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing.	DISCUSS
+175	184	structure	evidence	However, in the hNaa60(1-242) structure the N-terminus adopts an α-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing.	DISCUSS
+219	241	semi-helical structure	structure_element	However, in the hNaa60(1-242) structure the N-terminus adopts an α-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing.	DISCUSS
+276	291	crystal packing	evidence	However, in the hNaa60(1-242) structure the N-terminus adopts an α-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing.	DISCUSS
+47	56	wild-type	protein_state	Hence it is not clear if the N-terminal end of wild-type hNaa60 is an α-helix, and what roles the hydrophobic residues 4–6 play in structure and function of wild-type hNaa60.	DISCUSS
+57	63	hNaa60	protein	Hence it is not clear if the N-terminal end of wild-type hNaa60 is an α-helix, and what roles the hydrophobic residues 4–6 play in structure and function of wild-type hNaa60.	DISCUSS
+70	77	α-helix	structure_element	Hence it is not clear if the N-terminal end of wild-type hNaa60 is an α-helix, and what roles the hydrophobic residues 4–6 play in structure and function of wild-type hNaa60.	DISCUSS
+119	122	4–6	residue_range	Hence it is not clear if the N-terminal end of wild-type hNaa60 is an α-helix, and what roles the hydrophobic residues 4–6 play in structure and function of wild-type hNaa60.	DISCUSS
+157	166	wild-type	protein_state	Hence it is not clear if the N-terminal end of wild-type hNaa60 is an α-helix, and what roles the hydrophobic residues 4–6 play in structure and function of wild-type hNaa60.	DISCUSS
+167	173	hNaa60	protein	Hence it is not clear if the N-terminal end of wild-type hNaa60 is an α-helix, and what roles the hydrophobic residues 4–6 play in structure and function of wild-type hNaa60.	DISCUSS
+33	41	mutation	experimental_method	In addition to the three-residue mutation (VVP to EER), we also tried many other hNaa60 constructs, but only the full-length protein and the truncated variant 1-199 behaved well.	DISCUSS
+43	46	VVP	structure_element	In addition to the three-residue mutation (VVP to EER), we also tried many other hNaa60 constructs, but only the full-length protein and the truncated variant 1-199 behaved well.	DISCUSS
+50	53	EER	structure_element	In addition to the three-residue mutation (VVP to EER), we also tried many other hNaa60 constructs, but only the full-length protein and the truncated variant 1-199 behaved well.	DISCUSS
+81	87	hNaa60	protein	In addition to the three-residue mutation (VVP to EER), we also tried many other hNaa60 constructs, but only the full-length protein and the truncated variant 1-199 behaved well.	DISCUSS
+113	124	full-length	protein_state	In addition to the three-residue mutation (VVP to EER), we also tried many other hNaa60 constructs, but only the full-length protein and the truncated variant 1-199 behaved well.	DISCUSS
+141	150	truncated	protein_state	In addition to the three-residue mutation (VVP to EER), we also tried many other hNaa60 constructs, but only the full-length protein and the truncated variant 1-199 behaved well.	DISCUSS
+159	164	1-199	residue_range	In addition to the three-residue mutation (VVP to EER), we also tried many other hNaa60 constructs, but only the full-length protein and the truncated variant 1-199 behaved well.	DISCUSS
+43	49	hNaa60	protein	The finding that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) is intriguing.	DISCUSS
+50	55	1-242	residue_range	The finding that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) is intriguing.	DISCUSS
+84	97	hNaa60(1-199)	mutant	The finding that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) is intriguing.	DISCUSS
+38	49	full-length	protein_state	We speculate that low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies or there remains some undiscovered auto-inhibitory regulation in the full-length protein.	DISCUSS
+50	56	hNaa60	protein	We speculate that low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies or there remains some undiscovered auto-inhibitory regulation in the full-length protein.	DISCUSS
+211	222	full-length	protein_state	We speculate that low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies or there remains some undiscovered auto-inhibitory regulation in the full-length protein.	DISCUSS
+4	10	hNaa60	protein	The hNaa60 protein was proven to be localized on Golgi apparatus.	DISCUSS
+41	62	transmembrane domains	structure_element	Aksnes and colleagues predicted putative transmembrane domains and two putative sites of S-palmitoylation, by bioinformatics means, to account for Golgi localization of the protein.	DISCUSS
+89	105	S-palmitoylation	ptm	Aksnes and colleagues predicted putative transmembrane domains and two putative sites of S-palmitoylation, by bioinformatics means, to account for Golgi localization of the protein.	DISCUSS
+10	17	mutated	experimental_method	They then mutated all five cysteine residues of hNaa60’s to serine, including the two putative S-palmitoylation sites.	DISCUSS
+27	35	cysteine	residue_name	They then mutated all five cysteine residues of hNaa60’s to serine, including the two putative S-palmitoylation sites.	DISCUSS
+48	54	hNaa60	protein	They then mutated all five cysteine residues of hNaa60’s to serine, including the two putative S-palmitoylation sites.	DISCUSS
+60	66	serine	residue_name	They then mutated all five cysteine residues of hNaa60’s to serine, including the two putative S-palmitoylation sites.	DISCUSS
+95	117	S-palmitoylation sites	site	They then mutated all five cysteine residues of hNaa60’s to serine, including the two putative S-palmitoylation sites.	DISCUSS
+15	24	mutations	experimental_method	However, these mutations did not abolish Naa60 membrane localization, indicating that S-palmitoylation is unlikely to (solely) account for targeting hNaa60 on Golgi.	DISCUSS
+41	46	Naa60	protein	However, these mutations did not abolish Naa60 membrane localization, indicating that S-palmitoylation is unlikely to (solely) account for targeting hNaa60 on Golgi.	DISCUSS
+86	102	S-palmitoylation	ptm	However, these mutations did not abolish Naa60 membrane localization, indicating that S-palmitoylation is unlikely to (solely) account for targeting hNaa60 on Golgi.	DISCUSS
+149	155	hNaa60	protein	However, these mutations did not abolish Naa60 membrane localization, indicating that S-palmitoylation is unlikely to (solely) account for targeting hNaa60 on Golgi.	DISCUSS
+13	19	adding	experimental_method	Furthermore, adding residues 217–242 of hNaa60 (containing residues 217–236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182–216 are important for Golgi localization of hNaa60.	DISCUSS
+29	36	217–242	residue_range	Furthermore, adding residues 217–242 of hNaa60 (containing residues 217–236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182–216 are important for Golgi localization of hNaa60.	DISCUSS
+40	46	hNaa60	protein	Furthermore, adding residues 217–242 of hNaa60 (containing residues 217–236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182–216 are important for Golgi localization of hNaa60.	DISCUSS
+68	75	217–236	residue_range	Furthermore, adding residues 217–242 of hNaa60 (containing residues 217–236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182–216 are important for Golgi localization of hNaa60.	DISCUSS
+97	118	transmembrane domains	structure_element	Furthermore, adding residues 217–242 of hNaa60 (containing residues 217–236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182–216 are important for Golgi localization of hNaa60.	DISCUSS
+141	145	eGFP	experimental_method	Furthermore, adding residues 217–242 of hNaa60 (containing residues 217–236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182–216 are important for Golgi localization of hNaa60.	DISCUSS
+216	220	eGFP	experimental_method	Furthermore, adding residues 217–242 of hNaa60 (containing residues 217–236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182–216 are important for Golgi localization of hNaa60.	DISCUSS
+221	234	hNaa60182-242	mutant	Furthermore, adding residues 217–242 of hNaa60 (containing residues 217–236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182–216 are important for Golgi localization of hNaa60.	DISCUSS
+279	286	182–216	residue_range	Furthermore, adding residues 217–242 of hNaa60 (containing residues 217–236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182–216 are important for Golgi localization of hNaa60.	DISCUSS
+327	333	hNaa60	protein	Furthermore, adding residues 217–242 of hNaa60 (containing residues 217–236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182–216 are important for Golgi localization of hNaa60.	DISCUSS
+23	30	190–202	residue_range	We found that residues 190–202 formed an amphipathic helix with an array of hydrophobic residues located on one side.	DISCUSS
+41	58	amphipathic helix	structure_element	We found that residues 190–202 formed an amphipathic helix with an array of hydrophobic residues located on one side.	DISCUSS
+76	95	amphipathic helices	structure_element	This observation is reminiscent of the protein/membrane interaction through amphipathic helices in the cases of KalSec14, Atg3, PB1-F2 etc.	DISCUSS
+112	120	KalSec14	protein	This observation is reminiscent of the protein/membrane interaction through amphipathic helices in the cases of KalSec14, Atg3, PB1-F2 etc.	DISCUSS
+122	126	Atg3	protein	This observation is reminiscent of the protein/membrane interaction through amphipathic helices in the cases of KalSec14, Atg3, PB1-F2 etc.	DISCUSS
+128	134	PB1-F2	protein	This observation is reminiscent of the protein/membrane interaction through amphipathic helices in the cases of KalSec14, Atg3, PB1-F2 etc.	DISCUSS
+17	34	amphipathic helix	structure_element	In this model an amphipathic helix can immerse its hydrophobic side into the lipid bilayer through hydrophobic interactions.	DISCUSS
+99	123	hydrophobic interactions	bond_interaction	In this model an amphipathic helix can immerse its hydrophobic side into the lipid bilayer through hydrophobic interactions.	DISCUSS
+30	47	amphipathic helix	structure_element	Therefore we propose that the amphipathic helix α5 may contribute to Golgi localization of hNaa60.	DISCUSS
+48	50	α5	structure_element	Therefore we propose that the amphipathic helix α5 may contribute to Golgi localization of hNaa60.	DISCUSS
+91	97	hNaa60	protein	Therefore we propose that the amphipathic helix α5 may contribute to Golgi localization of hNaa60.	DISCUSS
+43	46	NAT	protein_type	Previous studies indicated that members of NAT family are bi-functional NAT and KAT enzymes.	DISCUSS
+72	75	NAT	protein_type	Previous studies indicated that members of NAT family are bi-functional NAT and KAT enzymes.	DISCUSS
+80	83	KAT	protein_type	Previous studies indicated that members of NAT family are bi-functional NAT and KAT enzymes.	DISCUSS
+15	25	structures	evidence	However, known structures of NATs do not well support this hypothesis, since the β6-β7 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the α1-α2 loop, which would be good for the NAT but not KAT activity of the enzyme.	DISCUSS
+29	33	NATs	protein_type	However, known structures of NATs do not well support this hypothesis, since the β6-β7 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the α1-α2 loop, which would be good for the NAT but not KAT activity of the enzyme.	DISCUSS
+81	94	β6-β7 hairpin	structure_element	However, known structures of NATs do not well support this hypothesis, since the β6-β7 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the α1-α2 loop, which would be good for the NAT but not KAT activity of the enzyme.	DISCUSS
+95	99	loop	structure_element	However, known structures of NATs do not well support this hypothesis, since the β6-β7 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the α1-α2 loop, which would be good for the NAT but not KAT activity of the enzyme.	DISCUSS
+111	115	NATs	protein_type	However, known structures of NATs do not well support this hypothesis, since the β6-β7 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the α1-α2 loop, which would be good for the NAT but not KAT activity of the enzyme.	DISCUSS
+150	184	tunnel-like substrate-binding site	site	However, known structures of NATs do not well support this hypothesis, since the β6-β7 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the α1-α2 loop, which would be good for the NAT but not KAT activity of the enzyme.	DISCUSS
+194	204	α1-α2 loop	structure_element	However, known structures of NATs do not well support this hypothesis, since the β6-β7 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the α1-α2 loop, which would be good for the NAT but not KAT activity of the enzyme.	DISCUSS
+234	237	NAT	protein_type	However, known structures of NATs do not well support this hypothesis, since the β6-β7 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the α1-α2 loop, which would be good for the NAT but not KAT activity of the enzyme.	DISCUSS
+246	249	KAT	protein_type	However, known structures of NATs do not well support this hypothesis, since the β6-β7 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the α1-α2 loop, which would be good for the NAT but not KAT activity of the enzyme.	DISCUSS
+0	15	Kinetic studies	experimental_method	Kinetic studies have been conducted to compare the NAT and KAT activity of hNaa50 in vitro, and indicate that the NAT activity of Naa50 is much higher than KAT activity.	DISCUSS
+51	54	NAT	protein_type	Kinetic studies have been conducted to compare the NAT and KAT activity of hNaa50 in vitro, and indicate that the NAT activity of Naa50 is much higher than KAT activity.	DISCUSS
+59	62	KAT	protein_type	Kinetic studies have been conducted to compare the NAT and KAT activity of hNaa50 in vitro, and indicate that the NAT activity of Naa50 is much higher than KAT activity.	DISCUSS
+75	81	hNaa50	protein	Kinetic studies have been conducted to compare the NAT and KAT activity of hNaa50 in vitro, and indicate that the NAT activity of Naa50 is much higher than KAT activity.	DISCUSS
+114	117	NAT	protein_type	Kinetic studies have been conducted to compare the NAT and KAT activity of hNaa50 in vitro, and indicate that the NAT activity of Naa50 is much higher than KAT activity.	DISCUSS
+130	135	Naa50	protein	Kinetic studies have been conducted to compare the NAT and KAT activity of hNaa50 in vitro, and indicate that the NAT activity of Naa50 is much higher than KAT activity.	DISCUSS
+156	159	KAT	protein_type	Kinetic studies have been conducted to compare the NAT and KAT activity of hNaa50 in vitro, and indicate that the NAT activity of Naa50 is much higher than KAT activity.	DISCUSS
+56	59	KAT	protein_type	However, the substrate used in this study for assessing KAT activity was a small peptide which could not really mimic the 3D structure of a folded protein substrate in vivo.	DISCUSS
+81	88	peptide	chemical	However, the substrate used in this study for assessing KAT activity was a small peptide which could not really mimic the 3D structure of a folded protein substrate in vivo.	DISCUSS
+122	134	3D structure	evidence	However, the substrate used in this study for assessing KAT activity was a small peptide which could not really mimic the 3D structure of a folded protein substrate in vivo.	DISCUSS
+140	146	folded	protein_state	However, the substrate used in this study for assessing KAT activity was a small peptide which could not really mimic the 3D structure of a folded protein substrate in vivo.	DISCUSS
+4	21	mass spectrometry	experimental_method	Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro.	DISCUSS
+22	26	data	evidence	Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro.	DISCUSS
+60	71	acetylation	ptm	Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro.	DISCUSS
+75	82	histone	protein_type	Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro.	DISCUSS
+83	88	H3-H4	complex_assembly	Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro.	DISCUSS
+89	97	tetramer	oligomeric_state	Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro.	DISCUSS
+98	105	lysines	residue_name	Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro.	DISCUSS
+115	137	N-terminal acetylation	ptm	Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro.	DISCUSS
+142	160	lysine acetylation	ptm	Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro.	DISCUSS
+168	175	peptide	chemical	Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro.	DISCUSS
+188	202	activity assay	experimental_method	Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro.	DISCUSS
+223	226	KAT	protein_type	Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro.	DISCUSS
+29	42	β7-β8 hairpin	structure_element	Conformational change of the β7-β8 hairpin (corresponding to the β6-β7 loop of other NATs) is noted in our structures (Figs 1D and 2C), which might provide an explanation to the NAT/KAT dual-activity in a structural biological view, but we were unable to rule out the possibility that the observed conformational change of this hairpin might be an artifact related to crystal packing or truncation of the C-terminal end of the protein.	DISCUSS
+65	75	β6-β7 loop	structure_element	Conformational change of the β7-β8 hairpin (corresponding to the β6-β7 loop of other NATs) is noted in our structures (Figs 1D and 2C), which might provide an explanation to the NAT/KAT dual-activity in a structural biological view, but we were unable to rule out the possibility that the observed conformational change of this hairpin might be an artifact related to crystal packing or truncation of the C-terminal end of the protein.	DISCUSS
+85	89	NATs	protein_type	Conformational change of the β7-β8 hairpin (corresponding to the β6-β7 loop of other NATs) is noted in our structures (Figs 1D and 2C), which might provide an explanation to the NAT/KAT dual-activity in a structural biological view, but we were unable to rule out the possibility that the observed conformational change of this hairpin might be an artifact related to crystal packing or truncation of the C-terminal end of the protein.	DISCUSS
+107	117	structures	evidence	Conformational change of the β7-β8 hairpin (corresponding to the β6-β7 loop of other NATs) is noted in our structures (Figs 1D and 2C), which might provide an explanation to the NAT/KAT dual-activity in a structural biological view, but we were unable to rule out the possibility that the observed conformational change of this hairpin might be an artifact related to crystal packing or truncation of the C-terminal end of the protein.	DISCUSS
+178	181	NAT	protein_type	Conformational change of the β7-β8 hairpin (corresponding to the β6-β7 loop of other NATs) is noted in our structures (Figs 1D and 2C), which might provide an explanation to the NAT/KAT dual-activity in a structural biological view, but we were unable to rule out the possibility that the observed conformational change of this hairpin might be an artifact related to crystal packing or truncation of the C-terminal end of the protein.	DISCUSS
+182	185	KAT	protein_type	Conformational change of the β7-β8 hairpin (corresponding to the β6-β7 loop of other NATs) is noted in our structures (Figs 1D and 2C), which might provide an explanation to the NAT/KAT dual-activity in a structural biological view, but we were unable to rule out the possibility that the observed conformational change of this hairpin might be an artifact related to crystal packing or truncation of the C-terminal end of the protein.	DISCUSS
+328	335	hairpin	structure_element	Conformational change of the β7-β8 hairpin (corresponding to the β6-β7 loop of other NATs) is noted in our structures (Figs 1D and 2C), which might provide an explanation to the NAT/KAT dual-activity in a structural biological view, but we were unable to rule out the possibility that the observed conformational change of this hairpin might be an artifact related to crystal packing or truncation of the C-terminal end of the protein.	DISCUSS
+368	383	crystal packing	evidence	Conformational change of the β7-β8 hairpin (corresponding to the β6-β7 loop of other NATs) is noted in our structures (Figs 1D and 2C), which might provide an explanation to the NAT/KAT dual-activity in a structural biological view, but we were unable to rule out the possibility that the observed conformational change of this hairpin might be an artifact related to crystal packing or truncation of the C-terminal end of the protein.	DISCUSS
+69	72	KAT	protein_type	Further studies are therefore needed to reveal the mechanism for the KAT activity of this enzyme.	DISCUSS
+48	78	GCN5 histone acetyltransferase	protein_type	In early years, researchers found adjustment of GCN5 histone acetyltransferase structure when it binds CoA molecule.	DISCUSS
+79	88	structure	evidence	In early years, researchers found adjustment of GCN5 histone acetyltransferase structure when it binds CoA molecule.	DISCUSS
+103	106	CoA	chemical	In early years, researchers found adjustment of GCN5 histone acetyltransferase structure when it binds CoA molecule.	DISCUSS
+4	13	complexed	protein_state	The complexed form of NatA is more suitable for catalytic activation, since the α1-α2 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA).	DISCUSS
+22	26	NatA	complex_assembly	The complexed form of NatA is more suitable for catalytic activation, since the α1-α2 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA).	DISCUSS
+80	90	α1-α2 loop	structure_element	The complexed form of NatA is more suitable for catalytic activation, since the α1-α2 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA).	DISCUSS
+158	180	substrate-binding site	site	The complexed form of NatA is more suitable for catalytic activation, since the α1-α2 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA).	DISCUSS
+208	213	Naa15	protein	The complexed form of NatA is more suitable for catalytic activation, since the α1-α2 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA).	DISCUSS
+229	234	Naa10	protein	The complexed form of NatA is more suitable for catalytic activation, since the α1-α2 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA).	DISCUSS
+240	249	catalytic	protein_state	The complexed form of NatA is more suitable for catalytic activation, since the α1-α2 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA).	DISCUSS
+250	257	subunit	structure_element	The complexed form of NatA is more suitable for catalytic activation, since the α1-α2 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA).	DISCUSS
+261	265	NatA	complex_assembly	The complexed form of NatA is more suitable for catalytic activation, since the α1-α2 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA).	DISCUSS
+7	16	structure	evidence	In the structure of hNaa50/CoA/peptide, Phe 27 in the α1-α2 loop appears to make hydrophobic interaction with the N-terminal Met of substrate peptide.	DISCUSS
+20	38	hNaa50/CoA/peptide	complex_assembly	In the structure of hNaa50/CoA/peptide, Phe 27 in the α1-α2 loop appears to make hydrophobic interaction with the N-terminal Met of substrate peptide.	DISCUSS
+40	46	Phe 27	residue_name_number	In the structure of hNaa50/CoA/peptide, Phe 27 in the α1-α2 loop appears to make hydrophobic interaction with the N-terminal Met of substrate peptide.	DISCUSS
+54	64	α1-α2 loop	structure_element	In the structure of hNaa50/CoA/peptide, Phe 27 in the α1-α2 loop appears to make hydrophobic interaction with the N-terminal Met of substrate peptide.	DISCUSS
+81	104	hydrophobic interaction	bond_interaction	In the structure of hNaa50/CoA/peptide, Phe 27 in the α1-α2 loop appears to make hydrophobic interaction with the N-terminal Met of substrate peptide.	DISCUSS
+125	128	Met	residue_name	In the structure of hNaa50/CoA/peptide, Phe 27 in the α1-α2 loop appears to make hydrophobic interaction with the N-terminal Met of substrate peptide.	DISCUSS
+142	149	peptide	chemical	In the structure of hNaa50/CoA/peptide, Phe 27 in the α1-α2 loop appears to make hydrophobic interaction with the N-terminal Met of substrate peptide.	DISCUSS
+13	33	hNaa60(1-242)/Ac-CoA	complex_assembly	However, the hNaa60(1-242)/Ac-CoA crystal structure indicated that its counterpart in hNaa60, Phe 34, could also accommodate the binding of a hydrophilic malonate that occupied the substrate binding site although it maintained the same conformation as that observed in hNaa50.	DISCUSS
+34	51	crystal structure	evidence	However, the hNaa60(1-242)/Ac-CoA crystal structure indicated that its counterpart in hNaa60, Phe 34, could also accommodate the binding of a hydrophilic malonate that occupied the substrate binding site although it maintained the same conformation as that observed in hNaa50.	DISCUSS
+86	92	hNaa60	protein	However, the hNaa60(1-242)/Ac-CoA crystal structure indicated that its counterpart in hNaa60, Phe 34, could also accommodate the binding of a hydrophilic malonate that occupied the substrate binding site although it maintained the same conformation as that observed in hNaa50.	DISCUSS
+94	100	Phe 34	residue_name_number	However, the hNaa60(1-242)/Ac-CoA crystal structure indicated that its counterpart in hNaa60, Phe 34, could also accommodate the binding of a hydrophilic malonate that occupied the substrate binding site although it maintained the same conformation as that observed in hNaa50.	DISCUSS
+154	162	malonate	chemical	However, the hNaa60(1-242)/Ac-CoA crystal structure indicated that its counterpart in hNaa60, Phe 34, could also accommodate the binding of a hydrophilic malonate that occupied the substrate binding site although it maintained the same conformation as that observed in hNaa50.	DISCUSS
+181	203	substrate binding site	site	However, the hNaa60(1-242)/Ac-CoA crystal structure indicated that its counterpart in hNaa60, Phe 34, could also accommodate the binding of a hydrophilic malonate that occupied the substrate binding site although it maintained the same conformation as that observed in hNaa50.	DISCUSS
+269	275	hNaa50	protein	However, the hNaa60(1-242)/Ac-CoA crystal structure indicated that its counterpart in hNaa60, Phe 34, could also accommodate the binding of a hydrophilic malonate that occupied the substrate binding site although it maintained the same conformation as that observed in hNaa50.	DISCUSS
+28	33	thiol	chemical	Interestingly, the terminal thiol of CoA adopted alternative conformations in the structure of hNaa60(1-199)/CoA. One was to approach the substrate amine; the other was to approach the α1-α2 loop and away from the substrate amine.	DISCUSS
+37	40	CoA	chemical	Interestingly, the terminal thiol of CoA adopted alternative conformations in the structure of hNaa60(1-199)/CoA. One was to approach the substrate amine; the other was to approach the α1-α2 loop and away from the substrate amine.	DISCUSS
+82	91	structure	evidence	Interestingly, the terminal thiol of CoA adopted alternative conformations in the structure of hNaa60(1-199)/CoA. One was to approach the substrate amine; the other was to approach the α1-α2 loop and away from the substrate amine.	DISCUSS
+95	112	hNaa60(1-199)/CoA	complex_assembly	Interestingly, the terminal thiol of CoA adopted alternative conformations in the structure of hNaa60(1-199)/CoA. One was to approach the substrate amine; the other was to approach the α1-α2 loop and away from the substrate amine.	DISCUSS
+185	195	α1-α2 loop	structure_element	Interestingly, the terminal thiol of CoA adopted alternative conformations in the structure of hNaa60(1-199)/CoA. One was to approach the substrate amine; the other was to approach the α1-α2 loop and away from the substrate amine.	DISCUSS
+34	37	CoA	chemical	Same alternative conformations of CoA were observed in the hNaa60(1-199)(F34A) crystal structure, and our kinetic data showed that the F34A mutation abolished the activity of the enzyme.	DISCUSS
+59	78	hNaa60(1-199)(F34A)	mutant	Same alternative conformations of CoA were observed in the hNaa60(1-199)(F34A) crystal structure, and our kinetic data showed that the F34A mutation abolished the activity of the enzyme.	DISCUSS
+79	96	crystal structure	evidence	Same alternative conformations of CoA were observed in the hNaa60(1-199)(F34A) crystal structure, and our kinetic data showed that the F34A mutation abolished the activity of the enzyme.	DISCUSS
+106	118	kinetic data	evidence	Same alternative conformations of CoA were observed in the hNaa60(1-199)(F34A) crystal structure, and our kinetic data showed that the F34A mutation abolished the activity of the enzyme.	DISCUSS
+135	139	F34A	mutant	Same alternative conformations of CoA were observed in the hNaa60(1-199)(F34A) crystal structure, and our kinetic data showed that the F34A mutation abolished the activity of the enzyme.	DISCUSS
+140	148	mutation	experimental_method	Same alternative conformations of CoA were observed in the hNaa60(1-199)(F34A) crystal structure, and our kinetic data showed that the F34A mutation abolished the activity of the enzyme.	DISCUSS
+40	46	Phe 34	residue_name_number	Taken together, our data indicated that Phe 34 in hNaa60 may play a role in placing co-enzyme at the right location to facilitate the acetyl-transfer.	DISCUSS
+50	56	hNaa60	protein	Taken together, our data indicated that Phe 34 in hNaa60 may play a role in placing co-enzyme at the right location to facilitate the acetyl-transfer.	DISCUSS
+134	140	acetyl	chemical	Taken together, our data indicated that Phe 34 in hNaa60 may play a role in placing co-enzyme at the right location to facilitate the acetyl-transfer.	DISCUSS
+59	65	Phe 34	residue_name_number	However, these data did not rule out that possibility that Phe 34 may coordinate the binding of the N-terminal Met through hydrophobic interaction as was proposed by previous studies.	DISCUSS
+111	114	Met	residue_name	However, these data did not rule out that possibility that Phe 34 may coordinate the binding of the N-terminal Met through hydrophobic interaction as was proposed by previous studies.	DISCUSS
+123	146	hydrophobic interaction	bond_interaction	However, these data did not rule out that possibility that Phe 34 may coordinate the binding of the N-terminal Met through hydrophobic interaction as was proposed by previous studies.	DISCUSS
+28	34	hNaa60	protein	Furthermore, we showed that hNaa60 adopts the classical two base mechanism to catalyze acetyl-transfer.	DISCUSS
+87	93	acetyl	chemical	Furthermore, we showed that hNaa60 adopts the classical two base mechanism to catalyze acetyl-transfer.	DISCUSS
+35	41	hNaa60	protein	Although sequence identity between hNaa60 and hNaa50 is low, key residues in the active site of both enzymes are highly conserved.	DISCUSS
+46	52	hNaa50	protein	Although sequence identity between hNaa60 and hNaa50 is low, key residues in the active site of both enzymes are highly conserved.	DISCUSS
+81	92	active site	site	Although sequence identity between hNaa60 and hNaa50 is low, key residues in the active site of both enzymes are highly conserved.	DISCUSS
+113	129	highly conserved	protein_state	Although sequence identity between hNaa60 and hNaa50 is low, key residues in the active site of both enzymes are highly conserved.	DISCUSS
+82	88	hNaa60	protein	This can reasonably explain the high overlapping substrates specificities between hNaa60 and hNaa50.	DISCUSS
+93	99	hNaa50	protein	This can reasonably explain the high overlapping substrates specificities between hNaa60 and hNaa50.	DISCUSS
+30	36	hNaa60	protein	Another structural feature of hNaa60 that distinguishes it from other NATs is the β3-β4 long loop which appears to inhibit the catalytic activity of hNaa60.	DISCUSS
+70	74	NATs	protein_type	Another structural feature of hNaa60 that distinguishes it from other NATs is the β3-β4 long loop which appears to inhibit the catalytic activity of hNaa60.	DISCUSS
+82	97	β3-β4 long loop	structure_element	Another structural feature of hNaa60 that distinguishes it from other NATs is the β3-β4 long loop which appears to inhibit the catalytic activity of hNaa60.	DISCUSS
+149	155	hNaa60	protein	Another structural feature of hNaa60 that distinguishes it from other NATs is the β3-β4 long loop which appears to inhibit the catalytic activity of hNaa60.	DISCUSS
+14	18	loop	structure_element	However, this loop also seems to stabilize the whole hNaa60 structure, because deletion mutations of this region led to protein precipitation and aggregation (Figure S7).	DISCUSS
+53	59	hNaa60	protein	However, this loop also seems to stabilize the whole hNaa60 structure, because deletion mutations of this region led to protein precipitation and aggregation (Figure S7).	DISCUSS
+60	69	structure	evidence	However, this loop also seems to stabilize the whole hNaa60 structure, because deletion mutations of this region led to protein precipitation and aggregation (Figure S7).	DISCUSS
+79	97	deletion mutations	experimental_method	However, this loop also seems to stabilize the whole hNaa60 structure, because deletion mutations of this region led to protein precipitation and aggregation (Figure S7).	DISCUSS
+36	52	auto-acetylation	ptm	A previous study suggested that the auto-acetylation of Lys 79 was important for hNaa60-activity, whereas the point mutation K79R did not decrease the activity of hNaa60 in our study.	DISCUSS
+56	62	Lys 79	residue_name_number	A previous study suggested that the auto-acetylation of Lys 79 was important for hNaa60-activity, whereas the point mutation K79R did not decrease the activity of hNaa60 in our study.	DISCUSS
+81	87	hNaa60	protein	A previous study suggested that the auto-acetylation of Lys 79 was important for hNaa60-activity, whereas the point mutation K79R did not decrease the activity of hNaa60 in our study.	DISCUSS
+110	124	point mutation	experimental_method	A previous study suggested that the auto-acetylation of Lys 79 was important for hNaa60-activity, whereas the point mutation K79R did not decrease the activity of hNaa60 in our study.	DISCUSS
+125	129	K79R	mutant	A previous study suggested that the auto-acetylation of Lys 79 was important for hNaa60-activity, whereas the point mutation K79R did not decrease the activity of hNaa60 in our study.	DISCUSS
+163	169	hNaa60	protein	A previous study suggested that the auto-acetylation of Lys 79 was important for hNaa60-activity, whereas the point mutation K79R did not decrease the activity of hNaa60 in our study.	DISCUSS
+14	30	electron density	evidence	Meanwhile, no electron density of acetyl group was found on Lys 79 in our structures and mass spectrometry analysis.	DISCUSS
+34	40	acetyl	chemical	Meanwhile, no electron density of acetyl group was found on Lys 79 in our structures and mass spectrometry analysis.	DISCUSS
+60	66	Lys 79	residue_name_number	Meanwhile, no electron density of acetyl group was found on Lys 79 in our structures and mass spectrometry analysis.	DISCUSS
+74	84	structures	evidence	Meanwhile, no electron density of acetyl group was found on Lys 79 in our structures and mass spectrometry analysis.	DISCUSS
+89	106	mass spectrometry	experimental_method	Meanwhile, no electron density of acetyl group was found on Lys 79 in our structures and mass spectrometry analysis.	DISCUSS
+27	43	auto-acetylation	ptm	Hence, it appears that the auto-acetylation of hNaa60 is not an essential modification for its activity for the protein we used here.	DISCUSS
+47	53	hNaa60	protein	Hence, it appears that the auto-acetylation of hNaa60 is not an essential modification for its activity for the protein we used here.	DISCUSS
+22	26	K79R	mutant	As for the reason why K79R in Yang’s previous studies reduced the activity of the enzyme, but in our studies it didn’t, we suspect that the stability of this mutant may play some role.	DISCUSS
+158	164	mutant	protein_state	As for the reason why K79R in Yang’s previous studies reduced the activity of the enzyme, but in our studies it didn’t, we suspect that the stability of this mutant may play some role.	DISCUSS
+0	4	K79R	mutant	K79R is less stable than the wild-type enzyme as was judged by its poorer gel-filtration behavior and tendency to precipitate.	DISCUSS
+13	19	stable	protein_state	K79R is less stable than the wild-type enzyme as was judged by its poorer gel-filtration behavior and tendency to precipitate.	DISCUSS
+29	38	wild-type	protein_state	K79R is less stable than the wild-type enzyme as was judged by its poorer gel-filtration behavior and tendency to precipitate.	DISCUSS
+74	88	gel-filtration	experimental_method	K79R is less stable than the wild-type enzyme as was judged by its poorer gel-filtration behavior and tendency to precipitate.	DISCUSS
+151	165	kinetic assays	experimental_method	In our studies we have paid special attention and carefully handled this protein to ensure that we did get enough of the protein in good condition for kinetic assays.	DISCUSS
+107	113	hNaa60	protein	The intracellular environment is more complicated than our in vitro assay and the substrate specificity of hNaa60 most focuses on transmembrane proteins.	DISCUSS
+24	30	hNaa60	protein	The interaction between hNaa60 and its substrates may involve the protein-membrane interaction which would further increase the complexity.	DISCUSS
+23	32	structure	evidence	It is not clear if the structure of hNaa60 is different in vivo or if other potential partner proteins may help to regulate its activity.	DISCUSS
+36	42	hNaa60	protein	It is not clear if the structure of hNaa60 is different in vivo or if other potential partner proteins may help to regulate its activity.	DISCUSS
+129	132	NAT	protein_type	Nevertheless, our study may be an inspiration for further studies on the functions and regulation of this youngest member of the NAT family.	DISCUSS
+8	17	structure	evidence	Overall structure of Naa60.	FIG
+21	26	Naa60	protein	Overall structure of Naa60.	FIG
+4	22	Sequence alignment	experimental_method	(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus).	FIG
+26	31	Naa60	protein	(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus).	FIG
+33	37	NatF	complex_assembly	(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus).	FIG
+39	43	HAT4	protein	(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus).	FIG
+78	90	Homo sapiens	species	(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus).	FIG
+92	96	Homo	species	(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus).	FIG
+99	108	Bos mutus	species	(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus).	FIG
+110	113	Bos	species	(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus).	FIG
+116	127	Salmo salar	species	(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus).	FIG
+129	134	Salmo	species	(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus).	FIG
+140	147	Xenopus	species	(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus).	FIG
+149	157	Silurana	species	(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus).	FIG
+159	169	tropicalis	species	(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus).	FIG
+171	178	Xenopus	species	(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus).	FIG
+0	9	Alignment	experimental_method	Alignment was generated using NPS@ and ESPript.3.0 (http://espript.ibcp.fr/ESPript/ESPript/).	FIG
+9	12	4–6	residue_range	Residues 4–6 are highlighted in red box.	FIG
+8	17	structure	evidence	(B) The structure of hNaa60(1-199)/CoA complex is shown as a yellow cartoon model.	FIG
+21	38	hNaa60(1-199)/CoA	complex_assembly	(B) The structure of hNaa60(1-199)/CoA complex is shown as a yellow cartoon model.	FIG
+4	7	CoA	chemical	The CoA molecule is shown as sticks. (C) The structure of hNaa60(1-242)/Ac-CoA complex is presented as a cartoon model in cyan.	FIG
+45	54	structure	evidence	The CoA molecule is shown as sticks. (C) The structure of hNaa60(1-242)/Ac-CoA complex is presented as a cartoon model in cyan.	FIG
+58	78	hNaa60(1-242)/Ac-CoA	complex_assembly	The CoA molecule is shown as sticks. (C) The structure of hNaa60(1-242)/Ac-CoA complex is presented as a cartoon model in cyan.	FIG
+4	10	Ac-CoA	chemical	The Ac-CoA and malonate molecules are shown as cyan and purple sticks, respectively.	FIG
+15	23	malonate	chemical	The Ac-CoA and malonate molecules are shown as cyan and purple sticks, respectively.	FIG
+51	53	α0	structure_element	The secondary structures are labeled starting with α0. (D) Superposition of hNaa60(1-242) (cyan), hNaa60(1-199) (yellow) and hNaa50 (pink, PDB 3TFY).	FIG
+59	72	Superposition	experimental_method	The secondary structures are labeled starting with α0. (D) Superposition of hNaa60(1-242) (cyan), hNaa60(1-199) (yellow) and hNaa50 (pink, PDB 3TFY).	FIG
+76	82	hNaa60	protein	The secondary structures are labeled starting with α0. (D) Superposition of hNaa60(1-242) (cyan), hNaa60(1-199) (yellow) and hNaa50 (pink, PDB 3TFY).	FIG
+83	88	1-242	residue_range	The secondary structures are labeled starting with α0. (D) Superposition of hNaa60(1-242) (cyan), hNaa60(1-199) (yellow) and hNaa50 (pink, PDB 3TFY).	FIG
+98	111	hNaa60(1-199)	mutant	The secondary structures are labeled starting with α0. (D) Superposition of hNaa60(1-242) (cyan), hNaa60(1-199) (yellow) and hNaa50 (pink, PDB 3TFY).	FIG
+125	131	hNaa50	protein	The secondary structures are labeled starting with α0. (D) Superposition of hNaa60(1-242) (cyan), hNaa60(1-199) (yellow) and hNaa50 (pink, PDB 3TFY).	FIG
+4	10	Ac-CoA	chemical	The Ac-CoA of hNaa60(1-242)/Ac-CoA complex is represented as cyan sticks.	FIG
+14	34	hNaa60(1-242)/Ac-CoA	complex_assembly	The Ac-CoA of hNaa60(1-242)/Ac-CoA complex is represented as cyan sticks.	FIG
+0	14	Amphipathicity	protein_state	Amphipathicity of the α5 helix and alternative conformations of the β7-β8 hairpin.	FIG
+22	30	α5 helix	structure_element	Amphipathicity of the α5 helix and alternative conformations of the β7-β8 hairpin.	FIG
+68	81	β7-β8 hairpin	structure_element	Amphipathicity of the α5 helix and alternative conformations of the β7-β8 hairpin.	FIG
+8	16	α5 helix	structure_element	(A) The α5 helix of hNaa60(1-242) in one asymmetric unit (slate) interacts with another hNaa60 molecule in a neighboring asymmetric unit (cyan).	FIG
+20	26	hNaa60	protein	(A) The α5 helix of hNaa60(1-242) in one asymmetric unit (slate) interacts with another hNaa60 molecule in a neighboring asymmetric unit (cyan).	FIG
+27	32	1-242	residue_range	(A) The α5 helix of hNaa60(1-242) in one asymmetric unit (slate) interacts with another hNaa60 molecule in a neighboring asymmetric unit (cyan).	FIG
+88	94	hNaa60	protein	(A) The α5 helix of hNaa60(1-242) in one asymmetric unit (slate) interacts with another hNaa60 molecule in a neighboring asymmetric unit (cyan).	FIG
+39	47	α5 helix	structure_element	Side-chains of hydrophobic residues on α5 helix and the neighboring molecule participating in the interaction are shown as yellow and green sticks, respectively. (B) The α5 helix of hNaa60(1-199) in one asymmetric unit (yellow) interacts with another hNaa60 molecule in the neighboring asymmetric units (green).	FIG
+170	178	α5 helix	structure_element	Side-chains of hydrophobic residues on α5 helix and the neighboring molecule participating in the interaction are shown as yellow and green sticks, respectively. (B) The α5 helix of hNaa60(1-199) in one asymmetric unit (yellow) interacts with another hNaa60 molecule in the neighboring asymmetric units (green).	FIG
+182	195	hNaa60(1-199)	mutant	Side-chains of hydrophobic residues on α5 helix and the neighboring molecule participating in the interaction are shown as yellow and green sticks, respectively. (B) The α5 helix of hNaa60(1-199) in one asymmetric unit (yellow) interacts with another hNaa60 molecule in the neighboring asymmetric units (green).	FIG
+251	257	hNaa60	protein	Side-chains of hydrophobic residues on α5 helix and the neighboring molecule participating in the interaction are shown as yellow and green sticks, respectively. (B) The α5 helix of hNaa60(1-199) in one asymmetric unit (yellow) interacts with another hNaa60 molecule in the neighboring asymmetric units (green).	FIG
+39	47	α5 helix	structure_element	Side-chains of hydrophobic residues on α5 helix and the neighboring molecule (green) participating in the interaction are shown as yellow and green sticks, respectively.	FIG
+62	70	α5 helix	structure_element	The third molecule (pink) does not directly interact with the α5 helix.	FIG
+4	17	Superposition	experimental_method	(C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E).	FIG
+21	34	hNaa60(1-199)	mutant	(C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E).	FIG
+48	54	hNaa60	protein	(C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E).	FIG
+55	60	1-242	residue_range	(C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E).	FIG
+106	119	β7-β8 hairpin	structure_element	(C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E).	FIG
+133	143	structures	evidence	(C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E).	FIG
+151	164	Superposition	experimental_method	(C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E).	FIG
+168	173	Hat1p	protein	(C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E).	FIG
+174	176	H4	protein_type	(C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E).	FIG
+210	216	hNaa60	protein	(C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E).	FIG
+217	222	1-242	residue_range	(C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E).	FIG
+237	250	hNaa60(1-199)	mutant	(C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E).	FIG
+4	11	histone	protein_type	The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D).	FIG
+12	14	H4	protein_type	The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D).	FIG
+15	22	peptide	chemical	The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D).	FIG
+26	29	KAT	protein_type	The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D).	FIG
+41	49	bound to	protein_state	The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D).	FIG
+50	55	Hat1p	protein	The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D).	FIG
+92	99	peptide	chemical	The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D).	FIG
+100	108	bound to	protein_state	The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D).	FIG
+109	115	hNaa50	protein	The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D).	FIG
+119	122	NAT	protein_type	The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D).	FIG
+175	185	Nt-peptide	chemical	The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D).	FIG
+193	206	superimposing	experimental_method	The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D).	FIG
+207	213	hNaa50	protein	The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D).	FIG
+239	245	hNaa60	protein	The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D).	FIG
+19	22	NAT	protein_type	The α-amine of the NAT substrate and ε-amine of the KAT substrate (along with the lysine side-chain) subject to acetylation are shown as sticks.	FIG
+52	55	KAT	protein_type	The α-amine of the NAT substrate and ε-amine of the KAT substrate (along with the lysine side-chain) subject to acetylation are shown as sticks.	FIG
+82	88	lysine	residue_name	The α-amine of the NAT substrate and ε-amine of the KAT substrate (along with the lysine side-chain) subject to acetylation are shown as sticks.	FIG
+112	123	acetylation	ptm	The α-amine of the NAT substrate and ε-amine of the KAT substrate (along with the lysine side-chain) subject to acetylation are shown as sticks.	FIG
+0	20	Electron density map	evidence	Electron density map of the active site.	FIG
+28	39	active site	site	Electron density map of the active site.	FIG
+4	15	2Fo-Fc maps	evidence	The 2Fo-Fc maps contoured at 1.0σ are shown for hNaa60(1-242)/Ac-CoA (A), hNaa60(1-199)/CoA (B) and hNaa60(1-199) F34A/CoA (C).	FIG
+48	68	hNaa60(1-242)/Ac-CoA	complex_assembly	The 2Fo-Fc maps contoured at 1.0σ are shown for hNaa60(1-242)/Ac-CoA (A), hNaa60(1-199)/CoA (B) and hNaa60(1-199) F34A/CoA (C).	FIG
+74	91	hNaa60(1-199)/CoA	complex_assembly	The 2Fo-Fc maps contoured at 1.0σ are shown for hNaa60(1-242)/Ac-CoA (A), hNaa60(1-199)/CoA (B) and hNaa60(1-199) F34A/CoA (C).	FIG
+100	122	hNaa60(1-199) F34A/CoA	complex_assembly	The 2Fo-Fc maps contoured at 1.0σ are shown for hNaa60(1-242)/Ac-CoA (A), hNaa60(1-199)/CoA (B) and hNaa60(1-199) F34A/CoA (C).	FIG
+13	43	substrate peptide binding site	site	The putative substrate peptide binding site is indicated by the peptide (shown as pink sticks) from the hNaa50/CoA/peptide complex structure after superimposing hNaa50 on the hNaa60 structures determined in this study.	FIG
+64	71	peptide	chemical	The putative substrate peptide binding site is indicated by the peptide (shown as pink sticks) from the hNaa50/CoA/peptide complex structure after superimposing hNaa50 on the hNaa60 structures determined in this study.	FIG
+104	122	hNaa50/CoA/peptide	complex_assembly	The putative substrate peptide binding site is indicated by the peptide (shown as pink sticks) from the hNaa50/CoA/peptide complex structure after superimposing hNaa50 on the hNaa60 structures determined in this study.	FIG
+131	140	structure	evidence	The putative substrate peptide binding site is indicated by the peptide (shown as pink sticks) from the hNaa50/CoA/peptide complex structure after superimposing hNaa50 on the hNaa60 structures determined in this study.	FIG
+147	160	superimposing	experimental_method	The putative substrate peptide binding site is indicated by the peptide (shown as pink sticks) from the hNaa50/CoA/peptide complex structure after superimposing hNaa50 on the hNaa60 structures determined in this study.	FIG
+161	167	hNaa50	protein	The putative substrate peptide binding site is indicated by the peptide (shown as pink sticks) from the hNaa50/CoA/peptide complex structure after superimposing hNaa50 on the hNaa60 structures determined in this study.	FIG
+175	181	hNaa60	protein	The putative substrate peptide binding site is indicated by the peptide (shown as pink sticks) from the hNaa50/CoA/peptide complex structure after superimposing hNaa50 on the hNaa60 structures determined in this study.	FIG
+182	192	structures	evidence	The putative substrate peptide binding site is indicated by the peptide (shown as pink sticks) from the hNaa50/CoA/peptide complex structure after superimposing hNaa50 on the hNaa60 structures determined in this study.	FIG
+45	54	first Met	residue_name_number	The black arrow indicates the α-amine of the first Met (M1) (all panels).	FIG
+56	58	M1	residue_name_number	The black arrow indicates the α-amine of the first Met (M1) (all panels).	FIG
+31	37	acetyl	chemical	The purple arrow indicates the acetyl moiety of Ac-CoA (A).	FIG
+48	54	Ac-CoA	chemical	The purple arrow indicates the acetyl moiety of Ac-CoA (A).	FIG
+95	101	Phe 34	residue_name_number	The red arrow indicates the alternative conformation of the thiol moiety of the co-enzyme when Phe 34 side-chain is displaced (B) or mutated to Ala (C).	FIG
+133	140	mutated	experimental_method	The red arrow indicates the alternative conformation of the thiol moiety of the co-enzyme when Phe 34 side-chain is displaced (B) or mutated to Ala (C).	FIG
+144	147	Ala	residue_name	The red arrow indicates the alternative conformation of the thiol moiety of the co-enzyme when Phe 34 side-chain is displaced (B) or mutated to Ala (C).	FIG
+21	27	hNaa60	protein	Structural basis for hNaa60 catalytic activity.	FIG
+4	17	Superposition	experimental_method	(A) Superposition of hNaa60 active site (cyan) on that of hNaa50 (pink, PDB 3TFY).	FIG
+21	27	hNaa60	protein	(A) Superposition of hNaa60 active site (cyan) on that of hNaa50 (pink, PDB 3TFY).	FIG
+28	39	active site	site	(A) Superposition of hNaa60 active site (cyan) on that of hNaa50 (pink, PDB 3TFY).	FIG
+58	64	hNaa50	protein	(A) Superposition of hNaa60 active site (cyan) on that of hNaa50 (pink, PDB 3TFY).	FIG
+19	59	catalytic and substrate-binding residues	site	Side-chains of key catalytic and substrate-binding residues are highlighted as sticks.	FIG
+4	12	malonate	chemical	The malonate molecule in the hNaa60(1-242)/Ac-CoA structure and the peptide in the hNaa50/CoA/peptide structure are shown as purple and yellow sticks respectively. (B) A close view of the active site of hNaa60.	FIG
+29	49	hNaa60(1-242)/Ac-CoA	complex_assembly	The malonate molecule in the hNaa60(1-242)/Ac-CoA structure and the peptide in the hNaa50/CoA/peptide structure are shown as purple and yellow sticks respectively. (B) A close view of the active site of hNaa60.	FIG
+50	59	structure	evidence	The malonate molecule in the hNaa60(1-242)/Ac-CoA structure and the peptide in the hNaa50/CoA/peptide structure are shown as purple and yellow sticks respectively. (B) A close view of the active site of hNaa60.	FIG
+68	75	peptide	chemical	The malonate molecule in the hNaa60(1-242)/Ac-CoA structure and the peptide in the hNaa50/CoA/peptide structure are shown as purple and yellow sticks respectively. (B) A close view of the active site of hNaa60.	FIG
+83	101	hNaa50/CoA/peptide	complex_assembly	The malonate molecule in the hNaa60(1-242)/Ac-CoA structure and the peptide in the hNaa50/CoA/peptide structure are shown as purple and yellow sticks respectively. (B) A close view of the active site of hNaa60.	FIG
+102	111	structure	evidence	The malonate molecule in the hNaa60(1-242)/Ac-CoA structure and the peptide in the hNaa50/CoA/peptide structure are shown as purple and yellow sticks respectively. (B) A close view of the active site of hNaa60.	FIG
+188	199	active site	site	The malonate molecule in the hNaa60(1-242)/Ac-CoA structure and the peptide in the hNaa50/CoA/peptide structure are shown as purple and yellow sticks respectively. (B) A close view of the active site of hNaa60.	FIG
+203	209	hNaa60	protein	The malonate molecule in the hNaa60(1-242)/Ac-CoA structure and the peptide in the hNaa50/CoA/peptide structure are shown as purple and yellow sticks respectively. (B) A close view of the active site of hNaa60.	FIG
+9	15	Glu 37	residue_name_number	Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks.	FIG
+17	23	Tyr 97	residue_name_number	Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks.	FIG
+28	35	His 138	residue_name_number	Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks.	FIG
+39	45	hNaa60	protein	Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks.	FIG
+81	87	Tyr 73	residue_name_number	Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks.	FIG
+92	99	His 112	residue_name_number	Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks.	FIG
+104	110	hNaa50	protein	Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks.	FIG
+171	177	Glu 24	residue_name_number	Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks.	FIG
+179	185	His 72	residue_name_number	Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks.	FIG
+190	197	His 111	residue_name_number	Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks.	FIG
+202	211	complexed	protein_state	Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks.	FIG
+219	226	hNaa10p	protein	Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks.	FIG
+4	9	water	chemical	The water molecules participating in catalysis in the hNaa60 and hNaa50 structures are showed as green and red spheres, separately. (C) The interaction between the malonate molecule and surrounding residues observed in the hNaa60(1-242)/Ac-CoA structure.	FIG
+54	60	hNaa60	protein	The water molecules participating in catalysis in the hNaa60 and hNaa50 structures are showed as green and red spheres, separately. (C) The interaction between the malonate molecule and surrounding residues observed in the hNaa60(1-242)/Ac-CoA structure.	FIG
+65	71	hNaa50	protein	The water molecules participating in catalysis in the hNaa60 and hNaa50 structures are showed as green and red spheres, separately. (C) The interaction between the malonate molecule and surrounding residues observed in the hNaa60(1-242)/Ac-CoA structure.	FIG
+72	82	structures	evidence	The water molecules participating in catalysis in the hNaa60 and hNaa50 structures are showed as green and red spheres, separately. (C) The interaction between the malonate molecule and surrounding residues observed in the hNaa60(1-242)/Ac-CoA structure.	FIG
+164	172	malonate	chemical	The water molecules participating in catalysis in the hNaa60 and hNaa50 structures are showed as green and red spheres, separately. (C) The interaction between the malonate molecule and surrounding residues observed in the hNaa60(1-242)/Ac-CoA structure.	FIG
+223	243	hNaa60(1-242)/Ac-CoA	complex_assembly	The water molecules participating in catalysis in the hNaa60 and hNaa50 structures are showed as green and red spheres, separately. (C) The interaction between the malonate molecule and surrounding residues observed in the hNaa60(1-242)/Ac-CoA structure.	FIG
+244	253	structure	evidence	The water molecules participating in catalysis in the hNaa60 and hNaa50 structures are showed as green and red spheres, separately. (C) The interaction between the malonate molecule and surrounding residues observed in the hNaa60(1-242)/Ac-CoA structure.	FIG
+37	51	hydrogen bonds	bond_interaction	The yellow dotted lines indicate the hydrogen bonds. (D) A zoomed view of β3-β4 loop of hNaa60.	FIG
+74	84	β3-β4 loop	structure_element	The yellow dotted lines indicate the hydrogen bonds. (D) A zoomed view of β3-β4 loop of hNaa60.	FIG
+88	94	hNaa60	protein	The yellow dotted lines indicate the hydrogen bonds. (D) A zoomed view of β3-β4 loop of hNaa60.	FIG
+47	55	malonate	chemical	Key residues discussed in the text (cyan), the malonate (purple) and Ac-CoA (gray) are shown as sticks.	FIG
+69	75	Ac-CoA	chemical	Key residues discussed in the text (cyan), the malonate (purple) and Ac-CoA (gray) are shown as sticks.	FIG
+37	49	salt bridges	bond_interaction	The yellow dotted lines indicate the salt bridges.	FIG
+22	28	hNaa60	protein	Catalytic activity of hNaa60 and mutant proteins.	FIG
+33	39	mutant	protein_state	Catalytic activity of hNaa60 and mutant proteins.	FIG
+4	24	Catalytic efficiency	evidence	(A) Catalytic efficiency (shown as kcat/Km values) of hNaa60 (1-199) WT and mutants.	FIG
+35	39	kcat	evidence	(A) Catalytic efficiency (shown as kcat/Km values) of hNaa60 (1-199) WT and mutants.	FIG
+40	42	Km	evidence	(A) Catalytic efficiency (shown as kcat/Km values) of hNaa60 (1-199) WT and mutants.	FIG
+54	68	hNaa60 (1-199)	mutant	(A) Catalytic efficiency (shown as kcat/Km values) of hNaa60 (1-199) WT and mutants.	FIG
+69	71	WT	protein_state	(A) Catalytic efficiency (shown as kcat/Km values) of hNaa60 (1-199) WT and mutants.	FIG
+76	83	mutants	protein_state	(A) Catalytic efficiency (shown as kcat/Km values) of hNaa60 (1-199) WT and mutants.	FIG
+4	6	CD	experimental_method	(B) CD spectra of wild-type and mutant proteins from 250 nm to 190 nm.	FIG
+7	14	spectra	evidence	(B) CD spectra of wild-type and mutant proteins from 250 nm to 190 nm.	FIG
+18	27	wild-type	protein_state	(B) CD spectra of wild-type and mutant proteins from 250 nm to 190 nm.	FIG
+32	38	mutant	protein_state	(B) CD spectra of wild-type and mutant proteins from 250 nm to 190 nm.	FIG
+93	97	TCEP	chemical	The sample concentration was 4.5 μM in 20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP at room temperature.	FIG
+0	41	Data collection and refinement statistics	evidence	Data collection and refinement statistics.	TABLE
+21	41	hNaa60(1-242)/Ac-CoA	complex_assembly	"Structure and PDB ID	hNaa60(1-242)/Ac-CoA 5HGZ	hNaa60(1-199)/CoA 5HH0	hNaa60(1-199)F34A/CoA 5HH1	 	Data collection*	 	 Space group	P212121	P21212	P21212	 	Cell dimensions	 	 a, b, c (Å)	53.3, 57.4, 68.8	67.8, 73.8, 43.2	66.7, 74.0, 43.5	 	 α,β,γ (°)	90.0, 90.0, 90.0	90.0, 90.0, 90.0	90.0, 90.0, 90.0	 	Resolution (Å)	50–1.38 (1.42–1.38)	50–1.60 (1.66–1.60)	50–1.80 (1.86–1.80)	 	Rp.i.m.(%)**	3.0 (34.4)	2.1 (32.5)	2.6 (47.8)	 	I/σ	21.5 (2.0)	31.8 (2.0)	28.0 (2.4)	 	Completeness (%)	99.8 (99.1)	99.6 (98.5)	99.9 (99.7)	 	Redundancy	6.9 (5.0)	6.9 (6.2)	6.3 (5.9)	 	Refinement	 	 Resolution (Å)	25.81–1.38	33.55–1.60	43.52–1.80	 	 No. reflections	43660	28588	20490	 	 Rwork/Rfree	0.182/0.192	0.181/0.184	0.189/0.209	 	No. atoms	 	 Protein	1717	1576	1566	 	 Ligand/ion	116	96	96	 	 Water	289	258	168	 	B-factors	 	 Protein	23.8	32.0	37.4	 	 Ligand/ion	22.2	34.6	43.7	 	 Water	35.1	46.4	49.1	 	R.m.s."	TABLE
+47	64	hNaa60(1-199)/CoA	complex_assembly	"Structure and PDB ID	hNaa60(1-242)/Ac-CoA 5HGZ	hNaa60(1-199)/CoA 5HH0	hNaa60(1-199)F34A/CoA 5HH1	 	Data collection*	 	 Space group	P212121	P21212	P21212	 	Cell dimensions	 	 a, b, c (Å)	53.3, 57.4, 68.8	67.8, 73.8, 43.2	66.7, 74.0, 43.5	 	 α,β,γ (°)	90.0, 90.0, 90.0	90.0, 90.0, 90.0	90.0, 90.0, 90.0	 	Resolution (Å)	50–1.38 (1.42–1.38)	50–1.60 (1.66–1.60)	50–1.80 (1.86–1.80)	 	Rp.i.m.(%)**	3.0 (34.4)	2.1 (32.5)	2.6 (47.8)	 	I/σ	21.5 (2.0)	31.8 (2.0)	28.0 (2.4)	 	Completeness (%)	99.8 (99.1)	99.6 (98.5)	99.9 (99.7)	 	Redundancy	6.9 (5.0)	6.9 (6.2)	6.3 (5.9)	 	Refinement	 	 Resolution (Å)	25.81–1.38	33.55–1.60	43.52–1.80	 	 No. reflections	43660	28588	20490	 	 Rwork/Rfree	0.182/0.192	0.181/0.184	0.189/0.209	 	No. atoms	 	 Protein	1717	1576	1566	 	 Ligand/ion	116	96	96	 	 Water	289	258	168	 	B-factors	 	 Protein	23.8	32.0	37.4	 	 Ligand/ion	22.2	34.6	43.7	 	 Water	35.1	46.4	49.1	 	R.m.s."	TABLE
+70	91	hNaa60(1-199)F34A/CoA	complex_assembly	"Structure and PDB ID	hNaa60(1-242)/Ac-CoA 5HGZ	hNaa60(1-199)/CoA 5HH0	hNaa60(1-199)F34A/CoA 5HH1	 	Data collection*	 	 Space group	P212121	P21212	P21212	 	Cell dimensions	 	 a, b, c (Å)	53.3, 57.4, 68.8	67.8, 73.8, 43.2	66.7, 74.0, 43.5	 	 α,β,γ (°)	90.0, 90.0, 90.0	90.0, 90.0, 90.0	90.0, 90.0, 90.0	 	Resolution (Å)	50–1.38 (1.42–1.38)	50–1.60 (1.66–1.60)	50–1.80 (1.86–1.80)	 	Rp.i.m.(%)**	3.0 (34.4)	2.1 (32.5)	2.6 (47.8)	 	I/σ	21.5 (2.0)	31.8 (2.0)	28.0 (2.4)	 	Completeness (%)	99.8 (99.1)	99.6 (98.5)	99.9 (99.7)	 	Redundancy	6.9 (5.0)	6.9 (6.2)	6.3 (5.9)	 	Refinement	 	 Resolution (Å)	25.81–1.38	33.55–1.60	43.52–1.80	 	 No. reflections	43660	28588	20490	 	 Rwork/Rfree	0.182/0.192	0.181/0.184	0.189/0.209	 	No. atoms	 	 Protein	1717	1576	1566	 	 Ligand/ion	116	96	96	 	 Water	289	258	168	 	B-factors	 	 Protein	23.8	32.0	37.4	 	 Ligand/ion	22.2	34.6	43.7	 	 Water	35.1	46.4	49.1	 	R.m.s."	TABLE
+780	785	Water	chemical	"Structure and PDB ID	hNaa60(1-242)/Ac-CoA 5HGZ	hNaa60(1-199)/CoA 5HH0	hNaa60(1-199)F34A/CoA 5HH1	 	Data collection*	 	 Space group	P212121	P21212	P21212	 	Cell dimensions	 	 a, b, c (Å)	53.3, 57.4, 68.8	67.8, 73.8, 43.2	66.7, 74.0, 43.5	 	 α,β,γ (°)	90.0, 90.0, 90.0	90.0, 90.0, 90.0	90.0, 90.0, 90.0	 	Resolution (Å)	50–1.38 (1.42–1.38)	50–1.60 (1.66–1.60)	50–1.80 (1.86–1.80)	 	Rp.i.m.(%)**	3.0 (34.4)	2.1 (32.5)	2.6 (47.8)	 	I/σ	21.5 (2.0)	31.8 (2.0)	28.0 (2.4)	 	Completeness (%)	99.8 (99.1)	99.6 (98.5)	99.9 (99.7)	 	Redundancy	6.9 (5.0)	6.9 (6.2)	6.3 (5.9)	 	Refinement	 	 Resolution (Å)	25.81–1.38	33.55–1.60	43.52–1.80	 	 No. reflections	43660	28588	20490	 	 Rwork/Rfree	0.182/0.192	0.181/0.184	0.189/0.209	 	No. atoms	 	 Protein	1717	1576	1566	 	 Ligand/ion	116	96	96	 	 Water	289	258	168	 	B-factors	 	 Protein	23.8	32.0	37.4	 	 Ligand/ion	22.2	34.6	43.7	 	 Water	35.1	46.4	49.1	 	R.m.s."	TABLE
+868	873	Water	chemical	"Structure and PDB ID	hNaa60(1-242)/Ac-CoA 5HGZ	hNaa60(1-199)/CoA 5HH0	hNaa60(1-199)F34A/CoA 5HH1	 	Data collection*	 	 Space group	P212121	P21212	P21212	 	Cell dimensions	 	 a, b, c (Å)	53.3, 57.4, 68.8	67.8, 73.8, 43.2	66.7, 74.0, 43.5	 	 α,β,γ (°)	90.0, 90.0, 90.0	90.0, 90.0, 90.0	90.0, 90.0, 90.0	 	Resolution (Å)	50–1.38 (1.42–1.38)	50–1.60 (1.66–1.60)	50–1.80 (1.86–1.80)	 	Rp.i.m.(%)**	3.0 (34.4)	2.1 (32.5)	2.6 (47.8)	 	I/σ	21.5 (2.0)	31.8 (2.0)	28.0 (2.4)	 	Completeness (%)	99.8 (99.1)	99.6 (98.5)	99.9 (99.7)	 	Redundancy	6.9 (5.0)	6.9 (6.2)	6.3 (5.9)	 	Refinement	 	 Resolution (Å)	25.81–1.38	33.55–1.60	43.52–1.80	 	 No. reflections	43660	28588	20490	 	 Rwork/Rfree	0.182/0.192	0.181/0.184	0.189/0.209	 	No. atoms	 	 Protein	1717	1576	1566	 	 Ligand/ion	116	96	96	 	 Water	289	258	168	 	B-factors	 	 Protein	23.8	32.0	37.4	 	 Ligand/ion	22.2	34.6	43.7	 	 Water	35.1	46.4	49.1	 	R.m.s."	TABLE
+4	11	crystal	evidence	One crystal was used for each data set.	TABLE
+36	44	R factor	evidence	**Rp.i.m., a redundancy-independent R factor was used to evaluate the diffraction data quality as was proposed by Evans.	TABLE
+70	86	diffraction data	evidence	**Rp.i.m., a redundancy-independent R factor was used to evaluate the diffraction data quality as was proposed by Evans.	TABLE
diff --git a/annotation_CSV/PMC5012862.csv b/annotation_CSV/PMC5012862.csv
new file mode 100644
index 0000000000000000000000000000000000000000..e5f29573c26fe28bbfd0b1339a5ce33b16a08059
--- /dev/null
+++ b/annotation_CSV/PMC5012862.csv
@@ -0,0 +1,2203 @@
+anno_start	anno_end	anno_text	entity_type	sentence	section
+0	27	Structural characterization	experimental_method	Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments	TITLE
+31	43	encapsulated	protein_state	Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments	TITLE
+44	52	ferritin	protein_type	Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments	TITLE
+75	79	iron	chemical	Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments	TITLE
+91	100	bacterial	taxonomy_domain	Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments	TITLE
+101	117	nanocompartments	complex_assembly	Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments	TITLE
+0	9	Ferritins	protein_type	Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage.	ABSTRACT
+57	61	iron	chemical	Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage.	ABSTRACT
+79	84	shell	structure_element	Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage.	ABSTRACT
+26	35	structure	evidence	We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid.	ABSTRACT
+72	80	ferritin	protein_type	We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid.	ABSTRACT
+123	133	encapsulin	protein	We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid.	ABSTRACT
+18	30	encapsulated	protein_state	We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface.	ABSTRACT
+31	39	ferritin	protein_type	We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface.	ABSTRACT
+41	47	EncFtn	protein	We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface.	ABSTRACT
+57	75	main alpha helices	structure_element	We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface.	ABSTRACT
+97	112	metal dependent	protein_state	We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface.	ABSTRACT
+130	148	ferroxidase center	site	We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface.	ABSTRACT
+154	169	dimer interface	site	We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface.	ABSTRACT
+0	6	EncFtn	protein	EncFtn adopts an open decameric structure that is topologically distinct from other ferritins.	ABSTRACT
+17	21	open	protein_state	EncFtn adopts an open decameric structure that is topologically distinct from other ferritins.	ABSTRACT
+22	31	decameric	oligomeric_state	EncFtn adopts an open decameric structure that is topologically distinct from other ferritins.	ABSTRACT
+32	41	structure	evidence	EncFtn adopts an open decameric structure that is topologically distinct from other ferritins.	ABSTRACT
+84	93	ferritins	protein_type	EncFtn adopts an open decameric structure that is topologically distinct from other ferritins.	ABSTRACT
+6	12	EncFtn	protein	While EncFtn acts as a ferroxidase, it cannot mineralize iron.	ABSTRACT
+23	34	ferroxidase	protein_type	While EncFtn acts as a ferroxidase, it cannot mineralize iron.	ABSTRACT
+57	61	iron	chemical	While EncFtn acts as a ferroxidase, it cannot mineralize iron.	ABSTRACT
+16	26	encapsulin	protein	Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage.	ABSTRACT
+27	32	shell	structure_element	Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage.	ABSTRACT
+49	53	iron	chemical	Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage.	ABSTRACT
+62	86	not enzymatically active	protein_state	Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage.	ABSTRACT
+112	118	EncFtn	protein	Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage.	ABSTRACT
+145	155	encapsulin	protein	Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage.	ABSTRACT
+160	164	iron	chemical	Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage.	ABSTRACT
+5	15	encapsulin	protein	This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins.	ABSTRACT
+16	31	nanocompartment	complex_assembly	This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins.	ABSTRACT
+57	65	bacteria	taxonomy_domain	This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins.	ABSTRACT
+70	77	archaea	taxonomy_domain	This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins.	ABSTRACT
+113	117	iron	chemical	This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins.	ABSTRACT
+176	180	iron	chemical	This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins.	ABSTRACT
+0	4	Iron	chemical	Iron is essential for life as it is a key component of many different enzymes that participate in processes such as energy production and metabolism.	ABSTRACT
+9	13	iron	chemical	However, iron can also be highly toxic to cells because it readily reacts with oxygen.	ABSTRACT
+79	85	oxygen	chemical	However, iron can also be highly toxic to cells because it readily reacts with oxygen.	ABSTRACT
+31	35	iron	chemical	To balance the cell’s need for iron against its potential damaging effects, organisms have evolved iron storage proteins known as ferritins that form cage-like structures.	ABSTRACT
+99	120	iron storage proteins	protein_type	To balance the cell’s need for iron against its potential damaging effects, organisms have evolved iron storage proteins known as ferritins that form cage-like structures.	ABSTRACT
+130	139	ferritins	protein_type	To balance the cell’s need for iron against its potential damaging effects, organisms have evolved iron storage proteins known as ferritins that form cage-like structures.	ABSTRACT
+150	170	cage-like structures	structure_element	To balance the cell’s need for iron against its potential damaging effects, organisms have evolved iron storage proteins known as ferritins that form cage-like structures.	ABSTRACT
+4	13	ferritins	protein_type	The ferritins convert iron into a less reactive form that is mineralised and safely stored in the central cavity of the ferritin cage and is available for cells when they need it.	ABSTRACT
+22	26	iron	chemical	The ferritins convert iron into a less reactive form that is mineralised and safely stored in the central cavity of the ferritin cage and is available for cells when they need it.	ABSTRACT
+98	112	central cavity	site	The ferritins convert iron into a less reactive form that is mineralised and safely stored in the central cavity of the ferritin cage and is available for cells when they need it.	ABSTRACT
+120	128	ferritin	protein_type	The ferritins convert iron into a less reactive form that is mineralised and safely stored in the central cavity of the ferritin cage and is available for cells when they need it.	ABSTRACT
+26	35	ferritins	protein_type	Recently, a new family of ferritins known as encapsulated ferritins have been found in some microorganisms.	ABSTRACT
+45	57	encapsulated	protein_state	Recently, a new family of ferritins known as encapsulated ferritins have been found in some microorganisms.	ABSTRACT
+58	67	ferritins	protein_type	Recently, a new family of ferritins known as encapsulated ferritins have been found in some microorganisms.	ABSTRACT
+92	106	microorganisms	taxonomy_domain	Recently, a new family of ferritins known as encapsulated ferritins have been found in some microorganisms.	ABSTRACT
+6	15	ferritins	protein_type	These ferritins are found in bacterial genomes with a gene that codes for a protein cage called an encapsulin.	ABSTRACT
+29	38	bacterial	taxonomy_domain	These ferritins are found in bacterial genomes with a gene that codes for a protein cage called an encapsulin.	ABSTRACT
+99	109	encapsulin	protein	These ferritins are found in bacterial genomes with a gene that codes for a protein cage called an encapsulin.	ABSTRACT
+13	22	structure	evidence	Although the structure of the encapsulin cage is known to look like the shell of a virus, the structure that the encapsulated ferritin itself forms is not known.	ABSTRACT
+30	40	encapsulin	protein	Although the structure of the encapsulin cage is known to look like the shell of a virus, the structure that the encapsulated ferritin itself forms is not known.	ABSTRACT
+72	77	shell	structure_element	Although the structure of the encapsulin cage is known to look like the shell of a virus, the structure that the encapsulated ferritin itself forms is not known.	ABSTRACT
+83	88	virus	taxonomy_domain	Although the structure of the encapsulin cage is known to look like the shell of a virus, the structure that the encapsulated ferritin itself forms is not known.	ABSTRACT
+94	103	structure	evidence	Although the structure of the encapsulin cage is known to look like the shell of a virus, the structure that the encapsulated ferritin itself forms is not known.	ABSTRACT
+113	125	encapsulated	protein_state	Although the structure of the encapsulin cage is known to look like the shell of a virus, the structure that the encapsulated ferritin itself forms is not known.	ABSTRACT
+126	134	ferritin	protein_type	Although the structure of the encapsulin cage is known to look like the shell of a virus, the structure that the encapsulated ferritin itself forms is not known.	ABSTRACT
+25	35	encapsulin	protein	It is also not clear how encapsulin and the encapsulated ferritin work together to store iron.	ABSTRACT
+44	56	encapsulated	protein_state	It is also not clear how encapsulin and the encapsulated ferritin work together to store iron.	ABSTRACT
+57	65	ferritin	protein_type	It is also not clear how encapsulin and the encapsulated ferritin work together to store iron.	ABSTRACT
+89	93	iron	chemical	It is also not clear how encapsulin and the encapsulated ferritin work together to store iron.	ABSTRACT
+42	63	X-ray crystallography	experimental_method	He et al. have now used the techniques of X-ray crystallography and mass spectrometry to determine the structure of the encapsulated ferritin found in some bacteria.	ABSTRACT
+68	85	mass spectrometry	experimental_method	He et al. have now used the techniques of X-ray crystallography and mass spectrometry to determine the structure of the encapsulated ferritin found in some bacteria.	ABSTRACT
+103	112	structure	evidence	He et al. have now used the techniques of X-ray crystallography and mass spectrometry to determine the structure of the encapsulated ferritin found in some bacteria.	ABSTRACT
+120	132	encapsulated	protein_state	He et al. have now used the techniques of X-ray crystallography and mass spectrometry to determine the structure of the encapsulated ferritin found in some bacteria.	ABSTRACT
+133	141	ferritin	protein_type	He et al. have now used the techniques of X-ray crystallography and mass spectrometry to determine the structure of the encapsulated ferritin found in some bacteria.	ABSTRACT
+156	164	bacteria	taxonomy_domain	He et al. have now used the techniques of X-ray crystallography and mass spectrometry to determine the structure of the encapsulated ferritin found in some bacteria.	ABSTRACT
+4	16	encapsulated	protein_state	The encapsulated ferritin forms a ring-shaped doughnut in which ten subunits of ferritin are arranged in a ring; this is totally different from the enclosed cages that other ferritins form.	ABSTRACT
+17	25	ferritin	protein_type	The encapsulated ferritin forms a ring-shaped doughnut in which ten subunits of ferritin are arranged in a ring; this is totally different from the enclosed cages that other ferritins form.	ABSTRACT
+34	45	ring-shaped	structure_element	The encapsulated ferritin forms a ring-shaped doughnut in which ten subunits of ferritin are arranged in a ring; this is totally different from the enclosed cages that other ferritins form.	ABSTRACT
+46	54	doughnut	structure_element	The encapsulated ferritin forms a ring-shaped doughnut in which ten subunits of ferritin are arranged in a ring; this is totally different from the enclosed cages that other ferritins form.	ABSTRACT
+68	76	subunits	structure_element	The encapsulated ferritin forms a ring-shaped doughnut in which ten subunits of ferritin are arranged in a ring; this is totally different from the enclosed cages that other ferritins form.	ABSTRACT
+80	88	ferritin	protein_type	The encapsulated ferritin forms a ring-shaped doughnut in which ten subunits of ferritin are arranged in a ring; this is totally different from the enclosed cages that other ferritins form.	ABSTRACT
+107	111	ring	structure_element	The encapsulated ferritin forms a ring-shaped doughnut in which ten subunits of ferritin are arranged in a ring; this is totally different from the enclosed cages that other ferritins form.	ABSTRACT
+157	162	cages	structure_element	The encapsulated ferritin forms a ring-shaped doughnut in which ten subunits of ferritin are arranged in a ring; this is totally different from the enclosed cages that other ferritins form.	ABSTRACT
+174	183	ferritins	protein_type	The encapsulated ferritin forms a ring-shaped doughnut in which ten subunits of ferritin are arranged in a ring; this is totally different from the enclosed cages that other ferritins form.	ABSTRACT
+0	19	Biochemical studies	experimental_method	Biochemical studies revealed that the encapsulated ferritin is able to convert iron into a less reactive form, but it cannot store iron on its own since it does not form a cage.	ABSTRACT
+38	50	encapsulated	protein_state	Biochemical studies revealed that the encapsulated ferritin is able to convert iron into a less reactive form, but it cannot store iron on its own since it does not form a cage.	ABSTRACT
+51	59	ferritin	protein_type	Biochemical studies revealed that the encapsulated ferritin is able to convert iron into a less reactive form, but it cannot store iron on its own since it does not form a cage.	ABSTRACT
+79	83	iron	chemical	Biochemical studies revealed that the encapsulated ferritin is able to convert iron into a less reactive form, but it cannot store iron on its own since it does not form a cage.	ABSTRACT
+131	135	iron	chemical	Biochemical studies revealed that the encapsulated ferritin is able to convert iron into a less reactive form, but it cannot store iron on its own since it does not form a cage.	ABSTRACT
+10	22	encapsulated	protein_state	Thus, the encapsulated ferritin needs to be housed within the encapsulin cage to store iron.	ABSTRACT
+23	31	ferritin	protein_type	Thus, the encapsulated ferritin needs to be housed within the encapsulin cage to store iron.	ABSTRACT
+62	72	encapsulin	protein	Thus, the encapsulated ferritin needs to be housed within the encapsulin cage to store iron.	ABSTRACT
+87	91	iron	chemical	Thus, the encapsulated ferritin needs to be housed within the encapsulin cage to store iron.	ABSTRACT
+42	46	iron	chemical	Further work is needed to investigate how iron moves into the encapsulin cage to reach the ferritin proteins.	ABSTRACT
+62	72	encapsulin	protein	Further work is needed to investigate how iron moves into the encapsulin cage to reach the ferritin proteins.	ABSTRACT
+91	99	ferritin	protein_type	Further work is needed to investigate how iron moves into the encapsulin cage to reach the ferritin proteins.	ABSTRACT
+34	42	ferritin	protein_type	Some organisms have both standard ferritin cages and encapsulated ferritins; why this is the case also remains to be discovered.	ABSTRACT
+53	65	encapsulated	protein_state	Some organisms have both standard ferritin cages and encapsulated ferritins; why this is the case also remains to be discovered.	ABSTRACT
+66	75	ferritins	protein_type	Some organisms have both standard ferritin cages and encapsulated ferritins; why this is the case also remains to be discovered.	ABSTRACT
+0	10	Encapsulin	protein_type	Encapsulin nanocompartments are a family of proteinaceous metabolic compartments that are widely distributed in bacteria and archaea.	INTRO
+11	27	nanocompartments	complex_assembly	Encapsulin nanocompartments are a family of proteinaceous metabolic compartments that are widely distributed in bacteria and archaea.	INTRO
+112	120	bacteria	taxonomy_domain	Encapsulin nanocompartments are a family of proteinaceous metabolic compartments that are widely distributed in bacteria and archaea.	INTRO
+125	132	archaea	taxonomy_domain	Encapsulin nanocompartments are a family of proteinaceous metabolic compartments that are widely distributed in bacteria and archaea.	INTRO
+48	59	icosahedral	protein_state	They share a common architecture, comprising an icosahedral shell formed by the oligomeric assembly of a protein, encapsulin, that is structurally related to the HK97 bacteriophage capsid protein gp5.	INTRO
+60	65	shell	structure_element	They share a common architecture, comprising an icosahedral shell formed by the oligomeric assembly of a protein, encapsulin, that is structurally related to the HK97 bacteriophage capsid protein gp5.	INTRO
+114	124	encapsulin	protein_type	They share a common architecture, comprising an icosahedral shell formed by the oligomeric assembly of a protein, encapsulin, that is structurally related to the HK97 bacteriophage capsid protein gp5.	INTRO
+162	180	HK97 bacteriophage	taxonomy_domain	They share a common architecture, comprising an icosahedral shell formed by the oligomeric assembly of a protein, encapsulin, that is structurally related to the HK97 bacteriophage capsid protein gp5.	INTRO
+196	199	gp5	protein	They share a common architecture, comprising an icosahedral shell formed by the oligomeric assembly of a protein, encapsulin, that is structurally related to the HK97 bacteriophage capsid protein gp5.	INTRO
+0	3	Gp5	protein	Gp5 is known to assemble as a 66 nm diameter icosahedral shell of 420 subunits.	INTRO
+45	56	icosahedral	protein_state	Gp5 is known to assemble as a 66 nm diameter icosahedral shell of 420 subunits.	INTRO
+57	62	shell	structure_element	Gp5 is known to assemble as a 66 nm diameter icosahedral shell of 420 subunits.	INTRO
+70	78	subunits	structure_element	Gp5 is known to assemble as a 66 nm diameter icosahedral shell of 420 subunits.	INTRO
+22	41	Pyrococcus furiosus	species	In contrast, both the Pyrococcus furiosus and Myxococcus xanthus encapsulin shell-proteins form 32 nm icosahedra with 180 subunits; while the Thermotoga maritima encapsulin is smaller still with a 25 nm, 60-subunit icosahedron.	INTRO
+46	64	Myxococcus xanthus	species	In contrast, both the Pyrococcus furiosus and Myxococcus xanthus encapsulin shell-proteins form 32 nm icosahedra with 180 subunits; while the Thermotoga maritima encapsulin is smaller still with a 25 nm, 60-subunit icosahedron.	INTRO
+65	75	encapsulin	protein	In contrast, both the Pyrococcus furiosus and Myxococcus xanthus encapsulin shell-proteins form 32 nm icosahedra with 180 subunits; while the Thermotoga maritima encapsulin is smaller still with a 25 nm, 60-subunit icosahedron.	INTRO
+76	81	shell	structure_element	In contrast, both the Pyrococcus furiosus and Myxococcus xanthus encapsulin shell-proteins form 32 nm icosahedra with 180 subunits; while the Thermotoga maritima encapsulin is smaller still with a 25 nm, 60-subunit icosahedron.	INTRO
+102	112	icosahedra	structure_element	In contrast, both the Pyrococcus furiosus and Myxococcus xanthus encapsulin shell-proteins form 32 nm icosahedra with 180 subunits; while the Thermotoga maritima encapsulin is smaller still with a 25 nm, 60-subunit icosahedron.	INTRO
+122	130	subunits	structure_element	In contrast, both the Pyrococcus furiosus and Myxococcus xanthus encapsulin shell-proteins form 32 nm icosahedra with 180 subunits; while the Thermotoga maritima encapsulin is smaller still with a 25 nm, 60-subunit icosahedron.	INTRO
+142	161	Thermotoga maritima	species	In contrast, both the Pyrococcus furiosus and Myxococcus xanthus encapsulin shell-proteins form 32 nm icosahedra with 180 subunits; while the Thermotoga maritima encapsulin is smaller still with a 25 nm, 60-subunit icosahedron.	INTRO
+162	172	encapsulin	protein	In contrast, both the Pyrococcus furiosus and Myxococcus xanthus encapsulin shell-proteins form 32 nm icosahedra with 180 subunits; while the Thermotoga maritima encapsulin is smaller still with a 25 nm, 60-subunit icosahedron.	INTRO
+215	226	icosahedron	structure_element	In contrast, both the Pyrococcus furiosus and Myxococcus xanthus encapsulin shell-proteins form 32 nm icosahedra with 180 subunits; while the Thermotoga maritima encapsulin is smaller still with a 25 nm, 60-subunit icosahedron.	INTRO
+38	48	encapsulin	protein_type	The high structural similarity of the encapsulin shell-proteins to gp5 suggests a common evolutionary origin for these proteins.	INTRO
+49	54	shell	structure_element	The high structural similarity of the encapsulin shell-proteins to gp5 suggests a common evolutionary origin for these proteins.	INTRO
+67	70	gp5	protein	The high structural similarity of the encapsulin shell-proteins to gp5 suggests a common evolutionary origin for these proteins.	INTRO
+19	29	encapsulin	protein_type	The genes encoding encapsulin proteins are found downstream of genes for dye-dependent peroxidase (DyP) family enzymes, or encapsulin-associated ferritins (EncFtn).	INTRO
+73	97	dye-dependent peroxidase	protein_type	The genes encoding encapsulin proteins are found downstream of genes for dye-dependent peroxidase (DyP) family enzymes, or encapsulin-associated ferritins (EncFtn).	INTRO
+99	102	DyP	protein_type	The genes encoding encapsulin proteins are found downstream of genes for dye-dependent peroxidase (DyP) family enzymes, or encapsulin-associated ferritins (EncFtn).	INTRO
+123	154	encapsulin-associated ferritins	protein_type	The genes encoding encapsulin proteins are found downstream of genes for dye-dependent peroxidase (DyP) family enzymes, or encapsulin-associated ferritins (EncFtn).	INTRO
+156	162	EncFtn	protein_type	The genes encoding encapsulin proteins are found downstream of genes for dye-dependent peroxidase (DyP) family enzymes, or encapsulin-associated ferritins (EncFtn).	INTRO
+15	25	DyP family	protein_type	Enzymes in the DyP family are active against polyphenolic compounds such as azo dyes and lignin breakdown products; although their physiological function and natural substrates are not known.	INTRO
+0	8	Ferritin	protein_type	Ferritin family proteins are found in all kingdoms and have a wide range of activities, including ribonucleotide reductase, protecting DNA from oxidative damage, and iron storage.	INTRO
+42	50	kingdoms	taxonomy_domain	Ferritin family proteins are found in all kingdoms and have a wide range of activities, including ribonucleotide reductase, protecting DNA from oxidative damage, and iron storage.	INTRO
+98	122	ribonucleotide reductase	protein_type	Ferritin family proteins are found in all kingdoms and have a wide range of activities, including ribonucleotide reductase, protecting DNA from oxidative damage, and iron storage.	INTRO
+166	170	iron	chemical	Ferritin family proteins are found in all kingdoms and have a wide range of activities, including ribonucleotide reductase, protecting DNA from oxidative damage, and iron storage.	INTRO
+4	13	classical	protein_state	The classical iron storage ferritin nanocages are found in all kingdoms and are essential in eukaryotes; they play a central role in iron homeostasis, where they protect the cell from toxic free Fe2+ by oxidizing it and storing the resulting Fe3+ as ferrihydrite minerals within their central cavity.	INTRO
+14	45	iron storage ferritin nanocages	complex_assembly	The classical iron storage ferritin nanocages are found in all kingdoms and are essential in eukaryotes; they play a central role in iron homeostasis, where they protect the cell from toxic free Fe2+ by oxidizing it and storing the resulting Fe3+ as ferrihydrite minerals within their central cavity.	INTRO
+63	71	kingdoms	taxonomy_domain	The classical iron storage ferritin nanocages are found in all kingdoms and are essential in eukaryotes; they play a central role in iron homeostasis, where they protect the cell from toxic free Fe2+ by oxidizing it and storing the resulting Fe3+ as ferrihydrite minerals within their central cavity.	INTRO
+93	103	eukaryotes	taxonomy_domain	The classical iron storage ferritin nanocages are found in all kingdoms and are essential in eukaryotes; they play a central role in iron homeostasis, where they protect the cell from toxic free Fe2+ by oxidizing it and storing the resulting Fe3+ as ferrihydrite minerals within their central cavity.	INTRO
+133	137	iron	chemical	The classical iron storage ferritin nanocages are found in all kingdoms and are essential in eukaryotes; they play a central role in iron homeostasis, where they protect the cell from toxic free Fe2+ by oxidizing it and storing the resulting Fe3+ as ferrihydrite minerals within their central cavity.	INTRO
+195	199	Fe2+	chemical	The classical iron storage ferritin nanocages are found in all kingdoms and are essential in eukaryotes; they play a central role in iron homeostasis, where they protect the cell from toxic free Fe2+ by oxidizing it and storing the resulting Fe3+ as ferrihydrite minerals within their central cavity.	INTRO
+242	246	Fe3+	chemical	The classical iron storage ferritin nanocages are found in all kingdoms and are essential in eukaryotes; they play a central role in iron homeostasis, where they protect the cell from toxic free Fe2+ by oxidizing it and storing the resulting Fe3+ as ferrihydrite minerals within their central cavity.	INTRO
+250	262	ferrihydrite	chemical	The classical iron storage ferritin nanocages are found in all kingdoms and are essential in eukaryotes; they play a central role in iron homeostasis, where they protect the cell from toxic free Fe2+ by oxidizing it and storing the resulting Fe3+ as ferrihydrite minerals within their central cavity.	INTRO
+285	299	central cavity	site	The classical iron storage ferritin nanocages are found in all kingdoms and are essential in eukaryotes; they play a central role in iron homeostasis, where they protect the cell from toxic free Fe2+ by oxidizing it and storing the resulting Fe3+ as ferrihydrite minerals within their central cavity.	INTRO
+4	14	encapsulin	protein_type	The encapsulin-associated enzymes are sequestered within the icosahedral shell through interactions between the shell’s inner surface and a short localization sequence (Gly-Ser-Leu-Lys) appended to their C-termini.	INTRO
+61	72	icosahedral	protein_state	The encapsulin-associated enzymes are sequestered within the icosahedral shell through interactions between the shell’s inner surface and a short localization sequence (Gly-Ser-Leu-Lys) appended to their C-termini.	INTRO
+73	78	shell	structure_element	The encapsulin-associated enzymes are sequestered within the icosahedral shell through interactions between the shell’s inner surface and a short localization sequence (Gly-Ser-Leu-Lys) appended to their C-termini.	INTRO
+112	117	shell	structure_element	The encapsulin-associated enzymes are sequestered within the icosahedral shell through interactions between the shell’s inner surface and a short localization sequence (Gly-Ser-Leu-Lys) appended to their C-termini.	INTRO
+140	167	short localization sequence	structure_element	The encapsulin-associated enzymes are sequestered within the icosahedral shell through interactions between the shell’s inner surface and a short localization sequence (Gly-Ser-Leu-Lys) appended to their C-termini.	INTRO
+169	184	Gly-Ser-Leu-Lys	structure_element	The encapsulin-associated enzymes are sequestered within the icosahedral shell through interactions between the shell’s inner surface and a short localization sequence (Gly-Ser-Leu-Lys) appended to their C-termini.	INTRO
+0	10	This motif	structure_element	This motif is well-conserved, and the addition of this sequence to heterologous proteins is sufficient to direct them to the interior of encapsulins.	INTRO
+14	28	well-conserved	protein_state	This motif is well-conserved, and the addition of this sequence to heterologous proteins is sufficient to direct them to the interior of encapsulins.	INTRO
+137	148	encapsulins	protein_type	This motif is well-conserved, and the addition of this sequence to heterologous proteins is sufficient to direct them to the interior of encapsulins.	INTRO
+22	40	Myxococcus xanthus	species	A recent study of the Myxococcus xanthus encapsulin showed that it sequesters a number of different EncFtn proteins and acts as an ‘iron-megastore’ to protect these bacteria from oxidative stress.	INTRO
+41	51	encapsulin	protein	A recent study of the Myxococcus xanthus encapsulin showed that it sequesters a number of different EncFtn proteins and acts as an ‘iron-megastore’ to protect these bacteria from oxidative stress.	INTRO
+100	106	EncFtn	protein_type	A recent study of the Myxococcus xanthus encapsulin showed that it sequesters a number of different EncFtn proteins and acts as an ‘iron-megastore’ to protect these bacteria from oxidative stress.	INTRO
+132	136	iron	chemical	A recent study of the Myxococcus xanthus encapsulin showed that it sequesters a number of different EncFtn proteins and acts as an ‘iron-megastore’ to protect these bacteria from oxidative stress.	INTRO
+165	173	bacteria	taxonomy_domain	A recent study of the Myxococcus xanthus encapsulin showed that it sequesters a number of different EncFtn proteins and acts as an ‘iron-megastore’ to protect these bacteria from oxidative stress.	INTRO
+66	74	ferritin	protein_type	At 32 nm in diameter, it is much larger than other members of the ferritin superfamily, such as the 12 nm 24-subunit classical ferritin nanocage and the 8 nm 12-subunit Dps (DNA-binding protein from starved cells) complex; and is thus capable of sequestering up to ten times more iron than these ferritins.	INTRO
+117	126	classical	protein_state	At 32 nm in diameter, it is much larger than other members of the ferritin superfamily, such as the 12 nm 24-subunit classical ferritin nanocage and the 8 nm 12-subunit Dps (DNA-binding protein from starved cells) complex; and is thus capable of sequestering up to ten times more iron than these ferritins.	INTRO
+127	135	ferritin	protein_type	At 32 nm in diameter, it is much larger than other members of the ferritin superfamily, such as the 12 nm 24-subunit classical ferritin nanocage and the 8 nm 12-subunit Dps (DNA-binding protein from starved cells) complex; and is thus capable of sequestering up to ten times more iron than these ferritins.	INTRO
+136	144	nanocage	complex_assembly	At 32 nm in diameter, it is much larger than other members of the ferritin superfamily, such as the 12 nm 24-subunit classical ferritin nanocage and the 8 nm 12-subunit Dps (DNA-binding protein from starved cells) complex; and is thus capable of sequestering up to ten times more iron than these ferritins.	INTRO
+169	172	Dps	protein_type	At 32 nm in diameter, it is much larger than other members of the ferritin superfamily, such as the 12 nm 24-subunit classical ferritin nanocage and the 8 nm 12-subunit Dps (DNA-binding protein from starved cells) complex; and is thus capable of sequestering up to ten times more iron than these ferritins.	INTRO
+174	193	DNA-binding protein	protein_type	At 32 nm in diameter, it is much larger than other members of the ferritin superfamily, such as the 12 nm 24-subunit classical ferritin nanocage and the 8 nm 12-subunit Dps (DNA-binding protein from starved cells) complex; and is thus capable of sequestering up to ten times more iron than these ferritins.	INTRO
+280	284	iron	chemical	At 32 nm in diameter, it is much larger than other members of the ferritin superfamily, such as the 12 nm 24-subunit classical ferritin nanocage and the 8 nm 12-subunit Dps (DNA-binding protein from starved cells) complex; and is thus capable of sequestering up to ten times more iron than these ferritins.	INTRO
+296	305	ferritins	protein_type	At 32 nm in diameter, it is much larger than other members of the ferritin superfamily, such as the 12 nm 24-subunit classical ferritin nanocage and the 8 nm 12-subunit Dps (DNA-binding protein from starved cells) complex; and is thus capable of sequestering up to ten times more iron than these ferritins.	INTRO
+25	31	EncFtn	protein_type	The primary sequences of EncFtn proteins have Glu-X-X-His metal coordination sites, which are shared features of the ferritin family proteins.	INTRO
+46	57	Glu-X-X-His	structure_element	The primary sequences of EncFtn proteins have Glu-X-X-His metal coordination sites, which are shared features of the ferritin family proteins.	INTRO
+58	82	metal coordination sites	site	The primary sequences of EncFtn proteins have Glu-X-X-His metal coordination sites, which are shared features of the ferritin family proteins.	INTRO
+117	125	ferritin	protein_type	The primary sequences of EncFtn proteins have Glu-X-X-His metal coordination sites, which are shared features of the ferritin family proteins.	INTRO
+0	30	Secondary structure prediction	experimental_method	Secondary structure prediction identifies two major α-helical regions in these proteins; this is in contrast to other members of the ferritin superfamily, which have four major α-helices (Supplementary file 1).	INTRO
+46	69	major α-helical regions	structure_element	Secondary structure prediction identifies two major α-helical regions in these proteins; this is in contrast to other members of the ferritin superfamily, which have four major α-helices (Supplementary file 1).	INTRO
+133	141	ferritin	protein_type	Secondary structure prediction identifies two major α-helical regions in these proteins; this is in contrast to other members of the ferritin superfamily, which have four major α-helices (Supplementary file 1).	INTRO
+171	186	major α-helices	structure_element	Secondary structure prediction identifies two major α-helical regions in these proteins; this is in contrast to other members of the ferritin superfamily, which have four major α-helices (Supplementary file 1).	INTRO
+10	18	ferritin	protein_type	The ‘half-ferritin’ primary sequence of the EncFtn family and their association with encapsulin nanocompartments suggests a distinct biochemical and structural organization to other ferritin family proteins.	INTRO
+44	50	EncFtn	protein_type	The ‘half-ferritin’ primary sequence of the EncFtn family and their association with encapsulin nanocompartments suggests a distinct biochemical and structural organization to other ferritin family proteins.	INTRO
+85	95	encapsulin	protein	The ‘half-ferritin’ primary sequence of the EncFtn family and their association with encapsulin nanocompartments suggests a distinct biochemical and structural organization to other ferritin family proteins.	INTRO
+96	112	nanocompartments	complex_assembly	The ‘half-ferritin’ primary sequence of the EncFtn family and their association with encapsulin nanocompartments suggests a distinct biochemical and structural organization to other ferritin family proteins.	INTRO
+182	190	ferritin	protein_type	The ‘half-ferritin’ primary sequence of the EncFtn family and their association with encapsulin nanocompartments suggests a distinct biochemical and structural organization to other ferritin family proteins.	INTRO
+4	25	Rhodospirillum rubrum	species	The Rhodospirillum rubrum EncFtn protein (Rru_A0973) shares 33% protein sequence identity with the M. xanthus (MXAN_4464), 53% with the T. maritima (Tmari_0787), and 29% with the P. furiosus (PF1192) homologues.	INTRO
+26	32	EncFtn	protein	The Rhodospirillum rubrum EncFtn protein (Rru_A0973) shares 33% protein sequence identity with the M. xanthus (MXAN_4464), 53% with the T. maritima (Tmari_0787), and 29% with the P. furiosus (PF1192) homologues.	INTRO
+42	51	Rru_A0973	gene	The Rhodospirillum rubrum EncFtn protein (Rru_A0973) shares 33% protein sequence identity with the M. xanthus (MXAN_4464), 53% with the T. maritima (Tmari_0787), and 29% with the P. furiosus (PF1192) homologues.	INTRO
+99	109	M. xanthus	species	The Rhodospirillum rubrum EncFtn protein (Rru_A0973) shares 33% protein sequence identity with the M. xanthus (MXAN_4464), 53% with the T. maritima (Tmari_0787), and 29% with the P. furiosus (PF1192) homologues.	INTRO
+111	120	MXAN_4464	gene	The Rhodospirillum rubrum EncFtn protein (Rru_A0973) shares 33% protein sequence identity with the M. xanthus (MXAN_4464), 53% with the T. maritima (Tmari_0787), and 29% with the P. furiosus (PF1192) homologues.	INTRO
+136	147	T. maritima	species	The Rhodospirillum rubrum EncFtn protein (Rru_A0973) shares 33% protein sequence identity with the M. xanthus (MXAN_4464), 53% with the T. maritima (Tmari_0787), and 29% with the P. furiosus (PF1192) homologues.	INTRO
+149	159	Tmari_0787	gene	The Rhodospirillum rubrum EncFtn protein (Rru_A0973) shares 33% protein sequence identity with the M. xanthus (MXAN_4464), 53% with the T. maritima (Tmari_0787), and 29% with the P. furiosus (PF1192) homologues.	INTRO
+179	190	P. furiosus	species	The Rhodospirillum rubrum EncFtn protein (Rru_A0973) shares 33% protein sequence identity with the M. xanthus (MXAN_4464), 53% with the T. maritima (Tmari_0787), and 29% with the P. furiosus (PF1192) homologues.	INTRO
+192	198	PF1192	gene	The Rhodospirillum rubrum EncFtn protein (Rru_A0973) shares 33% protein sequence identity with the M. xanthus (MXAN_4464), 53% with the T. maritima (Tmari_0787), and 29% with the P. furiosus (PF1192) homologues.	INTRO
+4	8	GXXH	structure_element	The GXXH motifs are strictly conserved in each of these species (Supplementary file 1).	INTRO
+20	38	strictly conserved	protein_state	The GXXH motifs are strictly conserved in each of these species (Supplementary file 1).	INTRO
+24	33	structure	evidence	Here we investigate the structure and biochemistry of EncFtn in order to understand iron storage within the encapsulin nanocompartment.	INTRO
+54	60	EncFtn	protein	Here we investigate the structure and biochemistry of EncFtn in order to understand iron storage within the encapsulin nanocompartment.	INTRO
+84	88	iron	chemical	Here we investigate the structure and biochemistry of EncFtn in order to understand iron storage within the encapsulin nanocompartment.	INTRO
+108	118	encapsulin	protein	Here we investigate the structure and biochemistry of EncFtn in order to understand iron storage within the encapsulin nanocompartment.	INTRO
+119	134	nanocompartment	complex_assembly	Here we investigate the structure and biochemistry of EncFtn in order to understand iron storage within the encapsulin nanocompartment.	INTRO
+29	39	encapsulin	protein	We have produced recombinant encapsulin (Enc) and EncFtn from the aquatic purple-sulfur bacterium R. rubrum, which serves as a model organism for the study of the control of the bacterial nitrogen fixation machinery, in Escherichia coli.	INTRO
+41	44	Enc	protein	We have produced recombinant encapsulin (Enc) and EncFtn from the aquatic purple-sulfur bacterium R. rubrum, which serves as a model organism for the study of the control of the bacterial nitrogen fixation machinery, in Escherichia coli.	INTRO
+50	56	EncFtn	protein	We have produced recombinant encapsulin (Enc) and EncFtn from the aquatic purple-sulfur bacterium R. rubrum, which serves as a model organism for the study of the control of the bacterial nitrogen fixation machinery, in Escherichia coli.	INTRO
+66	73	aquatic	taxonomy_domain	We have produced recombinant encapsulin (Enc) and EncFtn from the aquatic purple-sulfur bacterium R. rubrum, which serves as a model organism for the study of the control of the bacterial nitrogen fixation machinery, in Escherichia coli.	INTRO
+74	97	purple-sulfur bacterium	taxonomy_domain	We have produced recombinant encapsulin (Enc) and EncFtn from the aquatic purple-sulfur bacterium R. rubrum, which serves as a model organism for the study of the control of the bacterial nitrogen fixation machinery, in Escherichia coli.	INTRO
+98	107	R. rubrum	species	We have produced recombinant encapsulin (Enc) and EncFtn from the aquatic purple-sulfur bacterium R. rubrum, which serves as a model organism for the study of the control of the bacterial nitrogen fixation machinery, in Escherichia coli.	INTRO
+178	187	bacterial	taxonomy_domain	We have produced recombinant encapsulin (Enc) and EncFtn from the aquatic purple-sulfur bacterium R. rubrum, which serves as a model organism for the study of the control of the bacterial nitrogen fixation machinery, in Escherichia coli.	INTRO
+220	236	Escherichia coli	species	We have produced recombinant encapsulin (Enc) and EncFtn from the aquatic purple-sulfur bacterium R. rubrum, which serves as a model organism for the study of the control of the bacterial nitrogen fixation machinery, in Escherichia coli.	INTRO
+12	44	transmission electron microscopy	experimental_method	Analysis by transmission electron microscopy (TEM) indicates that their co-expression leads to the production of an icosahedral nanocompartment with encapsulated EncFtn.	INTRO
+46	49	TEM	experimental_method	Analysis by transmission electron microscopy (TEM) indicates that their co-expression leads to the production of an icosahedral nanocompartment with encapsulated EncFtn.	INTRO
+72	85	co-expression	experimental_method	Analysis by transmission electron microscopy (TEM) indicates that their co-expression leads to the production of an icosahedral nanocompartment with encapsulated EncFtn.	INTRO
+116	127	icosahedral	protein_state	Analysis by transmission electron microscopy (TEM) indicates that their co-expression leads to the production of an icosahedral nanocompartment with encapsulated EncFtn.	INTRO
+128	143	nanocompartment	complex_assembly	Analysis by transmission electron microscopy (TEM) indicates that their co-expression leads to the production of an icosahedral nanocompartment with encapsulated EncFtn.	INTRO
+149	161	encapsulated	protein_state	Analysis by transmission electron microscopy (TEM) indicates that their co-expression leads to the production of an icosahedral nanocompartment with encapsulated EncFtn.	INTRO
+162	168	EncFtn	protein	Analysis by transmission electron microscopy (TEM) indicates that their co-expression leads to the production of an icosahedral nanocompartment with encapsulated EncFtn.	INTRO
+4	21	crystal structure	evidence	The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins.	INTRO
+27	36	truncated	protein_state	The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins.	INTRO
+37	57	hexahistidine-tagged	protein_state	The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins.	INTRO
+73	79	EncFtn	protein	The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins.	INTRO
+89	97	EncFtnsH	protein	The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins.	INTRO
+121	130	decameric	oligomeric_state	The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins.	INTRO
+131	140	structure	evidence	The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins.	INTRO
+158	171	ring-doughnut	structure_element	The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins.	INTRO
+210	230	four-helical bundles	structure_element	The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins.	INTRO
+238	245	24meric	oligomeric_state	The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins.	INTRO
+246	255	ferritins	protein_type	The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins.	INTRO
+260	272	dodecahedral	oligomeric_state	The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins.	INTRO
+273	276	DPS	protein_type	The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins.	INTRO
+26	36	iron bound	protein_state	We identify a symmetrical iron bound ferroxidase center (FOC) formed between subunits in the decamer and additional metal-binding sites close to the center of the ring and on the outer surface.	INTRO
+37	55	ferroxidase center	site	We identify a symmetrical iron bound ferroxidase center (FOC) formed between subunits in the decamer and additional metal-binding sites close to the center of the ring and on the outer surface.	INTRO
+57	60	FOC	site	We identify a symmetrical iron bound ferroxidase center (FOC) formed between subunits in the decamer and additional metal-binding sites close to the center of the ring and on the outer surface.	INTRO
+77	85	subunits	structure_element	We identify a symmetrical iron bound ferroxidase center (FOC) formed between subunits in the decamer and additional metal-binding sites close to the center of the ring and on the outer surface.	INTRO
+93	100	decamer	oligomeric_state	We identify a symmetrical iron bound ferroxidase center (FOC) formed between subunits in the decamer and additional metal-binding sites close to the center of the ring and on the outer surface.	INTRO
+116	135	metal-binding sites	site	We identify a symmetrical iron bound ferroxidase center (FOC) formed between subunits in the decamer and additional metal-binding sites close to the center of the ring and on the outer surface.	INTRO
+163	167	ring	structure_element	We identify a symmetrical iron bound ferroxidase center (FOC) formed between subunits in the decamer and additional metal-binding sites close to the center of the ring and on the outer surface.	INTRO
+52	58	EncFtn	protein	We also demonstrate the metal-dependent assembly of EncFtn decamers using native PAGE, analytical gel-filtration, and native mass spectrometry.	INTRO
+59	67	decamers	oligomeric_state	We also demonstrate the metal-dependent assembly of EncFtn decamers using native PAGE, analytical gel-filtration, and native mass spectrometry.	INTRO
+74	85	native PAGE	experimental_method	We also demonstrate the metal-dependent assembly of EncFtn decamers using native PAGE, analytical gel-filtration, and native mass spectrometry.	INTRO
+87	112	analytical gel-filtration	experimental_method	We also demonstrate the metal-dependent assembly of EncFtn decamers using native PAGE, analytical gel-filtration, and native mass spectrometry.	INTRO
+118	142	native mass spectrometry	experimental_method	We also demonstrate the metal-dependent assembly of EncFtn decamers using native PAGE, analytical gel-filtration, and native mass spectrometry.	INTRO
+0	18	Biochemical assays	experimental_method	Biochemical assays show that EncFtn is active as a ferroxidase enzyme.	INTRO
+29	35	EncFtn	protein	Biochemical assays show that EncFtn is active as a ferroxidase enzyme.	INTRO
+39	45	active	protein_state	Biochemical assays show that EncFtn is active as a ferroxidase enzyme.	INTRO
+51	62	ferroxidase	protein_type	Biochemical assays show that EncFtn is active as a ferroxidase enzyme.	INTRO
+8	33	site-directed mutagenesis	experimental_method	Through site-directed mutagenesis we show that the conserved glutamic acid and histidine residues in the FOC influence protein assembly and activity.	INTRO
+51	60	conserved	protein_state	Through site-directed mutagenesis we show that the conserved glutamic acid and histidine residues in the FOC influence protein assembly and activity.	INTRO
+61	74	glutamic acid	residue_name	Through site-directed mutagenesis we show that the conserved glutamic acid and histidine residues in the FOC influence protein assembly and activity.	INTRO
+79	88	histidine	residue_name	Through site-directed mutagenesis we show that the conserved glutamic acid and histidine residues in the FOC influence protein assembly and activity.	INTRO
+105	108	FOC	site	Through site-directed mutagenesis we show that the conserved glutamic acid and histidine residues in the FOC influence protein assembly and activity.	INTRO
+20	51	structural and biochemical data	evidence	We use our combined structural and biochemical data to propose a model for the EncFtn-catalyzed sequestration of iron within the encapsulin shell.	INTRO
+79	85	EncFtn	protein	We use our combined structural and biochemical data to propose a model for the EncFtn-catalyzed sequestration of iron within the encapsulin shell.	INTRO
+113	117	iron	chemical	We use our combined structural and biochemical data to propose a model for the EncFtn-catalyzed sequestration of iron within the encapsulin shell.	INTRO
+129	139	encapsulin	protein	We use our combined structural and biochemical data to propose a model for the EncFtn-catalyzed sequestration of iron within the encapsulin shell.	INTRO
+140	145	shell	structure_element	We use our combined structural and biochemical data to propose a model for the EncFtn-catalyzed sequestration of iron within the encapsulin shell.	INTRO
+12	21	R. rubrum	species	Assembly of R. rubrum EncFtn encapsulin nanocompartments in E. coli	RESULTS
+22	28	EncFtn	protein	Assembly of R. rubrum EncFtn encapsulin nanocompartments in E. coli	RESULTS
+29	39	encapsulin	protein	Assembly of R. rubrum EncFtn encapsulin nanocompartments in E. coli	RESULTS
+40	56	nanocompartments	complex_assembly	Assembly of R. rubrum EncFtn encapsulin nanocompartments in E. coli	RESULTS
+60	67	E. coli	species	Assembly of R. rubrum EncFtn encapsulin nanocompartments in E. coli	RESULTS
+0	44	Full-frame transmission electron micrographs	evidence	Full-frame transmission electron micrographs of R. rubrum nanocompartments.	FIG
+48	57	R. rubrum	species	Full-frame transmission electron micrographs of R. rubrum nanocompartments.	FIG
+58	74	nanocompartments	complex_assembly	Full-frame transmission electron micrographs of R. rubrum nanocompartments.	FIG
+6	24	Negative stain TEM	experimental_method	(A/B) Negative stain TEM image of recombinant R. rubrum encapsulin and EncFtn-Enc nanocompartments.	FIG
+25	30	image	evidence	(A/B) Negative stain TEM image of recombinant R. rubrum encapsulin and EncFtn-Enc nanocompartments.	FIG
+46	55	R. rubrum	species	(A/B) Negative stain TEM image of recombinant R. rubrum encapsulin and EncFtn-Enc nanocompartments.	FIG
+56	66	encapsulin	protein	(A/B) Negative stain TEM image of recombinant R. rubrum encapsulin and EncFtn-Enc nanocompartments.	FIG
+71	81	EncFtn-Enc	complex_assembly	(A/B) Negative stain TEM image of recombinant R. rubrum encapsulin and EncFtn-Enc nanocompartments.	FIG
+82	98	nanocompartments	complex_assembly	(A/B) Negative stain TEM image of recombinant R. rubrum encapsulin and EncFtn-Enc nanocompartments.	FIG
+99	108	Histogram	evidence	All samples were imaged at 143,000 x magnification; the scale bar length corresponds to 50 nm. (C) Histogram showing the distribution of nanocompartment diameters.	FIG
+137	152	nanocompartment	complex_assembly	All samples were imaged at 143,000 x magnification; the scale bar length corresponds to 50 nm. (C) Histogram showing the distribution of nanocompartment diameters.	FIG
+8	48	Gaussian nonlinear least square function	experimental_method	A model Gaussian nonlinear least square function was fitted to the data to obtain a mean diameter of 24.6 nm with a standard deviation of 2.0 nm for encapsulin (grey) and a mean value of 23.9 nm with a standard deviation of 2.2 nm for co-expressed EncFtn and encapsulin (EncFtn-Enc, black).	FIG
+149	159	encapsulin	protein	A model Gaussian nonlinear least square function was fitted to the data to obtain a mean diameter of 24.6 nm with a standard deviation of 2.0 nm for encapsulin (grey) and a mean value of 23.9 nm with a standard deviation of 2.2 nm for co-expressed EncFtn and encapsulin (EncFtn-Enc, black).	FIG
+235	247	co-expressed	experimental_method	A model Gaussian nonlinear least square function was fitted to the data to obtain a mean diameter of 24.6 nm with a standard deviation of 2.0 nm for encapsulin (grey) and a mean value of 23.9 nm with a standard deviation of 2.2 nm for co-expressed EncFtn and encapsulin (EncFtn-Enc, black).	FIG
+248	254	EncFtn	protein	A model Gaussian nonlinear least square function was fitted to the data to obtain a mean diameter of 24.6 nm with a standard deviation of 2.0 nm for encapsulin (grey) and a mean value of 23.9 nm with a standard deviation of 2.2 nm for co-expressed EncFtn and encapsulin (EncFtn-Enc, black).	FIG
+259	269	encapsulin	protein	A model Gaussian nonlinear least square function was fitted to the data to obtain a mean diameter of 24.6 nm with a standard deviation of 2.0 nm for encapsulin (grey) and a mean value of 23.9 nm with a standard deviation of 2.2 nm for co-expressed EncFtn and encapsulin (EncFtn-Enc, black).	FIG
+271	281	EncFtn-Enc	complex_assembly	A model Gaussian nonlinear least square function was fitted to the data to obtain a mean diameter of 24.6 nm with a standard deviation of 2.0 nm for encapsulin (grey) and a mean value of 23.9 nm with a standard deviation of 2.2 nm for co-expressed EncFtn and encapsulin (EncFtn-Enc, black).	FIG
+28	37	R. rubrum	species	Purification of recombinant R. rubrum encapsulin nanocompartments.	FIG
+38	48	encapsulin	protein	Purification of recombinant R. rubrum encapsulin nanocompartments.	FIG
+49	65	nanocompartments	complex_assembly	Purification of recombinant R. rubrum encapsulin nanocompartments.	FIG
+4	27	Recombinantly expressed	experimental_method	(A) Recombinantly expressed encapsulin (Enc) and co-expressed EncFtn-Enc were purified by sucrose gradient ultracentrifugation from E. coli B834(DE3) grown in SeMet medium.	FIG
+28	38	encapsulin	protein	(A) Recombinantly expressed encapsulin (Enc) and co-expressed EncFtn-Enc were purified by sucrose gradient ultracentrifugation from E. coli B834(DE3) grown in SeMet medium.	FIG
+40	43	Enc	protein	(A) Recombinantly expressed encapsulin (Enc) and co-expressed EncFtn-Enc were purified by sucrose gradient ultracentrifugation from E. coli B834(DE3) grown in SeMet medium.	FIG
+49	61	co-expressed	experimental_method	(A) Recombinantly expressed encapsulin (Enc) and co-expressed EncFtn-Enc were purified by sucrose gradient ultracentrifugation from E. coli B834(DE3) grown in SeMet medium.	FIG
+62	72	EncFtn-Enc	complex_assembly	(A) Recombinantly expressed encapsulin (Enc) and co-expressed EncFtn-Enc were purified by sucrose gradient ultracentrifugation from E. coli B834(DE3) grown in SeMet medium.	FIG
+90	126	sucrose gradient ultracentrifugation	experimental_method	(A) Recombinantly expressed encapsulin (Enc) and co-expressed EncFtn-Enc were purified by sucrose gradient ultracentrifugation from E. coli B834(DE3) grown in SeMet medium.	FIG
+132	139	E. coli	species	(A) Recombinantly expressed encapsulin (Enc) and co-expressed EncFtn-Enc were purified by sucrose gradient ultracentrifugation from E. coli B834(DE3) grown in SeMet medium.	FIG
+159	164	SeMet	chemical	(A) Recombinantly expressed encapsulin (Enc) and co-expressed EncFtn-Enc were purified by sucrose gradient ultracentrifugation from E. coli B834(DE3) grown in SeMet medium.	FIG
+40	48	SDS-PAGE	experimental_method	Samples were resolved by 18% acrylamide SDS-PAGE; the position of the proteins found in the complexes as resolved on the gel are shown with arrows.	FIG
+6	24	Negative stain TEM	experimental_method	(B/C) Negative stain TEM image of recombinant encapsulin and EncFtn-Enc nanocompartments.	FIG
+46	56	encapsulin	protein	(B/C) Negative stain TEM image of recombinant encapsulin and EncFtn-Enc nanocompartments.	FIG
+61	71	EncFtn-Enc	complex_assembly	(B/C) Negative stain TEM image of recombinant encapsulin and EncFtn-Enc nanocompartments.	FIG
+72	88	nanocompartments	complex_assembly	(B/C) Negative stain TEM image of recombinant encapsulin and EncFtn-Enc nanocompartments.	FIG
+15	25	encapsulin	protein	Representative encapsulin and EncFtn-Enc complexes are indicated with red arrows.	FIG
+30	40	EncFtn-Enc	complex_assembly	Representative encapsulin and EncFtn-Enc complexes are indicated with red arrows.	FIG
+24	33	R. rubrum	species	We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A).	RESULTS
+34	44	encapsulin	protein	We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A).	RESULTS
+45	61	nanocompartments	complex_assembly	We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A).	RESULTS
+65	72	E. coli	species	We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A).	RESULTS
+76	89	co-expression	experimental_method	We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A).	RESULTS
+97	107	encapsulin	protein	We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A).	RESULTS
+109	118	Rru_A0974	gene	We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A).	RESULTS
+124	130	EncFtn	protein	We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A).	RESULTS
+132	141	Rru_A0973	gene	We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A).	RESULTS
+175	212	sucrose gradient ultra-centrifugation	experimental_method	We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A).	RESULTS
+0	3	TEM	experimental_method	TEM imaging of uranyl acetate-stained samples revealed that, when expressed in isolation, the encapsulin protein forms empty compartments with an average diameter of 24 nm (Figure 1B and Figure 1—figure supplement 1A/C), consistent with the appearance and size of the T. maritima encapsulin.	RESULTS
+66	88	expressed in isolation	experimental_method	TEM imaging of uranyl acetate-stained samples revealed that, when expressed in isolation, the encapsulin protein forms empty compartments with an average diameter of 24 nm (Figure 1B and Figure 1—figure supplement 1A/C), consistent with the appearance and size of the T. maritima encapsulin.	RESULTS
+94	104	encapsulin	protein	TEM imaging of uranyl acetate-stained samples revealed that, when expressed in isolation, the encapsulin protein forms empty compartments with an average diameter of 24 nm (Figure 1B and Figure 1—figure supplement 1A/C), consistent with the appearance and size of the T. maritima encapsulin.	RESULTS
+119	124	empty	protein_state	TEM imaging of uranyl acetate-stained samples revealed that, when expressed in isolation, the encapsulin protein forms empty compartments with an average diameter of 24 nm (Figure 1B and Figure 1—figure supplement 1A/C), consistent with the appearance and size of the T. maritima encapsulin.	RESULTS
+125	137	compartments	complex_assembly	TEM imaging of uranyl acetate-stained samples revealed that, when expressed in isolation, the encapsulin protein forms empty compartments with an average diameter of 24 nm (Figure 1B and Figure 1—figure supplement 1A/C), consistent with the appearance and size of the T. maritima encapsulin.	RESULTS
+268	279	T. maritima	species	TEM imaging of uranyl acetate-stained samples revealed that, when expressed in isolation, the encapsulin protein forms empty compartments with an average diameter of 24 nm (Figure 1B and Figure 1—figure supplement 1A/C), consistent with the appearance and size of the T. maritima encapsulin.	RESULTS
+280	290	encapsulin	protein	TEM imaging of uranyl acetate-stained samples revealed that, when expressed in isolation, the encapsulin protein forms empty compartments with an average diameter of 24 nm (Figure 1B and Figure 1—figure supplement 1A/C), consistent with the appearance and size of the T. maritima encapsulin.	RESULTS
+59	65	EncFtn	protein	We were not able to resolve any higher-order structures of EncFtn by TEM.	RESULTS
+69	72	TEM	experimental_method	We were not able to resolve any higher-order structures of EncFtn by TEM.	RESULTS
+22	35	co-expression	experimental_method	Protein purified from co-expression of the encapsulin and EncFtn resulted in 24 nm compartments with regions in the center that exclude stain, consistent with the presence of the EncFtn within the encapsulin shell (Figure 1C and Figure 1—figure supplement 1B/C).	RESULTS
+43	53	encapsulin	protein	Protein purified from co-expression of the encapsulin and EncFtn resulted in 24 nm compartments with regions in the center that exclude stain, consistent with the presence of the EncFtn within the encapsulin shell (Figure 1C and Figure 1—figure supplement 1B/C).	RESULTS
+58	64	EncFtn	protein	Protein purified from co-expression of the encapsulin and EncFtn resulted in 24 nm compartments with regions in the center that exclude stain, consistent with the presence of the EncFtn within the encapsulin shell (Figure 1C and Figure 1—figure supplement 1B/C).	RESULTS
+163	174	presence of	protein_state	Protein purified from co-expression of the encapsulin and EncFtn resulted in 24 nm compartments with regions in the center that exclude stain, consistent with the presence of the EncFtn within the encapsulin shell (Figure 1C and Figure 1—figure supplement 1B/C).	RESULTS
+179	185	EncFtn	protein	Protein purified from co-expression of the encapsulin and EncFtn resulted in 24 nm compartments with regions in the center that exclude stain, consistent with the presence of the EncFtn within the encapsulin shell (Figure 1C and Figure 1—figure supplement 1B/C).	RESULTS
+197	207	encapsulin	protein	Protein purified from co-expression of the encapsulin and EncFtn resulted in 24 nm compartments with regions in the center that exclude stain, consistent with the presence of the EncFtn within the encapsulin shell (Figure 1C and Figure 1—figure supplement 1B/C).	RESULTS
+208	213	shell	structure_element	Protein purified from co-expression of the encapsulin and EncFtn resulted in 24 nm compartments with regions in the center that exclude stain, consistent with the presence of the EncFtn within the encapsulin shell (Figure 1C and Figure 1—figure supplement 1B/C).	RESULTS
+0	9	R. rubrum	species	R. rubrum EncFtn forms a metal-ion stabilized decamer in solution	RESULTS
+10	16	EncFtn	protein	R. rubrum EncFtn forms a metal-ion stabilized decamer in solution	RESULTS
+46	53	decamer	oligomeric_state	R. rubrum EncFtn forms a metal-ion stabilized decamer in solution	RESULTS
+0	27	Purification of recombinant	experimental_method	Purification of recombinant R. rubrum EncFtnsH.	FIG
+28	37	R. rubrum	species	Purification of recombinant R. rubrum EncFtnsH.	FIG
+38	46	EncFtnsH	protein	Purification of recombinant R. rubrum EncFtnsH.	FIG
+16	29	SeMet-labeled	protein_state	(A) Recombinant SeMet-labeled EncFtnsH produced with 1 mM Fe(NH4)2(SO4)2 in the growth medium was purified by nickel affinity chromatography and size-exclusion chromatography using a Superdex 200 16/60 column (GE Healthcare).	FIG
+30	38	EncFtnsH	protein	(A) Recombinant SeMet-labeled EncFtnsH produced with 1 mM Fe(NH4)2(SO4)2 in the growth medium was purified by nickel affinity chromatography and size-exclusion chromatography using a Superdex 200 16/60 column (GE Healthcare).	FIG
+58	72	Fe(NH4)2(SO4)2	chemical	(A) Recombinant SeMet-labeled EncFtnsH produced with 1 mM Fe(NH4)2(SO4)2 in the growth medium was purified by nickel affinity chromatography and size-exclusion chromatography using a Superdex 200 16/60 column (GE Healthcare).	FIG
+110	140	nickel affinity chromatography	experimental_method	(A) Recombinant SeMet-labeled EncFtnsH produced with 1 mM Fe(NH4)2(SO4)2 in the growth medium was purified by nickel affinity chromatography and size-exclusion chromatography using a Superdex 200 16/60 column (GE Healthcare).	FIG
+145	174	size-exclusion chromatography	experimental_method	(A) Recombinant SeMet-labeled EncFtnsH produced with 1 mM Fe(NH4)2(SO4)2 in the growth medium was purified by nickel affinity chromatography and size-exclusion chromatography using a Superdex 200 16/60 column (GE Healthcare).	FIG
+0	12	Chromatogram	evidence	Chromatogram traces measured at 280 nm and 315 nm are shown with the results from ICP-MS analysis of the iron content of the fractions collected during the experiment.	FIG
+82	88	ICP-MS	experimental_method	Chromatogram traces measured at 280 nm and 315 nm are shown with the results from ICP-MS analysis of the iron content of the fractions collected during the experiment.	FIG
+105	109	iron	chemical	Chromatogram traces measured at 280 nm and 315 nm are shown with the results from ICP-MS analysis of the iron content of the fractions collected during the experiment.	FIG
+39	55	molecular weight	evidence	The peak around 73 ml corresponds to a molecular weight of around 130 kDa when compared to calibration standards; this is consistent with a decamer of EncFtnsH. The small peak at 85 ml corresponds to the 13 kDa monomer compared to the standards.	FIG
+140	147	decamer	oligomeric_state	The peak around 73 ml corresponds to a molecular weight of around 130 kDa when compared to calibration standards; this is consistent with a decamer of EncFtnsH. The small peak at 85 ml corresponds to the 13 kDa monomer compared to the standards.	FIG
+151	159	EncFtnsH	protein	The peak around 73 ml corresponds to a molecular weight of around 130 kDa when compared to calibration standards; this is consistent with a decamer of EncFtnsH. The small peak at 85 ml corresponds to the 13 kDa monomer compared to the standards.	FIG
+211	218	monomer	oligomeric_state	The peak around 73 ml corresponds to a molecular weight of around 130 kDa when compared to calibration standards; this is consistent with a decamer of EncFtnsH. The small peak at 85 ml corresponds to the 13 kDa monomer compared to the standards.	FIG
+9	16	decamer	oligomeric_state	Only the decamer peak contains significant amounts of iron as indicated by the ICP-MS analysis.	FIG
+54	58	iron	chemical	Only the decamer peak contains significant amounts of iron as indicated by the ICP-MS analysis.	FIG
+79	85	ICP-MS	experimental_method	Only the decamer peak contains significant amounts of iron as indicated by the ICP-MS analysis.	FIG
+28	42	gel filtration	experimental_method	(B) Peak fractions from the gel filtration run were resolved by 15% acrylamide SDS-PAGE and stained with Coomassie blue stain.	FIG
+79	87	SDS-PAGE	experimental_method	(B) Peak fractions from the gel filtration run were resolved by 15% acrylamide SDS-PAGE and stained with Coomassie blue stain.	FIG
+49	57	EncFtnsH	protein	The bands around 13 kDa and 26 kDa correspond to EncFtnsH, as identified by MALDI peptide mass fingerprinting.	FIG
+76	109	MALDI peptide mass fingerprinting	experimental_method	The bands around 13 kDa and 26 kDa correspond to EncFtnsH, as identified by MALDI peptide mass fingerprinting.	FIG
+42	49	monomer	oligomeric_state	The band at 13 kDa is consistent with the monomer mass, while the band at 26 kDa is consistent with a dimer of EncFtnsH. The dimer species only appears in the decamer fractions.	FIG
+102	107	dimer	oligomeric_state	The band at 13 kDa is consistent with the monomer mass, while the band at 26 kDa is consistent with a dimer of EncFtnsH. The dimer species only appears in the decamer fractions.	FIG
+111	119	EncFtnsH	protein	The band at 13 kDa is consistent with the monomer mass, while the band at 26 kDa is consistent with a dimer of EncFtnsH. The dimer species only appears in the decamer fractions.	FIG
+125	130	dimer	oligomeric_state	The band at 13 kDa is consistent with the monomer mass, while the band at 26 kDa is consistent with a dimer of EncFtnsH. The dimer species only appears in the decamer fractions.	FIG
+159	166	decamer	oligomeric_state	The band at 13 kDa is consistent with the monomer mass, while the band at 26 kDa is consistent with a dimer of EncFtnsH. The dimer species only appears in the decamer fractions.	FIG
+4	13	SEC-MALLS	experimental_method	(C) SEC-MALLS analysis of EncFtnsH from decamer fractions and monomer fractions allows assignment of an average mass of 132 kDa to decamer fractions and 13 kDa to monomer fractions, consistent with decamer and monomer species (Table 2).	FIG
+26	34	EncFtnsH	protein	(C) SEC-MALLS analysis of EncFtnsH from decamer fractions and monomer fractions allows assignment of an average mass of 132 kDa to decamer fractions and 13 kDa to monomer fractions, consistent with decamer and monomer species (Table 2).	FIG
+40	47	decamer	oligomeric_state	(C) SEC-MALLS analysis of EncFtnsH from decamer fractions and monomer fractions allows assignment of an average mass of 132 kDa to decamer fractions and 13 kDa to monomer fractions, consistent with decamer and monomer species (Table 2).	FIG
+62	69	monomer	oligomeric_state	(C) SEC-MALLS analysis of EncFtnsH from decamer fractions and monomer fractions allows assignment of an average mass of 132 kDa to decamer fractions and 13 kDa to monomer fractions, consistent with decamer and monomer species (Table 2).	FIG
+131	138	decamer	oligomeric_state	(C) SEC-MALLS analysis of EncFtnsH from decamer fractions and monomer fractions allows assignment of an average mass of 132 kDa to decamer fractions and 13 kDa to monomer fractions, consistent with decamer and monomer species (Table 2).	FIG
+163	170	monomer	oligomeric_state	(C) SEC-MALLS analysis of EncFtnsH from decamer fractions and monomer fractions allows assignment of an average mass of 132 kDa to decamer fractions and 13 kDa to monomer fractions, consistent with decamer and monomer species (Table 2).	FIG
+198	205	decamer	oligomeric_state	(C) SEC-MALLS analysis of EncFtnsH from decamer fractions and monomer fractions allows assignment of an average mass of 132 kDa to decamer fractions and 13 kDa to monomer fractions, consistent with decamer and monomer species (Table 2).	FIG
+210	217	monomer	oligomeric_state	(C) SEC-MALLS analysis of EncFtnsH from decamer fractions and monomer fractions allows assignment of an average mass of 132 kDa to decamer fractions and 13 kDa to monomer fractions, consistent with decamer and monomer species (Table 2).	FIG
+21	23	Fe	chemical	Determination of the Fe/EncFtnsH protein ratio by ICP-MS.	TABLE
+24	32	EncFtnsH	protein	Determination of the Fe/EncFtnsH protein ratio by ICP-MS.	TABLE
+50	56	ICP-MS	experimental_method	Determination of the Fe/EncFtnsH protein ratio by ICP-MS.	TABLE
+0	8	EncFtnsH	protein	EncFtnsH was purified as a SeMet derivative from E. coli B834(DE3) cells grown in SeMet medium with 1 mM Fe(NH4)2(SO4)2.	TABLE
+27	32	SeMet	chemical	EncFtnsH was purified as a SeMet derivative from E. coli B834(DE3) cells grown in SeMet medium with 1 mM Fe(NH4)2(SO4)2.	TABLE
+49	66	E. coli B834(DE3)	species	EncFtnsH was purified as a SeMet derivative from E. coli B834(DE3) cells grown in SeMet medium with 1 mM Fe(NH4)2(SO4)2.	TABLE
+82	87	SeMet	chemical	EncFtnsH was purified as a SeMet derivative from E. coli B834(DE3) cells grown in SeMet medium with 1 mM Fe(NH4)2(SO4)2.	TABLE
+105	119	Fe(NH4)2(SO4)2	chemical	EncFtnsH was purified as a SeMet derivative from E. coli B834(DE3) cells grown in SeMet medium with 1 mM Fe(NH4)2(SO4)2.	TABLE
+15	18	SEC	experimental_method	Fractions from SEC were collected, acidified and analysed by ICP-MS.	TABLE
+61	67	ICP-MS	experimental_method	Fractions from SEC were collected, acidified and analysed by ICP-MS.	TABLE
+0	8	EncFtnsH	protein	EncFtnsH concentration was calculated based on the presence of two SeMet per mature monomer.	TABLE
+51	62	presence of	protein_state	EncFtnsH concentration was calculated based on the presence of two SeMet per mature monomer.	TABLE
+67	72	SeMet	chemical	EncFtnsH concentration was calculated based on the presence of two SeMet per mature monomer.	TABLE
+77	83	mature	protein_state	EncFtnsH concentration was calculated based on the presence of two SeMet per mature monomer.	TABLE
+84	91	monomer	oligomeric_state	EncFtnsH concentration was calculated based on the presence of two SeMet per mature monomer.	TABLE
+31	39	EncFtnsH	protein	These data were collected from EncFtnsH fractions from a single gel-filtration run.	TABLE
+64	78	gel-filtration	experimental_method	These data were collected from EncFtnsH fractions from a single gel-filtration run.	TABLE
+5	13	EncFtnsH	protein	"Peak	EncFtnsHretention volume (ml)	Element concentration (µM)	Derived EncFtnsHconcentration (µM)	Derived Fe/ EncFtnsH monomer	 	Ca	Fe	Zn	Se	 	Decamer	66.5	n.d."	TABLE
+70	78	EncFtnsH	protein	"Peak	EncFtnsHretention volume (ml)	Element concentration (µM)	Derived EncFtnsHconcentration (µM)	Derived Fe/ EncFtnsH monomer	 	Ca	Fe	Zn	Se	 	Decamer	66.5	n.d."	TABLE
+105	107	Fe	chemical	"Peak	EncFtnsHretention volume (ml)	Element concentration (µM)	Derived EncFtnsHconcentration (µM)	Derived Fe/ EncFtnsH monomer	 	Ca	Fe	Zn	Se	 	Decamer	66.5	n.d."	TABLE
+109	117	EncFtnsH	protein	"Peak	EncFtnsHretention volume (ml)	Element concentration (µM)	Derived EncFtnsHconcentration (µM)	Derived Fe/ EncFtnsH monomer	 	Ca	Fe	Zn	Se	 	Decamer	66.5	n.d."	TABLE
+118	125	monomer	oligomeric_state	"Peak	EncFtnsHretention volume (ml)	Element concentration (µM)	Derived EncFtnsHconcentration (µM)	Derived Fe/ EncFtnsH monomer	 	Ca	Fe	Zn	Se	 	Decamer	66.5	n.d."	TABLE
+128	130	Ca	chemical	"Peak	EncFtnsHretention volume (ml)	Element concentration (µM)	Derived EncFtnsHconcentration (µM)	Derived Fe/ EncFtnsH monomer	 	Ca	Fe	Zn	Se	 	Decamer	66.5	n.d."	TABLE
+131	133	Fe	chemical	"Peak	EncFtnsHretention volume (ml)	Element concentration (µM)	Derived EncFtnsHconcentration (µM)	Derived Fe/ EncFtnsH monomer	 	Ca	Fe	Zn	Se	 	Decamer	66.5	n.d."	TABLE
+134	136	Zn	chemical	"Peak	EncFtnsHretention volume (ml)	Element concentration (µM)	Derived EncFtnsHconcentration (µM)	Derived Fe/ EncFtnsH monomer	 	Ca	Fe	Zn	Se	 	Decamer	66.5	n.d."	TABLE
+137	139	Se	chemical	"Peak	EncFtnsHretention volume (ml)	Element concentration (µM)	Derived EncFtnsHconcentration (µM)	Derived Fe/ EncFtnsH monomer	 	Ca	Fe	Zn	Se	 	Decamer	66.5	n.d."	TABLE
+142	149	Decamer	oligomeric_state	"Peak	EncFtnsHretention volume (ml)	Element concentration (µM)	Derived EncFtnsHconcentration (µM)	Derived Fe/ EncFtnsH monomer	 	Ca	Fe	Zn	Se	 	Decamer	66.5	n.d."	TABLE
+13	21	EncFtnsH	protein	Estimates of EncFtnsH molecular weight from SEC-MALLS analysis.	TABLE
+22	38	molecular weight	evidence	Estimates of EncFtnsH molecular weight from SEC-MALLS analysis.	TABLE
+44	53	SEC-MALLS	experimental_method	Estimates of EncFtnsH molecular weight from SEC-MALLS analysis.	TABLE
+0	8	EncFtnsH	protein	EncFtnsH was purified from E. coli BL21(DE3) grown in minimal medium (MM) by nickel affinity chromatography and size-exclusion chromatography.	TABLE
+27	44	E. coli BL21(DE3)	species	EncFtnsH was purified from E. coli BL21(DE3) grown in minimal medium (MM) by nickel affinity chromatography and size-exclusion chromatography.	TABLE
+54	68	minimal medium	experimental_method	EncFtnsH was purified from E. coli BL21(DE3) grown in minimal medium (MM) by nickel affinity chromatography and size-exclusion chromatography.	TABLE
+70	72	MM	experimental_method	EncFtnsH was purified from E. coli BL21(DE3) grown in minimal medium (MM) by nickel affinity chromatography and size-exclusion chromatography.	TABLE
+77	107	nickel affinity chromatography	experimental_method	EncFtnsH was purified from E. coli BL21(DE3) grown in minimal medium (MM) by nickel affinity chromatography and size-exclusion chromatography.	TABLE
+112	141	size-exclusion chromatography	experimental_method	EncFtnsH was purified from E. coli BL21(DE3) grown in minimal medium (MM) by nickel affinity chromatography and size-exclusion chromatography.	TABLE
+19	24	peaks	evidence	Fractions from two peaks (decamer and monomer) were pooled separately (Figure 1C) and analysed by SEC-MALLS using a Superdex 200 10/300 GL column (GE Healthcare) and Viscotek SEC-MALLS instruments (Malvern Instruments) (Figure 2C).	TABLE
+26	33	decamer	oligomeric_state	Fractions from two peaks (decamer and monomer) were pooled separately (Figure 1C) and analysed by SEC-MALLS using a Superdex 200 10/300 GL column (GE Healthcare) and Viscotek SEC-MALLS instruments (Malvern Instruments) (Figure 2C).	TABLE
+38	45	monomer	oligomeric_state	Fractions from two peaks (decamer and monomer) were pooled separately (Figure 1C) and analysed by SEC-MALLS using a Superdex 200 10/300 GL column (GE Healthcare) and Viscotek SEC-MALLS instruments (Malvern Instruments) (Figure 2C).	TABLE
+98	107	SEC-MALLS	experimental_method	Fractions from two peaks (decamer and monomer) were pooled separately (Figure 1C) and analysed by SEC-MALLS using a Superdex 200 10/300 GL column (GE Healthcare) and Viscotek SEC-MALLS instruments (Malvern Instruments) (Figure 2C).	TABLE
+175	184	SEC-MALLS	experimental_method	Fractions from two peaks (decamer and monomer) were pooled separately (Figure 1C) and analysed by SEC-MALLS using a Superdex 200 10/300 GL column (GE Healthcare) and Viscotek SEC-MALLS instruments (Malvern Instruments) (Figure 2C).	TABLE
+4	11	decamer	oligomeric_state	The decamer and monomer peaks were both symmetric and monodisperse, allowing the estimation of the molecular weight of the species in these fractions.	TABLE
+16	23	monomer	oligomeric_state	The decamer and monomer peaks were both symmetric and monodisperse, allowing the estimation of the molecular weight of the species in these fractions.	TABLE
+24	29	peaks	evidence	The decamer and monomer peaks were both symmetric and monodisperse, allowing the estimation of the molecular weight of the species in these fractions.	TABLE
+99	115	molecular weight	evidence	The decamer and monomer peaks were both symmetric and monodisperse, allowing the estimation of the molecular weight of the species in these fractions.	TABLE
+25	34	SEC-MALLS	experimental_method	The proteins analyzed by SEC-MALLS came from single protein preparation.	TABLE
+0	16	Molecular Weight	evidence	"Molecular Weight (kDa)	Decamer peak	Monomer peak	 	Theoretical	133	13	 	EncFtnsH-decamer fractions	132	15	 	EncFtnsH-monomer fractions	126	13	 	"	TABLE
+23	30	Decamer	oligomeric_state	"Molecular Weight (kDa)	Decamer peak	Monomer peak	 	Theoretical	133	13	 	EncFtnsH-decamer fractions	132	15	 	EncFtnsH-monomer fractions	126	13	 	"	TABLE
+36	43	Monomer	oligomeric_state	"Molecular Weight (kDa)	Decamer peak	Monomer peak	 	Theoretical	133	13	 	EncFtnsH-decamer fractions	132	15	 	EncFtnsH-monomer fractions	126	13	 	"	TABLE
+72	80	EncFtnsH	protein	"Molecular Weight (kDa)	Decamer peak	Monomer peak	 	Theoretical	133	13	 	EncFtnsH-decamer fractions	132	15	 	EncFtnsH-monomer fractions	126	13	 	"	TABLE
+81	88	decamer	oligomeric_state	"Molecular Weight (kDa)	Decamer peak	Monomer peak	 	Theoretical	133	13	 	EncFtnsH-decamer fractions	132	15	 	EncFtnsH-monomer fractions	126	13	 	"	TABLE
+108	116	EncFtnsH	protein	"Molecular Weight (kDa)	Decamer peak	Monomer peak	 	Theoretical	133	13	 	EncFtnsH-decamer fractions	132	15	 	EncFtnsH-monomer fractions	126	13	 	"	TABLE
+117	124	monomer	oligomeric_state	"Molecular Weight (kDa)	Decamer peak	Monomer peak	 	Theoretical	133	13	 	EncFtnsH-decamer fractions	132	15	 	EncFtnsH-monomer fractions	126	13	 	"	TABLE
+24	33	R. rubrum	species	We purified recombinant R. rubrum EncFtn as both the full-length sequence (140 amino acids) and a truncated C-terminal hexahistidine-tagged variant (amino acids 1–96 plus the tag; herein EncFtnsH).	RESULTS
+34	40	EncFtn	protein	We purified recombinant R. rubrum EncFtn as both the full-length sequence (140 amino acids) and a truncated C-terminal hexahistidine-tagged variant (amino acids 1–96 plus the tag; herein EncFtnsH).	RESULTS
+53	64	full-length	protein_state	We purified recombinant R. rubrum EncFtn as both the full-length sequence (140 amino acids) and a truncated C-terminal hexahistidine-tagged variant (amino acids 1–96 plus the tag; herein EncFtnsH).	RESULTS
+75	90	140 amino acids	residue_range	We purified recombinant R. rubrum EncFtn as both the full-length sequence (140 amino acids) and a truncated C-terminal hexahistidine-tagged variant (amino acids 1–96 plus the tag; herein EncFtnsH).	RESULTS
+98	107	truncated	protein_state	We purified recombinant R. rubrum EncFtn as both the full-length sequence (140 amino acids) and a truncated C-terminal hexahistidine-tagged variant (amino acids 1–96 plus the tag; herein EncFtnsH).	RESULTS
+119	139	hexahistidine-tagged	protein_state	We purified recombinant R. rubrum EncFtn as both the full-length sequence (140 amino acids) and a truncated C-terminal hexahistidine-tagged variant (amino acids 1–96 plus the tag; herein EncFtnsH).	RESULTS
+161	165	1–96	residue_range	We purified recombinant R. rubrum EncFtn as both the full-length sequence (140 amino acids) and a truncated C-terminal hexahistidine-tagged variant (amino acids 1–96 plus the tag; herein EncFtnsH).	RESULTS
+187	195	EncFtnsH	protein	We purified recombinant R. rubrum EncFtn as both the full-length sequence (140 amino acids) and a truncated C-terminal hexahistidine-tagged variant (amino acids 1–96 plus the tag; herein EncFtnsH).	RESULTS
+18	33	elution profile	evidence	In both cases the elution profile from size-exclusion chromatography (SEC) displayed two peaks (Figure 2A).	RESULTS
+39	68	size-exclusion chromatography	experimental_method	In both cases the elution profile from size-exclusion chromatography (SEC) displayed two peaks (Figure 2A).	RESULTS
+70	73	SEC	experimental_method	In both cases the elution profile from size-exclusion chromatography (SEC) displayed two peaks (Figure 2A).	RESULTS
+89	94	peaks	evidence	In both cases the elution profile from size-exclusion chromatography (SEC) displayed two peaks (Figure 2A).	RESULTS
+0	8	SDS-PAGE	experimental_method	SDS-PAGE analysis of fractions from these peaks showed that the high molecular weight peak was partially resistant to SDS and heat-induced denaturation; in contrast, the low molecular weight peak was consistent with monomeric mass of 13 kDa (Figure 2B).	RESULTS
+42	47	peaks	evidence	SDS-PAGE analysis of fractions from these peaks showed that the high molecular weight peak was partially resistant to SDS and heat-induced denaturation; in contrast, the low molecular weight peak was consistent with monomeric mass of 13 kDa (Figure 2B).	RESULTS
+69	85	molecular weight	evidence	SDS-PAGE analysis of fractions from these peaks showed that the high molecular weight peak was partially resistant to SDS and heat-induced denaturation; in contrast, the low molecular weight peak was consistent with monomeric mass of 13 kDa (Figure 2B).	RESULTS
+174	190	molecular weight	evidence	SDS-PAGE analysis of fractions from these peaks showed that the high molecular weight peak was partially resistant to SDS and heat-induced denaturation; in contrast, the low molecular weight peak was consistent with monomeric mass of 13 kDa (Figure 2B).	RESULTS
+216	225	monomeric	oligomeric_state	SDS-PAGE analysis of fractions from these peaks showed that the high molecular weight peak was partially resistant to SDS and heat-induced denaturation; in contrast, the low molecular weight peak was consistent with monomeric mass of 13 kDa (Figure 2B).	RESULTS
+0	33	MALDI peptide mass fingerprinting	experimental_method	MALDI peptide mass fingerprinting of these bands confirmed the identity of both as EncFtn.	RESULTS
+83	89	EncFtn	protein	MALDI peptide mass fingerprinting of these bands confirmed the identity of both as EncFtn.	RESULTS
+0	44	Inductively coupled plasma mass spectrometry	experimental_method	Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the SEC fractions showed 100 times more iron in the oligomeric fraction than the monomer (Figure 2A, blue scatter points; Table 1), suggesting that EncFtn oligomerization is associated with iron binding.	RESULTS
+46	52	ICP-MS	experimental_method	Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the SEC fractions showed 100 times more iron in the oligomeric fraction than the monomer (Figure 2A, blue scatter points; Table 1), suggesting that EncFtn oligomerization is associated with iron binding.	RESULTS
+70	73	SEC	experimental_method	Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the SEC fractions showed 100 times more iron in the oligomeric fraction than the monomer (Figure 2A, blue scatter points; Table 1), suggesting that EncFtn oligomerization is associated with iron binding.	RESULTS
+106	110	iron	chemical	Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the SEC fractions showed 100 times more iron in the oligomeric fraction than the monomer (Figure 2A, blue scatter points; Table 1), suggesting that EncFtn oligomerization is associated with iron binding.	RESULTS
+147	154	monomer	oligomeric_state	Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the SEC fractions showed 100 times more iron in the oligomeric fraction than the monomer (Figure 2A, blue scatter points; Table 1), suggesting that EncFtn oligomerization is associated with iron binding.	RESULTS
+214	220	EncFtn	protein	Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the SEC fractions showed 100 times more iron in the oligomeric fraction than the monomer (Figure 2A, blue scatter points; Table 1), suggesting that EncFtn oligomerization is associated with iron binding.	RESULTS
+256	260	iron	chemical	Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the SEC fractions showed 100 times more iron in the oligomeric fraction than the monomer (Figure 2A, blue scatter points; Table 1), suggesting that EncFtn oligomerization is associated with iron binding.	RESULTS
+26	30	iron	chemical	In order to determine the iron-loading stoichiometry in the EncFtn complex, further ICP-MS experiments were performed using selenomethionine (SeMet)-labelled protein EncFtn (Table 1).	RESULTS
+60	66	EncFtn	protein	In order to determine the iron-loading stoichiometry in the EncFtn complex, further ICP-MS experiments were performed using selenomethionine (SeMet)-labelled protein EncFtn (Table 1).	RESULTS
+84	90	ICP-MS	experimental_method	In order to determine the iron-loading stoichiometry in the EncFtn complex, further ICP-MS experiments were performed using selenomethionine (SeMet)-labelled protein EncFtn (Table 1).	RESULTS
+124	140	selenomethionine	chemical	In order to determine the iron-loading stoichiometry in the EncFtn complex, further ICP-MS experiments were performed using selenomethionine (SeMet)-labelled protein EncFtn (Table 1).	RESULTS
+142	147	SeMet	chemical	In order to determine the iron-loading stoichiometry in the EncFtn complex, further ICP-MS experiments were performed using selenomethionine (SeMet)-labelled protein EncFtn (Table 1).	RESULTS
+166	172	EncFtn	protein	In order to determine the iron-loading stoichiometry in the EncFtn complex, further ICP-MS experiments were performed using selenomethionine (SeMet)-labelled protein EncFtn (Table 1).	RESULTS
+96	105	classical	protein_state	In these experiments, we observed sub-stoichiometric metal binding, which is in contrast to the classical ferritins.	RESULTS
+106	115	ferritins	protein_type	In these experiments, we observed sub-stoichiometric metal binding, which is in contrast to the classical ferritins.	RESULTS
+0	29	Size-exclusion chromatography	experimental_method	Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) analysis of samples taken from each peak gave calculated molecular weights consistent with a decamer for the high molecular weight peak and a monomer for the low molecular weight peak (Figure 2C, Table 2).	RESULTS
+35	69	multi-angle laser light scattering	experimental_method	Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) analysis of samples taken from each peak gave calculated molecular weights consistent with a decamer for the high molecular weight peak and a monomer for the low molecular weight peak (Figure 2C, Table 2).	RESULTS
+71	80	SEC-MALLS	experimental_method	Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) analysis of samples taken from each peak gave calculated molecular weights consistent with a decamer for the high molecular weight peak and a monomer for the low molecular weight peak (Figure 2C, Table 2).	RESULTS
+175	182	decamer	oligomeric_state	Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) analysis of samples taken from each peak gave calculated molecular weights consistent with a decamer for the high molecular weight peak and a monomer for the low molecular weight peak (Figure 2C, Table 2).	RESULTS
+196	212	molecular weight	evidence	Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) analysis of samples taken from each peak gave calculated molecular weights consistent with a decamer for the high molecular weight peak and a monomer for the low molecular weight peak (Figure 2C, Table 2).	RESULTS
+224	231	monomer	oligomeric_state	Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) analysis of samples taken from each peak gave calculated molecular weights consistent with a decamer for the high molecular weight peak and a monomer for the low molecular weight peak (Figure 2C, Table 2).	RESULTS
+244	260	molecular weight	evidence	Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) analysis of samples taken from each peak gave calculated molecular weights consistent with a decamer for the high molecular weight peak and a monomer for the low molecular weight peak (Figure 2C, Table 2).	RESULTS
+48	56	EncFtnsH	protein	Effect of metal ions on the oligomeric state of EncFtnsH in solution.	FIG
+6	14	EncFtnsH	protein	(A/B) EncFtnsH-monomer was incubated with one mole equivalent of various metal salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column.	FIG
+15	22	monomer	oligomeric_state	(A/B) EncFtnsH-monomer was incubated with one mole equivalent of various metal salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column.	FIG
+27	36	incubated	experimental_method	(A/B) EncFtnsH-monomer was incubated with one mole equivalent of various metal salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column.	FIG
+108	133	analytical gel-filtration	experimental_method	(A/B) EncFtnsH-monomer was incubated with one mole equivalent of various metal salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column.	FIG
+0	4	Co2+	chemical	Co2+ and Zn2+ induced the formation of the decameric form of EncFtnsH; while Mn2+, Mg2+ and Fe3+ did not significantly alter the oligomeric state of EncFtnsH.	FIG
+9	13	Zn2+	chemical	Co2+ and Zn2+ induced the formation of the decameric form of EncFtnsH; while Mn2+, Mg2+ and Fe3+ did not significantly alter the oligomeric state of EncFtnsH.	FIG
+43	52	decameric	oligomeric_state	Co2+ and Zn2+ induced the formation of the decameric form of EncFtnsH; while Mn2+, Mg2+ and Fe3+ did not significantly alter the oligomeric state of EncFtnsH.	FIG
+61	69	EncFtnsH	protein	Co2+ and Zn2+ induced the formation of the decameric form of EncFtnsH; while Mn2+, Mg2+ and Fe3+ did not significantly alter the oligomeric state of EncFtnsH.	FIG
+77	81	Mn2+	chemical	Co2+ and Zn2+ induced the formation of the decameric form of EncFtnsH; while Mn2+, Mg2+ and Fe3+ did not significantly alter the oligomeric state of EncFtnsH.	FIG
+83	87	Mg2+	chemical	Co2+ and Zn2+ induced the formation of the decameric form of EncFtnsH; while Mn2+, Mg2+ and Fe3+ did not significantly alter the oligomeric state of EncFtnsH.	FIG
+92	96	Fe3+	chemical	Co2+ and Zn2+ induced the formation of the decameric form of EncFtnsH; while Mn2+, Mg2+ and Fe3+ did not significantly alter the oligomeric state of EncFtnsH.	FIG
+149	157	EncFtnsH	protein	Co2+ and Zn2+ induced the formation of the decameric form of EncFtnsH; while Mn2+, Mg2+ and Fe3+ did not significantly alter the oligomeric state of EncFtnsH.	FIG
+0	4	PAGE	experimental_method	PAGE analysis of the effect of metal ions on the oligomeric state of EncFtnsH.	FIG
+69	77	EncFtnsH	protein	PAGE analysis of the effect of metal ions on the oligomeric state of EncFtnsH.	FIG
+6	14	EncFtnsH	protein	50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE.	FIG
+15	22	monomer	oligomeric_state	50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE.	FIG
+26	33	decamer	oligomeric_state	50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE.	FIG
+91	96	Fe2+,	chemical	50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE.	FIG
+97	102	Co2+,	chemical	50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE.	FIG
+103	108	Zn2+,	chemical	50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE.	FIG
+109	114	Mn2+,	chemical	50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE.	FIG
+115	120	Ca2+,	chemical	50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE.	FIG
+121	125	Mg2+	chemical	50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE.	FIG
+130	135	Fe3+,	chemical	50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE.	FIG
+159	170	Native PAGE	experimental_method	50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE.	FIG
+181	189	SDS-PAGE	experimental_method	50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE.	FIG
+9	20	Native PAGE	experimental_method	 (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.	FIG
+33	41	EncFtnsH	protein	 (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.	FIG
+42	49	monomer	oligomeric_state	 (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.	FIG
+104	115	Native PAGE	experimental_method	 (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.	FIG
+128	136	EncFtnsH	protein	 (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.	FIG
+137	144	decamer	oligomeric_state	 (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.	FIG
+199	207	SDS-PAGE	experimental_method	 (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.	FIG
+236	244	EncFtnsH	protein	 (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.	FIG
+245	252	monomer	oligomeric_state	 (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.	FIG
+292	300	SDS-PAGE	experimental_method	 (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.	FIG
+329	337	EncFtnsH	protein	 (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.	FIG
+338	345	decamer	oligomeric_state	 (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.	FIG
+10	14	Fe2+	chemical	Effect of Fe2+ and protein concentration on the oligomeric state of EncFtnsH in solution.	FIG
+68	76	EncFtnsH	protein	Effect of Fe2+ and protein concentration on the oligomeric state of EncFtnsH in solution.	FIG
+16	24	EncFtnsH	protein	(A) Recombinant EncFtnsH was purified by Gel filtration Superdex 200 chromatography from E. coli BL21(DE3) grown in MM or in MM supplemented with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+).	FIG
+41	55	Gel filtration	experimental_method	(A) Recombinant EncFtnsH was purified by Gel filtration Superdex 200 chromatography from E. coli BL21(DE3) grown in MM or in MM supplemented with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+).	FIG
+89	106	E. coli BL21(DE3)	species	(A) Recombinant EncFtnsH was purified by Gel filtration Superdex 200 chromatography from E. coli BL21(DE3) grown in MM or in MM supplemented with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+).	FIG
+116	118	MM	experimental_method	(A) Recombinant EncFtnsH was purified by Gel filtration Superdex 200 chromatography from E. coli BL21(DE3) grown in MM or in MM supplemented with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+).	FIG
+125	127	MM	experimental_method	(A) Recombinant EncFtnsH was purified by Gel filtration Superdex 200 chromatography from E. coli BL21(DE3) grown in MM or in MM supplemented with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+).	FIG
+151	165	Fe(NH4)2(SO4)2	chemical	(A) Recombinant EncFtnsH was purified by Gel filtration Superdex 200 chromatography from E. coli BL21(DE3) grown in MM or in MM supplemented with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+).	FIG
+167	169	MM	experimental_method	(A) Recombinant EncFtnsH was purified by Gel filtration Superdex 200 chromatography from E. coli BL21(DE3) grown in MM or in MM supplemented with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+).	FIG
+170	174	Fe2+	chemical	(A) Recombinant EncFtnsH was purified by Gel filtration Superdex 200 chromatography from E. coli BL21(DE3) grown in MM or in MM supplemented with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+).	FIG
+23	30	decamer	oligomeric_state	A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column (GE Healthcare).	FIG
+95	97	MM	experimental_method	A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column (GE Healthcare).	FIG
+98	102	Fe2+	chemical	A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column (GE Healthcare).	FIG
+115	123	EncFtnsH	protein	A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column (GE Healthcare).	FIG
+124	126	MM	experimental_method	A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column (GE Healthcare).	FIG
+144	148	Fe2+	chemical	A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column (GE Healthcare).	FIG
+184	192	EncFtnsH	protein	A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column (GE Healthcare).	FIG
+206	214	EncFtnsH	protein	A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column (GE Healthcare).	FIG
+215	222	monomer	oligomeric_state	A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column (GE Healthcare).	FIG
+266	270	Fe2+	chemical	A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column (GE Healthcare).	FIG
+300	325	analytical gel-filtration	experimental_method	A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column (GE Healthcare).	FIG
+5	9	Fe2+	chemical	Both Fe2+ salts tested induced the formation of decamer indicated by the peak between 1.2 and 1.6 ml.	FIG
+48	55	decamer	oligomeric_state	Both Fe2+ salts tested induced the formation of decamer indicated by the peak between 1.2 and 1.6 ml.	FIG
+0	9	Monomeric	oligomeric_state	Monomeric and decameric samples of EncFtnsH are shown as controls.	FIG
+14	23	decameric	oligomeric_state	Monomeric and decameric samples of EncFtnsH are shown as controls.	FIG
+35	43	EncFtnsH	protein	Monomeric and decameric samples of EncFtnsH are shown as controls.	FIG
+0	5	Peaks	evidence	Peaks around 0.8 ml were seen as protein aggregation.	FIG
+4	29	Analytical gel filtration	experimental_method	(C) Analytical gel filtration of EncFtn monomer at different concentrations to illustrate the effect of protein concentration on multimerization.	FIG
+33	39	EncFtn	protein	(C) Analytical gel filtration of EncFtn monomer at different concentrations to illustrate the effect of protein concentration on multimerization.	FIG
+40	47	monomer	oligomeric_state	(C) Analytical gel filtration of EncFtn monomer at different concentrations to illustrate the effect of protein concentration on multimerization.	FIG
+39	44	dimer	oligomeric_state	The major peak shows a shift towards a dimer species at high concentration of protein, but the ratio of this peak (1.5–1.8 ml) to the decamer peak (1.2–1.5 ml) does not change when compared to the low concentration sample.	FIG
+134	141	decamer	oligomeric_state	The major peak shows a shift towards a dimer species at high concentration of protein, but the ratio of this peak (1.5–1.8 ml) to the decamer peak (1.2–1.5 ml) does not change when compared to the low concentration sample.	FIG
+0	14	Gel-filtration	experimental_method	Gel-filtration peak area ratios for EncFtnsH decamer and monomer on addition of different metal ions.	TABLE
+15	31	peak area ratios	evidence	Gel-filtration peak area ratios for EncFtnsH decamer and monomer on addition of different metal ions.	TABLE
+36	44	EncFtnsH	protein	Gel-filtration peak area ratios for EncFtnsH decamer and monomer on addition of different metal ions.	TABLE
+45	52	decamer	oligomeric_state	Gel-filtration peak area ratios for EncFtnsH decamer and monomer on addition of different metal ions.	TABLE
+57	64	monomer	oligomeric_state	Gel-filtration peak area ratios for EncFtnsH decamer and monomer on addition of different metal ions.	TABLE
+0	8	EncFtnsH	protein	EncFtnsH was produced in E. coli BL21(DE3) cultured in MM and MM with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+) and purified by gel-filtration chromatography using an Superdex 200 16/60 column (GE Healthcare).	TABLE
+25	42	E. coli BL21(DE3)	species	EncFtnsH was produced in E. coli BL21(DE3) cultured in MM and MM with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+) and purified by gel-filtration chromatography using an Superdex 200 16/60 column (GE Healthcare).	TABLE
+55	57	MM	experimental_method	EncFtnsH was produced in E. coli BL21(DE3) cultured in MM and MM with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+) and purified by gel-filtration chromatography using an Superdex 200 16/60 column (GE Healthcare).	TABLE
+62	64	MM	experimental_method	EncFtnsH was produced in E. coli BL21(DE3) cultured in MM and MM with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+) and purified by gel-filtration chromatography using an Superdex 200 16/60 column (GE Healthcare).	TABLE
+75	89	Fe(NH4)2(SO4)2	chemical	EncFtnsH was produced in E. coli BL21(DE3) cultured in MM and MM with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+) and purified by gel-filtration chromatography using an Superdex 200 16/60 column (GE Healthcare).	TABLE
+91	93	MM	experimental_method	EncFtnsH was produced in E. coli BL21(DE3) cultured in MM and MM with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+) and purified by gel-filtration chromatography using an Superdex 200 16/60 column (GE Healthcare).	TABLE
+94	98	Fe2+	chemical	EncFtnsH was produced in E. coli BL21(DE3) cultured in MM and MM with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+) and purified by gel-filtration chromatography using an Superdex 200 16/60 column (GE Healthcare).	TABLE
+116	145	gel-filtration chromatography	experimental_method	EncFtnsH was produced in E. coli BL21(DE3) cultured in MM and MM with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+) and purified by gel-filtration chromatography using an Superdex 200 16/60 column (GE Healthcare).	TABLE
+0	7	Monomer	oligomeric_state	Monomer fractions of EncFtnsH purified from MM were pooled and run in subsequent analytical gel-filtration runs over the course of three days.	TABLE
+21	29	EncFtnsH	protein	Monomer fractions of EncFtnsH purified from MM were pooled and run in subsequent analytical gel-filtration runs over the course of three days.	TABLE
+44	46	MM	experimental_method	Monomer fractions of EncFtnsH purified from MM were pooled and run in subsequent analytical gel-filtration runs over the course of three days.	TABLE
+81	106	analytical gel-filtration	experimental_method	Monomer fractions of EncFtnsH purified from MM were pooled and run in subsequent analytical gel-filtration runs over the course of three days.	TABLE
+11	19	EncFtnsH	protein	Samples of EncFtnsH monomer were incubated with one molar equivalent of metal ion salts at room temperature for two hours before analysis by analytical gel filtration chromatography (AGF) using a Superdex 200 10/300 GL column.	TABLE
+20	27	monomer	oligomeric_state	Samples of EncFtnsH monomer were incubated with one molar equivalent of metal ion salts at room temperature for two hours before analysis by analytical gel filtration chromatography (AGF) using a Superdex 200 10/300 GL column.	TABLE
+141	181	analytical gel filtration chromatography	experimental_method	Samples of EncFtnsH monomer were incubated with one molar equivalent of metal ion salts at room temperature for two hours before analysis by analytical gel filtration chromatography (AGF) using a Superdex 200 10/300 GL column.	TABLE
+183	186	AGF	experimental_method	Samples of EncFtnsH monomer were incubated with one molar equivalent of metal ion salts at room temperature for two hours before analysis by analytical gel filtration chromatography (AGF) using a Superdex 200 10/300 GL column.	TABLE
+31	36	peaks	evidence	The area for resulting protein peaks were calculated using the Unicorn software (GE Healthcare); peak ratios were calculated to quantify the propensity of EncFtnsH to multimerize in the presence of the different metal ions.	TABLE
+97	108	peak ratios	evidence	The area for resulting protein peaks were calculated using the Unicorn software (GE Healthcare); peak ratios were calculated to quantify the propensity of EncFtnsH to multimerize in the presence of the different metal ions.	TABLE
+155	163	EncFtnsH	protein	The area for resulting protein peaks were calculated using the Unicorn software (GE Healthcare); peak ratios were calculated to quantify the propensity of EncFtnsH to multimerize in the presence of the different metal ions.	TABLE
+186	197	presence of	protein_state	The area for resulting protein peaks were calculated using the Unicorn software (GE Healthcare); peak ratios were calculated to quantify the propensity of EncFtnsH to multimerize in the presence of the different metal ions.	TABLE
+28	35	monomer	oligomeric_state	The change in the ratios of monomer to decamer over the three days of experiments may be a consequence of experimental variability, or the propensity of this protein to equilibrate towards decamer over time.	TABLE
+39	46	decamer	oligomeric_state	The change in the ratios of monomer to decamer over the three days of experiments may be a consequence of experimental variability, or the propensity of this protein to equilibrate towards decamer over time.	TABLE
+189	196	decamer	oligomeric_state	The change in the ratios of monomer to decamer over the three days of experiments may be a consequence of experimental variability, or the propensity of this protein to equilibrate towards decamer over time.	TABLE
+14	21	decamer	oligomeric_state	The increased decamer: monomer ratio seen in the presence of Fe2+, Co2+, and Zn2+ indicates that these metal ions facilitate multimerization of the EncFtnsH protein, while the other metal ions tested do not appear to induce multimerization.	TABLE
+23	30	monomer	oligomeric_state	The increased decamer: monomer ratio seen in the presence of Fe2+, Co2+, and Zn2+ indicates that these metal ions facilitate multimerization of the EncFtnsH protein, while the other metal ions tested do not appear to induce multimerization.	TABLE
+49	60	presence of	protein_state	The increased decamer: monomer ratio seen in the presence of Fe2+, Co2+, and Zn2+ indicates that these metal ions facilitate multimerization of the EncFtnsH protein, while the other metal ions tested do not appear to induce multimerization.	TABLE
+61	65	Fe2+	chemical	The increased decamer: monomer ratio seen in the presence of Fe2+, Co2+, and Zn2+ indicates that these metal ions facilitate multimerization of the EncFtnsH protein, while the other metal ions tested do not appear to induce multimerization.	TABLE
+67	71	Co2+	chemical	The increased decamer: monomer ratio seen in the presence of Fe2+, Co2+, and Zn2+ indicates that these metal ions facilitate multimerization of the EncFtnsH protein, while the other metal ions tested do not appear to induce multimerization.	TABLE
+77	81	Zn2+	chemical	The increased decamer: monomer ratio seen in the presence of Fe2+, Co2+, and Zn2+ indicates that these metal ions facilitate multimerization of the EncFtnsH protein, while the other metal ions tested do not appear to induce multimerization.	TABLE
+148	156	EncFtnsH	protein	The increased decamer: monomer ratio seen in the presence of Fe2+, Co2+, and Zn2+ indicates that these metal ions facilitate multimerization of the EncFtnsH protein, while the other metal ions tested do not appear to induce multimerization.	TABLE
+4	29	analytical gel filtration	experimental_method	The analytical gel filtration experiment was repeated twice using two independent preparations of protein, of which values calculated from one sample are presented here.	TABLE
+14	21	Monomer	oligomeric_state	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+27	34	Decamer	oligomeric_state	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+40	47	Decamer	oligomeric_state	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+48	55	Monomer	oligomeric_state	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+58	72	Gel filtration	experimental_method	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+101	109	EncFtnsH	protein	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+110	112	MM	experimental_method	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+130	138	EncFtnsH	protein	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+139	141	MM	experimental_method	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+142	146	Fe2+	chemical	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+166	191	Analytical Gel filtration	experimental_method	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+197	205	EncFtnsH	protein	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+206	213	decamer	oligomeric_state	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+240	248	EncFtnsH	protein	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+249	256	monomer	oligomeric_state	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+282	296	Fe(NH4)2(SO4)2	chemical	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+297	305	EncFtnsH	protein	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+306	313	monomer	oligomeric_state	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+330	339	FeSO4-HCl	chemical	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+340	348	EncFtnsH	protein	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+349	356	monomer	oligomeric_state	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+373	398	Analytical Gel filtration	experimental_method	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+404	412	EncFtnsH	protein	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+413	420	monomer	oligomeric_state	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+446	451	CoCl2	chemical	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+452	460	EncFtnsH	protein	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+461	468	monomer	oligomeric_state	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+485	490	MnCl2	chemical	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+491	499	EncFtnsH	protein	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+500	507	monomer	oligomeric_state	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+523	528	ZnSO4	chemical	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+529	537	EncFtnsH	protein	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+538	545	monomer	oligomeric_state	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+561	566	FeCl3	chemical	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+567	575	EncFtnsH	protein	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+576	583	monomer	oligomeric_state	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+599	624	Analytical Gel filtration	experimental_method	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+630	638	EncFtnsH	protein	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+639	646	monomer	oligomeric_state	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+672	677	MgSO4	chemical	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+678	686	EncFtnsH	protein	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+687	694	monomer	oligomeric_state	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+710	720	Ca acetate	chemical	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+721	729	EncFtnsH	protein	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+730	737	monomer	oligomeric_state	"Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	"	TABLE
+12	20	EncFtnsH	protein	We purified EncFtnsH from E. coli grown in MM with or without the addition of 1 mM Fe(NH4)2(SO4)2.	RESULTS
+26	33	E. coli	species	We purified EncFtnsH from E. coli grown in MM with or without the addition of 1 mM Fe(NH4)2(SO4)2.	RESULTS
+43	45	MM	experimental_method	We purified EncFtnsH from E. coli grown in MM with or without the addition of 1 mM Fe(NH4)2(SO4)2.	RESULTS
+83	97	Fe(NH4)2(SO4)2	chemical	We purified EncFtnsH from E. coli grown in MM with or without the addition of 1 mM Fe(NH4)2(SO4)2.	RESULTS
+4	11	decamer	oligomeric_state	The decamer to monomer ratio in the sample purified from cells grown in iron-supplemented media was 4.5, while that from the iron-free media was 0.11, suggesting that iron induces the oligomerization of EncFtnsH in vivo (Figure 3A, Table 3).	RESULTS
+15	22	monomer	oligomeric_state	The decamer to monomer ratio in the sample purified from cells grown in iron-supplemented media was 4.5, while that from the iron-free media was 0.11, suggesting that iron induces the oligomerization of EncFtnsH in vivo (Figure 3A, Table 3).	RESULTS
+72	76	iron	chemical	The decamer to monomer ratio in the sample purified from cells grown in iron-supplemented media was 4.5, while that from the iron-free media was 0.11, suggesting that iron induces the oligomerization of EncFtnsH in vivo (Figure 3A, Table 3).	RESULTS
+125	134	iron-free	protein_state	The decamer to monomer ratio in the sample purified from cells grown in iron-supplemented media was 4.5, while that from the iron-free media was 0.11, suggesting that iron induces the oligomerization of EncFtnsH in vivo (Figure 3A, Table 3).	RESULTS
+167	171	iron	chemical	The decamer to monomer ratio in the sample purified from cells grown in iron-supplemented media was 4.5, while that from the iron-free media was 0.11, suggesting that iron induces the oligomerization of EncFtnsH in vivo (Figure 3A, Table 3).	RESULTS
+203	211	EncFtnsH	protein	The decamer to monomer ratio in the sample purified from cells grown in iron-supplemented media was 4.5, while that from the iron-free media was 0.11, suggesting that iron induces the oligomerization of EncFtnsH in vivo (Figure 3A, Table 3).	RESULTS
+47	55	EncFtnsH	protein	To test the metal-dependent oligomerization of EncFtnsH in vitro, we incubated the protein with various metal cations and subjected samples to analytical SEC and non-denaturing PAGE.	RESULTS
+69	78	incubated	experimental_method	To test the metal-dependent oligomerization of EncFtnsH in vitro, we incubated the protein with various metal cations and subjected samples to analytical SEC and non-denaturing PAGE.	RESULTS
+143	157	analytical SEC	experimental_method	To test the metal-dependent oligomerization of EncFtnsH in vitro, we incubated the protein with various metal cations and subjected samples to analytical SEC and non-denaturing PAGE.	RESULTS
+162	181	non-denaturing PAGE	experimental_method	To test the metal-dependent oligomerization of EncFtnsH in vitro, we incubated the protein with various metal cations and subjected samples to analytical SEC and non-denaturing PAGE.	RESULTS
+27	32	Fe2+,	chemical	Of the metals tested, only Fe2+, Zn2+ and Co2+ induced the formation of significant amounts of the decamer (Figure 3B, Figure 3—figure supplement 1/2).	RESULTS
+33	37	Zn2+	chemical	Of the metals tested, only Fe2+, Zn2+ and Co2+ induced the formation of significant amounts of the decamer (Figure 3B, Figure 3—figure supplement 1/2).	RESULTS
+42	46	Co2+	chemical	Of the metals tested, only Fe2+, Zn2+ and Co2+ induced the formation of significant amounts of the decamer (Figure 3B, Figure 3—figure supplement 1/2).	RESULTS
+99	106	decamer	oligomeric_state	Of the metals tested, only Fe2+, Zn2+ and Co2+ induced the formation of significant amounts of the decamer (Figure 3B, Figure 3—figure supplement 1/2).	RESULTS
+6	10	Fe2+	chemical	While Fe2+ induces the multimerization of EncFtnsH, Fe3+ in the form of FeCl3 does not have this effect on the protein, highlighting the apparent preference this protein has for the ferrous form of iron.	RESULTS
+42	50	EncFtnsH	protein	While Fe2+ induces the multimerization of EncFtnsH, Fe3+ in the form of FeCl3 does not have this effect on the protein, highlighting the apparent preference this protein has for the ferrous form of iron.	RESULTS
+52	56	Fe3+	chemical	While Fe2+ induces the multimerization of EncFtnsH, Fe3+ in the form of FeCl3 does not have this effect on the protein, highlighting the apparent preference this protein has for the ferrous form of iron.	RESULTS
+72	77	FeCl3	chemical	While Fe2+ induces the multimerization of EncFtnsH, Fe3+ in the form of FeCl3 does not have this effect on the protein, highlighting the apparent preference this protein has for the ferrous form of iron.	RESULTS
+182	202	ferrous form of iron	chemical	While Fe2+ induces the multimerization of EncFtnsH, Fe3+ in the form of FeCl3 does not have this effect on the protein, highlighting the apparent preference this protein has for the ferrous form of iron.	RESULTS
+39	47	EncFtnsH	protein	To determine if the oligomerization of EncFtnsH was concentration dependent we performed analytical SEC at 90 and 700 µM protein concentration (Figure 3C).	RESULTS
+89	103	analytical SEC	experimental_method	To determine if the oligomerization of EncFtnsH was concentration dependent we performed analytical SEC at 90 and 700 µM protein concentration (Figure 3C).	RESULTS
+48	57	decameric	oligomeric_state	At the higher concentration, no increase in the decameric form of EncFtn was observed; however, the shift in the major peak from the position of the monomer species indicated a tendency to dimerize at high concentration.	RESULTS
+66	72	EncFtn	protein	At the higher concentration, no increase in the decameric form of EncFtn was observed; however, the shift in the major peak from the position of the monomer species indicated a tendency to dimerize at high concentration.	RESULTS
+149	156	monomer	oligomeric_state	At the higher concentration, no increase in the decameric form of EncFtn was observed; however, the shift in the major peak from the position of the monomer species indicated a tendency to dimerize at high concentration.	RESULTS
+189	197	dimerize	oligomeric_state	At the higher concentration, no increase in the decameric form of EncFtn was observed; however, the shift in the major peak from the position of the monomer species indicated a tendency to dimerize at high concentration.	RESULTS
+0	17	Crystal structure	evidence	Crystal structure of EncFtnsH	RESULTS
+21	29	EncFtnsH	protein	Crystal structure of EncFtnsH	RESULTS
+25	33	EncFtnsH	protein	Electrostatic surface of EncFtnsH.	FIG
+34	42	EncFtnsH	protein	The solvent accessible surface of EncFtnsH is shown, colored by electrostatic potential as calculated using the APBS plugin in PyMOL.	FIG
+129	137	EncFtnsH	protein	Negatively charged regions are colored red and positive regions in blue, neutral regions in grey. (A) View of the surface of the EncFtnsH decamer looking down the central axis.	FIG
+138	145	decamer	oligomeric_state	Negatively charged regions are colored red and positive regions in blue, neutral regions in grey. (A) View of the surface of the EncFtnsH decamer looking down the central axis.	FIG
+95	109	central cavity	site	(B) Orthogonal view of (A). (C) Cutaway view of (B) showing the charge distribution within the central cavity.	FIG
+0	17	Crystal structure	evidence	Crystal structure of EncFtnsH.	FIG
+21	29	EncFtnsH	protein	Crystal structure of EncFtnsH.	FIG
+28	36	EncFtnsH	protein	(A) Overall architecture of EncFtnsH. Transparent solvent accessible surface view with α-helices shown as tubes and bound metal ions as spheres.	FIG
+87	96	α-helices	structure_element	(A) Overall architecture of EncFtnsH. Transparent solvent accessible surface view with α-helices shown as tubes and bound metal ions as spheres.	FIG
+12	20	subunits	structure_element	Alternating subunits are colored blue and green for clarity.	FIG
+4	17	doughnut-like	structure_element	The doughnut-like decamer is 7 nm in diameter and 4.5 nm thick. (B) Monomer of EncFtnsH shown as a secondary structure cartoon. (C/D) Dimer interfaces formed in the decameric ring of EncFtnsH. Subunits are shown as secondary structure cartoons and colored blue and green for clarity.	FIG
+18	25	decamer	oligomeric_state	The doughnut-like decamer is 7 nm in diameter and 4.5 nm thick. (B) Monomer of EncFtnsH shown as a secondary structure cartoon. (C/D) Dimer interfaces formed in the decameric ring of EncFtnsH. Subunits are shown as secondary structure cartoons and colored blue and green for clarity.	FIG
+68	75	Monomer	oligomeric_state	The doughnut-like decamer is 7 nm in diameter and 4.5 nm thick. (B) Monomer of EncFtnsH shown as a secondary structure cartoon. (C/D) Dimer interfaces formed in the decameric ring of EncFtnsH. Subunits are shown as secondary structure cartoons and colored blue and green for clarity.	FIG
+79	87	EncFtnsH	protein	The doughnut-like decamer is 7 nm in diameter and 4.5 nm thick. (B) Monomer of EncFtnsH shown as a secondary structure cartoon. (C/D) Dimer interfaces formed in the decameric ring of EncFtnsH. Subunits are shown as secondary structure cartoons and colored blue and green for clarity.	FIG
+134	150	Dimer interfaces	site	The doughnut-like decamer is 7 nm in diameter and 4.5 nm thick. (B) Monomer of EncFtnsH shown as a secondary structure cartoon. (C/D) Dimer interfaces formed in the decameric ring of EncFtnsH. Subunits are shown as secondary structure cartoons and colored blue and green for clarity.	FIG
+165	174	decameric	oligomeric_state	The doughnut-like decamer is 7 nm in diameter and 4.5 nm thick. (B) Monomer of EncFtnsH shown as a secondary structure cartoon. (C/D) Dimer interfaces formed in the decameric ring of EncFtnsH. Subunits are shown as secondary structure cartoons and colored blue and green for clarity.	FIG
+175	179	ring	structure_element	The doughnut-like decamer is 7 nm in diameter and 4.5 nm thick. (B) Monomer of EncFtnsH shown as a secondary structure cartoon. (C/D) Dimer interfaces formed in the decameric ring of EncFtnsH. Subunits are shown as secondary structure cartoons and colored blue and green for clarity.	FIG
+183	191	EncFtnsH	protein	The doughnut-like decamer is 7 nm in diameter and 4.5 nm thick. (B) Monomer of EncFtnsH shown as a secondary structure cartoon. (C/D) Dimer interfaces formed in the decameric ring of EncFtnsH. Subunits are shown as secondary structure cartoons and colored blue and green for clarity.	FIG
+193	201	Subunits	structure_element	The doughnut-like decamer is 7 nm in diameter and 4.5 nm thick. (B) Monomer of EncFtnsH shown as a secondary structure cartoon. (C/D) Dimer interfaces formed in the decameric ring of EncFtnsH. Subunits are shown as secondary structure cartoons and colored blue and green for clarity.	FIG
+49	53	Fe3+	chemical	Bound metal ions are shown as orange spheres for Fe3+ and grey and white spheres for Ca2+.	FIG
+85	89	Ca2+	chemical	Bound metal ions are shown as orange spheres for Fe3+ and grey and white spheres for Ca2+.	FIG
+18	35	crystal structure	evidence	We determined the crystal structure of EncFtnsH by molecular replacement to 2.0 Å resolution (see Table 1 for X-ray data collection and refinement statistics).	RESULTS
+39	47	EncFtnsH	protein	We determined the crystal structure of EncFtnsH by molecular replacement to 2.0 Å resolution (see Table 1 for X-ray data collection and refinement statistics).	RESULTS
+51	72	molecular replacement	experimental_method	We determined the crystal structure of EncFtnsH by molecular replacement to 2.0 Å resolution (see Table 1 for X-ray data collection and refinement statistics).	RESULTS
+110	157	X-ray data collection and refinement statistics	evidence	We determined the crystal structure of EncFtnsH by molecular replacement to 2.0 Å resolution (see Table 1 for X-ray data collection and refinement statistics).	RESULTS
+54	62	monomers	oligomeric_state	The crystallographic asymmetric unit contained thirty monomers of EncFtn with visible electron density for residues 7 – 96 in each chain.	RESULTS
+66	72	EncFtn	protein	The crystallographic asymmetric unit contained thirty monomers of EncFtn with visible electron density for residues 7 – 96 in each chain.	RESULTS
+86	102	electron density	evidence	The crystallographic asymmetric unit contained thirty monomers of EncFtn with visible electron density for residues 7 – 96 in each chain.	RESULTS
+116	122	7 – 96	residue_range	The crystallographic asymmetric unit contained thirty monomers of EncFtn with visible electron density for residues 7 – 96 in each chain.	RESULTS
+52	59	annular	structure_element	The protein chains were arranged as three identical annular decamers, each with D5 symmetry.	RESULTS
+60	68	decamers	oligomeric_state	The protein chains were arranged as three identical annular decamers, each with D5 symmetry.	RESULTS
+4	11	decamer	oligomeric_state	The decamer has a diameter of 7 nm and thickness of 4 nm (Figure 4A).	RESULTS
+4	11	monomer	oligomeric_state	The monomer of EncFtn has an N-terminal 310-helix that precedes two 4 nm long antiparallel α-helices arranged with their long axes at 25° to each other; these helices are followed by a shorter 1.4 nm helix projecting at 70° from α2 (Figure 4B).	RESULTS
+15	21	EncFtn	protein	The monomer of EncFtn has an N-terminal 310-helix that precedes two 4 nm long antiparallel α-helices arranged with their long axes at 25° to each other; these helices are followed by a shorter 1.4 nm helix projecting at 70° from α2 (Figure 4B).	RESULTS
+40	49	310-helix	structure_element	The monomer of EncFtn has an N-terminal 310-helix that precedes two 4 nm long antiparallel α-helices arranged with their long axes at 25° to each other; these helices are followed by a shorter 1.4 nm helix projecting at 70° from α2 (Figure 4B).	RESULTS
+78	100	antiparallel α-helices	structure_element	The monomer of EncFtn has an N-terminal 310-helix that precedes two 4 nm long antiparallel α-helices arranged with their long axes at 25° to each other; these helices are followed by a shorter 1.4 nm helix projecting at 70° from α2 (Figure 4B).	RESULTS
+159	166	helices	structure_element	The monomer of EncFtn has an N-terminal 310-helix that precedes two 4 nm long antiparallel α-helices arranged with their long axes at 25° to each other; these helices are followed by a shorter 1.4 nm helix projecting at 70° from α2 (Figure 4B).	RESULTS
+200	205	helix	structure_element	The monomer of EncFtn has an N-terminal 310-helix that precedes two 4 nm long antiparallel α-helices arranged with their long axes at 25° to each other; these helices are followed by a shorter 1.4 nm helix projecting at 70° from α2 (Figure 4B).	RESULTS
+229	231	α2	structure_element	The monomer of EncFtn has an N-terminal 310-helix that precedes two 4 nm long antiparallel α-helices arranged with their long axes at 25° to each other; these helices are followed by a shorter 1.4 nm helix projecting at 70° from α2 (Figure 4B).	RESULTS
+4	21	C-terminal region	structure_element	The C-terminal region of the crystallized construct extends from the outer circumference of the ring, indicating that the encapsulin localization sequence in the full-length protein is on the exterior of the ring and is thus free to interact with its binding site on the encapsulin shell protein.	RESULTS
+96	100	ring	structure_element	The C-terminal region of the crystallized construct extends from the outer circumference of the ring, indicating that the encapsulin localization sequence in the full-length protein is on the exterior of the ring and is thus free to interact with its binding site on the encapsulin shell protein.	RESULTS
+122	154	encapsulin localization sequence	site	The C-terminal region of the crystallized construct extends from the outer circumference of the ring, indicating that the encapsulin localization sequence in the full-length protein is on the exterior of the ring and is thus free to interact with its binding site on the encapsulin shell protein.	RESULTS
+162	173	full-length	protein_state	The C-terminal region of the crystallized construct extends from the outer circumference of the ring, indicating that the encapsulin localization sequence in the full-length protein is on the exterior of the ring and is thus free to interact with its binding site on the encapsulin shell protein.	RESULTS
+208	212	ring	structure_element	The C-terminal region of the crystallized construct extends from the outer circumference of the ring, indicating that the encapsulin localization sequence in the full-length protein is on the exterior of the ring and is thus free to interact with its binding site on the encapsulin shell protein.	RESULTS
+251	263	binding site	site	The C-terminal region of the crystallized construct extends from the outer circumference of the ring, indicating that the encapsulin localization sequence in the full-length protein is on the exterior of the ring and is thus free to interact with its binding site on the encapsulin shell protein.	RESULTS
+271	281	encapsulin	protein	The C-terminal region of the crystallized construct extends from the outer circumference of the ring, indicating that the encapsulin localization sequence in the full-length protein is on the exterior of the ring and is thus free to interact with its binding site on the encapsulin shell protein.	RESULTS
+282	287	shell	structure_element	The C-terminal region of the crystallized construct extends from the outer circumference of the ring, indicating that the encapsulin localization sequence in the full-length protein is on the exterior of the ring and is thus free to interact with its binding site on the encapsulin shell protein.	RESULTS
+4	11	monomer	oligomeric_state	The monomer of EncFtnsH forms two distinct dimer interfaces within the decamer (Figure 4 C/D).	RESULTS
+15	23	EncFtnsH	protein	The monomer of EncFtnsH forms two distinct dimer interfaces within the decamer (Figure 4 C/D).	RESULTS
+43	59	dimer interfaces	site	The monomer of EncFtnsH forms two distinct dimer interfaces within the decamer (Figure 4 C/D).	RESULTS
+71	78	decamer	oligomeric_state	The monomer of EncFtnsH forms two distinct dimer interfaces within the decamer (Figure 4 C/D).	RESULTS
+10	15	dimer	oligomeric_state	The first dimer is formed from two monomers arranged antiparallel to each other, with α1 from each monomer interacting along their lengths and α3 interdigitating with α2 and α3 of the partner chain.	RESULTS
+35	43	monomers	oligomeric_state	The first dimer is formed from two monomers arranged antiparallel to each other, with α1 from each monomer interacting along their lengths and α3 interdigitating with α2 and α3 of the partner chain.	RESULTS
+86	88	α1	structure_element	The first dimer is formed from two monomers arranged antiparallel to each other, with α1 from each monomer interacting along their lengths and α3 interdigitating with α2 and α3 of the partner chain.	RESULTS
+99	106	monomer	oligomeric_state	The first dimer is formed from two monomers arranged antiparallel to each other, with α1 from each monomer interacting along their lengths and α3 interdigitating with α2 and α3 of the partner chain.	RESULTS
+143	145	α3	structure_element	The first dimer is formed from two monomers arranged antiparallel to each other, with α1 from each monomer interacting along their lengths and α3 interdigitating with α2 and α3 of the partner chain.	RESULTS
+167	169	α2	structure_element	The first dimer is formed from two monomers arranged antiparallel to each other, with α1 from each monomer interacting along their lengths and α3 interdigitating with α2 and α3 of the partner chain.	RESULTS
+174	176	α3	structure_element	The first dimer is formed from two monomers arranged antiparallel to each other, with α1 from each monomer interacting along their lengths and α3 interdigitating with α2 and α3 of the partner chain.	RESULTS
+5	14	interface	site	This interface buries one third of the surface area from each partner and is stabilized by thirty hydrogen bonds and fourteen salt bridges (Figure 4C).	RESULTS
+98	112	hydrogen bonds	bond_interaction	This interface buries one third of the surface area from each partner and is stabilized by thirty hydrogen bonds and fourteen salt bridges (Figure 4C).	RESULTS
+126	138	salt bridges	bond_interaction	This interface buries one third of the surface area from each partner and is stabilized by thirty hydrogen bonds and fourteen salt bridges (Figure 4C).	RESULTS
+11	26	dimer interface	site	The second dimer interface forms an antiparallel four-helix bundle between helices 1 and 2 from each monomer (Figure 4D).	RESULTS
+36	66	antiparallel four-helix bundle	structure_element	The second dimer interface forms an antiparallel four-helix bundle between helices 1 and 2 from each monomer (Figure 4D).	RESULTS
+75	90	helices 1 and 2	structure_element	The second dimer interface forms an antiparallel four-helix bundle between helices 1 and 2 from each monomer (Figure 4D).	RESULTS
+101	108	monomer	oligomeric_state	The second dimer interface forms an antiparallel four-helix bundle between helices 1 and 2 from each monomer (Figure 4D).	RESULTS
+5	14	interface	site	This interface is less extensive than the first and is stabilized by twenty-one hydrogen bonds, six salt bridges, and a number of metal ions.	RESULTS
+80	94	hydrogen bonds	bond_interaction	This interface is less extensive than the first and is stabilized by twenty-one hydrogen bonds, six salt bridges, and a number of metal ions.	RESULTS
+100	112	salt bridges	bond_interaction	This interface is less extensive than the first and is stabilized by twenty-one hydrogen bonds, six salt bridges, and a number of metal ions.	RESULTS
+23	31	monomers	oligomeric_state	The arrangement of ten monomers in alternating orientation forms the decamer of EncFtn, which assembles as a pentamer of dimers (Figure 4A).	RESULTS
+69	76	decamer	oligomeric_state	The arrangement of ten monomers in alternating orientation forms the decamer of EncFtn, which assembles as a pentamer of dimers (Figure 4A).	RESULTS
+80	86	EncFtn	protein	The arrangement of ten monomers in alternating orientation forms the decamer of EncFtn, which assembles as a pentamer of dimers (Figure 4A).	RESULTS
+109	117	pentamer	oligomeric_state	The arrangement of ten monomers in alternating orientation forms the decamer of EncFtn, which assembles as a pentamer of dimers (Figure 4A).	RESULTS
+121	127	dimers	oligomeric_state	The arrangement of ten monomers in alternating orientation forms the decamer of EncFtn, which assembles as a pentamer of dimers (Figure 4A).	RESULTS
+5	12	monomer	oligomeric_state	Each monomer lies at 45° relative to the vertical central-axis of the ring, with the N-termini of alternating subunits capping the center of the ring at each end, while the C-termini are arranged around the circumference.	RESULTS
+70	74	ring	structure_element	Each monomer lies at 45° relative to the vertical central-axis of the ring, with the N-termini of alternating subunits capping the center of the ring at each end, while the C-termini are arranged around the circumference.	RESULTS
+110	118	subunits	structure_element	Each monomer lies at 45° relative to the vertical central-axis of the ring, with the N-termini of alternating subunits capping the center of the ring at each end, while the C-termini are arranged around the circumference.	RESULTS
+145	149	ring	structure_element	Each monomer lies at 45° relative to the vertical central-axis of the ring, with the N-termini of alternating subunits capping the center of the ring at each end, while the C-termini are arranged around the circumference.	RESULTS
+4	16	central hole	site	The central hole in the ring is 2.5 nm at its widest in the center of the complex, and 1.5 nm at its narrowest point near the outer surface, although it should be noted that a number of residues at the N-terminus are not visible in the crystallographic electron density and these may occupy the central channel.	RESULTS
+24	28	ring	structure_element	The central hole in the ring is 2.5 nm at its widest in the center of the complex, and 1.5 nm at its narrowest point near the outer surface, although it should be noted that a number of residues at the N-terminus are not visible in the crystallographic electron density and these may occupy the central channel.	RESULTS
+236	269	crystallographic electron density	evidence	The central hole in the ring is 2.5 nm at its widest in the center of the complex, and 1.5 nm at its narrowest point near the outer surface, although it should be noted that a number of residues at the N-terminus are not visible in the crystallographic electron density and these may occupy the central channel.	RESULTS
+295	310	central channel	site	The central hole in the ring is 2.5 nm at its widest in the center of the complex, and 1.5 nm at its narrowest point near the outer surface, although it should be noted that a number of residues at the N-terminus are not visible in the crystallographic electron density and these may occupy the central channel.	RESULTS
+19	26	decamer	oligomeric_state	The surface of the decamer has distinct negatively charged patches, both within the central hole and on the outer circumference, which form spokes through the radius of the complex (Figure 4—figure supplement 1).	RESULTS
+40	66	negatively charged patches	site	The surface of the decamer has distinct negatively charged patches, both within the central hole and on the outer circumference, which form spokes through the radius of the complex (Figure 4—figure supplement 1).	RESULTS
+84	96	central hole	site	The surface of the decamer has distinct negatively charged patches, both within the central hole and on the outer circumference, which form spokes through the radius of the complex (Figure 4—figure supplement 1).	RESULTS
+140	146	spokes	structure_element	The surface of the decamer has distinct negatively charged patches, both within the central hole and on the outer circumference, which form spokes through the radius of the complex (Figure 4—figure supplement 1).	RESULTS
+0	6	EncFtn	protein	EncFtn ferroxidase center	RESULTS
+7	25	ferroxidase center	site	EncFtn ferroxidase center	RESULTS
+9	28	ligand-binding site	site	Putative ligand-binding site in EncFtnsH.	FIG
+32	40	EncFtnsH	protein	Putative ligand-binding site in EncFtnsH.	FIG
+33	48	dimer interface	site	(A) Wall-eyed stereo view of the dimer interface of EncFtn.	FIG
+52	58	EncFtn	protein	(A) Wall-eyed stereo view of the dimer interface of EncFtn.	FIG
+41	66	2mFo-DFc electron density	evidence	Protein chains are shown as sticks, with 2mFo-DFc electron density shown in blue mesh and contoured at 1.5 σ and mFo-DFc shown in green mesh and contoured at 3 σ. (B) Wall-eyed stereo view of putative metal binding site at the external surface of EncFtnsH. Protein chains and electron density maps are shown as in (A).	FIG
+113	120	mFo-DFc	evidence	Protein chains are shown as sticks, with 2mFo-DFc electron density shown in blue mesh and contoured at 1.5 σ and mFo-DFc shown in green mesh and contoured at 3 σ. (B) Wall-eyed stereo view of putative metal binding site at the external surface of EncFtnsH. Protein chains and electron density maps are shown as in (A).	FIG
+201	219	metal binding site	site	Protein chains are shown as sticks, with 2mFo-DFc electron density shown in blue mesh and contoured at 1.5 σ and mFo-DFc shown in green mesh and contoured at 3 σ. (B) Wall-eyed stereo view of putative metal binding site at the external surface of EncFtnsH. Protein chains and electron density maps are shown as in (A).	FIG
+247	255	EncFtnsH	protein	Protein chains are shown as sticks, with 2mFo-DFc electron density shown in blue mesh and contoured at 1.5 σ and mFo-DFc shown in green mesh and contoured at 3 σ. (B) Wall-eyed stereo view of putative metal binding site at the external surface of EncFtnsH. Protein chains and electron density maps are shown as in (A).	FIG
+276	297	electron density maps	evidence	Protein chains are shown as sticks, with 2mFo-DFc electron density shown in blue mesh and contoured at 1.5 σ and mFo-DFc shown in green mesh and contoured at 3 σ. (B) Wall-eyed stereo view of putative metal binding site at the external surface of EncFtnsH. Protein chains and electron density maps are shown as in (A).	FIG
+0	8	EncFtnsH	protein	EncFtnsH metal binding sites.	FIG
+9	28	metal binding sites	site	EncFtnsH metal binding sites.	FIG
+33	69	metal-binding dimerization interface	site	(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH. Protein residues are shown as sticks with blue and green carbons for the different subunits, iron ions are shown as orange spheres and calcium as grey spheres, and the glycolic acid ligand is shown with yellow carbon atoms coordinated above the di-iron center.	FIG
+73	81	EncFtnsH	protein	(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH. Protein residues are shown as sticks with blue and green carbons for the different subunits, iron ions are shown as orange spheres and calcium as grey spheres, and the glycolic acid ligand is shown with yellow carbon atoms coordinated above the di-iron center.	FIG
+166	174	subunits	structure_element	(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH. Protein residues are shown as sticks with blue and green carbons for the different subunits, iron ions are shown as orange spheres and calcium as grey spheres, and the glycolic acid ligand is shown with yellow carbon atoms coordinated above the di-iron center.	FIG
+176	180	iron	chemical	(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH. Protein residues are shown as sticks with blue and green carbons for the different subunits, iron ions are shown as orange spheres and calcium as grey spheres, and the glycolic acid ligand is shown with yellow carbon atoms coordinated above the di-iron center.	FIG
+218	225	calcium	chemical	(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH. Protein residues are shown as sticks with blue and green carbons for the different subunits, iron ions are shown as orange spheres and calcium as grey spheres, and the glycolic acid ligand is shown with yellow carbon atoms coordinated above the di-iron center.	FIG
+251	264	glycolic acid	chemical	(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH. Protein residues are shown as sticks with blue and green carbons for the different subunits, iron ions are shown as orange spheres and calcium as grey spheres, and the glycolic acid ligand is shown with yellow carbon atoms coordinated above the di-iron center.	FIG
+328	342	di-iron center	site	(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH. Protein residues are shown as sticks with blue and green carbons for the different subunits, iron ions are shown as orange spheres and calcium as grey spheres, and the glycolic acid ligand is shown with yellow carbon atoms coordinated above the di-iron center.	FIG
+4	33	2mFo-DFc electron density map	evidence	The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ and the NCS-averaged anomalous difference map is shown as an orange mesh and contoured at 10 σ. (B) Iron coordination within the FOC including residues Glu32, Glu62, His65 and Tyr39 from two chains.	FIG
+85	122	NCS-averaged anomalous difference map	evidence	The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ and the NCS-averaged anomalous difference map is shown as an orange mesh and contoured at 10 σ. (B) Iron coordination within the FOC including residues Glu32, Glu62, His65 and Tyr39 from two chains.	FIG
+177	181	Iron	chemical	The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ and the NCS-averaged anomalous difference map is shown as an orange mesh and contoured at 10 σ. (B) Iron coordination within the FOC including residues Glu32, Glu62, His65 and Tyr39 from two chains.	FIG
+182	194	coordination	bond_interaction	The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ and the NCS-averaged anomalous difference map is shown as an orange mesh and contoured at 10 σ. (B) Iron coordination within the FOC including residues Glu32, Glu62, His65 and Tyr39 from two chains.	FIG
+206	209	FOC	site	The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ and the NCS-averaged anomalous difference map is shown as an orange mesh and contoured at 10 σ. (B) Iron coordination within the FOC including residues Glu32, Glu62, His65 and Tyr39 from two chains.	FIG
+229	234	Glu32	residue_name_number	The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ and the NCS-averaged anomalous difference map is shown as an orange mesh and contoured at 10 σ. (B) Iron coordination within the FOC including residues Glu32, Glu62, His65 and Tyr39 from two chains.	FIG
+236	241	Glu62	residue_name_number	The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ and the NCS-averaged anomalous difference map is shown as an orange mesh and contoured at 10 σ. (B) Iron coordination within the FOC including residues Glu32, Glu62, His65 and Tyr39 from two chains.	FIG
+243	248	His65	residue_name_number	The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ and the NCS-averaged anomalous difference map is shown as an orange mesh and contoured at 10 σ. (B) Iron coordination within the FOC including residues Glu32, Glu62, His65 and Tyr39 from two chains.	FIG
+253	258	Tyr39	residue_name_number	The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ and the NCS-averaged anomalous difference map is shown as an orange mesh and contoured at 10 σ. (B) Iron coordination within the FOC including residues Glu32, Glu62, His65 and Tyr39 from two chains.	FIG
+42	54	Coordination	bond_interaction	Protein and metal ions are shown as in A. Coordination between the protein and iron ions is shown as yellow dashed lines with distances indicated. (C) Coordination of calcium within the dimer interface by four glutamic acid residues (E31 and E34 from two chains).	FIG
+79	83	iron	chemical	Protein and metal ions are shown as in A. Coordination between the protein and iron ions is shown as yellow dashed lines with distances indicated. (C) Coordination of calcium within the dimer interface by four glutamic acid residues (E31 and E34 from two chains).	FIG
+151	163	Coordination	bond_interaction	Protein and metal ions are shown as in A. Coordination between the protein and iron ions is shown as yellow dashed lines with distances indicated. (C) Coordination of calcium within the dimer interface by four glutamic acid residues (E31 and E34 from two chains).	FIG
+167	174	calcium	chemical	Protein and metal ions are shown as in A. Coordination between the protein and iron ions is shown as yellow dashed lines with distances indicated. (C) Coordination of calcium within the dimer interface by four glutamic acid residues (E31 and E34 from two chains).	FIG
+186	201	dimer interface	site	Protein and metal ions are shown as in A. Coordination between the protein and iron ions is shown as yellow dashed lines with distances indicated. (C) Coordination of calcium within the dimer interface by four glutamic acid residues (E31 and E34 from two chains).	FIG
+210	223	glutamic acid	residue_name	Protein and metal ions are shown as in A. Coordination between the protein and iron ions is shown as yellow dashed lines with distances indicated. (C) Coordination of calcium within the dimer interface by four glutamic acid residues (E31 and E34 from two chains).	FIG
+234	237	E31	residue_name_number	Protein and metal ions are shown as in A. Coordination between the protein and iron ions is shown as yellow dashed lines with distances indicated. (C) Coordination of calcium within the dimer interface by four glutamic acid residues (E31 and E34 from two chains).	FIG
+242	245	E34	residue_name_number	Protein and metal ions are shown as in A. Coordination between the protein and iron ions is shown as yellow dashed lines with distances indicated. (C) Coordination of calcium within the dimer interface by four glutamic acid residues (E31 and E34 from two chains).	FIG
+4	11	calcium	chemical	The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.	FIG
+46	51	water	chemical	The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.	FIG
+78	90	coordination	bond_interaction	The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.	FIG
+98	105	calcium	chemical	The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.	FIG
+136	159	Metal coordination site	site	The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.	FIG
+184	192	EncFtnsH	protein	The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.	FIG
+202	209	calcium	chemical	The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.	FIG
+219	233	coordinated by	bond_interaction	The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.	FIG
+243	248	His57	residue_name_number	The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.	FIG
+250	255	Glu61	residue_name_number	The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.	FIG
+260	265	Glu64	residue_name_number	The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.	FIG
+293	296	FOC	site	The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.	FIG
+297	302	dimer	oligomeric_state	The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.	FIG
+387	390	FOC	site	The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.	FIG
+391	395	iron	chemical	The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.	FIG
+4	25	electron density maps	evidence	The electron density maps of the initial EncFtnsH model displayed significant positive peaks in the mFo-DFc map at the center of the 4-helix bundle dimer (Figure 5—figure supplement 1).	RESULTS
+41	49	EncFtnsH	protein	The electron density maps of the initial EncFtnsH model displayed significant positive peaks in the mFo-DFc map at the center of the 4-helix bundle dimer (Figure 5—figure supplement 1).	RESULTS
+100	111	mFo-DFc map	evidence	The electron density maps of the initial EncFtnsH model displayed significant positive peaks in the mFo-DFc map at the center of the 4-helix bundle dimer (Figure 5—figure supplement 1).	RESULTS
+133	147	4-helix bundle	structure_element	The electron density maps of the initial EncFtnsH model displayed significant positive peaks in the mFo-DFc map at the center of the 4-helix bundle dimer (Figure 5—figure supplement 1).	RESULTS
+148	153	dimer	oligomeric_state	The electron density maps of the initial EncFtnsH model displayed significant positive peaks in the mFo-DFc map at the center of the 4-helix bundle dimer (Figure 5—figure supplement 1).	RESULTS
+16	22	ICP-MS	experimental_method	Informed by the ICP-MS data indicating the presence of iron in the protein we collected diffraction data at the experimentally determined iron absorption edge (1.74 Å) and calculated an anomalous difference Fourier map using this data.	RESULTS
+43	54	presence of	protein_state	Informed by the ICP-MS data indicating the presence of iron in the protein we collected diffraction data at the experimentally determined iron absorption edge (1.74 Å) and calculated an anomalous difference Fourier map using this data.	RESULTS
+55	59	iron	chemical	Informed by the ICP-MS data indicating the presence of iron in the protein we collected diffraction data at the experimentally determined iron absorption edge (1.74 Å) and calculated an anomalous difference Fourier map using this data.	RESULTS
+88	104	diffraction data	evidence	Informed by the ICP-MS data indicating the presence of iron in the protein we collected diffraction data at the experimentally determined iron absorption edge (1.74 Å) and calculated an anomalous difference Fourier map using this data.	RESULTS
+138	142	iron	chemical	Informed by the ICP-MS data indicating the presence of iron in the protein we collected diffraction data at the experimentally determined iron absorption edge (1.74 Å) and calculated an anomalous difference Fourier map using this data.	RESULTS
+186	218	anomalous difference Fourier map	evidence	Informed by the ICP-MS data indicating the presence of iron in the protein we collected diffraction data at the experimentally determined iron absorption edge (1.74 Å) and calculated an anomalous difference Fourier map using this data.	RESULTS
+19	22	map	evidence	Inspection of this map showed two 10-sigma peaks between residues Glu32, Glu62 and His65 of two adjacent chains, and a statistically smaller 5-sigma peak between residues Glu31 and Glu34 of the two chains.	RESULTS
+43	48	peaks	evidence	Inspection of this map showed two 10-sigma peaks between residues Glu32, Glu62 and His65 of two adjacent chains, and a statistically smaller 5-sigma peak between residues Glu31 and Glu34 of the two chains.	RESULTS
+66	71	Glu32	residue_name_number	Inspection of this map showed two 10-sigma peaks between residues Glu32, Glu62 and His65 of two adjacent chains, and a statistically smaller 5-sigma peak between residues Glu31 and Glu34 of the two chains.	RESULTS
+73	78	Glu62	residue_name_number	Inspection of this map showed two 10-sigma peaks between residues Glu32, Glu62 and His65 of two adjacent chains, and a statistically smaller 5-sigma peak between residues Glu31 and Glu34 of the two chains.	RESULTS
+83	88	His65	residue_name_number	Inspection of this map showed two 10-sigma peaks between residues Glu32, Glu62 and His65 of two adjacent chains, and a statistically smaller 5-sigma peak between residues Glu31 and Glu34 of the two chains.	RESULTS
+171	176	Glu31	residue_name_number	Inspection of this map showed two 10-sigma peaks between residues Glu32, Glu62 and His65 of two adjacent chains, and a statistically smaller 5-sigma peak between residues Glu31 and Glu34 of the two chains.	RESULTS
+181	186	Glu34	residue_name_number	Inspection of this map showed two 10-sigma peaks between residues Glu32, Glu62 and His65 of two adjacent chains, and a statistically smaller 5-sigma peak between residues Glu31 and Glu34 of the two chains.	RESULTS
+41	51	refinement	experimental_method	Modeling metal ions into these peaks and refinement of the anomalous scattering parameters allowed us to identify these as two iron ions and a calcium ion respectively (Figure 5A).	RESULTS
+59	90	anomalous scattering parameters	evidence	Modeling metal ions into these peaks and refinement of the anomalous scattering parameters allowed us to identify these as two iron ions and a calcium ion respectively (Figure 5A).	RESULTS
+127	131	iron	chemical	Modeling metal ions into these peaks and refinement of the anomalous scattering parameters allowed us to identify these as two iron ions and a calcium ion respectively (Figure 5A).	RESULTS
+143	150	calcium	chemical	Modeling metal ions into these peaks and refinement of the anomalous scattering parameters allowed us to identify these as two iron ions and a calcium ion respectively (Figure 5A).	RESULTS
+35	51	electron density	evidence	An additional region of asymmetric electron density near the di-iron binding site in the mFo-DFc map was modeled as glycolic acid, presumably a breakdown product of the PEG 3350 used for crystallization.	RESULTS
+61	81	di-iron binding site	site	An additional region of asymmetric electron density near the di-iron binding site in the mFo-DFc map was modeled as glycolic acid, presumably a breakdown product of the PEG 3350 used for crystallization.	RESULTS
+89	100	mFo-DFc map	evidence	An additional region of asymmetric electron density near the di-iron binding site in the mFo-DFc map was modeled as glycolic acid, presumably a breakdown product of the PEG 3350 used for crystallization.	RESULTS
+116	129	glycolic acid	chemical	An additional region of asymmetric electron density near the di-iron binding site in the mFo-DFc map was modeled as glycolic acid, presumably a breakdown product of the PEG 3350 used for crystallization.	RESULTS
+169	177	PEG 3350	chemical	An additional region of asymmetric electron density near the di-iron binding site in the mFo-DFc map was modeled as glycolic acid, presumably a breakdown product of the PEG 3350 used for crystallization.	RESULTS
+5	19	di-iron center	site	This di-iron center has an Fe-Fe distance of 3.5 Å, Fe-Glu-O distances between 2.3 and 2.5 Å, and Fe-His-N distances of 2.5 Å (Figure 5B).	RESULTS
+27	41	Fe-Fe distance	evidence	This di-iron center has an Fe-Fe distance of 3.5 Å, Fe-Glu-O distances between 2.3 and 2.5 Å, and Fe-His-N distances of 2.5 Å (Figure 5B).	RESULTS
+52	70	Fe-Glu-O distances	evidence	This di-iron center has an Fe-Fe distance of 3.5 Å, Fe-Glu-O distances between 2.3 and 2.5 Å, and Fe-His-N distances of 2.5 Å (Figure 5B).	RESULTS
+98	116	Fe-His-N distances	evidence	This di-iron center has an Fe-Fe distance of 3.5 Å, Fe-Glu-O distances between 2.3 and 2.5 Å, and Fe-His-N distances of 2.5 Å (Figure 5B).	RESULTS
+5	17	coordination	bond_interaction	This coordination geometry is consistent with the di-nuclear ferroxidase center (FOC) found in ferritin.	RESULTS
+50	79	di-nuclear ferroxidase center	site	This coordination geometry is consistent with the di-nuclear ferroxidase center (FOC) found in ferritin.	RESULTS
+81	84	FOC	site	This coordination geometry is consistent with the di-nuclear ferroxidase center (FOC) found in ferritin.	RESULTS
+95	103	ferritin	protein_type	This coordination geometry is consistent with the di-nuclear ferroxidase center (FOC) found in ferritin.	RESULTS
+70	74	iron	chemical	It is interesting to note that although we did not add any additional iron to the crystallization trials, the FOC was fully occupied with iron in the final structure, implying that this site has a very high affinity for iron.	RESULTS
+82	104	crystallization trials	experimental_method	It is interesting to note that although we did not add any additional iron to the crystallization trials, the FOC was fully occupied with iron in the final structure, implying that this site has a very high affinity for iron.	RESULTS
+110	113	FOC	site	It is interesting to note that although we did not add any additional iron to the crystallization trials, the FOC was fully occupied with iron in the final structure, implying that this site has a very high affinity for iron.	RESULTS
+138	142	iron	chemical	It is interesting to note that although we did not add any additional iron to the crystallization trials, the FOC was fully occupied with iron in the final structure, implying that this site has a very high affinity for iron.	RESULTS
+156	165	structure	evidence	It is interesting to note that although we did not add any additional iron to the crystallization trials, the FOC was fully occupied with iron in the final structure, implying that this site has a very high affinity for iron.	RESULTS
+207	215	affinity	evidence	It is interesting to note that although we did not add any additional iron to the crystallization trials, the FOC was fully occupied with iron in the final structure, implying that this site has a very high affinity for iron.	RESULTS
+220	224	iron	chemical	It is interesting to note that although we did not add any additional iron to the crystallization trials, the FOC was fully occupied with iron in the final structure, implying that this site has a very high affinity for iron.	RESULTS
+4	11	calcium	chemical	The calcium ion coordinated by Glu31 and Glu34 adopts heptacoordinate geometry, with coordination distances of 2.5 Å between the metal ion and carboxylate oxygens of Glu31 and Glu34 (E31/34-site).	RESULTS
+16	30	coordinated by	bond_interaction	The calcium ion coordinated by Glu31 and Glu34 adopts heptacoordinate geometry, with coordination distances of 2.5 Å between the metal ion and carboxylate oxygens of Glu31 and Glu34 (E31/34-site).	RESULTS
+31	36	Glu31	residue_name_number	The calcium ion coordinated by Glu31 and Glu34 adopts heptacoordinate geometry, with coordination distances of 2.5 Å between the metal ion and carboxylate oxygens of Glu31 and Glu34 (E31/34-site).	RESULTS
+41	46	Glu34	residue_name_number	The calcium ion coordinated by Glu31 and Glu34 adopts heptacoordinate geometry, with coordination distances of 2.5 Å between the metal ion and carboxylate oxygens of Glu31 and Glu34 (E31/34-site).	RESULTS
+54	69	heptacoordinate	protein_state	The calcium ion coordinated by Glu31 and Glu34 adopts heptacoordinate geometry, with coordination distances of 2.5 Å between the metal ion and carboxylate oxygens of Glu31 and Glu34 (E31/34-site).	RESULTS
+85	97	coordination	bond_interaction	The calcium ion coordinated by Glu31 and Glu34 adopts heptacoordinate geometry, with coordination distances of 2.5 Å between the metal ion and carboxylate oxygens of Glu31 and Glu34 (E31/34-site).	RESULTS
+166	171	Glu31	residue_name_number	The calcium ion coordinated by Glu31 and Glu34 adopts heptacoordinate geometry, with coordination distances of 2.5 Å between the metal ion and carboxylate oxygens of Glu31 and Glu34 (E31/34-site).	RESULTS
+176	181	Glu34	residue_name_number	The calcium ion coordinated by Glu31 and Glu34 adopts heptacoordinate geometry, with coordination distances of 2.5 Å between the metal ion and carboxylate oxygens of Glu31 and Glu34 (E31/34-site).	RESULTS
+183	194	E31/34-site	site	The calcium ion coordinated by Glu31 and Glu34 adopts heptacoordinate geometry, with coordination distances of 2.5 Å between the metal ion and carboxylate oxygens of Glu31 and Glu34 (E31/34-site).	RESULTS
+47	58	coordinated	bond_interaction	A number of ordered solvent molecules are also coordinated to this metal ion at a distance of 2.5 Å. This heptacoordinate geometry is common in crystal structures with calcium ions (Figure 5C).	RESULTS
+106	121	heptacoordinate	protein_state	A number of ordered solvent molecules are also coordinated to this metal ion at a distance of 2.5 Å. This heptacoordinate geometry is common in crystal structures with calcium ions (Figure 5C).	RESULTS
+144	162	crystal structures	evidence	A number of ordered solvent molecules are also coordinated to this metal ion at a distance of 2.5 Å. This heptacoordinate geometry is common in crystal structures with calcium ions (Figure 5C).	RESULTS
+168	175	calcium	chemical	A number of ordered solvent molecules are also coordinated to this metal ion at a distance of 2.5 Å. This heptacoordinate geometry is common in crystal structures with calcium ions (Figure 5C).	RESULTS
+6	12	ICP-MS	experimental_method	While ICP-MS indicated that there were negligible amounts of calcium in the purified protein, the presence of 140 mM calcium acetate in the crystallization mother liquor favors the coordination of calcium at this site.	RESULTS
+61	68	calcium	chemical	While ICP-MS indicated that there were negligible amounts of calcium in the purified protein, the presence of 140 mM calcium acetate in the crystallization mother liquor favors the coordination of calcium at this site.	RESULTS
+98	109	presence of	protein_state	While ICP-MS indicated that there were negligible amounts of calcium in the purified protein, the presence of 140 mM calcium acetate in the crystallization mother liquor favors the coordination of calcium at this site.	RESULTS
+117	132	calcium acetate	chemical	While ICP-MS indicated that there were negligible amounts of calcium in the purified protein, the presence of 140 mM calcium acetate in the crystallization mother liquor favors the coordination of calcium at this site.	RESULTS
+181	193	coordination	bond_interaction	While ICP-MS indicated that there were negligible amounts of calcium in the purified protein, the presence of 140 mM calcium acetate in the crystallization mother liquor favors the coordination of calcium at this site.	RESULTS
+197	204	calcium	chemical	While ICP-MS indicated that there were negligible amounts of calcium in the purified protein, the presence of 140 mM calcium acetate in the crystallization mother liquor favors the coordination of calcium at this site.	RESULTS
+66	77	presence of	protein_state	The fact that the protein does not multimerize in solution in the presence of Fe3+ may indicate that these metal binding sites have a lower affinity for the ferric form of iron, which is the product of the ferroxidase reaction.	RESULTS
+78	82	Fe3+	chemical	The fact that the protein does not multimerize in solution in the presence of Fe3+ may indicate that these metal binding sites have a lower affinity for the ferric form of iron, which is the product of the ferroxidase reaction.	RESULTS
+107	126	metal binding sites	site	The fact that the protein does not multimerize in solution in the presence of Fe3+ may indicate that these metal binding sites have a lower affinity for the ferric form of iron, which is the product of the ferroxidase reaction.	RESULTS
+172	176	iron	chemical	The fact that the protein does not multimerize in solution in the presence of Fe3+ may indicate that these metal binding sites have a lower affinity for the ferric form of iron, which is the product of the ferroxidase reaction.	RESULTS
+206	217	ferroxidase	protein_type	The fact that the protein does not multimerize in solution in the presence of Fe3+ may indicate that these metal binding sites have a lower affinity for the ferric form of iron, which is the product of the ferroxidase reaction.	RESULTS
+90	97	decamer	oligomeric_state	A number of additional metal-ions were present at the outer circumference of at least one decamer in the asymmetric unit (Figure 5D).	RESULTS
+15	29	coordinated by	bond_interaction	These ions are coordinated by His57, Glu61 and Glu64 from both chains in the FOC dimer and are 4.5 Å apart; Fe-Glu-O distances are between 2.5 and 3.5 Å and the Fe-His-N distances are 4 and 4.5 Å.	RESULTS
+30	35	His57	residue_name_number	These ions are coordinated by His57, Glu61 and Glu64 from both chains in the FOC dimer and are 4.5 Å apart; Fe-Glu-O distances are between 2.5 and 3.5 Å and the Fe-His-N distances are 4 and 4.5 Å.	RESULTS
+37	42	Glu61	residue_name_number	These ions are coordinated by His57, Glu61 and Glu64 from both chains in the FOC dimer and are 4.5 Å apart; Fe-Glu-O distances are between 2.5 and 3.5 Å and the Fe-His-N distances are 4 and 4.5 Å.	RESULTS
+47	52	Glu64	residue_name_number	These ions are coordinated by His57, Glu61 and Glu64 from both chains in the FOC dimer and are 4.5 Å apart; Fe-Glu-O distances are between 2.5 and 3.5 Å and the Fe-His-N distances are 4 and 4.5 Å.	RESULTS
+77	80	FOC	site	These ions are coordinated by His57, Glu61 and Glu64 from both chains in the FOC dimer and are 4.5 Å apart; Fe-Glu-O distances are between 2.5 and 3.5 Å and the Fe-His-N distances are 4 and 4.5 Å.	RESULTS
+81	86	dimer	oligomeric_state	These ions are coordinated by His57, Glu61 and Glu64 from both chains in the FOC dimer and are 4.5 Å apart; Fe-Glu-O distances are between 2.5 and 3.5 Å and the Fe-His-N distances are 4 and 4.5 Å.	RESULTS
+108	116	Fe-Glu-O	evidence	These ions are coordinated by His57, Glu61 and Glu64 from both chains in the FOC dimer and are 4.5 Å apart; Fe-Glu-O distances are between 2.5 and 3.5 Å and the Fe-His-N distances are 4 and 4.5 Å.	RESULTS
+161	179	Fe-His-N distances	evidence	These ions are coordinated by His57, Glu61 and Glu64 from both chains in the FOC dimer and are 4.5 Å apart; Fe-Glu-O distances are between 2.5 and 3.5 Å and the Fe-His-N distances are 4 and 4.5 Å.	RESULTS
+38	46	EncFtnsH	protein	Comparison of quaternary structure of EncFtnsH and ferritin.	FIG
+51	59	ferritin	protein_type	Comparison of quaternary structure of EncFtnsH and ferritin.	FIG
+4	11	Aligned	experimental_method	(A) Aligned FOC of EncFtnsH and Pseudo-nitzschia multiseries ferritin (PmFtn).	FIG
+12	15	FOC	site	(A) Aligned FOC of EncFtnsH and Pseudo-nitzschia multiseries ferritin (PmFtn).	FIG
+19	27	EncFtnsH	protein	(A) Aligned FOC of EncFtnsH and Pseudo-nitzschia multiseries ferritin (PmFtn).	FIG
+32	60	Pseudo-nitzschia multiseries	species	(A) Aligned FOC of EncFtnsH and Pseudo-nitzschia multiseries ferritin (PmFtn).	FIG
+61	69	ferritin	protein	(A) Aligned FOC of EncFtnsH and Pseudo-nitzschia multiseries ferritin (PmFtn).	FIG
+71	76	PmFtn	protein	(A) Aligned FOC of EncFtnsH and Pseudo-nitzschia multiseries ferritin (PmFtn).	FIG
+4	22	metal binding site	site	The metal binding site residues from two EncFtnsH chains are shown in green and blue, while the PmFtn is shown in orange.	FIG
+41	49	EncFtnsH	protein	The metal binding site residues from two EncFtnsH chains are shown in green and blue, while the PmFtn is shown in orange.	FIG
+96	101	PmFtn	protein	The metal binding site residues from two EncFtnsH chains are shown in green and blue, while the PmFtn is shown in orange.	FIG
+0	4	Fe2+	chemical	Fe2+ in the FOC is shown as orange spheres and Ca2+ in EncFtnsH is shown as a grey sphere.	FIG
+12	15	FOC	site	Fe2+ in the FOC is shown as orange spheres and Ca2+ in EncFtnsH is shown as a grey sphere.	FIG
+47	51	Ca2+	chemical	Fe2+ in the FOC is shown as orange spheres and Ca2+ in EncFtnsH is shown as a grey sphere.	FIG
+55	63	EncFtnsH	protein	Fe2+ in the FOC is shown as orange spheres and Ca2+ in EncFtnsH is shown as a grey sphere.	FIG
+34	40	EncFtn	protein	The two-fold symmetry axis of the EncFtn FOC is shown with a grey arrow (B) Cross-section surface view of quaternary structure of EncFtnsH and PmFtn as aligned in (A) (dashed black box).	FIG
+41	44	FOC	site	The two-fold symmetry axis of the EncFtn FOC is shown with a grey arrow (B) Cross-section surface view of quaternary structure of EncFtnsH and PmFtn as aligned in (A) (dashed black box).	FIG
+130	138	EncFtnsH	protein	The two-fold symmetry axis of the EncFtn FOC is shown with a grey arrow (B) Cross-section surface view of quaternary structure of EncFtnsH and PmFtn as aligned in (A) (dashed black box).	FIG
+143	148	PmFtn	protein	The two-fold symmetry axis of the EncFtn FOC is shown with a grey arrow (B) Cross-section surface view of quaternary structure of EncFtnsH and PmFtn as aligned in (A) (dashed black box).	FIG
+4	19	central channel	site	The central channel of EncFtnsH is spatially equivalent to the outer surface of ferritin and its outer surface corresponds to the mineralization surface within ferritin.	FIG
+23	31	EncFtnsH	protein	The central channel of EncFtnsH is spatially equivalent to the outer surface of ferritin and its outer surface corresponds to the mineralization surface within ferritin.	FIG
+80	88	ferritin	protein_type	The central channel of EncFtnsH is spatially equivalent to the outer surface of ferritin and its outer surface corresponds to the mineralization surface within ferritin.	FIG
+130	152	mineralization surface	site	The central channel of EncFtnsH is spatially equivalent to the outer surface of ferritin and its outer surface corresponds to the mineralization surface within ferritin.	FIG
+160	168	ferritin	protein_type	The central channel of EncFtnsH is spatially equivalent to the outer surface of ferritin and its outer surface corresponds to the mineralization surface within ferritin.	FIG
+0	10	Comparison	experimental_method	Comparison of the symmetric metal ion binding site of EncFtnsH and the ferritin FOC.	FIG
+28	50	metal ion binding site	site	Comparison of the symmetric metal ion binding site of EncFtnsH and the ferritin FOC.	FIG
+54	62	EncFtnsH	protein	Comparison of the symmetric metal ion binding site of EncFtnsH and the ferritin FOC.	FIG
+71	79	ferritin	protein_type	Comparison of the symmetric metal ion binding site of EncFtnsH and the ferritin FOC.	FIG
+80	83	FOC	site	Comparison of the symmetric metal ion binding site of EncFtnsH and the ferritin FOC.	FIG
+4	24	Structural alignment	experimental_method	(A) Structural alignment of the FOC residues in a dimer of EncFtnsH (green/blue) with a monomer of Pseudo-nitzschia multiseries ferritin (PmFtn) (PDBID: 4ITW) (orange).	FIG
+32	35	FOC	site	(A) Structural alignment of the FOC residues in a dimer of EncFtnsH (green/blue) with a monomer of Pseudo-nitzschia multiseries ferritin (PmFtn) (PDBID: 4ITW) (orange).	FIG
+50	55	dimer	oligomeric_state	(A) Structural alignment of the FOC residues in a dimer of EncFtnsH (green/blue) with a monomer of Pseudo-nitzschia multiseries ferritin (PmFtn) (PDBID: 4ITW) (orange).	FIG
+59	67	EncFtnsH	protein	(A) Structural alignment of the FOC residues in a dimer of EncFtnsH (green/blue) with a monomer of Pseudo-nitzschia multiseries ferritin (PmFtn) (PDBID: 4ITW) (orange).	FIG
+88	95	monomer	oligomeric_state	(A) Structural alignment of the FOC residues in a dimer of EncFtnsH (green/blue) with a monomer of Pseudo-nitzschia multiseries ferritin (PmFtn) (PDBID: 4ITW) (orange).	FIG
+99	127	Pseudo-nitzschia multiseries	species	(A) Structural alignment of the FOC residues in a dimer of EncFtnsH (green/blue) with a monomer of Pseudo-nitzschia multiseries ferritin (PmFtn) (PDBID: 4ITW) (orange).	FIG
+128	136	ferritin	protein	(A) Structural alignment of the FOC residues in a dimer of EncFtnsH (green/blue) with a monomer of Pseudo-nitzschia multiseries ferritin (PmFtn) (PDBID: 4ITW) (orange).	FIG
+138	143	PmFtn	protein	(A) Structural alignment of the FOC residues in a dimer of EncFtnsH (green/blue) with a monomer of Pseudo-nitzschia multiseries ferritin (PmFtn) (PDBID: 4ITW) (orange).	FIG
+0	4	Iron	chemical	Iron ions are shown as orange spheres and a single calcium ion as a grey sphere.	FIG
+51	58	calcium	chemical	Iron ions are shown as orange spheres and a single calcium ion as a grey sphere.	FIG
+20	23	FOC	site	Residues within the FOC are conserved between EncFtn and ferritin PmFtn, with the exception of residues in the position equivalent to H65’ in the second subunit in the dimer (blue).	FIG
+28	37	conserved	protein_state	Residues within the FOC are conserved between EncFtn and ferritin PmFtn, with the exception of residues in the position equivalent to H65’ in the second subunit in the dimer (blue).	FIG
+46	52	EncFtn	protein	Residues within the FOC are conserved between EncFtn and ferritin PmFtn, with the exception of residues in the position equivalent to H65’ in the second subunit in the dimer (blue).	FIG
+57	65	ferritin	protein_type	Residues within the FOC are conserved between EncFtn and ferritin PmFtn, with the exception of residues in the position equivalent to H65’ in the second subunit in the dimer (blue).	FIG
+66	71	PmFtn	protein	Residues within the FOC are conserved between EncFtn and ferritin PmFtn, with the exception of residues in the position equivalent to H65’ in the second subunit in the dimer (blue).	FIG
+134	137	H65	residue_name_number	Residues within the FOC are conserved between EncFtn and ferritin PmFtn, with the exception of residues in the position equivalent to H65’ in the second subunit in the dimer (blue).	FIG
+153	160	subunit	oligomeric_state	Residues within the FOC are conserved between EncFtn and ferritin PmFtn, with the exception of residues in the position equivalent to H65’ in the second subunit in the dimer (blue).	FIG
+168	173	dimer	oligomeric_state	Residues within the FOC are conserved between EncFtn and ferritin PmFtn, with the exception of residues in the position equivalent to H65’ in the second subunit in the dimer (blue).	FIG
+12	18	EncFtn	protein	The site in EncFtn with bound calcium is not present in other family members.	FIG
+24	29	bound	protein_state	The site in EncFtn with bound calcium is not present in other family members.	FIG
+30	37	calcium	chemical	The site in EncFtn with bound calcium is not present in other family members.	FIG
+27	34	aligned	experimental_method	(B) Secondary structure of aligned dimeric EncFtnsH and monomeric ferritin highlighting the conserved four-helix bundle.	FIG
+35	42	dimeric	oligomeric_state	(B) Secondary structure of aligned dimeric EncFtnsH and monomeric ferritin highlighting the conserved four-helix bundle.	FIG
+43	51	EncFtnsH	protein	(B) Secondary structure of aligned dimeric EncFtnsH and monomeric ferritin highlighting the conserved four-helix bundle.	FIG
+56	65	monomeric	oligomeric_state	(B) Secondary structure of aligned dimeric EncFtnsH and monomeric ferritin highlighting the conserved four-helix bundle.	FIG
+66	74	ferritin	protein_type	(B) Secondary structure of aligned dimeric EncFtnsH and monomeric ferritin highlighting the conserved four-helix bundle.	FIG
+92	101	conserved	protein_state	(B) Secondary structure of aligned dimeric EncFtnsH and monomeric ferritin highlighting the conserved four-helix bundle.	FIG
+102	119	four-helix bundle	structure_element	(B) Secondary structure of aligned dimeric EncFtnsH and monomeric ferritin highlighting the conserved four-helix bundle.	FIG
+0	8	EncFtnsH	protein	EncFtnsH monomers are shown in green and blue and aligned PmFtn monomer in orange as in A. (C) Cartoon of secondary structure elements in EncFtn dimer and ferritin.	FIG
+9	17	monomers	oligomeric_state	EncFtnsH monomers are shown in green and blue and aligned PmFtn monomer in orange as in A. (C) Cartoon of secondary structure elements in EncFtn dimer and ferritin.	FIG
+50	57	aligned	experimental_method	EncFtnsH monomers are shown in green and blue and aligned PmFtn monomer in orange as in A. (C) Cartoon of secondary structure elements in EncFtn dimer and ferritin.	FIG
+58	63	PmFtn	protein	EncFtnsH monomers are shown in green and blue and aligned PmFtn monomer in orange as in A. (C) Cartoon of secondary structure elements in EncFtn dimer and ferritin.	FIG
+64	71	monomer	oligomeric_state	EncFtnsH monomers are shown in green and blue and aligned PmFtn monomer in orange as in A. (C) Cartoon of secondary structure elements in EncFtn dimer and ferritin.	FIG
+138	144	EncFtn	protein	EncFtnsH monomers are shown in green and blue and aligned PmFtn monomer in orange as in A. (C) Cartoon of secondary structure elements in EncFtn dimer and ferritin.	FIG
+145	150	dimer	oligomeric_state	EncFtnsH monomers are shown in green and blue and aligned PmFtn monomer in orange as in A. (C) Cartoon of secondary structure elements in EncFtn dimer and ferritin.	FIG
+155	163	ferritin	protein_type	EncFtnsH monomers are shown in green and blue and aligned PmFtn monomer in orange as in A. (C) Cartoon of secondary structure elements in EncFtn dimer and ferritin.	FIG
+7	12	dimer	oligomeric_state	In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between α2 and α3 in ferritin PmFtn.	FIG
+16	22	EncFtn	protein	In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between α2 and α3 in ferritin PmFtn.	FIG
+38	41	FOC	site	In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between α2 and α3 in ferritin PmFtn.	FIG
+71	78	monomer	oligomeric_state	In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between α2 and α3 in ferritin PmFtn.	FIG
+116	123	monomer	oligomeric_state	In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between α2 and α3 in ferritin PmFtn.	FIG
+165	176	long linker	structure_element	In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between α2 and α3 in ferritin PmFtn.	FIG
+185	187	α2	structure_element	In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between α2 and α3 in ferritin PmFtn.	FIG
+192	194	α3	structure_element	In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between α2 and α3 in ferritin PmFtn.	FIG
+198	206	ferritin	protein_type	In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between α2 and α3 in ferritin PmFtn.	FIG
+207	212	PmFtn	protein	In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between α2 and α3 in ferritin PmFtn.	FIG
+0	20	Structural alignment	experimental_method	Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins  (Figure 6A).	RESULTS
+28	48	di-iron binding site	site	Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins  (Figure 6A).	RESULTS
+52	60	EncFtnsH	protein	Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins  (Figure 6A).	RESULTS
+68	71	FOC	site	Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins  (Figure 6A).	RESULTS
+75	103	Pseudo-nitzschia multiseries	species	Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins  (Figure 6A).	RESULTS
+104	112	ferritin	protein_type	Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins  (Figure 6A).	RESULTS
+114	119	PmFtn	protein	Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins  (Figure 6A).	RESULTS
+177	196	metal binding sites	site	Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins  (Figure 6A).	RESULTS
+200	208	EncFtnsH	protein	Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins  (Figure 6A).	RESULTS
+217	226	classical	protein_state	Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins  (Figure 6A).	RESULTS
+227	236	ferritins	protein_type	Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins  (Figure 6A).	RESULTS
+4	16	di-iron site	site	The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65’) (Figure 6A).	RESULTS
+20	28	EncFtnsH	protein	The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65’) (Figure 6A).	RESULTS
+84	99	dimer interface	site	The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65’) (Figure 6A).	RESULTS
+111	114	FOC	site	The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65’) (Figure 6A).	RESULTS
+118	126	ferritin	protein_type	The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65’) (Figure 6A).	RESULTS
+219	224	His65	residue_name_number	The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65’) (Figure 6A).	RESULTS
+239	245	EncFtn	protein	The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65’) (Figure 6A).	RESULTS
+246	253	monomer	oligomeric_state	The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65’) (Figure 6A).	RESULTS
+261	274	FOC interface	site	The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65’) (Figure 6A).	RESULTS
+276	281	His65	residue_name_number	The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65’) (Figure 6A).	RESULTS
+0	26	Structural superimposition	experimental_method	Structural superimposition of the FOCs of ferritin and EncFtn brings the four-helix bundle of the ferritin fold into close alignment with the EncFtn dimer, showing that the two families of proteins have essentially the same architecture around the di-iron center (Figure 6B).	RESULTS
+34	38	FOCs	site	Structural superimposition of the FOCs of ferritin and EncFtn brings the four-helix bundle of the ferritin fold into close alignment with the EncFtn dimer, showing that the two families of proteins have essentially the same architecture around the di-iron center (Figure 6B).	RESULTS
+42	50	ferritin	protein_type	Structural superimposition of the FOCs of ferritin and EncFtn brings the four-helix bundle of the ferritin fold into close alignment with the EncFtn dimer, showing that the two families of proteins have essentially the same architecture around the di-iron center (Figure 6B).	RESULTS
+55	61	EncFtn	protein	Structural superimposition of the FOCs of ferritin and EncFtn brings the four-helix bundle of the ferritin fold into close alignment with the EncFtn dimer, showing that the two families of proteins have essentially the same architecture around the di-iron center (Figure 6B).	RESULTS
+73	90	four-helix bundle	structure_element	Structural superimposition of the FOCs of ferritin and EncFtn brings the four-helix bundle of the ferritin fold into close alignment with the EncFtn dimer, showing that the two families of proteins have essentially the same architecture around the di-iron center (Figure 6B).	RESULTS
+98	106	ferritin	protein_type	Structural superimposition of the FOCs of ferritin and EncFtn brings the four-helix bundle of the ferritin fold into close alignment with the EncFtn dimer, showing that the two families of proteins have essentially the same architecture around the di-iron center (Figure 6B).	RESULTS
+142	148	EncFtn	protein	Structural superimposition of the FOCs of ferritin and EncFtn brings the four-helix bundle of the ferritin fold into close alignment with the EncFtn dimer, showing that the two families of proteins have essentially the same architecture around the di-iron center (Figure 6B).	RESULTS
+149	154	dimer	oligomeric_state	Structural superimposition of the FOCs of ferritin and EncFtn brings the four-helix bundle of the ferritin fold into close alignment with the EncFtn dimer, showing that the two families of proteins have essentially the same architecture around the di-iron center (Figure 6B).	RESULTS
+248	262	di-iron center	site	Structural superimposition of the FOCs of ferritin and EncFtn brings the four-helix bundle of the ferritin fold into close alignment with the EncFtn dimer, showing that the two families of proteins have essentially the same architecture around the di-iron center (Figure 6B).	RESULTS
+4	10	linker	structure_element	The linker connecting helices 2 and 3 of ferritin is congruent with the start of the C-terminal helix of one EncFtn monomer and the N-terminal 310 helix of the second monomer (Figure 6C).	RESULTS
+22	37	helices 2 and 3	structure_element	The linker connecting helices 2 and 3 of ferritin is congruent with the start of the C-terminal helix of one EncFtn monomer and the N-terminal 310 helix of the second monomer (Figure 6C).	RESULTS
+41	49	ferritin	protein_type	The linker connecting helices 2 and 3 of ferritin is congruent with the start of the C-terminal helix of one EncFtn monomer and the N-terminal 310 helix of the second monomer (Figure 6C).	RESULTS
+96	101	helix	structure_element	The linker connecting helices 2 and 3 of ferritin is congruent with the start of the C-terminal helix of one EncFtn monomer and the N-terminal 310 helix of the second monomer (Figure 6C).	RESULTS
+109	115	EncFtn	protein	The linker connecting helices 2 and 3 of ferritin is congruent with the start of the C-terminal helix of one EncFtn monomer and the N-terminal 310 helix of the second monomer (Figure 6C).	RESULTS
+116	123	monomer	oligomeric_state	The linker connecting helices 2 and 3 of ferritin is congruent with the start of the C-terminal helix of one EncFtn monomer and the N-terminal 310 helix of the second monomer (Figure 6C).	RESULTS
+143	152	310 helix	structure_element	The linker connecting helices 2 and 3 of ferritin is congruent with the start of the C-terminal helix of one EncFtn monomer and the N-terminal 310 helix of the second monomer (Figure 6C).	RESULTS
+167	174	monomer	oligomeric_state	The linker connecting helices 2 and 3 of ferritin is congruent with the start of the C-terminal helix of one EncFtn monomer and the N-terminal 310 helix of the second monomer (Figure 6C).	RESULTS
+0	17	Mass spectrometry	experimental_method	Mass spectrometry of the EncFtn assembly	RESULTS
+25	31	EncFtn	protein	Mass spectrometry of the EncFtn assembly	RESULTS
+0	12	Native IM-MS	experimental_method	Native IM-MS analysis of the apo-EncFtnsH monomer.	FIG
+29	32	apo	protein_state	Native IM-MS analysis of the apo-EncFtnsH monomer.	FIG
+33	41	EncFtnsH	protein	Native IM-MS analysis of the apo-EncFtnsH monomer.	FIG
+42	49	monomer	oligomeric_state	Native IM-MS analysis of the apo-EncFtnsH monomer.	FIG
+4	17	Mass spectrum	evidence	(A) Mass spectrum of apo-EncFtnsH acquired from 100 mM ammonium acetate pH 8.0 under native MS conditions.	FIG
+21	24	apo	protein_state	(A) Mass spectrum of apo-EncFtnsH acquired from 100 mM ammonium acetate pH 8.0 under native MS conditions.	FIG
+25	33	EncFtnsH	protein	(A) Mass spectrum of apo-EncFtnsH acquired from 100 mM ammonium acetate pH 8.0 under native MS conditions.	FIG
+85	94	native MS	experimental_method	(A) Mass spectrum of apo-EncFtnsH acquired from 100 mM ammonium acetate pH 8.0 under native MS conditions.	FIG
+4	16	charge state	evidence	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+56	61	peaks	evidence	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+93	106	charge states	evidence	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+110	113	apo	protein_state	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+114	121	monomer	oligomeric_state	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+122	130	EncFtnsH	protein	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+175	201	arrival time distributions	evidence	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+203	220	ion mobility data	evidence	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+241	244	apo	protein_state	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+245	253	EncFtnsH	protein	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+254	266	charge state	evidence	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+355	380	arrival time distribution	evidence	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+415	427	charge state	evidence	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+485	511	arrival time distributions	evidence	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+567	590	collision cross section	evidence	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+592	593	Ω	evidence	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+609	632	collision cross section	evidence	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+645	652	monomer	oligomeric_state	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+667	684	crystal structure	evidence	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+692	701	Fe-loaded	protein_state	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+702	710	EncFtnsH	protein	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+711	718	decamer	oligomeric_state	The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1.	FIG
+22	35	charge states	evidence	The +8 to +15 protein charge states have observed CCS between 20–26 nm2, which is significantly higher than the calculated CCS for an EncFtnsH monomer taken from the decameric assembly crystal structure (15.8 nm2).	FIG
+50	53	CCS	evidence	The +8 to +15 protein charge states have observed CCS between 20–26 nm2, which is significantly higher than the calculated CCS for an EncFtnsH monomer taken from the decameric assembly crystal structure (15.8 nm2).	FIG
+123	126	CCS	evidence	The +8 to +15 protein charge states have observed CCS between 20–26 nm2, which is significantly higher than the calculated CCS for an EncFtnsH monomer taken from the decameric assembly crystal structure (15.8 nm2).	FIG
+134	142	EncFtnsH	protein	The +8 to +15 protein charge states have observed CCS between 20–26 nm2, which is significantly higher than the calculated CCS for an EncFtnsH monomer taken from the decameric assembly crystal structure (15.8 nm2).	FIG
+143	150	monomer	oligomeric_state	The +8 to +15 protein charge states have observed CCS between 20–26 nm2, which is significantly higher than the calculated CCS for an EncFtnsH monomer taken from the decameric assembly crystal structure (15.8 nm2).	FIG
+166	175	decameric	oligomeric_state	The +8 to +15 protein charge states have observed CCS between 20–26 nm2, which is significantly higher than the calculated CCS for an EncFtnsH monomer taken from the decameric assembly crystal structure (15.8 nm2).	FIG
+185	202	crystal structure	evidence	The +8 to +15 protein charge states have observed CCS between 20–26 nm2, which is significantly higher than the calculated CCS for an EncFtnsH monomer taken from the decameric assembly crystal structure (15.8 nm2).	FIG
+4	12	mobility	evidence	The mobility of the +7 charge state displays broad drift-time distribution with maxima consistent with CCS of 15.9 and 17.9 nm2.	FIG
+23	35	charge state	evidence	The mobility of the +7 charge state displays broad drift-time distribution with maxima consistent with CCS of 15.9 and 17.9 nm2.	FIG
+51	74	drift-time distribution	evidence	The mobility of the +7 charge state displays broad drift-time distribution with maxima consistent with CCS of 15.9 and 17.9 nm2.	FIG
+103	106	CCS	evidence	The mobility of the +7 charge state displays broad drift-time distribution with maxima consistent with CCS of 15.9 and 17.9 nm2.	FIG
+16	28	charge state	evidence	Finally, the 6+ charge state of EncFtnsH has mobility consistent with a CCS of 12.3 nm2, indicating a more compact/collapsed structure.	FIG
+32	40	EncFtnsH	protein	Finally, the 6+ charge state of EncFtnsH has mobility consistent with a CCS of 12.3 nm2, indicating a more compact/collapsed structure.	FIG
+45	53	mobility	evidence	Finally, the 6+ charge state of EncFtnsH has mobility consistent with a CCS of 12.3 nm2, indicating a more compact/collapsed structure.	FIG
+72	75	CCS	evidence	Finally, the 6+ charge state of EncFtnsH has mobility consistent with a CCS of 12.3 nm2, indicating a more compact/collapsed structure.	FIG
+107	114	compact	protein_state	Finally, the 6+ charge state of EncFtnsH has mobility consistent with a CCS of 12.3 nm2, indicating a more compact/collapsed structure.	FIG
+115	124	collapsed	protein_state	Finally, the 6+ charge state of EncFtnsH has mobility consistent with a CCS of 12.3 nm2, indicating a more compact/collapsed structure.	FIG
+32	35	apo	protein_state	It is clear from this data that apo-EncFtnsH exists in several gas phase conformations.	FIG
+36	44	EncFtnsH	protein	It is clear from this data that apo-EncFtnsH exists in several gas phase conformations.	FIG
+13	26	charge states	evidence	The range of charge states occupied by the protein (6+ to 15+) and the range of CCS in which the protein is observed (12.3 nm2 – 26 nm2) are both large.	FIG
+80	83	CCS	evidence	The range of charge states occupied by the protein (6+ to 15+) and the range of CCS in which the protein is observed (12.3 nm2 – 26 nm2) are both large.	FIG
+25	38	charge states	evidence	In addition, many of the charge states observed have higher charge than the theoretical maximal charge on spherical globular protein, as determined by the De La Mora relationship (ZR = 0.0778m; for the EncFtnsH monomer ZR = 8.9) Fernandez.	FIG
+116	124	globular	protein_state	In addition, many of the charge states observed have higher charge than the theoretical maximal charge on spherical globular protein, as determined by the De La Mora relationship (ZR = 0.0778m; for the EncFtnsH monomer ZR = 8.9) Fernandez.	FIG
+155	178	De La Mora relationship	experimental_method	In addition, many of the charge states observed have higher charge than the theoretical maximal charge on spherical globular protein, as determined by the De La Mora relationship (ZR = 0.0778m; for the EncFtnsH monomer ZR = 8.9) Fernandez.	FIG
+180	182	ZR	evidence	In addition, many of the charge states observed have higher charge than the theoretical maximal charge on spherical globular protein, as determined by the De La Mora relationship (ZR = 0.0778m; for the EncFtnsH monomer ZR = 8.9) Fernandez.	FIG
+202	210	EncFtnsH	protein	In addition, many of the charge states observed have higher charge than the theoretical maximal charge on spherical globular protein, as determined by the De La Mora relationship (ZR = 0.0778m; for the EncFtnsH monomer ZR = 8.9) Fernandez.	FIG
+211	218	monomer	oligomeric_state	In addition, many of the charge states observed have higher charge than the theoretical maximal charge on spherical globular protein, as determined by the De La Mora relationship (ZR = 0.0778m; for the EncFtnsH monomer ZR = 8.9) Fernandez.	FIG
+219	221	ZR	evidence	In addition, many of the charge states observed have higher charge than the theoretical maximal charge on spherical globular protein, as determined by the De La Mora relationship (ZR = 0.0778m; for the EncFtnsH monomer ZR = 8.9) Fernandez.	FIG
+72	82	disordered	protein_state	As described by Beveridge et al., all these factors are indicative of a disordered protein.	FIG
+29	33	holo	protein_state	Gas-phase disassembly of the holo-EncFtnsH decameric assembly.	FIG
+34	42	EncFtnsH	protein	Gas-phase disassembly of the holo-EncFtnsH decameric assembly.	FIG
+43	52	decameric	oligomeric_state	Gas-phase disassembly of the holo-EncFtnsH decameric assembly.	FIG
+11	23	charge state	evidence	The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles).	FIG
+44	53	Fe-loaded	protein_state	The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles).	FIG
+54	58	holo	protein_state	The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles).	FIG
+60	68	EncFtnsH	protein	The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles).	FIG
+109	141	collisional-induced dissociation	experimental_method	The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles).	FIG
+143	146	CID	experimental_method	The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles).	FIG
+239	242	CID	experimental_method	The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles).	FIG
+243	256	mass spectrum	evidence	The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles).	FIG
+295	299	holo	protein_state	The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles).	FIG
+301	309	EncFtnsH	protein	The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles).	FIG
+310	317	decamer	oligomeric_state	The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles).	FIG
+370	377	monomer	oligomeric_state	The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles).	FIG
+407	415	stripped	protein_state	The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles).	FIG
+428	432	9mer	oligomeric_state	The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles).	FIG
+24	31	monomer	oligomeric_state	The mass of the ejected-monomer is consistent with apo- EncFtnsH (13.2 kDa), suggesting unfolding of the monomer (and loss of Fe) occurs during ejection from the complex.	FIG
+51	54	apo	protein_state	The mass of the ejected-monomer is consistent with apo- EncFtnsH (13.2 kDa), suggesting unfolding of the monomer (and loss of Fe) occurs during ejection from the complex.	FIG
+56	64	EncFtnsH	protein	The mass of the ejected-monomer is consistent with apo- EncFtnsH (13.2 kDa), suggesting unfolding of the monomer (and loss of Fe) occurs during ejection from the complex.	FIG
+105	112	monomer	oligomeric_state	The mass of the ejected-monomer is consistent with apo- EncFtnsH (13.2 kDa), suggesting unfolding of the monomer (and loss of Fe) occurs during ejection from the complex.	FIG
+118	125	loss of	protein_state	The mass of the ejected-monomer is consistent with apo- EncFtnsH (13.2 kDa), suggesting unfolding of the monomer (and loss of Fe) occurs during ejection from the complex.	FIG
+126	128	Fe	chemical	The mass of the ejected-monomer is consistent with apo- EncFtnsH (13.2 kDa), suggesting unfolding of the monomer (and loss of Fe) occurs during ejection from the complex.	FIG
+194	197	CID	experimental_method	This observation of asymmetric charge partitioning of the sub-complexes with respect to the mass of the complex is consistent with the 'typical' pathway of dissociation of protein assemblies by CID, as described by.	FIG
+39	51	charge state	evidence	In addition, a third, lower abundance, charge state distribution is observed which overlaps the EncFtn ejected monomer charge state distribution; this region of the spectrum is highlighted in (B).	FIG
+96	102	EncFtn	protein	In addition, a third, lower abundance, charge state distribution is observed which overlaps the EncFtn ejected monomer charge state distribution; this region of the spectrum is highlighted in (B).	FIG
+111	118	monomer	oligomeric_state	In addition, a third, lower abundance, charge state distribution is observed which overlaps the EncFtn ejected monomer charge state distribution; this region of the spectrum is highlighted in (B).	FIG
+119	131	charge state	evidence	In addition, a third, lower abundance, charge state distribution is observed which overlaps the EncFtn ejected monomer charge state distribution; this region of the spectrum is highlighted in (B).	FIG
+48	56	EncFtnsH	protein	This distribution is consistent with an ejected EncFtnsH dimer (orange circles).	FIG
+57	62	dimer	oligomeric_state	This distribution is consistent with an ejected EncFtnsH dimer (orange circles).	FIG
+49	61	charge state	evidence	Interestingly, closer analysis of the individual charge state of this dimeric CID product shows that this sub-complex exists in three forms – displaying mass consistent with an EncFtnsH dimer binding 0, 1, and 2 Fe ions.	FIG
+70	77	dimeric	oligomeric_state	Interestingly, closer analysis of the individual charge state of this dimeric CID product shows that this sub-complex exists in three forms – displaying mass consistent with an EncFtnsH dimer binding 0, 1, and 2 Fe ions.	FIG
+78	81	CID	experimental_method	Interestingly, closer analysis of the individual charge state of this dimeric CID product shows that this sub-complex exists in three forms – displaying mass consistent with an EncFtnsH dimer binding 0, 1, and 2 Fe ions.	FIG
+177	185	EncFtnsH	protein	Interestingly, closer analysis of the individual charge state of this dimeric CID product shows that this sub-complex exists in three forms – displaying mass consistent with an EncFtnsH dimer binding 0, 1, and 2 Fe ions.	FIG
+186	191	dimer	oligomeric_state	Interestingly, closer analysis of the individual charge state of this dimeric CID product shows that this sub-complex exists in three forms – displaying mass consistent with an EncFtnsH dimer binding 0, 1, and 2 Fe ions.	FIG
+212	214	Fe	chemical	Interestingly, closer analysis of the individual charge state of this dimeric CID product shows that this sub-complex exists in three forms – displaying mass consistent with an EncFtnsH dimer binding 0, 1, and 2 Fe ions.	FIG
+42	54	charge state	evidence	This is highlighted in (C), where the 15+ charge state of the EncFtnsH dimer is shown; 3 peaks are observed with m/z 1760.5, 1763.8, and 1767.0 Th – the lowest peak corresponds to neutral masses of 26392.5 Da [predicted EncFtnsH dimer, (C572H884N172O185S2)2; 26388.6 Da].	FIG
+62	70	EncFtnsH	protein	This is highlighted in (C), where the 15+ charge state of the EncFtnsH dimer is shown; 3 peaks are observed with m/z 1760.5, 1763.8, and 1767.0 Th – the lowest peak corresponds to neutral masses of 26392.5 Da [predicted EncFtnsH dimer, (C572H884N172O185S2)2; 26388.6 Da].	FIG
+71	76	dimer	oligomeric_state	This is highlighted in (C), where the 15+ charge state of the EncFtnsH dimer is shown; 3 peaks are observed with m/z 1760.5, 1763.8, and 1767.0 Th – the lowest peak corresponds to neutral masses of 26392.5 Da [predicted EncFtnsH dimer, (C572H884N172O185S2)2; 26388.6 Da].	FIG
+89	94	peaks	evidence	This is highlighted in (C), where the 15+ charge state of the EncFtnsH dimer is shown; 3 peaks are observed with m/z 1760.5, 1763.8, and 1767.0 Th – the lowest peak corresponds to neutral masses of 26392.5 Da [predicted EncFtnsH dimer, (C572H884N172O185S2)2; 26388.6 Da].	FIG
+220	228	EncFtnsH	protein	This is highlighted in (C), where the 15+ charge state of the EncFtnsH dimer is shown; 3 peaks are observed with m/z 1760.5, 1763.8, and 1767.0 Th – the lowest peak corresponds to neutral masses of 26392.5 Da [predicted EncFtnsH dimer, (C572H884N172O185S2)2; 26388.6 Da].	FIG
+229	234	dimer	oligomeric_state	This is highlighted in (C), where the 15+ charge state of the EncFtnsH dimer is shown; 3 peaks are observed with m/z 1760.5, 1763.8, and 1767.0 Th – the lowest peak corresponds to neutral masses of 26392.5 Da [predicted EncFtnsH dimer, (C572H884N172O185S2)2; 26388.6 Da].	FIG
+16	21	peaks	evidence	The two further peaks have a delta-mass of ~+50 Da, consistent with Fe binding.	FIG
+68	70	Fe	chemical	The two further peaks have a delta-mass of ~+50 Da, consistent with Fe binding.	FIG
+54	57	CID	experimental_method	We interpret these observations as partial ‘atypical’ CID fragmentation of the decameric complex – i.e. fragmentation of the initial complex with retention of subunit and ligand interactions.	FIG
+79	88	decameric	oligomeric_state	We interpret these observations as partial ‘atypical’ CID fragmentation of the decameric complex – i.e. fragmentation of the initial complex with retention of subunit and ligand interactions.	FIG
+40	50	iron-bound	protein_state	We postulate the high stability of this iron-bound dimer sub-complex is due to the metal coordination at the dimer interface, increasing the strength of the dimer interface.	FIG
+51	56	dimer	oligomeric_state	We postulate the high stability of this iron-bound dimer sub-complex is due to the metal coordination at the dimer interface, increasing the strength of the dimer interface.	FIG
+83	88	metal	chemical	We postulate the high stability of this iron-bound dimer sub-complex is due to the metal coordination at the dimer interface, increasing the strength of the dimer interface.	FIG
+89	101	coordination	bond_interaction	We postulate the high stability of this iron-bound dimer sub-complex is due to the metal coordination at the dimer interface, increasing the strength of the dimer interface.	FIG
+109	124	dimer interface	site	We postulate the high stability of this iron-bound dimer sub-complex is due to the metal coordination at the dimer interface, increasing the strength of the dimer interface.	FIG
+157	172	dimer interface	site	We postulate the high stability of this iron-bound dimer sub-complex is due to the metal coordination at the dimer interface, increasing the strength of the dimer interface.	FIG
+81	90	decameric	oligomeric_state	Taken together, these observations support our findings that the topology of the decameric EncFtnsH assembly is arranged as a pentamer of dimers, with two Fe ions at each dimer interface.	FIG
+91	99	EncFtnsH	protein	Taken together, these observations support our findings that the topology of the decameric EncFtnsH assembly is arranged as a pentamer of dimers, with two Fe ions at each dimer interface.	FIG
+126	134	pentamer	oligomeric_state	Taken together, these observations support our findings that the topology of the decameric EncFtnsH assembly is arranged as a pentamer of dimers, with two Fe ions at each dimer interface.	FIG
+138	144	dimers	oligomeric_state	Taken together, these observations support our findings that the topology of the decameric EncFtnsH assembly is arranged as a pentamer of dimers, with two Fe ions at each dimer interface.	FIG
+155	157	Fe	chemical	Taken together, these observations support our findings that the topology of the decameric EncFtnsH assembly is arranged as a pentamer of dimers, with two Fe ions at each dimer interface.	FIG
+171	186	dimer interface	site	Taken together, these observations support our findings that the topology of the decameric EncFtnsH assembly is arranged as a pentamer of dimers, with two Fe ions at each dimer interface.	FIG
+0	24	Native mass spectrometry	experimental_method	Native mass spectrometry and ion mobility analysis of iron loading in EncFtnsH.	FIG
+29	50	ion mobility analysis	experimental_method	Native mass spectrometry and ion mobility analysis of iron loading in EncFtnsH.	FIG
+54	58	iron	chemical	Native mass spectrometry and ion mobility analysis of iron loading in EncFtnsH.	FIG
+70	78	EncFtnsH	protein	Native mass spectrometry and ion mobility analysis of iron loading in EncFtnsH.	FIG
+4	11	spectra	evidence	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Native nanoelectrospray ionization (nESI) mass spectrometry of EncFtnsH at varying iron concentrations.	FIG
+45	52	acetate	chemical	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Native nanoelectrospray ionization (nESI) mass spectrometry of EncFtnsH at varying iron concentrations.	FIG
+103	137	Native nanoelectrospray ionization	experimental_method	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Native nanoelectrospray ionization (nESI) mass spectrometry of EncFtnsH at varying iron concentrations.	FIG
+139	143	nESI	experimental_method	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Native nanoelectrospray ionization (nESI) mass spectrometry of EncFtnsH at varying iron concentrations.	FIG
+145	162	mass spectrometry	experimental_method	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Native nanoelectrospray ionization (nESI) mass spectrometry of EncFtnsH at varying iron concentrations.	FIG
+166	174	EncFtnsH	protein	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Native nanoelectrospray ionization (nESI) mass spectrometry of EncFtnsH at varying iron concentrations.	FIG
+186	190	iron	chemical	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Native nanoelectrospray ionization (nESI) mass spectrometry of EncFtnsH at varying iron concentrations.	FIG
+4	8	nESI	experimental_method	A1, nESI spectrum of iron-free EncFtnsH displays a charge state distribution consistent with EncFtnsH monomer (blue circles, 13,194 Da).	FIG
+9	17	spectrum	evidence	A1, nESI spectrum of iron-free EncFtnsH displays a charge state distribution consistent with EncFtnsH monomer (blue circles, 13,194 Da).	FIG
+21	30	iron-free	protein_state	A1, nESI spectrum of iron-free EncFtnsH displays a charge state distribution consistent with EncFtnsH monomer (blue circles, 13,194 Da).	FIG
+31	39	EncFtnsH	protein	A1, nESI spectrum of iron-free EncFtnsH displays a charge state distribution consistent with EncFtnsH monomer (blue circles, 13,194 Da).	FIG
+51	63	charge state	evidence	A1, nESI spectrum of iron-free EncFtnsH displays a charge state distribution consistent with EncFtnsH monomer (blue circles, 13,194 Da).	FIG
+93	101	EncFtnsH	protein	A1, nESI spectrum of iron-free EncFtnsH displays a charge state distribution consistent with EncFtnsH monomer (blue circles, 13,194 Da).	FIG
+102	109	monomer	oligomeric_state	A1, nESI spectrum of iron-free EncFtnsH displays a charge state distribution consistent with EncFtnsH monomer (blue circles, 13,194 Da).	FIG
+40	44	Fe2+	chemical	Addition of 100 µM (A2) and 300 µM (A3) Fe2+ results in the appearance of a second higher molecular weight charge state distribution consistent with a decameric assembly of EncFtnsH (green circles, 132.6 kDa).	FIG
+90	106	molecular weight	evidence	Addition of 100 µM (A2) and 300 µM (A3) Fe2+ results in the appearance of a second higher molecular weight charge state distribution consistent with a decameric assembly of EncFtnsH (green circles, 132.6 kDa).	FIG
+107	119	charge state	evidence	Addition of 100 µM (A2) and 300 µM (A3) Fe2+ results in the appearance of a second higher molecular weight charge state distribution consistent with a decameric assembly of EncFtnsH (green circles, 132.6 kDa).	FIG
+151	160	decameric	oligomeric_state	Addition of 100 µM (A2) and 300 µM (A3) Fe2+ results in the appearance of a second higher molecular weight charge state distribution consistent with a decameric assembly of EncFtnsH (green circles, 132.6 kDa).	FIG
+173	181	EncFtnsH	protein	Addition of 100 µM (A2) and 300 µM (A3) Fe2+ results in the appearance of a second higher molecular weight charge state distribution consistent with a decameric assembly of EncFtnsH (green circles, 132.6 kDa).	FIG
+4	24	Ion mobility (IM)-MS	experimental_method	(B) Ion mobility (IM)-MS of the iron-bound holo-EncFtnsH decamer.	FIG
+32	42	iron-bound	protein_state	(B) Ion mobility (IM)-MS of the iron-bound holo-EncFtnsH decamer.	FIG
+43	47	holo	protein_state	(B) Ion mobility (IM)-MS of the iron-bound holo-EncFtnsH decamer.	FIG
+48	56	EncFtnsH	protein	(B) Ion mobility (IM)-MS of the iron-bound holo-EncFtnsH decamer.	FIG
+57	64	decamer	oligomeric_state	(B) Ion mobility (IM)-MS of the iron-bound holo-EncFtnsH decamer.	FIG
+5	10	Peaks	evidence	Top, Peaks corresponding to the 22+ to 26+ charge states of a homo-decameric assembly of EncFtnsH are observed (132.6 kDa).	FIG
+43	56	charge states	evidence	Top, Peaks corresponding to the 22+ to 26+ charge states of a homo-decameric assembly of EncFtnsH are observed (132.6 kDa).	FIG
+62	76	homo-decameric	oligomeric_state	Top, Peaks corresponding to the 22+ to 26+ charge states of a homo-decameric assembly of EncFtnsH are observed (132.6 kDa).	FIG
+89	97	EncFtnsH	protein	Top, Peaks corresponding to the 22+ to 26+ charge states of a homo-decameric assembly of EncFtnsH are observed (132.6 kDa).	FIG
+32	44	charge state	evidence	Top Insert, Analysis of the 24+ charge state of the assembly at m/z 5528.2 Th.	FIG
+39	51	charge state	evidence	The theoretical average m/z of the 24+ charge state with no additional metals bound is marked by a red line (5498.7 Th); the observed m/z of the 24+ charge state indicates that the EncFtnsH assembly binds between 10 (green line, 5521.1 Th) and 15 Fe ions (blue line, 5532.4 Th) per decamer.	FIG
+149	161	charge state	evidence	The theoretical average m/z of the 24+ charge state with no additional metals bound is marked by a red line (5498.7 Th); the observed m/z of the 24+ charge state indicates that the EncFtnsH assembly binds between 10 (green line, 5521.1 Th) and 15 Fe ions (blue line, 5532.4 Th) per decamer.	FIG
+181	189	EncFtnsH	protein	The theoretical average m/z of the 24+ charge state with no additional metals bound is marked by a red line (5498.7 Th); the observed m/z of the 24+ charge state indicates that the EncFtnsH assembly binds between 10 (green line, 5521.1 Th) and 15 Fe ions (blue line, 5532.4 Th) per decamer.	FIG
+247	249	Fe	chemical	The theoretical average m/z of the 24+ charge state with no additional metals bound is marked by a red line (5498.7 Th); the observed m/z of the 24+ charge state indicates that the EncFtnsH assembly binds between 10 (green line, 5521.1 Th) and 15 Fe ions (blue line, 5532.4 Th) per decamer.	FIG
+282	289	decamer	oligomeric_state	The theoretical average m/z of the 24+ charge state with no additional metals bound is marked by a red line (5498.7 Th); the observed m/z of the 24+ charge state indicates that the EncFtnsH assembly binds between 10 (green line, 5521.1 Th) and 15 Fe ions (blue line, 5532.4 Th) per decamer.	FIG
+12	38	arrival time distributions	evidence	Bottom, The arrival time distributions (ion mobility data) of all ions in the EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale).	FIG
+40	57	ion mobility data	evidence	Bottom, The arrival time distributions (ion mobility data) of all ions in the EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale).	FIG
+78	86	EncFtnsH	protein	Bottom, The arrival time distributions (ion mobility data) of all ions in the EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale).	FIG
+87	99	charge state	evidence	Bottom, The arrival time distributions (ion mobility data) of all ions in the EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale).	FIG
+18	43	arrival time distribution	evidence	Bottom right, The arrival time distribution of the 24+ charge state (dashed blue box) has been extracted and plotted.	FIG
+55	67	charge state	evidence	Bottom right, The arrival time distribution of the 24+ charge state (dashed blue box) has been extracted and plotted.	FIG
+4	14	drift time	evidence	The drift time for this ion is shown (ms), along with the calibrated collision cross section (CCS), Ω (nm2).	FIG
+69	92	collision cross section	evidence	The drift time for this ion is shown (ms), along with the calibrated collision cross section (CCS), Ω (nm2).	FIG
+94	97	CCS	evidence	The drift time for this ion is shown (ms), along with the calibrated collision cross section (CCS), Ω (nm2).	FIG
+100	101	Ω	evidence	The drift time for this ion is shown (ms), along with the calibrated collision cross section (CCS), Ω (nm2).	FIG
+62	70	EncFtnsH	protein	In order to confirm the assignment of the oligomeric state of EncFtnsH and investigate further the Fe2+-dependent assembly, we used native nano-electrospray ionization (nESI) and ion-mobility mass spectrometry (IM-MS).	RESULTS
+99	103	Fe2+	chemical	In order to confirm the assignment of the oligomeric state of EncFtnsH and investigate further the Fe2+-dependent assembly, we used native nano-electrospray ionization (nESI) and ion-mobility mass spectrometry (IM-MS).	RESULTS
+132	167	native nano-electrospray ionization	experimental_method	In order to confirm the assignment of the oligomeric state of EncFtnsH and investigate further the Fe2+-dependent assembly, we used native nano-electrospray ionization (nESI) and ion-mobility mass spectrometry (IM-MS).	RESULTS
+169	173	nESI	experimental_method	In order to confirm the assignment of the oligomeric state of EncFtnsH and investigate further the Fe2+-dependent assembly, we used native nano-electrospray ionization (nESI) and ion-mobility mass spectrometry (IM-MS).	RESULTS
+179	209	ion-mobility mass spectrometry	experimental_method	In order to confirm the assignment of the oligomeric state of EncFtnsH and investigate further the Fe2+-dependent assembly, we used native nano-electrospray ionization (nESI) and ion-mobility mass spectrometry (IM-MS).	RESULTS
+211	216	IM-MS	experimental_method	In order to confirm the assignment of the oligomeric state of EncFtnsH and investigate further the Fe2+-dependent assembly, we used native nano-electrospray ionization (nESI) and ion-mobility mass spectrometry (IM-MS).	RESULTS
+23	45	recombinant production	experimental_method	As described above, by recombinant production of EncFtnsH in minimal media we were able to limit the bioavailability of iron.	RESULTS
+49	57	EncFtnsH	protein	As described above, by recombinant production of EncFtnsH in minimal media we were able to limit the bioavailability of iron.	RESULTS
+120	124	iron	chemical	As described above, by recombinant production of EncFtnsH in minimal media we were able to limit the bioavailability of iron.	RESULTS
+0	9	Native MS	experimental_method	Native MS analysis of EncFtnsH produced in this way displayed a charge state distribution consistent with an EncFtnsH monomer (blue circles, Figure 7A1) with an average neutral mass of 13,194 Da, in agreement with the predicted mass of the EncFtnsH protein (13,194.53 Da).	RESULTS
+22	30	EncFtnsH	protein	Native MS analysis of EncFtnsH produced in this way displayed a charge state distribution consistent with an EncFtnsH monomer (blue circles, Figure 7A1) with an average neutral mass of 13,194 Da, in agreement with the predicted mass of the EncFtnsH protein (13,194.53 Da).	RESULTS
+64	76	charge state	evidence	Native MS analysis of EncFtnsH produced in this way displayed a charge state distribution consistent with an EncFtnsH monomer (blue circles, Figure 7A1) with an average neutral mass of 13,194 Da, in agreement with the predicted mass of the EncFtnsH protein (13,194.53 Da).	RESULTS
+109	117	EncFtnsH	protein	Native MS analysis of EncFtnsH produced in this way displayed a charge state distribution consistent with an EncFtnsH monomer (blue circles, Figure 7A1) with an average neutral mass of 13,194 Da, in agreement with the predicted mass of the EncFtnsH protein (13,194.53 Da).	RESULTS
+118	125	monomer	oligomeric_state	Native MS analysis of EncFtnsH produced in this way displayed a charge state distribution consistent with an EncFtnsH monomer (blue circles, Figure 7A1) with an average neutral mass of 13,194 Da, in agreement with the predicted mass of the EncFtnsH protein (13,194.53 Da).	RESULTS
+240	248	EncFtnsH	protein	Native MS analysis of EncFtnsH produced in this way displayed a charge state distribution consistent with an EncFtnsH monomer (blue circles, Figure 7A1) with an average neutral mass of 13,194 Da, in agreement with the predicted mass of the EncFtnsH protein (13,194.53 Da).	RESULTS
+0	9	Titration	experimental_method	Titration with Fe2+ directly before native MS analysis resulted in the appearance of a new charge state distribution, consistent with an EncFtnsH decameric assembly (+22 to +26; 132.65 kDa) (Figure 7A2/3).	RESULTS
+15	19	Fe2+	chemical	Titration with Fe2+ directly before native MS analysis resulted in the appearance of a new charge state distribution, consistent with an EncFtnsH decameric assembly (+22 to +26; 132.65 kDa) (Figure 7A2/3).	RESULTS
+36	45	native MS	experimental_method	Titration with Fe2+ directly before native MS analysis resulted in the appearance of a new charge state distribution, consistent with an EncFtnsH decameric assembly (+22 to +26; 132.65 kDa) (Figure 7A2/3).	RESULTS
+91	103	charge state	evidence	Titration with Fe2+ directly before native MS analysis resulted in the appearance of a new charge state distribution, consistent with an EncFtnsH decameric assembly (+22 to +26; 132.65 kDa) (Figure 7A2/3).	RESULTS
+137	145	EncFtnsH	protein	Titration with Fe2+ directly before native MS analysis resulted in the appearance of a new charge state distribution, consistent with an EncFtnsH decameric assembly (+22 to +26; 132.65 kDa) (Figure 7A2/3).	RESULTS
+146	155	decameric	oligomeric_state	Titration with Fe2+ directly before native MS analysis resulted in the appearance of a new charge state distribution, consistent with an EncFtnsH decameric assembly (+22 to +26; 132.65 kDa) (Figure 7A2/3).	RESULTS
+90	94	iron	chemical	After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa).	RESULTS
+139	141	Fe	chemical	After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa).	RESULTS
+151	158	decamer	oligomeric_state	After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa).	RESULTS
+209	220	presence of	protein_state	After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa).	RESULTS
+224	229	irons	chemical	After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa).	RESULTS
+237	240	FOC	site	After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa).	RESULTS
+249	261	coordination	bond_interaction	After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa).	RESULTS
+265	269	iron	chemical	After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa).	RESULTS
+277	290	Glu31/34-site	site	After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa).	RESULTS
+303	310	calcium	chemical	After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa).	RESULTS
+318	335	crystal structure	evidence	After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa).	RESULTS
+337	342	Δmass	evidence	After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa).	RESULTS
+0	2	MS	experimental_method	MS analysis of EncFtnsH after addition of further Fe2+ did not result in iron loading above this stoichiometry.	RESULTS
+15	23	EncFtnsH	protein	MS analysis of EncFtnsH after addition of further Fe2+ did not result in iron loading above this stoichiometry.	RESULTS
+50	54	Fe2+	chemical	MS analysis of EncFtnsH after addition of further Fe2+ did not result in iron loading above this stoichiometry.	RESULTS
+73	77	iron	chemical	MS analysis of EncFtnsH after addition of further Fe2+ did not result in iron loading above this stoichiometry.	RESULTS
+25	29	iron	chemical	Therefore, the extent of iron binding seen is limited to the FOC and Glu31/34 secondary metal binding site.	RESULTS
+61	64	FOC	site	Therefore, the extent of iron binding seen is limited to the FOC and Glu31/34 secondary metal binding site.	RESULTS
+69	106	Glu31/34 secondary metal binding site	site	Therefore, the extent of iron binding seen is limited to the FOC and Glu31/34 secondary metal binding site.	RESULTS
+28	37	decameric	oligomeric_state	These data suggest that the decameric assembly of EncFtnsH does not accrue iron in the same manner as classical ferritin, which is able to sequester around 4500 iron ions within its nanocage.	RESULTS
+50	58	EncFtnsH	protein	These data suggest that the decameric assembly of EncFtnsH does not accrue iron in the same manner as classical ferritin, which is able to sequester around 4500 iron ions within its nanocage.	RESULTS
+75	79	iron	chemical	These data suggest that the decameric assembly of EncFtnsH does not accrue iron in the same manner as classical ferritin, which is able to sequester around 4500 iron ions within its nanocage.	RESULTS
+102	111	classical	protein_state	These data suggest that the decameric assembly of EncFtnsH does not accrue iron in the same manner as classical ferritin, which is able to sequester around 4500 iron ions within its nanocage.	RESULTS
+112	120	ferritin	protein_type	These data suggest that the decameric assembly of EncFtnsH does not accrue iron in the same manner as classical ferritin, which is able to sequester around 4500 iron ions within its nanocage.	RESULTS
+161	165	iron	chemical	These data suggest that the decameric assembly of EncFtnsH does not accrue iron in the same manner as classical ferritin, which is able to sequester around 4500 iron ions within its nanocage.	RESULTS
+182	190	nanocage	complex_assembly	These data suggest that the decameric assembly of EncFtnsH does not accrue iron in the same manner as classical ferritin, which is able to sequester around 4500 iron ions within its nanocage.	RESULTS
+0	21	Ion mobility analysis	experimental_method	Ion mobility analysis of the EncFtnsH decameric assembly, collected with minimal collisional activation, suggested that it consists of a single conformation with a collision cross section (CCS) of 58.2 nm2 (Figure 7B).	RESULTS
+29	37	EncFtnsH	protein	Ion mobility analysis of the EncFtnsH decameric assembly, collected with minimal collisional activation, suggested that it consists of a single conformation with a collision cross section (CCS) of 58.2 nm2 (Figure 7B).	RESULTS
+38	47	decameric	oligomeric_state	Ion mobility analysis of the EncFtnsH decameric assembly, collected with minimal collisional activation, suggested that it consists of a single conformation with a collision cross section (CCS) of 58.2 nm2 (Figure 7B).	RESULTS
+164	187	collision cross section	evidence	Ion mobility analysis of the EncFtnsH decameric assembly, collected with minimal collisional activation, suggested that it consists of a single conformation with a collision cross section (CCS) of 58.2 nm2 (Figure 7B).	RESULTS
+189	192	CCS	evidence	Ion mobility analysis of the EncFtnsH decameric assembly, collected with minimal collisional activation, suggested that it consists of a single conformation with a collision cross section (CCS) of 58.2 nm2 (Figure 7B).	RESULTS
+53	56	CCS	evidence	This observation is in agreement with the calculated CCS of 58.7 nm2derived from our crystal structure of the EncFtnsH decamer.	RESULTS
+85	102	crystal structure	evidence	This observation is in agreement with the calculated CCS of 58.7 nm2derived from our crystal structure of the EncFtnsH decamer.	RESULTS
+110	118	EncFtnsH	protein	This observation is in agreement with the calculated CCS of 58.7 nm2derived from our crystal structure of the EncFtnsH decamer.	RESULTS
+119	126	decamer	oligomeric_state	This observation is in agreement with the calculated CCS of 58.7 nm2derived from our crystal structure of the EncFtnsH decamer.	RESULTS
+13	18	IM-MS	experimental_method	By contrast, IM-MS measurements of the monomeric EncFtnsH at pH 8.0 under the same instrumental conditions revealed that the metal-free protein monomer exists in a wide range of charge states (+6 to +16) and adopts many conformations in the gas phase with collision cross sections ranging from 12 nm2 to 26 nm2 (Figure 7—figure supplement 1).	RESULTS
+39	48	monomeric	oligomeric_state	By contrast, IM-MS measurements of the monomeric EncFtnsH at pH 8.0 under the same instrumental conditions revealed that the metal-free protein monomer exists in a wide range of charge states (+6 to +16) and adopts many conformations in the gas phase with collision cross sections ranging from 12 nm2 to 26 nm2 (Figure 7—figure supplement 1).	RESULTS
+49	57	EncFtnsH	protein	By contrast, IM-MS measurements of the monomeric EncFtnsH at pH 8.0 under the same instrumental conditions revealed that the metal-free protein monomer exists in a wide range of charge states (+6 to +16) and adopts many conformations in the gas phase with collision cross sections ranging from 12 nm2 to 26 nm2 (Figure 7—figure supplement 1).	RESULTS
+61	67	pH 8.0	protein_state	By contrast, IM-MS measurements of the monomeric EncFtnsH at pH 8.0 under the same instrumental conditions revealed that the metal-free protein monomer exists in a wide range of charge states (+6 to +16) and adopts many conformations in the gas phase with collision cross sections ranging from 12 nm2 to 26 nm2 (Figure 7—figure supplement 1).	RESULTS
+125	135	metal-free	protein_state	By contrast, IM-MS measurements of the monomeric EncFtnsH at pH 8.0 under the same instrumental conditions revealed that the metal-free protein monomer exists in a wide range of charge states (+6 to +16) and adopts many conformations in the gas phase with collision cross sections ranging from 12 nm2 to 26 nm2 (Figure 7—figure supplement 1).	RESULTS
+136	143	protein	protein	By contrast, IM-MS measurements of the monomeric EncFtnsH at pH 8.0 under the same instrumental conditions revealed that the metal-free protein monomer exists in a wide range of charge states (+6 to +16) and adopts many conformations in the gas phase with collision cross sections ranging from 12 nm2 to 26 nm2 (Figure 7—figure supplement 1).	RESULTS
+144	151	monomer	oligomeric_state	By contrast, IM-MS measurements of the monomeric EncFtnsH at pH 8.0 under the same instrumental conditions revealed that the metal-free protein monomer exists in a wide range of charge states (+6 to +16) and adopts many conformations in the gas phase with collision cross sections ranging from 12 nm2 to 26 nm2 (Figure 7—figure supplement 1).	RESULTS
+178	191	charge states	evidence	By contrast, IM-MS measurements of the monomeric EncFtnsH at pH 8.0 under the same instrumental conditions revealed that the metal-free protein monomer exists in a wide range of charge states (+6 to +16) and adopts many conformations in the gas phase with collision cross sections ranging from 12 nm2 to 26 nm2 (Figure 7—figure supplement 1).	RESULTS
+6	11	IM-MS	experimental_method	Thus, IM-MS studies highlight that higher order structure in EncFtnsH is mediated/stabilized by metal binding, an observation that is in agreement with our solution studies.	RESULTS
+61	69	EncFtnsH	protein	Thus, IM-MS studies highlight that higher order structure in EncFtnsH is mediated/stabilized by metal binding, an observation that is in agreement with our solution studies.	RESULTS
+46	50	iron	chemical	Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure.	RESULTS
+72	75	FOC	site	Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure.	RESULTS
+79	87	EncFtnsH	protein	Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure.	RESULTS
+118	132	4-helix bundle	structure_element	Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure.	RESULTS
+133	148	dimer interface	site	Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure.	RESULTS
+181	190	classical	protein_state	Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure.	RESULTS
+191	199	ferritin	protein_type	Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure.	RESULTS
+234	240	dimers	oligomeric_state	Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure.	RESULTS
+309	318	decameric	oligomeric_state	Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure.	RESULTS
+352	369	crystal structure	evidence	Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure.	RESULTS
+55	64	decameric	oligomeric_state	We subsequently performed gas phase disassembly of the decameric EncFtnsH using collision-induced dissociation (CID) tandem mass spectrometry.	RESULTS
+65	73	EncFtnsH	protein	We subsequently performed gas phase disassembly of the decameric EncFtnsH using collision-induced dissociation (CID) tandem mass spectrometry.	RESULTS
+80	110	collision-induced dissociation	experimental_method	We subsequently performed gas phase disassembly of the decameric EncFtnsH using collision-induced dissociation (CID) tandem mass spectrometry.	RESULTS
+112	115	CID	experimental_method	We subsequently performed gas phase disassembly of the decameric EncFtnsH using collision-induced dissociation (CID) tandem mass spectrometry.	RESULTS
+117	141	tandem mass spectrometry	experimental_method	We subsequently performed gas phase disassembly of the decameric EncFtnsH using collision-induced dissociation (CID) tandem mass spectrometry.	RESULTS
+18	21	CID	experimental_method	Under the correct CID conditions, protein assemblies can dissociate with retention of subunit and ligand interactions, and thus provide structurally-informative evidence as to the topology of the original assembly; this has been termed ‘atypical’ dissociation.	RESULTS
+4	12	EncFtnsH	protein	For EncFtnsH, this atypical dissociation pathway was clearly evident; CID of the EncFtnsH decamer resulted in the appearance of a dimeric EncFtnsH subcomplex containing 0, 1, or 2 iron ions (Figure 7—figure supplement 2).	RESULTS
+70	73	CID	experimental_method	For EncFtnsH, this atypical dissociation pathway was clearly evident; CID of the EncFtnsH decamer resulted in the appearance of a dimeric EncFtnsH subcomplex containing 0, 1, or 2 iron ions (Figure 7—figure supplement 2).	RESULTS
+81	89	EncFtnsH	protein	For EncFtnsH, this atypical dissociation pathway was clearly evident; CID of the EncFtnsH decamer resulted in the appearance of a dimeric EncFtnsH subcomplex containing 0, 1, or 2 iron ions (Figure 7—figure supplement 2).	RESULTS
+90	97	decamer	oligomeric_state	For EncFtnsH, this atypical dissociation pathway was clearly evident; CID of the EncFtnsH decamer resulted in the appearance of a dimeric EncFtnsH subcomplex containing 0, 1, or 2 iron ions (Figure 7—figure supplement 2).	RESULTS
+130	137	dimeric	oligomeric_state	For EncFtnsH, this atypical dissociation pathway was clearly evident; CID of the EncFtnsH decamer resulted in the appearance of a dimeric EncFtnsH subcomplex containing 0, 1, or 2 iron ions (Figure 7—figure supplement 2).	RESULTS
+138	146	EncFtnsH	protein	For EncFtnsH, this atypical dissociation pathway was clearly evident; CID of the EncFtnsH decamer resulted in the appearance of a dimeric EncFtnsH subcomplex containing 0, 1, or 2 iron ions (Figure 7—figure supplement 2).	RESULTS
+180	184	iron	chemical	For EncFtnsH, this atypical dissociation pathway was clearly evident; CID of the EncFtnsH decamer resulted in the appearance of a dimeric EncFtnsH subcomplex containing 0, 1, or 2 iron ions (Figure 7—figure supplement 2).	RESULTS
+16	33	crystal structure	evidence	In light of the crystal structure, this observation can be rationalized as dissociation of the EncFtnsH decamer by disruption of the non-FOC interface with at least partial retention of the FOC interface and the FOC-Fe.	RESULTS
+95	103	EncFtnsH	protein	In light of the crystal structure, this observation can be rationalized as dissociation of the EncFtnsH decamer by disruption of the non-FOC interface with at least partial retention of the FOC interface and the FOC-Fe.	RESULTS
+104	111	decamer	oligomeric_state	In light of the crystal structure, this observation can be rationalized as dissociation of the EncFtnsH decamer by disruption of the non-FOC interface with at least partial retention of the FOC interface and the FOC-Fe.	RESULTS
+133	150	non-FOC interface	site	In light of the crystal structure, this observation can be rationalized as dissociation of the EncFtnsH decamer by disruption of the non-FOC interface with at least partial retention of the FOC interface and the FOC-Fe.	RESULTS
+190	203	FOC interface	site	In light of the crystal structure, this observation can be rationalized as dissociation of the EncFtnsH decamer by disruption of the non-FOC interface with at least partial retention of the FOC interface and the FOC-Fe.	RESULTS
+212	215	FOC	site	In light of the crystal structure, this observation can be rationalized as dissociation of the EncFtnsH decamer by disruption of the non-FOC interface with at least partial retention of the FOC interface and the FOC-Fe.	RESULTS
+216	218	Fe	chemical	In light of the crystal structure, this observation can be rationalized as dissociation of the EncFtnsH decamer by disruption of the non-FOC interface with at least partial retention of the FOC interface and the FOC-Fe.	RESULTS
+95	103	EncFtnsH	protein	Thus, this observation supports our crystallographic assignment of the overall topology of the EncFtnsH assembly as a pentameric assembly of dimers with two iron ions located at the FOC dimer interface.	RESULTS
+118	128	pentameric	oligomeric_state	Thus, this observation supports our crystallographic assignment of the overall topology of the EncFtnsH assembly as a pentameric assembly of dimers with two iron ions located at the FOC dimer interface.	RESULTS
+141	147	dimers	oligomeric_state	Thus, this observation supports our crystallographic assignment of the overall topology of the EncFtnsH assembly as a pentameric assembly of dimers with two iron ions located at the FOC dimer interface.	RESULTS
+157	161	iron	chemical	Thus, this observation supports our crystallographic assignment of the overall topology of the EncFtnsH assembly as a pentameric assembly of dimers with two iron ions located at the FOC dimer interface.	RESULTS
+182	201	FOC dimer interface	site	Thus, this observation supports our crystallographic assignment of the overall topology of the EncFtnsH assembly as a pentameric assembly of dimers with two iron ions located at the FOC dimer interface.	RESULTS
+111	118	crystal	evidence	In addition, this analysis provides evidence that the overall architecture of the complex is consistent in the crystal, solution and gas phases.	RESULTS
+0	11	Ferroxidase	protein_type	Ferroxidase activity	RESULTS
+0	3	TEM	experimental_method	TEM visualization of iron-loaded bacterial nanocompartments and ferritin.	FIG
+21	32	iron-loaded	protein_state	TEM visualization of iron-loaded bacterial nanocompartments and ferritin.	FIG
+33	42	bacterial	taxonomy_domain	TEM visualization of iron-loaded bacterial nanocompartments and ferritin.	FIG
+43	59	nanocompartments	complex_assembly	TEM visualization of iron-loaded bacterial nanocompartments and ferritin.	FIG
+64	72	ferritin	protein_type	TEM visualization of iron-loaded bacterial nanocompartments and ferritin.	FIG
+0	9	Decameric	oligomeric_state	Decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin, at 8.5 µM, were mixed with 147 µM, 1 mM, 1 mM and 215 µM acidic Fe(NH4)2(SO4)2, respectively.	FIG
+10	18	EncFtnsH	protein	Decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin, at 8.5 µM, were mixed with 147 µM, 1 mM, 1 mM and 215 µM acidic Fe(NH4)2(SO4)2, respectively.	FIG
+20	30	encapsulin	protein	Decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin, at 8.5 µM, were mixed with 147 µM, 1 mM, 1 mM and 215 µM acidic Fe(NH4)2(SO4)2, respectively.	FIG
+32	42	EncFtn-Enc	complex_assembly	Decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin, at 8.5 µM, were mixed with 147 µM, 1 mM, 1 mM and 215 µM acidic Fe(NH4)2(SO4)2, respectively.	FIG
+47	58	apoferritin	protein_state	Decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin, at 8.5 µM, were mixed with 147 µM, 1 mM, 1 mM and 215 µM acidic Fe(NH4)2(SO4)2, respectively.	FIG
+124	138	Fe(NH4)2(SO4)2	chemical	Decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin, at 8.5 µM, were mixed with 147 µM, 1 mM, 1 mM and 215 µM acidic Fe(NH4)2(SO4)2, respectively.	FIG
+70	73	TEM	experimental_method	Protein mixtures were incubated at room temperature for 1 hr prior to TEM analysis with or without uranyl acetate stain.	FIG
+99	113	uranyl acetate	chemical	Protein mixtures were incubated at room temperature for 1 hr prior to TEM analysis with or without uranyl acetate stain.	FIG
+16	24	EncFtnsH	protein	(A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.	FIG
+26	36	encapsulin	protein	(A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.	FIG
+38	48	EncFtn-Enc	complex_assembly	(A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.	FIG
+50	61	apoferritin	protein_state	(A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.	FIG
+62	73	loaded with	protein_state	(A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.	FIG
+74	78	Fe2+	chemical	(A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.	FIG
+198	205	Stained	experimental_method	(A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.	FIG
+206	214	EncFtnsH	protein	(A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.	FIG
+216	226	encapsulin	protein	(A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.	FIG
+228	238	EncFtn-Enc	complex_assembly	(A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.	FIG
+240	251	apoferritin	protein_state	(A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.	FIG
+252	263	loaded with	protein_state	(A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.	FIG
+264	268	Fe2+	chemical	(A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.	FIG
+31	42	ferroxidase	protein_type	Spectroscopic evidence for the ferroxidase activity and comparison of iron loading capacity of apoferritin, EncFtnsH, encapsulin, and EncFtn-Enc.	FIG
+70	74	iron	chemical	Spectroscopic evidence for the ferroxidase activity and comparison of iron loading capacity of apoferritin, EncFtnsH, encapsulin, and EncFtn-Enc.	FIG
+95	106	apoferritin	protein_state	Spectroscopic evidence for the ferroxidase activity and comparison of iron loading capacity of apoferritin, EncFtnsH, encapsulin, and EncFtn-Enc.	FIG
+108	116	EncFtnsH	protein	Spectroscopic evidence for the ferroxidase activity and comparison of iron loading capacity of apoferritin, EncFtnsH, encapsulin, and EncFtn-Enc.	FIG
+118	128	encapsulin	protein	Spectroscopic evidence for the ferroxidase activity and comparison of iron loading capacity of apoferritin, EncFtnsH, encapsulin, and EncFtn-Enc.	FIG
+134	144	EncFtn-Enc	complex_assembly	Spectroscopic evidence for the ferroxidase activity and comparison of iron loading capacity of apoferritin, EncFtnsH, encapsulin, and EncFtn-Enc.	FIG
+4	15	Apoferritin	protein_state	(A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer.	FIG
+23	30	monomer	oligomeric_state	(A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer.	FIG
+50	58	EncFtnsH	protein	(A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer.	FIG
+59	66	decamer	oligomeric_state	(A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer.	FIG
+84	91	monomer	oligomeric_state	(A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer.	FIG
+113	116	FOC	site	(A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer.	FIG
+166	170	iron	chemical	(A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer.	FIG
+204	208	Fe2+	chemical	(A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer.	FIG
+213	216	FOC	site	(A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer.	FIG
+222	237	progress curves	evidence	(A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer.	FIG
+258	262	Fe2+	chemical	(A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer.	FIG
+266	270	Fe3+	chemical	(A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer.	FIG
+28	32	iron	chemical	The background oxidation of iron at 20 and 100 μM in enzyme-free controls are shown for reference. (B) Encapsulin and EncFtn-Enc complexes at 10 μM asymmetric unit concentration were incubated with Fe2+ at 20 and 100 μM and progress curves for iron oxidation at A315 were measured in a UV/visible spectrophotometer.	FIG
+103	113	Encapsulin	protein	The background oxidation of iron at 20 and 100 μM in enzyme-free controls are shown for reference. (B) Encapsulin and EncFtn-Enc complexes at 10 μM asymmetric unit concentration were incubated with Fe2+ at 20 and 100 μM and progress curves for iron oxidation at A315 were measured in a UV/visible spectrophotometer.	FIG
+118	128	EncFtn-Enc	complex_assembly	The background oxidation of iron at 20 and 100 μM in enzyme-free controls are shown for reference. (B) Encapsulin and EncFtn-Enc complexes at 10 μM asymmetric unit concentration were incubated with Fe2+ at 20 and 100 μM and progress curves for iron oxidation at A315 were measured in a UV/visible spectrophotometer.	FIG
+183	192	incubated	experimental_method	The background oxidation of iron at 20 and 100 μM in enzyme-free controls are shown for reference. (B) Encapsulin and EncFtn-Enc complexes at 10 μM asymmetric unit concentration were incubated with Fe2+ at 20 and 100 μM and progress curves for iron oxidation at A315 were measured in a UV/visible spectrophotometer.	FIG
+198	202	Fe2+	chemical	The background oxidation of iron at 20 and 100 μM in enzyme-free controls are shown for reference. (B) Encapsulin and EncFtn-Enc complexes at 10 μM asymmetric unit concentration were incubated with Fe2+ at 20 and 100 μM and progress curves for iron oxidation at A315 were measured in a UV/visible spectrophotometer.	FIG
+224	239	progress curves	evidence	The background oxidation of iron at 20 and 100 μM in enzyme-free controls are shown for reference. (B) Encapsulin and EncFtn-Enc complexes at 10 μM asymmetric unit concentration were incubated with Fe2+ at 20 and 100 μM and progress curves for iron oxidation at A315 were measured in a UV/visible spectrophotometer.	FIG
+244	248	iron	chemical	The background oxidation of iron at 20 and 100 μM in enzyme-free controls are shown for reference. (B) Encapsulin and EncFtn-Enc complexes at 10 μM asymmetric unit concentration were incubated with Fe2+ at 20 and 100 μM and progress curves for iron oxidation at A315 were measured in a UV/visible spectrophotometer.	FIG
+286	314	UV/visible spectrophotometer	experimental_method	The background oxidation of iron at 20 and 100 μM in enzyme-free controls are shown for reference. (B) Encapsulin and EncFtn-Enc complexes at 10 μM asymmetric unit concentration were incubated with Fe2+ at 20 and 100 μM and progress curves for iron oxidation at A315 were measured in a UV/visible spectrophotometer.	FIG
+49	53	Fe2+	chemical	Enzyme free controls for background oxidation of Fe2+ are shown for reference. (C) Histogram of the iron loading capacity per biological assembly of EncFtnsH, encapsulin, EncFtn-Enc and apoferritin.	FIG
+100	104	iron	chemical	Enzyme free controls for background oxidation of Fe2+ are shown for reference. (C) Histogram of the iron loading capacity per biological assembly of EncFtnsH, encapsulin, EncFtn-Enc and apoferritin.	FIG
+149	157	EncFtnsH	protein	Enzyme free controls for background oxidation of Fe2+ are shown for reference. (C) Histogram of the iron loading capacity per biological assembly of EncFtnsH, encapsulin, EncFtn-Enc and apoferritin.	FIG
+159	169	encapsulin	protein	Enzyme free controls for background oxidation of Fe2+ are shown for reference. (C) Histogram of the iron loading capacity per biological assembly of EncFtnsH, encapsulin, EncFtn-Enc and apoferritin.	FIG
+171	181	EncFtn-Enc	complex_assembly	Enzyme free controls for background oxidation of Fe2+ are shown for reference. (C) Histogram of the iron loading capacity per biological assembly of EncFtnsH, encapsulin, EncFtn-Enc and apoferritin.	FIG
+186	197	apoferritin	protein_state	Enzyme free controls for background oxidation of Fe2+ are shown for reference. (C) Histogram of the iron loading capacity per biological assembly of EncFtnsH, encapsulin, EncFtn-Enc and apoferritin.	FIG
+79	83	iron	chemical	The results shown are for three technical replicates and represent the optimal iron loading by the complexes after three hours when incubated with Fe2+.	FIG
+147	151	Fe2+	chemical	The results shown are for three technical replicates and represent the optimal iron loading by the complexes after three hours when incubated with Fe2+.	FIG
+37	48	iron-loaded	protein_state	In light of the identification of an iron-loaded FOC in the crystal structure of EncFtn and our native mass spectrometry data, we performed ferroxidase and peroxidase assays to demonstrate the catalytic activity of this protein.	RESULTS
+49	52	FOC	site	In light of the identification of an iron-loaded FOC in the crystal structure of EncFtn and our native mass spectrometry data, we performed ferroxidase and peroxidase assays to demonstrate the catalytic activity of this protein.	RESULTS
+60	77	crystal structure	evidence	In light of the identification of an iron-loaded FOC in the crystal structure of EncFtn and our native mass spectrometry data, we performed ferroxidase and peroxidase assays to demonstrate the catalytic activity of this protein.	RESULTS
+81	87	EncFtn	protein	In light of the identification of an iron-loaded FOC in the crystal structure of EncFtn and our native mass spectrometry data, we performed ferroxidase and peroxidase assays to demonstrate the catalytic activity of this protein.	RESULTS
+96	120	native mass spectrometry	experimental_method	In light of the identification of an iron-loaded FOC in the crystal structure of EncFtn and our native mass spectrometry data, we performed ferroxidase and peroxidase assays to demonstrate the catalytic activity of this protein.	RESULTS
+140	173	ferroxidase and peroxidase assays	experimental_method	In light of the identification of an iron-loaded FOC in the crystal structure of EncFtn and our native mass spectrometry data, we performed ferroxidase and peroxidase assays to demonstrate the catalytic activity of this protein.	RESULTS
+29	35	equine	taxonomy_domain	In addition, we also assayed equine apoferritin, an example of a classical ferritin enzyme, as a positive control.	RESULTS
+36	47	apoferritin	protein_state	In addition, we also assayed equine apoferritin, an example of a classical ferritin enzyme, as a positive control.	RESULTS
+65	74	classical	protein_state	In addition, we also assayed equine apoferritin, an example of a classical ferritin enzyme, as a positive control.	RESULTS
+75	83	ferritin	protein_type	In addition, we also assayed equine apoferritin, an example of a classical ferritin enzyme, as a positive control.	RESULTS
+11	21	Dps family	protein_type	Unlike the Dps family of ferritin-like proteins, EncFtn showed no peroxidase activity when assayed with the substrate ortho-phenylenediamine.	RESULTS
+25	47	ferritin-like proteins	protein_type	Unlike the Dps family of ferritin-like proteins, EncFtn showed no peroxidase activity when assayed with the substrate ortho-phenylenediamine.	RESULTS
+49	55	EncFtn	protein	Unlike the Dps family of ferritin-like proteins, EncFtn showed no peroxidase activity when assayed with the substrate ortho-phenylenediamine.	RESULTS
+118	140	ortho-phenylenediamine	chemical	Unlike the Dps family of ferritin-like proteins, EncFtn showed no peroxidase activity when assayed with the substrate ortho-phenylenediamine.	RESULTS
+4	15	ferroxidase	protein_type	The ferroxidase activity of EncFtnsH was measured by recording the progress curve of Fe2+ oxidation to Fe3+ at 315 nm after addition of 20 and 100 µM Fe2+ (2 and 10 times molar ratio Fe2+/FOC).	RESULTS
+28	36	EncFtnsH	protein	The ferroxidase activity of EncFtnsH was measured by recording the progress curve of Fe2+ oxidation to Fe3+ at 315 nm after addition of 20 and 100 µM Fe2+ (2 and 10 times molar ratio Fe2+/FOC).	RESULTS
+67	81	progress curve	evidence	The ferroxidase activity of EncFtnsH was measured by recording the progress curve of Fe2+ oxidation to Fe3+ at 315 nm after addition of 20 and 100 µM Fe2+ (2 and 10 times molar ratio Fe2+/FOC).	RESULTS
+85	89	Fe2+	chemical	The ferroxidase activity of EncFtnsH was measured by recording the progress curve of Fe2+ oxidation to Fe3+ at 315 nm after addition of 20 and 100 µM Fe2+ (2 and 10 times molar ratio Fe2+/FOC).	RESULTS
+103	107	Fe3+	chemical	The ferroxidase activity of EncFtnsH was measured by recording the progress curve of Fe2+ oxidation to Fe3+ at 315 nm after addition of 20 and 100 µM Fe2+ (2 and 10 times molar ratio Fe2+/FOC).	RESULTS
+150	154	Fe2+	chemical	The ferroxidase activity of EncFtnsH was measured by recording the progress curve of Fe2+ oxidation to Fe3+ at 315 nm after addition of 20 and 100 µM Fe2+ (2 and 10 times molar ratio Fe2+/FOC).	RESULTS
+183	187	Fe2+	chemical	The ferroxidase activity of EncFtnsH was measured by recording the progress curve of Fe2+ oxidation to Fe3+ at 315 nm after addition of 20 and 100 µM Fe2+ (2 and 10 times molar ratio Fe2+/FOC).	RESULTS
+188	191	FOC	site	The ferroxidase activity of EncFtnsH was measured by recording the progress curve of Fe2+ oxidation to Fe3+ at 315 nm after addition of 20 and 100 µM Fe2+ (2 and 10 times molar ratio Fe2+/FOC).	RESULTS
+82	86	Fe2+	chemical	In both experiments the rate of oxidation was faster than background oxidation of Fe2+ by molecular oxygen, and was highest for 100 µM Fe2+ (Figure 8A).	RESULTS
+100	106	oxygen	chemical	In both experiments the rate of oxidation was faster than background oxidation of Fe2+ by molecular oxygen, and was highest for 100 µM Fe2+ (Figure 8A).	RESULTS
+135	139	Fe2+	chemical	In both experiments the rate of oxidation was faster than background oxidation of Fe2+ by molecular oxygen, and was highest for 100 µM Fe2+ (Figure 8A).	RESULTS
+33	41	EncFtnsH	protein	These data show that recombinant EncFtnsH acts as an active ferroxidase enzyme.	RESULTS
+53	59	active	protein_state	These data show that recombinant EncFtnsH acts as an active ferroxidase enzyme.	RESULTS
+60	71	ferroxidase	protein_type	These data show that recombinant EncFtnsH acts as an active ferroxidase enzyme.	RESULTS
+17	28	apoferritin	protein_state	When compared to apoferritin, EncFtnsH oxidized Fe2+ at a slower rate and the reaction did not run to completion over the 1800 s of the experiment.	RESULTS
+30	38	EncFtnsH	protein	When compared to apoferritin, EncFtnsH oxidized Fe2+ at a slower rate and the reaction did not run to completion over the 1800 s of the experiment.	RESULTS
+48	52	Fe2+	chemical	When compared to apoferritin, EncFtnsH oxidized Fe2+ at a slower rate and the reaction did not run to completion over the 1800 s of the experiment.	RESULTS
+33	37	iron	chemical	Addition of higher quantities of iron resulted in the formation of a yellow/red precipitate at the end of the reaction.	RESULTS
+55	65	encapsulin	protein	We also performed these assays on purified recombinant encapsulin; which, when assayed alone, did not display ferroxidase activity above background Fe2+ oxidation (Figure 8B).	RESULTS
+110	121	ferroxidase	protein_type	We also performed these assays on purified recombinant encapsulin; which, when assayed alone, did not display ferroxidase activity above background Fe2+ oxidation (Figure 8B).	RESULTS
+148	152	Fe2+	chemical	We also performed these assays on purified recombinant encapsulin; which, when assayed alone, did not display ferroxidase activity above background Fe2+ oxidation (Figure 8B).	RESULTS
+30	34	full	protein_state	In contrast, complexes of the full EncFtn encapsulin nanocompartment (i.e. the EncFtn-Enc protein complex) displayed ferroxidase activity comparable to apoferritin without the formation of precipitates (Figure 8B).	RESULTS
+35	41	EncFtn	protein	In contrast, complexes of the full EncFtn encapsulin nanocompartment (i.e. the EncFtn-Enc protein complex) displayed ferroxidase activity comparable to apoferritin without the formation of precipitates (Figure 8B).	RESULTS
+42	52	encapsulin	protein	In contrast, complexes of the full EncFtn encapsulin nanocompartment (i.e. the EncFtn-Enc protein complex) displayed ferroxidase activity comparable to apoferritin without the formation of precipitates (Figure 8B).	RESULTS
+53	68	nanocompartment	complex_assembly	In contrast, complexes of the full EncFtn encapsulin nanocompartment (i.e. the EncFtn-Enc protein complex) displayed ferroxidase activity comparable to apoferritin without the formation of precipitates (Figure 8B).	RESULTS
+79	89	EncFtn-Enc	complex_assembly	In contrast, complexes of the full EncFtn encapsulin nanocompartment (i.e. the EncFtn-Enc protein complex) displayed ferroxidase activity comparable to apoferritin without the formation of precipitates (Figure 8B).	RESULTS
+117	128	ferroxidase	protein_type	In contrast, complexes of the full EncFtn encapsulin nanocompartment (i.e. the EncFtn-Enc protein complex) displayed ferroxidase activity comparable to apoferritin without the formation of precipitates (Figure 8B).	RESULTS
+152	163	apoferritin	protein_state	In contrast, complexes of the full EncFtn encapsulin nanocompartment (i.e. the EncFtn-Enc protein complex) displayed ferroxidase activity comparable to apoferritin without the formation of precipitates (Figure 8B).	RESULTS
+47	55	EncFtnsH	protein	We attributed the precipitates observed in the EncFtnsH ferroxidase assay to the production of insoluble Fe3+ complexes, which led us to propose that EncFtn does not directly store Fe3+ in a mineral form.	RESULTS
+56	73	ferroxidase assay	experimental_method	We attributed the precipitates observed in the EncFtnsH ferroxidase assay to the production of insoluble Fe3+ complexes, which led us to propose that EncFtn does not directly store Fe3+ in a mineral form.	RESULTS
+105	109	Fe3+	chemical	We attributed the precipitates observed in the EncFtnsH ferroxidase assay to the production of insoluble Fe3+ complexes, which led us to propose that EncFtn does not directly store Fe3+ in a mineral form.	RESULTS
+150	156	EncFtn	protein	We attributed the precipitates observed in the EncFtnsH ferroxidase assay to the production of insoluble Fe3+ complexes, which led us to propose that EncFtn does not directly store Fe3+ in a mineral form.	RESULTS
+181	185	Fe3+	chemical	We attributed the precipitates observed in the EncFtnsH ferroxidase assay to the production of insoluble Fe3+ complexes, which led us to propose that EncFtn does not directly store Fe3+ in a mineral form.	RESULTS
+29	38	native MS	experimental_method	This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.	RESULTS
+74	78	iron	chemical	This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.	RESULTS
+96	100	iron	chemical	This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.	RESULTS
+110	119	decameric	oligomeric_state	This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.	RESULTS
+120	126	EncFtn	protein	This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.	RESULTS
+136	145	structure	evidence	This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.	RESULTS
+183	202	iron-storage cavity	site	This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.	RESULTS
+221	230	classical	protein_state	This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.	RESULTS
+231	240	ferritins	protein_type	This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.	RESULTS
+245	264	Dps family proteins	protein_type	This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.	RESULTS
+302	306	Fe3+	chemical	This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.	RESULTS
+320	335	nanocompartment	complex_assembly	This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.	RESULTS
+336	346	structures	evidence	This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.	RESULTS
+69	75	EncFtn	protein	To analyze the products of these reactions and determine whether the EncFtn and encapsulin were able to store iron in a mineral form, we performed TEM on the reaction mixtures from the ferroxidase assay.	RESULTS
+80	90	encapsulin	protein	To analyze the products of these reactions and determine whether the EncFtn and encapsulin were able to store iron in a mineral form, we performed TEM on the reaction mixtures from the ferroxidase assay.	RESULTS
+110	114	iron	chemical	To analyze the products of these reactions and determine whether the EncFtn and encapsulin were able to store iron in a mineral form, we performed TEM on the reaction mixtures from the ferroxidase assay.	RESULTS
+147	150	TEM	experimental_method	To analyze the products of these reactions and determine whether the EncFtn and encapsulin were able to store iron in a mineral form, we performed TEM on the reaction mixtures from the ferroxidase assay.	RESULTS
+185	202	ferroxidase assay	experimental_method	To analyze the products of these reactions and determine whether the EncFtn and encapsulin were able to store iron in a mineral form, we performed TEM on the reaction mixtures from the ferroxidase assay.	RESULTS
+4	12	EncFtnsH	protein	The EncFtnsH reaction mixture showed the formation of large, irregular electron-dense precipitates (Figure 8—figure supplement 1A).	RESULTS
+67	71	Fe2+	chemical	A similar distribution of particles was observed after addition of Fe2+ to the encapsulin protein (Figure 8—figure supplement 1B).	RESULTS
+79	89	encapsulin	protein	A similar distribution of particles was observed after addition of Fe2+ to the encapsulin protein (Figure 8—figure supplement 1B).	RESULTS
+25	29	Fe2+	chemical	In contrast, addition of Fe2+ to the EncFtn-Enc nanocompartment resulted in small, highly regular, electron dense particles of approximately 5 nm in diameter (Figure 8—figure supplement 1C); we interpret these observations as controlled mineralization of iron within the nanocompartment.	RESULTS
+37	47	EncFtn-Enc	complex_assembly	In contrast, addition of Fe2+ to the EncFtn-Enc nanocompartment resulted in small, highly regular, electron dense particles of approximately 5 nm in diameter (Figure 8—figure supplement 1C); we interpret these observations as controlled mineralization of iron within the nanocompartment.	RESULTS
+48	63	nanocompartment	complex_assembly	In contrast, addition of Fe2+ to the EncFtn-Enc nanocompartment resulted in small, highly regular, electron dense particles of approximately 5 nm in diameter (Figure 8—figure supplement 1C); we interpret these observations as controlled mineralization of iron within the nanocompartment.	RESULTS
+255	259	iron	chemical	In contrast, addition of Fe2+ to the EncFtn-Enc nanocompartment resulted in small, highly regular, electron dense particles of approximately 5 nm in diameter (Figure 8—figure supplement 1C); we interpret these observations as controlled mineralization of iron within the nanocompartment.	RESULTS
+271	286	nanocompartment	complex_assembly	In contrast, addition of Fe2+ to the EncFtn-Enc nanocompartment resulted in small, highly regular, electron dense particles of approximately 5 nm in diameter (Figure 8—figure supplement 1C); we interpret these observations as controlled mineralization of iron within the nanocompartment.	RESULTS
+12	16	Fe2+	chemical	Addition of Fe2+ to apoferritin resulted in a mixture of large particles and small (~2 nm) particles consistent with partial mineralization by the ferritin and some background oxidation of the iron (Figure 8—figure supplement 1D).	RESULTS
+20	31	apoferritin	protein_state	Addition of Fe2+ to apoferritin resulted in a mixture of large particles and small (~2 nm) particles consistent with partial mineralization by the ferritin and some background oxidation of the iron (Figure 8—figure supplement 1D).	RESULTS
+147	155	ferritin	protein_type	Addition of Fe2+ to apoferritin resulted in a mixture of large particles and small (~2 nm) particles consistent with partial mineralization by the ferritin and some background oxidation of the iron (Figure 8—figure supplement 1D).	RESULTS
+193	197	iron	chemical	Addition of Fe2+ to apoferritin resulted in a mixture of large particles and small (~2 nm) particles consistent with partial mineralization by the ferritin and some background oxidation of the iron (Figure 8—figure supplement 1D).	RESULTS
+0	18	Negative stain TEM	experimental_method	Negative stain TEM of these samples revealed that upon addition of iron, the EncFtnsH protein showed significant aggregation (Figure 8—figure supplement 1F); while the encapsulin, EncFtn-Enc system, and apoferritin are present as distinct nanocompartments without significant protein aggregation (Figure 8—figure supplement 1G–I).	RESULTS
+67	71	iron	chemical	Negative stain TEM of these samples revealed that upon addition of iron, the EncFtnsH protein showed significant aggregation (Figure 8—figure supplement 1F); while the encapsulin, EncFtn-Enc system, and apoferritin are present as distinct nanocompartments without significant protein aggregation (Figure 8—figure supplement 1G–I).	RESULTS
+77	85	EncFtnsH	protein	Negative stain TEM of these samples revealed that upon addition of iron, the EncFtnsH protein showed significant aggregation (Figure 8—figure supplement 1F); while the encapsulin, EncFtn-Enc system, and apoferritin are present as distinct nanocompartments without significant protein aggregation (Figure 8—figure supplement 1G–I).	RESULTS
+168	178	encapsulin	protein	Negative stain TEM of these samples revealed that upon addition of iron, the EncFtnsH protein showed significant aggregation (Figure 8—figure supplement 1F); while the encapsulin, EncFtn-Enc system, and apoferritin are present as distinct nanocompartments without significant protein aggregation (Figure 8—figure supplement 1G–I).	RESULTS
+180	190	EncFtn-Enc	complex_assembly	Negative stain TEM of these samples revealed that upon addition of iron, the EncFtnsH protein showed significant aggregation (Figure 8—figure supplement 1F); while the encapsulin, EncFtn-Enc system, and apoferritin are present as distinct nanocompartments without significant protein aggregation (Figure 8—figure supplement 1G–I).	RESULTS
+203	214	apoferritin	protein_state	Negative stain TEM of these samples revealed that upon addition of iron, the EncFtnsH protein showed significant aggregation (Figure 8—figure supplement 1F); while the encapsulin, EncFtn-Enc system, and apoferritin are present as distinct nanocompartments without significant protein aggregation (Figure 8—figure supplement 1G–I).	RESULTS
+239	255	nanocompartments	complex_assembly	Negative stain TEM of these samples revealed that upon addition of iron, the EncFtnsH protein showed significant aggregation (Figure 8—figure supplement 1F); while the encapsulin, EncFtn-Enc system, and apoferritin are present as distinct nanocompartments without significant protein aggregation (Figure 8—figure supplement 1G–I).	RESULTS
+0	4	Iron	chemical	Iron storage in encapsulin nanocompartments	RESULTS
+16	26	encapsulin	protein	Iron storage in encapsulin nanocompartments	RESULTS
+27	43	nanocompartments	complex_assembly	Iron storage in encapsulin nanocompartments	RESULTS
+19	36	ferroxidase assay	experimental_method	The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage.	RESULTS
+41	52	micrographs	evidence	The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage.	RESULTS
+140	149	classical	protein_state	The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage.	RESULTS
+150	159	ferritins	protein_type	The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage.	RESULTS
+182	188	EncFtn	protein	The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage.	RESULTS
+193	203	encapsulin	protein	The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage.	RESULTS
+223	229	EncFtn	protein	The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage.	RESULTS
+242	253	ferroxidase	protein_type	The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage.	RESULTS
+262	272	encapsulin	protein	The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage.	RESULTS
+273	278	shell	structure_element	The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage.	RESULTS
+321	325	iron	chemical	The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage.	RESULTS
+38	42	Fe2+	chemical	To investigate this further, we added Fe2+ at various concentrations to samples of apo-ferritin, EncFtn, isolated encapsulin, and the EncFtn-Enc protein complex, and subjected these samples to a ferrozine assay to quantify the amount of iron associated with the proteins after three hours of incubation.	RESULTS
+83	86	apo	protein_state	To investigate this further, we added Fe2+ at various concentrations to samples of apo-ferritin, EncFtn, isolated encapsulin, and the EncFtn-Enc protein complex, and subjected these samples to a ferrozine assay to quantify the amount of iron associated with the proteins after three hours of incubation.	RESULTS
+87	95	ferritin	protein_type	To investigate this further, we added Fe2+ at various concentrations to samples of apo-ferritin, EncFtn, isolated encapsulin, and the EncFtn-Enc protein complex, and subjected these samples to a ferrozine assay to quantify the amount of iron associated with the proteins after three hours of incubation.	RESULTS
+97	103	EncFtn	protein	To investigate this further, we added Fe2+ at various concentrations to samples of apo-ferritin, EncFtn, isolated encapsulin, and the EncFtn-Enc protein complex, and subjected these samples to a ferrozine assay to quantify the amount of iron associated with the proteins after three hours of incubation.	RESULTS
+114	124	encapsulin	protein	To investigate this further, we added Fe2+ at various concentrations to samples of apo-ferritin, EncFtn, isolated encapsulin, and the EncFtn-Enc protein complex, and subjected these samples to a ferrozine assay to quantify the amount of iron associated with the proteins after three hours of incubation.	RESULTS
+134	144	EncFtn-Enc	complex_assembly	To investigate this further, we added Fe2+ at various concentrations to samples of apo-ferritin, EncFtn, isolated encapsulin, and the EncFtn-Enc protein complex, and subjected these samples to a ferrozine assay to quantify the amount of iron associated with the proteins after three hours of incubation.	RESULTS
+195	210	ferrozine assay	experimental_method	To investigate this further, we added Fe2+ at various concentrations to samples of apo-ferritin, EncFtn, isolated encapsulin, and the EncFtn-Enc protein complex, and subjected these samples to a ferrozine assay to quantify the amount of iron associated with the proteins after three hours of incubation.	RESULTS
+237	241	iron	chemical	To investigate this further, we added Fe2+ at various concentrations to samples of apo-ferritin, EncFtn, isolated encapsulin, and the EncFtn-Enc protein complex, and subjected these samples to a ferrozine assay to quantify the amount of iron associated with the proteins after three hours of incubation.	RESULTS
+12	16	iron	chemical	The maximum iron loading capacity of these systems was calculated as the quantity of iron per biological assembly (Figure 8C).	RESULTS
+85	89	iron	chemical	The maximum iron loading capacity of these systems was calculated as the quantity of iron per biological assembly (Figure 8C).	RESULTS
+19	27	EncFtnsH	protein	In this assay, the EncFtnsH decamer binds a maximum of around 48 iron ions before excess iron induces protein precipitation.	RESULTS
+28	35	decamer	oligomeric_state	In this assay, the EncFtnsH decamer binds a maximum of around 48 iron ions before excess iron induces protein precipitation.	RESULTS
+65	69	iron	chemical	In this assay, the EncFtnsH decamer binds a maximum of around 48 iron ions before excess iron induces protein precipitation.	RESULTS
+89	93	iron	chemical	In this assay, the EncFtnsH decamer binds a maximum of around 48 iron ions before excess iron induces protein precipitation.	RESULTS
+4	14	encapsulin	protein	The encapsulin shell protein can sequester about 2200 iron ions before significant protein loss occurs, and the reconstituted EncFtn-Enc nanocompartment sequestered about 4150 iron ions.	RESULTS
+15	20	shell	structure_element	The encapsulin shell protein can sequester about 2200 iron ions before significant protein loss occurs, and the reconstituted EncFtn-Enc nanocompartment sequestered about 4150 iron ions.	RESULTS
+54	58	iron	chemical	The encapsulin shell protein can sequester about 2200 iron ions before significant protein loss occurs, and the reconstituted EncFtn-Enc nanocompartment sequestered about 4150 iron ions.	RESULTS
+126	136	EncFtn-Enc	complex_assembly	The encapsulin shell protein can sequester about 2200 iron ions before significant protein loss occurs, and the reconstituted EncFtn-Enc nanocompartment sequestered about 4150 iron ions.	RESULTS
+137	152	nanocompartment	complex_assembly	The encapsulin shell protein can sequester about 2200 iron ions before significant protein loss occurs, and the reconstituted EncFtn-Enc nanocompartment sequestered about 4150 iron ions.	RESULTS
+176	180	iron	chemical	The encapsulin shell protein can sequester about 2200 iron ions before significant protein loss occurs, and the reconstituted EncFtn-Enc nanocompartment sequestered about 4150 iron ions.	RESULTS
+50	61	apoferritin	protein_state	This latter result is significantly more than the apoferritin used in our assay, which sequesters approximately 570 iron ions in this assay (Figure 8C, Table 5).	RESULTS
+116	120	iron	chemical	This latter result is significantly more than the apoferritin used in our assay, which sequesters approximately 570 iron ions in this assay (Figure 8C, Table 5).	RESULTS
+75	81	EncFtn	protein	Consideration of the functional oligomeric states of these proteins, where EncFtn is a decamer and encapsulin forms an icosahedral cage, and estimation of the iron loading capacity of these complexes gives insight into the role of the two proteins in iron storage and mineralization.	RESULTS
+87	94	decamer	oligomeric_state	Consideration of the functional oligomeric states of these proteins, where EncFtn is a decamer and encapsulin forms an icosahedral cage, and estimation of the iron loading capacity of these complexes gives insight into the role of the two proteins in iron storage and mineralization.	RESULTS
+99	109	encapsulin	protein	Consideration of the functional oligomeric states of these proteins, where EncFtn is a decamer and encapsulin forms an icosahedral cage, and estimation of the iron loading capacity of these complexes gives insight into the role of the two proteins in iron storage and mineralization.	RESULTS
+119	130	icosahedral	protein_state	Consideration of the functional oligomeric states of these proteins, where EncFtn is a decamer and encapsulin forms an icosahedral cage, and estimation of the iron loading capacity of these complexes gives insight into the role of the two proteins in iron storage and mineralization.	RESULTS
+131	135	cage	complex_assembly	Consideration of the functional oligomeric states of these proteins, where EncFtn is a decamer and encapsulin forms an icosahedral cage, and estimation of the iron loading capacity of these complexes gives insight into the role of the two proteins in iron storage and mineralization.	RESULTS
+159	163	iron	chemical	Consideration of the functional oligomeric states of these proteins, where EncFtn is a decamer and encapsulin forms an icosahedral cage, and estimation of the iron loading capacity of these complexes gives insight into the role of the two proteins in iron storage and mineralization.	RESULTS
+251	255	iron	chemical	Consideration of the functional oligomeric states of these proteins, where EncFtn is a decamer and encapsulin forms an icosahedral cage, and estimation of the iron loading capacity of these complexes gives insight into the role of the two proteins in iron storage and mineralization.	RESULTS
+0	6	EncFtn	protein	EncFtn decamers bind up to 48 iron ions (Figure 8C), which is significantly higher than the stoichiometry of fifteen metal ions visible in the FOC and E31/34-site of the crystal structure of the EncFtnsH decamer and our MS analysis.	RESULTS
+7	15	decamers	oligomeric_state	EncFtn decamers bind up to 48 iron ions (Figure 8C), which is significantly higher than the stoichiometry of fifteen metal ions visible in the FOC and E31/34-site of the crystal structure of the EncFtnsH decamer and our MS analysis.	RESULTS
+30	34	iron	chemical	EncFtn decamers bind up to 48 iron ions (Figure 8C), which is significantly higher than the stoichiometry of fifteen metal ions visible in the FOC and E31/34-site of the crystal structure of the EncFtnsH decamer and our MS analysis.	RESULTS
+143	146	FOC	site	EncFtn decamers bind up to 48 iron ions (Figure 8C), which is significantly higher than the stoichiometry of fifteen metal ions visible in the FOC and E31/34-site of the crystal structure of the EncFtnsH decamer and our MS analysis.	RESULTS
+151	162	E31/34-site	site	EncFtn decamers bind up to 48 iron ions (Figure 8C), which is significantly higher than the stoichiometry of fifteen metal ions visible in the FOC and E31/34-site of the crystal structure of the EncFtnsH decamer and our MS analysis.	RESULTS
+170	187	crystal structure	evidence	EncFtn decamers bind up to 48 iron ions (Figure 8C), which is significantly higher than the stoichiometry of fifteen metal ions visible in the FOC and E31/34-site of the crystal structure of the EncFtnsH decamer and our MS analysis.	RESULTS
+195	203	EncFtnsH	protein	EncFtn decamers bind up to 48 iron ions (Figure 8C), which is significantly higher than the stoichiometry of fifteen metal ions visible in the FOC and E31/34-site of the crystal structure of the EncFtnsH decamer and our MS analysis.	RESULTS
+204	211	decamer	oligomeric_state	EncFtn decamers bind up to 48 iron ions (Figure 8C), which is significantly higher than the stoichiometry of fifteen metal ions visible in the FOC and E31/34-site of the crystal structure of the EncFtnsH decamer and our MS analysis.	RESULTS
+220	222	MS	experimental_method	EncFtn decamers bind up to 48 iron ions (Figure 8C), which is significantly higher than the stoichiometry of fifteen metal ions visible in the FOC and E31/34-site of the crystal structure of the EncFtnsH decamer and our MS analysis.	RESULTS
+30	51	solution measurements	experimental_method	The discrepancy between these solution measurements and our MS analysis may indicate that there are additional metal-binding sites on the interior channel and exterior faces of the protein; this is consistent with our identification of a number of weak metal-binding sites at the surface of the protein in the crystal structure (Figure 5D).	RESULTS
+60	62	MS	experimental_method	The discrepancy between these solution measurements and our MS analysis may indicate that there are additional metal-binding sites on the interior channel and exterior faces of the protein; this is consistent with our identification of a number of weak metal-binding sites at the surface of the protein in the crystal structure (Figure 5D).	RESULTS
+111	130	metal-binding sites	site	The discrepancy between these solution measurements and our MS analysis may indicate that there are additional metal-binding sites on the interior channel and exterior faces of the protein; this is consistent with our identification of a number of weak metal-binding sites at the surface of the protein in the crystal structure (Figure 5D).	RESULTS
+147	154	channel	site	The discrepancy between these solution measurements and our MS analysis may indicate that there are additional metal-binding sites on the interior channel and exterior faces of the protein; this is consistent with our identification of a number of weak metal-binding sites at the surface of the protein in the crystal structure (Figure 5D).	RESULTS
+253	272	metal-binding sites	site	The discrepancy between these solution measurements and our MS analysis may indicate that there are additional metal-binding sites on the interior channel and exterior faces of the protein; this is consistent with our identification of a number of weak metal-binding sites at the surface of the protein in the crystal structure (Figure 5D).	RESULTS
+310	327	crystal structure	evidence	The discrepancy between these solution measurements and our MS analysis may indicate that there are additional metal-binding sites on the interior channel and exterior faces of the protein; this is consistent with our identification of a number of weak metal-binding sites at the surface of the protein in the crystal structure (Figure 5D).	RESULTS
+48	52	Fe2+	chemical	These observations are consistent with hydrated Fe2+ ions being channeled to the active site from the E31/34-site and the subsequent exit of Fe3+ products on the outer surface, as is seen in other ferritin family proteins.	RESULTS
+81	92	active site	site	These observations are consistent with hydrated Fe2+ ions being channeled to the active site from the E31/34-site and the subsequent exit of Fe3+ products on the outer surface, as is seen in other ferritin family proteins.	RESULTS
+102	113	E31/34-site	site	These observations are consistent with hydrated Fe2+ ions being channeled to the active site from the E31/34-site and the subsequent exit of Fe3+ products on the outer surface, as is seen in other ferritin family proteins.	RESULTS
+141	145	Fe3+	chemical	These observations are consistent with hydrated Fe2+ ions being channeled to the active site from the E31/34-site and the subsequent exit of Fe3+ products on the outer surface, as is seen in other ferritin family proteins.	RESULTS
+197	205	ferritin	protein_type	These observations are consistent with hydrated Fe2+ ions being channeled to the active site from the E31/34-site and the subsequent exit of Fe3+ products on the outer surface, as is seen in other ferritin family proteins.	RESULTS
+19	29	encapsulin	protein	While the isolated encapsulin shell does not display any ferroxidase activity, it binds around 2200 iron ions in our assay (Table 5).	RESULTS
+30	35	shell	structure_element	While the isolated encapsulin shell does not display any ferroxidase activity, it binds around 2200 iron ions in our assay (Table 5).	RESULTS
+57	68	ferroxidase	protein_type	While the isolated encapsulin shell does not display any ferroxidase activity, it binds around 2200 iron ions in our assay (Table 5).	RESULTS
+100	104	iron	chemical	While the isolated encapsulin shell does not display any ferroxidase activity, it binds around 2200 iron ions in our assay (Table 5).	RESULTS
+22	27	shell	structure_element	This implies that the shell can bind a significant amount of iron on its outer and inner surfaces.	RESULTS
+61	65	iron	chemical	This implies that the shell can bind a significant amount of iron on its outer and inner surfaces.	RESULTS
+47	56	classical	protein_state	While the maximum reported loading capacity of classical ferritins is approximately 4500 iron ions, in our assay system we were only able to load apoferritin with around 570 iron ions.	RESULTS
+57	66	ferritins	protein_type	While the maximum reported loading capacity of classical ferritins is approximately 4500 iron ions, in our assay system we were only able to load apoferritin with around 570 iron ions.	RESULTS
+89	93	iron	chemical	While the maximum reported loading capacity of classical ferritins is approximately 4500 iron ions, in our assay system we were only able to load apoferritin with around 570 iron ions.	RESULTS
+146	157	apoferritin	protein_state	While the maximum reported loading capacity of classical ferritins is approximately 4500 iron ions, in our assay system we were only able to load apoferritin with around 570 iron ions.	RESULTS
+174	178	iron	chemical	While the maximum reported loading capacity of classical ferritins is approximately 4500 iron ions, in our assay system we were only able to load apoferritin with around 570 iron ions.	RESULTS
+25	35	EncFtn-Enc	complex_assembly	However, the recombinant EncFtn-Enc nanocompartment was able to bind over 4100 iron ions in the same time period, over seven times the amount seen for the apoferritin.	RESULTS
+36	51	nanocompartment	complex_assembly	However, the recombinant EncFtn-Enc nanocompartment was able to bind over 4100 iron ions in the same time period, over seven times the amount seen for the apoferritin.	RESULTS
+79	83	iron	chemical	However, the recombinant EncFtn-Enc nanocompartment was able to bind over 4100 iron ions in the same time period, over seven times the amount seen for the apoferritin.	RESULTS
+155	166	apoferritin	protein_state	However, the recombinant EncFtn-Enc nanocompartment was able to bind over 4100 iron ions in the same time period, over seven times the amount seen for the apoferritin.	RESULTS
+49	53	iron	chemical	We note we do not reach the experimental maximum iron loading for apoferritin and therefore the total iron-loading capacity of our system may be significantly higher than in this experimental system.	RESULTS
+66	77	apoferritin	protein_state	We note we do not reach the experimental maximum iron loading for apoferritin and therefore the total iron-loading capacity of our system may be significantly higher than in this experimental system.	RESULTS
+102	106	iron	chemical	We note we do not reach the experimental maximum iron loading for apoferritin and therefore the total iron-loading capacity of our system may be significantly higher than in this experimental system.	RESULTS
+35	41	EncFtn	protein	Taken together, our data show that EncFtn can catalytically oxidize Fe2+ to Fe3+; however, iron binding in EncFtn is limited to the FOC and several surface metal binding sites.	RESULTS
+68	72	Fe2+	chemical	Taken together, our data show that EncFtn can catalytically oxidize Fe2+ to Fe3+; however, iron binding in EncFtn is limited to the FOC and several surface metal binding sites.	RESULTS
+76	80	Fe3+	chemical	Taken together, our data show that EncFtn can catalytically oxidize Fe2+ to Fe3+; however, iron binding in EncFtn is limited to the FOC and several surface metal binding sites.	RESULTS
+91	95	iron	chemical	Taken together, our data show that EncFtn can catalytically oxidize Fe2+ to Fe3+; however, iron binding in EncFtn is limited to the FOC and several surface metal binding sites.	RESULTS
+107	113	EncFtn	protein	Taken together, our data show that EncFtn can catalytically oxidize Fe2+ to Fe3+; however, iron binding in EncFtn is limited to the FOC and several surface metal binding sites.	RESULTS
+132	135	FOC	site	Taken together, our data show that EncFtn can catalytically oxidize Fe2+ to Fe3+; however, iron binding in EncFtn is limited to the FOC and several surface metal binding sites.	RESULTS
+156	175	metal binding sites	site	Taken together, our data show that EncFtn can catalytically oxidize Fe2+ to Fe3+; however, iron binding in EncFtn is limited to the FOC and several surface metal binding sites.	RESULTS
+17	27	encapsulin	protein	In contrast, the encapsulin protein displays no catalytic activity, but has the ability to bind a considerable amount of iron.	RESULTS
+121	125	iron	chemical	In contrast, the encapsulin protein displays no catalytic activity, but has the ability to bind a considerable amount of iron.	RESULTS
+13	23	EncFtn-Enc	complex_assembly	Finally, the EncFtn-Enc nanocompartment complex retains the catalytic activity of EncFtn, and sequesters iron within the encapsulin shell at a higher level than the isolated components of the system, and at a significantly higher level than the classical ferritins.	RESULTS
+24	39	nanocompartment	complex_assembly	Finally, the EncFtn-Enc nanocompartment complex retains the catalytic activity of EncFtn, and sequesters iron within the encapsulin shell at a higher level than the isolated components of the system, and at a significantly higher level than the classical ferritins.	RESULTS
+82	88	EncFtn	protein	Finally, the EncFtn-Enc nanocompartment complex retains the catalytic activity of EncFtn, and sequesters iron within the encapsulin shell at a higher level than the isolated components of the system, and at a significantly higher level than the classical ferritins.	RESULTS
+105	109	iron	chemical	Finally, the EncFtn-Enc nanocompartment complex retains the catalytic activity of EncFtn, and sequesters iron within the encapsulin shell at a higher level than the isolated components of the system, and at a significantly higher level than the classical ferritins.	RESULTS
+121	131	encapsulin	protein	Finally, the EncFtn-Enc nanocompartment complex retains the catalytic activity of EncFtn, and sequesters iron within the encapsulin shell at a higher level than the isolated components of the system, and at a significantly higher level than the classical ferritins.	RESULTS
+132	137	shell	structure_element	Finally, the EncFtn-Enc nanocompartment complex retains the catalytic activity of EncFtn, and sequesters iron within the encapsulin shell at a higher level than the isolated components of the system, and at a significantly higher level than the classical ferritins.	RESULTS
+245	254	classical	protein_state	Finally, the EncFtn-Enc nanocompartment complex retains the catalytic activity of EncFtn, and sequesters iron within the encapsulin shell at a higher level than the isolated components of the system, and at a significantly higher level than the classical ferritins.	RESULTS
+255	264	ferritins	protein_type	Finally, the EncFtn-Enc nanocompartment complex retains the catalytic activity of EncFtn, and sequesters iron within the encapsulin shell at a higher level than the isolated components of the system, and at a significantly higher level than the classical ferritins.	RESULTS
+30	46	nanocompartments	complex_assembly	 Furthermore, our recombinant nanocompartments may not have the physiological subunit stoichiometry, and the iron-loading capacity of native nanocompartments is potentially much higher than the level we have observed.	RESULTS
+109	113	iron	chemical	 Furthermore, our recombinant nanocompartments may not have the physiological subunit stoichiometry, and the iron-loading capacity of native nanocompartments is potentially much higher than the level we have observed.	RESULTS
+134	140	native	protein_state	 Furthermore, our recombinant nanocompartments may not have the physiological subunit stoichiometry, and the iron-loading capacity of native nanocompartments is potentially much higher than the level we have observed.	RESULTS
+141	157	nanocompartments	complex_assembly	 Furthermore, our recombinant nanocompartments may not have the physiological subunit stoichiometry, and the iron-loading capacity of native nanocompartments is potentially much higher than the level we have observed.	RESULTS
+0	11	Mutagenesis	experimental_method	Mutagenesis of the EncFtnsHferroxidase center	RESULTS
+19	27	EncFtnsH	protein	Mutagenesis of the EncFtnsHferroxidase center	RESULTS
+27	45	ferroxidase center	site	Mutagenesis of the EncFtnsHferroxidase center	RESULTS
+28	37	R. rubrum	species	Purification of recombinant R. rubrum EncFtnsH FOC mutants.	FIG
+38	46	EncFtnsH	protein	Purification of recombinant R. rubrum EncFtnsH FOC mutants.	FIG
+47	50	FOC	site	Purification of recombinant R. rubrum EncFtnsH FOC mutants.	FIG
+51	58	mutants	protein_state	Purification of recombinant R. rubrum EncFtnsH FOC mutants.	FIG
+7	14	mutants	protein_state	Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex 200 size-exclusion chromatography.	FIG
+15	19	E32A	mutant	Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex 200 size-exclusion chromatography.	FIG
+21	25	E62A	mutant	Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex 200 size-exclusion chromatography.	FIG
+31	35	H65A	mutant	Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex 200 size-exclusion chromatography.	FIG
+39	47	EncFtnsH	protein	Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex 200 size-exclusion chromatography.	FIG
+62	79	E. coli BL21(DE3)	species	Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex 200 size-exclusion chromatography.	FIG
+95	97	MM	experimental_method	Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex 200 size-exclusion chromatography.	FIG
+102	104	MM	experimental_method	Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex 200 size-exclusion chromatography.	FIG
+123	127	iron	chemical	Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex 200 size-exclusion chromatography.	FIG
+159	188	size-exclusion chromatography	experimental_method	Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex 200 size-exclusion chromatography.	FIG
+4	31	Gel-filtration chromatogram	evidence	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+39	43	E32A	mutant	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+44	50	mutant	protein_state	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+59	67	EncFtnsH	protein	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+83	98	elution profile	evidence	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+145	154	decameric	oligomeric_state	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+201	208	monomer	oligomeric_state	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+214	242	Gel-filtration chromatograhy	experimental_method	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+250	254	E62A	mutant	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+255	261	mutant	protein_state	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+270	278	EncFtnsH	protein	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+294	309	elution profile	evidence	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+330	339	decameric	oligomeric_state	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+350	379	Gel-filtration chromatography	experimental_method	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+387	391	H65A	mutant	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+392	398	mutant	protein_state	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+407	415	EncFtnsH	protein	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+471	478	monomer	oligomeric_state	(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.	FIG
+65	87	metal binding residues	site	To investigate the structural and biochemical role played by the metal binding residues in the di-iron FOC of EncFtnsH we produced alanine mutations in each of these residues: Glu32, Glu62, and His65.	RESULTS
+95	106	di-iron FOC	site	To investigate the structural and biochemical role played by the metal binding residues in the di-iron FOC of EncFtnsH we produced alanine mutations in each of these residues: Glu32, Glu62, and His65.	RESULTS
+110	118	EncFtnsH	protein	To investigate the structural and biochemical role played by the metal binding residues in the di-iron FOC of EncFtnsH we produced alanine mutations in each of these residues: Glu32, Glu62, and His65.	RESULTS
+131	148	alanine mutations	experimental_method	To investigate the structural and biochemical role played by the metal binding residues in the di-iron FOC of EncFtnsH we produced alanine mutations in each of these residues: Glu32, Glu62, and His65.	RESULTS
+176	181	Glu32	residue_name_number	To investigate the structural and biochemical role played by the metal binding residues in the di-iron FOC of EncFtnsH we produced alanine mutations in each of these residues: Glu32, Glu62, and His65.	RESULTS
+183	188	Glu62	residue_name_number	To investigate the structural and biochemical role played by the metal binding residues in the di-iron FOC of EncFtnsH we produced alanine mutations in each of these residues: Glu32, Glu62, and His65.	RESULTS
+194	199	His65	residue_name_number	To investigate the structural and biochemical role played by the metal binding residues in the di-iron FOC of EncFtnsH we produced alanine mutations in each of these residues: Glu32, Glu62, and His65.	RESULTS
+6	14	EncFtnsH	protein	These EncFtnsH mutants were produced in E. coli cells grown in MM, both in the absence and presence of additional iron.	RESULTS
+15	22	mutants	protein_state	These EncFtnsH mutants were produced in E. coli cells grown in MM, both in the absence and presence of additional iron.	RESULTS
+40	47	E. coli	species	These EncFtnsH mutants were produced in E. coli cells grown in MM, both in the absence and presence of additional iron.	RESULTS
+63	65	MM	experimental_method	These EncFtnsH mutants were produced in E. coli cells grown in MM, both in the absence and presence of additional iron.	RESULTS
+79	86	absence	protein_state	These EncFtnsH mutants were produced in E. coli cells grown in MM, both in the absence and presence of additional iron.	RESULTS
+91	102	presence of	protein_state	These EncFtnsH mutants were produced in E. coli cells grown in MM, both in the absence and presence of additional iron.	RESULTS
+114	118	iron	chemical	These EncFtnsH mutants were produced in E. coli cells grown in MM, both in the absence and presence of additional iron.	RESULTS
+4	8	E32A	mutant	The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9).	RESULTS
+13	17	E62A	mutant	The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9).	RESULTS
+18	25	mutants	protein_state	The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9).	RESULTS
+38	41	SEC	experimental_method	The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9).	RESULTS
+74	83	decameric	oligomeric_state	The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9).	RESULTS
+92	100	EncFtnsH	protein	The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9).	RESULTS
+129	136	monomer	oligomeric_state	The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9).	RESULTS
+142	146	H65A	mutant	The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9).	RESULTS
+147	153	mutant	protein_state	The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9).	RESULTS
+193	202	monomeric	oligomeric_state	The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9).	RESULTS
+211	219	EncFtnsH	protein	The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9).	RESULTS
+15	22	mutants	protein_state	For all of the mutants studied, no change in oligomerization state was apparent upon addition of Fe2+ in vitro.	RESULTS
+97	101	Fe2+	chemical	For all of the mutants studied, no change in oligomerization state was apparent upon addition of Fe2+ in vitro.	RESULTS
+0	24	Native mass spectrometry	experimental_method	Native mass spectrometry of EncFtnsH mutants.	FIG
+28	36	EncFtnsH	protein	Native mass spectrometry of EncFtnsH mutants.	FIG
+37	44	mutants	protein_state	Native mass spectrometry of EncFtnsH mutants.	FIG
+4	11	spectra	evidence	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles).	FIG
+45	52	acetate	chemical	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles).	FIG
+103	112	Wild-type	protein_state	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles).	FIG
+113	121	EncFtnsH	protein	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles).	FIG
+129	139	absence of	protein_state	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles).	FIG
+140	144	iron	chemical	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles).	FIG
+156	181	charge state distribution	evidence	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles).	FIG
+200	207	monomer	oligomeric_state	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles).	FIG
+233	237	E32A	mutant	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles).	FIG
+238	246	EncFtnsH	protein	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles).	FIG
+258	271	charge states	evidence	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles).	FIG
+290	297	decamer	oligomeric_state	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles).	FIG
+352	359	monomer	oligomeric_state	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles).	FIG
+363	367	E32A	mutant	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles).	FIG
+368	374	mutant	protein_state	All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles).	FIG
+4	8	E62A	mutant	(C) E62A EncFtnsH displays charge states consistent with a decamer (green circles). (D) H65A EncFtnsH displays charge states consistent with both monomer (blue circles) and dimer (purple circles).	FIG
+9	17	EncFtnsH	protein	(C) E62A EncFtnsH displays charge states consistent with a decamer (green circles). (D) H65A EncFtnsH displays charge states consistent with both monomer (blue circles) and dimer (purple circles).	FIG
+27	40	charge states	evidence	(C) E62A EncFtnsH displays charge states consistent with a decamer (green circles). (D) H65A EncFtnsH displays charge states consistent with both monomer (blue circles) and dimer (purple circles).	FIG
+59	66	decamer	oligomeric_state	(C) E62A EncFtnsH displays charge states consistent with a decamer (green circles). (D) H65A EncFtnsH displays charge states consistent with both monomer (blue circles) and dimer (purple circles).	FIG
+88	92	H65A	mutant	(C) E62A EncFtnsH displays charge states consistent with a decamer (green circles). (D) H65A EncFtnsH displays charge states consistent with both monomer (blue circles) and dimer (purple circles).	FIG
+93	101	EncFtnsH	protein	(C) E62A EncFtnsH displays charge states consistent with a decamer (green circles). (D) H65A EncFtnsH displays charge states consistent with both monomer (blue circles) and dimer (purple circles).	FIG
+111	124	charge states	evidence	(C) E62A EncFtnsH displays charge states consistent with a decamer (green circles). (D) H65A EncFtnsH displays charge states consistent with both monomer (blue circles) and dimer (purple circles).	FIG
+146	153	monomer	oligomeric_state	(C) E62A EncFtnsH displays charge states consistent with a decamer (green circles). (D) H65A EncFtnsH displays charge states consistent with both monomer (blue circles) and dimer (purple circles).	FIG
+173	178	dimer	oligomeric_state	(C) E62A EncFtnsH displays charge states consistent with a decamer (green circles). (D) H65A EncFtnsH displays charge states consistent with both monomer (blue circles) and dimer (purple circles).	FIG
+15	18	SEC	experimental_method	In addition to SEC studies, native mass spectrometry of the apo-EncFtnsH mutants was performed and compared with the wild-type apo-EncFtnsH protein (Figure 10).	RESULTS
+28	52	native mass spectrometry	experimental_method	In addition to SEC studies, native mass spectrometry of the apo-EncFtnsH mutants was performed and compared with the wild-type apo-EncFtnsH protein (Figure 10).	RESULTS
+60	63	apo	protein_state	In addition to SEC studies, native mass spectrometry of the apo-EncFtnsH mutants was performed and compared with the wild-type apo-EncFtnsH protein (Figure 10).	RESULTS
+64	72	EncFtnsH	protein	In addition to SEC studies, native mass spectrometry of the apo-EncFtnsH mutants was performed and compared with the wild-type apo-EncFtnsH protein (Figure 10).	RESULTS
+73	80	mutants	protein_state	In addition to SEC studies, native mass spectrometry of the apo-EncFtnsH mutants was performed and compared with the wild-type apo-EncFtnsH protein (Figure 10).	RESULTS
+117	126	wild-type	protein_state	In addition to SEC studies, native mass spectrometry of the apo-EncFtnsH mutants was performed and compared with the wild-type apo-EncFtnsH protein (Figure 10).	RESULTS
+127	130	apo	protein_state	In addition to SEC studies, native mass spectrometry of the apo-EncFtnsH mutants was performed and compared with the wild-type apo-EncFtnsH protein (Figure 10).	RESULTS
+131	139	EncFtnsH	protein	In addition to SEC studies, native mass spectrometry of the apo-EncFtnsH mutants was performed and compared with the wild-type apo-EncFtnsH protein (Figure 10).	RESULTS
+24	27	apo	protein_state	As described above, the apo-EncFtnsH has a charge state distribution consistent with an unstructured monomer, and decamer formation is only initiated upon addition of ferrous iron.	RESULTS
+28	36	EncFtnsH	protein	As described above, the apo-EncFtnsH has a charge state distribution consistent with an unstructured monomer, and decamer formation is only initiated upon addition of ferrous iron.	RESULTS
+43	55	charge state	evidence	As described above, the apo-EncFtnsH has a charge state distribution consistent with an unstructured monomer, and decamer formation is only initiated upon addition of ferrous iron.	RESULTS
+88	100	unstructured	protein_state	As described above, the apo-EncFtnsH has a charge state distribution consistent with an unstructured monomer, and decamer formation is only initiated upon addition of ferrous iron.	RESULTS
+101	108	monomer	oligomeric_state	As described above, the apo-EncFtnsH has a charge state distribution consistent with an unstructured monomer, and decamer formation is only initiated upon addition of ferrous iron.	RESULTS
+114	121	decamer	oligomeric_state	As described above, the apo-EncFtnsH has a charge state distribution consistent with an unstructured monomer, and decamer formation is only initiated upon addition of ferrous iron.	RESULTS
+175	179	iron	chemical	As described above, the apo-EncFtnsH has a charge state distribution consistent with an unstructured monomer, and decamer formation is only initiated upon addition of ferrous iron.	RESULTS
+9	13	E32A	mutant	Both the E32A mutant and E62A mutant displayed charge state distributions consistent with decamers, even in the absence of Fe2+.	RESULTS
+14	20	mutant	protein_state	Both the E32A mutant and E62A mutant displayed charge state distributions consistent with decamers, even in the absence of Fe2+.	RESULTS
+25	29	E62A	mutant	Both the E32A mutant and E62A mutant displayed charge state distributions consistent with decamers, even in the absence of Fe2+.	RESULTS
+30	36	mutant	protein_state	Both the E32A mutant and E62A mutant displayed charge state distributions consistent with decamers, even in the absence of Fe2+.	RESULTS
+47	59	charge state	evidence	Both the E32A mutant and E62A mutant displayed charge state distributions consistent with decamers, even in the absence of Fe2+.	RESULTS
+90	98	decamers	oligomeric_state	Both the E32A mutant and E62A mutant displayed charge state distributions consistent with decamers, even in the absence of Fe2+.	RESULTS
+112	122	absence of	protein_state	Both the E32A mutant and E62A mutant displayed charge state distributions consistent with decamers, even in the absence of Fe2+.	RESULTS
+123	127	Fe2+	chemical	Both the E32A mutant and E62A mutant displayed charge state distributions consistent with decamers, even in the absence of Fe2+.	RESULTS
+46	49	SEC	experimental_method	This gas-phase observation is consistent with SEC measurements, which indicate both of these variants were also decamers in solution.	RESULTS
+112	120	decamers	oligomeric_state	This gas-phase observation is consistent with SEC measurements, which indicate both of these variants were also decamers in solution.	RESULTS
+45	52	decamer	oligomeric_state	Thus it seems that these mutations allow the decamer to form in the absence of iron in the FOC.	RESULTS
+68	78	absence of	protein_state	Thus it seems that these mutations allow the decamer to form in the absence of iron in the FOC.	RESULTS
+79	83	iron	chemical	Thus it seems that these mutations allow the decamer to form in the absence of iron in the FOC.	RESULTS
+91	94	FOC	site	Thus it seems that these mutations allow the decamer to form in the absence of iron in the FOC.	RESULTS
+19	32	glutamic acid	residue_name	In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.	RESULTS
+33	40	mutants	protein_state	In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.	RESULTS
+42	44	MS	experimental_method	In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.	RESULTS
+61	65	H65A	mutant	In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.	RESULTS
+66	72	mutant	protein_state	In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.	RESULTS
+87	96	wild-type	protein_state	In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.	RESULTS
+97	100	apo	protein_state	In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.	RESULTS
+101	109	EncFtnsH	protein	In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.	RESULTS
+130	137	monomer	oligomeric_state	In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.	RESULTS
+175	182	dimeric	oligomeric_state	In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.	RESULTS
+183	187	H65A	mutant	In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.	RESULTS
+77	81	E32A	mutant	We propose that the observed differences in the oligomerization state of the E32A and E62A mutants compared to wild-type are due to the changes in the electrostatic environment within the FOC.	RESULTS
+86	90	E62A	mutant	We propose that the observed differences in the oligomerization state of the E32A and E62A mutants compared to wild-type are due to the changes in the electrostatic environment within the FOC.	RESULTS
+91	98	mutants	protein_state	We propose that the observed differences in the oligomerization state of the E32A and E62A mutants compared to wild-type are due to the changes in the electrostatic environment within the FOC.	RESULTS
+111	120	wild-type	protein_state	We propose that the observed differences in the oligomerization state of the E32A and E62A mutants compared to wild-type are due to the changes in the electrostatic environment within the FOC.	RESULTS
+188	191	FOC	site	We propose that the observed differences in the oligomerization state of the E32A and E62A mutants compared to wild-type are due to the changes in the electrostatic environment within the FOC.	RESULTS
+3	13	neutral pH	protein_state	At neutral pH the glutamic acid residues are negatively charged, while the histidine residues are predominantly in their uncharged state.	RESULTS
+18	31	glutamic acid	residue_name	At neutral pH the glutamic acid residues are negatively charged, while the histidine residues are predominantly in their uncharged state.	RESULTS
+75	84	histidine	residue_name	At neutral pH the glutamic acid residues are negatively charged, while the histidine residues are predominantly in their uncharged state.	RESULTS
+7	16	wild-type	protein_state	In the wild-type (WT) EncFtnsH this leads to electrostatic repulsion between subunits in the absence of iron.	RESULTS
+18	20	WT	protein_state	In the wild-type (WT) EncFtnsH this leads to electrostatic repulsion between subunits in the absence of iron.	RESULTS
+22	30	EncFtnsH	protein	In the wild-type (WT) EncFtnsH this leads to electrostatic repulsion between subunits in the absence of iron.	RESULTS
+77	85	subunits	structure_element	In the wild-type (WT) EncFtnsH this leads to electrostatic repulsion between subunits in the absence of iron.	RESULTS
+93	103	absence of	protein_state	In the wild-type (WT) EncFtnsH this leads to electrostatic repulsion between subunits in the absence of iron.	RESULTS
+104	108	iron	chemical	In the wild-type (WT) EncFtnsH this leads to electrostatic repulsion between subunits in the absence of iron.	RESULTS
+0	12	Coordination	bond_interaction	Coordination of Fe2+ in this site stabilizes the dimer and reconstitutes the active FOC.	RESULTS
+16	20	Fe2+	chemical	Coordination of Fe2+ in this site stabilizes the dimer and reconstitutes the active FOC.	RESULTS
+49	54	dimer	oligomeric_state	Coordination of Fe2+ in this site stabilizes the dimer and reconstitutes the active FOC.	RESULTS
+77	83	active	protein_state	Coordination of Fe2+ in this site stabilizes the dimer and reconstitutes the active FOC.	RESULTS
+84	87	FOC	site	Coordination of Fe2+ in this site stabilizes the dimer and reconstitutes the active FOC.	RESULTS
+29	34	Glu32	residue_name_number	The geometric arrangement of Glu32 and Glu62 in the FOC explains their behavior in solution and the gas phase, where they both favor the formation of decamers due to the loss of a repulsive negative charge.	RESULTS
+39	44	Glu62	residue_name_number	The geometric arrangement of Glu32 and Glu62 in the FOC explains their behavior in solution and the gas phase, where they both favor the formation of decamers due to the loss of a repulsive negative charge.	RESULTS
+52	55	FOC	site	The geometric arrangement of Glu32 and Glu62 in the FOC explains their behavior in solution and the gas phase, where they both favor the formation of decamers due to the loss of a repulsive negative charge.	RESULTS
+150	158	decamers	oligomeric_state	The geometric arrangement of Glu32 and Glu62 in the FOC explains their behavior in solution and the gas phase, where they both favor the formation of decamers due to the loss of a repulsive negative charge.	RESULTS
+4	7	FOC	site	The FOC in the H65A mutant is destabilized through the loss of this metal coordinating residue and potential positive charge carrier, thus favoring the monomer in solution and the gas phase.	RESULTS
+15	19	H65A	mutant	The FOC in the H65A mutant is destabilized through the loss of this metal coordinating residue and potential positive charge carrier, thus favoring the monomer in solution and the gas phase.	RESULTS
+20	26	mutant	protein_state	The FOC in the H65A mutant is destabilized through the loss of this metal coordinating residue and potential positive charge carrier, thus favoring the monomer in solution and the gas phase.	RESULTS
+55	62	loss of	protein_state	The FOC in the H65A mutant is destabilized through the loss of this metal coordinating residue and potential positive charge carrier, thus favoring the monomer in solution and the gas phase.	RESULTS
+68	94	metal coordinating residue	site	The FOC in the H65A mutant is destabilized through the loss of this metal coordinating residue and potential positive charge carrier, thus favoring the monomer in solution and the gas phase.	RESULTS
+152	159	monomer	oligomeric_state	The FOC in the H65A mutant is destabilized through the loss of this metal coordinating residue and potential positive charge carrier, thus favoring the monomer in solution and the gas phase.	RESULTS
+0	41	Data collection and refinement statistics	evidence	Data collection and refinement statistics.	TABLE
+1	3	WT	protein_state	"	WT	E32A	E62A	H65A	 	Data collection					 	Wavelength (Å)	1.74	1.73	1.73	1.74	 	Resolution range (Å)	49.63 - 2.06 (2.10 - 2.06)	48.84 - 2.59 (2.683 - 2.59)	48.87 - 2.21 (2.29 - 2.21)	48.86 - 2.97 (3.08 - 2.97)	 	Space group	P 1 21 1	P 1 21 1	P 1 21 1	P 1 21 1	 	Unit cell (Å) a b  c β (°)	98.18 120.53 140.30 95.36	97.78 120.28 140.53 95.41	98.09 120.23 140.36 95.50	98.03 120.29 140.43 95.39	 	Total reflections	1,264,922 (41,360)	405,488 (36,186)	1,069,345 (95,716)	323,853 (32,120)	 	Unique reflections	197,873 (8,766)	100,067 (9,735)	162,379 (15,817)	66,658 (6,553)	 	Multiplicity	6.4 (4.7)	4.1 (3.7)	6.6 (6.1)	4.9 (4.9)	 	Anomalous multiplicity	3.2 (2.6)	N/A	N/A	N/A	 	Completeness (%)	99.2 (88.6)	99.0 (97.0)	100 (97.0)	100 (99.0)	 	Anomalous completeness (%)	96.7 (77.2)	N/A	N/A	N/A	 	Mean I/sigma(I)	10.6 (1.60)	8.46 (1.79)	13.74 (1.80)	8.09 (1.74)	 	Wilson B-factor	26.98	40.10	33.97	52.20	 	Rmerge	0.123 (0.790)	0.171 (0.792)	0.0979 (1.009)	0.177 (0.863)	 	Rmeas	0.147 (0.973)	0.196 (0.923)	0.1064 (1.107)	0.199 (0.966)	 	CC1/2	0.995 (0.469)	0.985 (0.557)	0.998 (0.642)	0.989 (0.627)	 	CC*	0.999 (0.846)	0.996 (0.846)	0.999 (0.884)	0.997 (0.878)	 	Image DOI	10.7488/ds/1342	10.7488/ds/1419	10.7488/ds/1420	10.7488/ds/1421	 	Refinement					 	Rwork	0.171 (0.318)	0.183 (0.288)	0.165 (0.299)	0.186 (0.273)	 	Rfree	0.206 (0.345)	0.225 (0351)	0.216 (0.364)	0.237 (0.325)	 	Number of non-hydrogen atoms	23,222	22,366	22,691	22,145	 	macromolecules	22,276	22,019	21,965	22,066	 	ligands	138	8	24	74	 	water	808	339	702	5	 	Protein residues	2,703	2,686	2,675	2,700	 	RMS(bonds) (Å)	0.012	0.005	0.011	0.002	 	RMS(angles) (°)	1.26	0.58	1.02	0.40	 	Ramachandran favored (%)	100	99	100	99	 	Ramachandran allowed (%)	0	1	0	1	 	Ramachandran outliers (%)	0	0	0	0	 	Clash score	1.42	1.42	1.79	0.97	 	Average B-factor (Å2)	33.90	42.31	41.34	47.68	 	macromolecules	33.80	42.35	41.31	47.60	 	ligands	40.40	72.80	65.55	72.34	 	solvent	36.20	38.95	41.46	33.85	 	PDB ID	5DA5	5L89	5L8B	5L8G	 	"	TABLE
+4	8	E32A	mutant	"	WT	E32A	E62A	H65A	 	Data collection					 	Wavelength (Å)	1.74	1.73	1.73	1.74	 	Resolution range (Å)	49.63 - 2.06 (2.10 - 2.06)	48.84 - 2.59 (2.683 - 2.59)	48.87 - 2.21 (2.29 - 2.21)	48.86 - 2.97 (3.08 - 2.97)	 	Space group	P 1 21 1	P 1 21 1	P 1 21 1	P 1 21 1	 	Unit cell (Å) a b  c β (°)	98.18 120.53 140.30 95.36	97.78 120.28 140.53 95.41	98.09 120.23 140.36 95.50	98.03 120.29 140.43 95.39	 	Total reflections	1,264,922 (41,360)	405,488 (36,186)	1,069,345 (95,716)	323,853 (32,120)	 	Unique reflections	197,873 (8,766)	100,067 (9,735)	162,379 (15,817)	66,658 (6,553)	 	Multiplicity	6.4 (4.7)	4.1 (3.7)	6.6 (6.1)	4.9 (4.9)	 	Anomalous multiplicity	3.2 (2.6)	N/A	N/A	N/A	 	Completeness (%)	99.2 (88.6)	99.0 (97.0)	100 (97.0)	100 (99.0)	 	Anomalous completeness (%)	96.7 (77.2)	N/A	N/A	N/A	 	Mean I/sigma(I)	10.6 (1.60)	8.46 (1.79)	13.74 (1.80)	8.09 (1.74)	 	Wilson B-factor	26.98	40.10	33.97	52.20	 	Rmerge	0.123 (0.790)	0.171 (0.792)	0.0979 (1.009)	0.177 (0.863)	 	Rmeas	0.147 (0.973)	0.196 (0.923)	0.1064 (1.107)	0.199 (0.966)	 	CC1/2	0.995 (0.469)	0.985 (0.557)	0.998 (0.642)	0.989 (0.627)	 	CC*	0.999 (0.846)	0.996 (0.846)	0.999 (0.884)	0.997 (0.878)	 	Image DOI	10.7488/ds/1342	10.7488/ds/1419	10.7488/ds/1420	10.7488/ds/1421	 	Refinement					 	Rwork	0.171 (0.318)	0.183 (0.288)	0.165 (0.299)	0.186 (0.273)	 	Rfree	0.206 (0.345)	0.225 (0351)	0.216 (0.364)	0.237 (0.325)	 	Number of non-hydrogen atoms	23,222	22,366	22,691	22,145	 	macromolecules	22,276	22,019	21,965	22,066	 	ligands	138	8	24	74	 	water	808	339	702	5	 	Protein residues	2,703	2,686	2,675	2,700	 	RMS(bonds) (Å)	0.012	0.005	0.011	0.002	 	RMS(angles) (°)	1.26	0.58	1.02	0.40	 	Ramachandran favored (%)	100	99	100	99	 	Ramachandran allowed (%)	0	1	0	1	 	Ramachandran outliers (%)	0	0	0	0	 	Clash score	1.42	1.42	1.79	0.97	 	Average B-factor (Å2)	33.90	42.31	41.34	47.68	 	macromolecules	33.80	42.35	41.31	47.60	 	ligands	40.40	72.80	65.55	72.34	 	solvent	36.20	38.95	41.46	33.85	 	PDB ID	5DA5	5L89	5L8B	5L8G	 	"	TABLE
+9	13	E62A	mutant	"	WT	E32A	E62A	H65A	 	Data collection					 	Wavelength (Å)	1.74	1.73	1.73	1.74	 	Resolution range (Å)	49.63 - 2.06 (2.10 - 2.06)	48.84 - 2.59 (2.683 - 2.59)	48.87 - 2.21 (2.29 - 2.21)	48.86 - 2.97 (3.08 - 2.97)	 	Space group	P 1 21 1	P 1 21 1	P 1 21 1	P 1 21 1	 	Unit cell (Å) a b  c β (°)	98.18 120.53 140.30 95.36	97.78 120.28 140.53 95.41	98.09 120.23 140.36 95.50	98.03 120.29 140.43 95.39	 	Total reflections	1,264,922 (41,360)	405,488 (36,186)	1,069,345 (95,716)	323,853 (32,120)	 	Unique reflections	197,873 (8,766)	100,067 (9,735)	162,379 (15,817)	66,658 (6,553)	 	Multiplicity	6.4 (4.7)	4.1 (3.7)	6.6 (6.1)	4.9 (4.9)	 	Anomalous multiplicity	3.2 (2.6)	N/A	N/A	N/A	 	Completeness (%)	99.2 (88.6)	99.0 (97.0)	100 (97.0)	100 (99.0)	 	Anomalous completeness (%)	96.7 (77.2)	N/A	N/A	N/A	 	Mean I/sigma(I)	10.6 (1.60)	8.46 (1.79)	13.74 (1.80)	8.09 (1.74)	 	Wilson B-factor	26.98	40.10	33.97	52.20	 	Rmerge	0.123 (0.790)	0.171 (0.792)	0.0979 (1.009)	0.177 (0.863)	 	Rmeas	0.147 (0.973)	0.196 (0.923)	0.1064 (1.107)	0.199 (0.966)	 	CC1/2	0.995 (0.469)	0.985 (0.557)	0.998 (0.642)	0.989 (0.627)	 	CC*	0.999 (0.846)	0.996 (0.846)	0.999 (0.884)	0.997 (0.878)	 	Image DOI	10.7488/ds/1342	10.7488/ds/1419	10.7488/ds/1420	10.7488/ds/1421	 	Refinement					 	Rwork	0.171 (0.318)	0.183 (0.288)	0.165 (0.299)	0.186 (0.273)	 	Rfree	0.206 (0.345)	0.225 (0351)	0.216 (0.364)	0.237 (0.325)	 	Number of non-hydrogen atoms	23,222	22,366	22,691	22,145	 	macromolecules	22,276	22,019	21,965	22,066	 	ligands	138	8	24	74	 	water	808	339	702	5	 	Protein residues	2,703	2,686	2,675	2,700	 	RMS(bonds) (Å)	0.012	0.005	0.011	0.002	 	RMS(angles) (°)	1.26	0.58	1.02	0.40	 	Ramachandran favored (%)	100	99	100	99	 	Ramachandran allowed (%)	0	1	0	1	 	Ramachandran outliers (%)	0	0	0	0	 	Clash score	1.42	1.42	1.79	0.97	 	Average B-factor (Å2)	33.90	42.31	41.34	47.68	 	macromolecules	33.80	42.35	41.31	47.60	 	ligands	40.40	72.80	65.55	72.34	 	solvent	36.20	38.95	41.46	33.85	 	PDB ID	5DA5	5L89	5L8B	5L8G	 	"	TABLE
+14	18	H65A	mutant	"	WT	E32A	E62A	H65A	 	Data collection					 	Wavelength (Å)	1.74	1.73	1.73	1.74	 	Resolution range (Å)	49.63 - 2.06 (2.10 - 2.06)	48.84 - 2.59 (2.683 - 2.59)	48.87 - 2.21 (2.29 - 2.21)	48.86 - 2.97 (3.08 - 2.97)	 	Space group	P 1 21 1	P 1 21 1	P 1 21 1	P 1 21 1	 	Unit cell (Å) a b  c β (°)	98.18 120.53 140.30 95.36	97.78 120.28 140.53 95.41	98.09 120.23 140.36 95.50	98.03 120.29 140.43 95.39	 	Total reflections	1,264,922 (41,360)	405,488 (36,186)	1,069,345 (95,716)	323,853 (32,120)	 	Unique reflections	197,873 (8,766)	100,067 (9,735)	162,379 (15,817)	66,658 (6,553)	 	Multiplicity	6.4 (4.7)	4.1 (3.7)	6.6 (6.1)	4.9 (4.9)	 	Anomalous multiplicity	3.2 (2.6)	N/A	N/A	N/A	 	Completeness (%)	99.2 (88.6)	99.0 (97.0)	100 (97.0)	100 (99.0)	 	Anomalous completeness (%)	96.7 (77.2)	N/A	N/A	N/A	 	Mean I/sigma(I)	10.6 (1.60)	8.46 (1.79)	13.74 (1.80)	8.09 (1.74)	 	Wilson B-factor	26.98	40.10	33.97	52.20	 	Rmerge	0.123 (0.790)	0.171 (0.792)	0.0979 (1.009)	0.177 (0.863)	 	Rmeas	0.147 (0.973)	0.196 (0.923)	0.1064 (1.107)	0.199 (0.966)	 	CC1/2	0.995 (0.469)	0.985 (0.557)	0.998 (0.642)	0.989 (0.627)	 	CC*	0.999 (0.846)	0.996 (0.846)	0.999 (0.884)	0.997 (0.878)	 	Image DOI	10.7488/ds/1342	10.7488/ds/1419	10.7488/ds/1420	10.7488/ds/1421	 	Refinement					 	Rwork	0.171 (0.318)	0.183 (0.288)	0.165 (0.299)	0.186 (0.273)	 	Rfree	0.206 (0.345)	0.225 (0351)	0.216 (0.364)	0.237 (0.325)	 	Number of non-hydrogen atoms	23,222	22,366	22,691	22,145	 	macromolecules	22,276	22,019	21,965	22,066	 	ligands	138	8	24	74	 	water	808	339	702	5	 	Protein residues	2,703	2,686	2,675	2,700	 	RMS(bonds) (Å)	0.012	0.005	0.011	0.002	 	RMS(angles) (°)	1.26	0.58	1.02	0.40	 	Ramachandran favored (%)	100	99	100	99	 	Ramachandran allowed (%)	0	1	0	1	 	Ramachandran outliers (%)	0	0	0	0	 	Clash score	1.42	1.42	1.79	0.97	 	Average B-factor (Å2)	33.90	42.31	41.34	47.68	 	macromolecules	33.80	42.35	41.31	47.60	 	ligands	40.40	72.80	65.55	72.34	 	solvent	36.20	38.95	41.46	33.85	 	PDB ID	5DA5	5L89	5L8B	5L8G	 	"	TABLE
+1504	1509	water	chemical	"	WT	E32A	E62A	H65A	 	Data collection					 	Wavelength (Å)	1.74	1.73	1.73	1.74	 	Resolution range (Å)	49.63 - 2.06 (2.10 - 2.06)	48.84 - 2.59 (2.683 - 2.59)	48.87 - 2.21 (2.29 - 2.21)	48.86 - 2.97 (3.08 - 2.97)	 	Space group	P 1 21 1	P 1 21 1	P 1 21 1	P 1 21 1	 	Unit cell (Å) a b  c β (°)	98.18 120.53 140.30 95.36	97.78 120.28 140.53 95.41	98.09 120.23 140.36 95.50	98.03 120.29 140.43 95.39	 	Total reflections	1,264,922 (41,360)	405,488 (36,186)	1,069,345 (95,716)	323,853 (32,120)	 	Unique reflections	197,873 (8,766)	100,067 (9,735)	162,379 (15,817)	66,658 (6,553)	 	Multiplicity	6.4 (4.7)	4.1 (3.7)	6.6 (6.1)	4.9 (4.9)	 	Anomalous multiplicity	3.2 (2.6)	N/A	N/A	N/A	 	Completeness (%)	99.2 (88.6)	99.0 (97.0)	100 (97.0)	100 (99.0)	 	Anomalous completeness (%)	96.7 (77.2)	N/A	N/A	N/A	 	Mean I/sigma(I)	10.6 (1.60)	8.46 (1.79)	13.74 (1.80)	8.09 (1.74)	 	Wilson B-factor	26.98	40.10	33.97	52.20	 	Rmerge	0.123 (0.790)	0.171 (0.792)	0.0979 (1.009)	0.177 (0.863)	 	Rmeas	0.147 (0.973)	0.196 (0.923)	0.1064 (1.107)	0.199 (0.966)	 	CC1/2	0.995 (0.469)	0.985 (0.557)	0.998 (0.642)	0.989 (0.627)	 	CC*	0.999 (0.846)	0.996 (0.846)	0.999 (0.884)	0.997 (0.878)	 	Image DOI	10.7488/ds/1342	10.7488/ds/1419	10.7488/ds/1420	10.7488/ds/1421	 	Refinement					 	Rwork	0.171 (0.318)	0.183 (0.288)	0.165 (0.299)	0.186 (0.273)	 	Rfree	0.206 (0.345)	0.225 (0351)	0.216 (0.364)	0.237 (0.325)	 	Number of non-hydrogen atoms	23,222	22,366	22,691	22,145	 	macromolecules	22,276	22,019	21,965	22,066	 	ligands	138	8	24	74	 	water	808	339	702	5	 	Protein residues	2,703	2,686	2,675	2,700	 	RMS(bonds) (Å)	0.012	0.005	0.011	0.002	 	RMS(angles) (°)	1.26	0.58	1.02	0.40	 	Ramachandran favored (%)	100	99	100	99	 	Ramachandran allowed (%)	0	1	0	1	 	Ramachandran outliers (%)	0	0	0	0	 	Clash score	1.42	1.42	1.79	0.97	 	Average B-factor (Å2)	33.90	42.31	41.34	47.68	 	macromolecules	33.80	42.35	41.31	47.60	 	ligands	40.40	72.80	65.55	72.34	 	solvent	36.20	38.95	41.46	33.85	 	PDB ID	5DA5	5L89	5L8B	5L8G	 	"	TABLE
+0	4	Iron	chemical	Iron loading capacity of EncFtn, encapsulin and ferritin.	TABLE
+25	31	EncFtn	protein	Iron loading capacity of EncFtn, encapsulin and ferritin.	TABLE
+33	43	encapsulin	protein	Iron loading capacity of EncFtn, encapsulin and ferritin.	TABLE
+48	56	ferritin	protein_type	Iron loading capacity of EncFtn, encapsulin and ferritin.	TABLE
+38	47	decameric	oligomeric_state	Protein samples (at 8.5 µM) including decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were mixed with Fe(NH4)2(SO4) (in 0.1% (v/v) HCl) of different concentrations in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl buffer at room temperature for 3 hrs in the air.	TABLE
+48	56	EncFtnsH	protein	Protein samples (at 8.5 µM) including decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were mixed with Fe(NH4)2(SO4) (in 0.1% (v/v) HCl) of different concentrations in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl buffer at room temperature for 3 hrs in the air.	TABLE
+58	68	encapsulin	protein	Protein samples (at 8.5 µM) including decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were mixed with Fe(NH4)2(SO4) (in 0.1% (v/v) HCl) of different concentrations in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl buffer at room temperature for 3 hrs in the air.	TABLE
+70	80	EncFtn-Enc	complex_assembly	Protein samples (at 8.5 µM) including decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were mixed with Fe(NH4)2(SO4) (in 0.1% (v/v) HCl) of different concentrations in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl buffer at room temperature for 3 hrs in the air.	TABLE
+85	96	apoferritin	protein_state	Protein samples (at 8.5 µM) including decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were mixed with Fe(NH4)2(SO4) (in 0.1% (v/v) HCl) of different concentrations in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl buffer at room temperature for 3 hrs in the air.	TABLE
+113	126	Fe(NH4)2(SO4)	chemical	Protein samples (at 8.5 µM) including decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were mixed with Fe(NH4)2(SO4) (in 0.1% (v/v) HCl) of different concentrations in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl buffer at room temperature for 3 hrs in the air.	TABLE
+142	145	HCl	chemical	Protein samples (at 8.5 µM) including decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were mixed with Fe(NH4)2(SO4) (in 0.1% (v/v) HCl) of different concentrations in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl buffer at room temperature for 3 hrs in the air.	TABLE
+210	214	NaCl	chemical	Protein samples (at 8.5 µM) including decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were mixed with Fe(NH4)2(SO4) (in 0.1% (v/v) HCl) of different concentrations in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl buffer at room temperature for 3 hrs in the air.	TABLE
+8	10	Fe	chemical	Protein-Fe mixtures were centrifuged at 13,000 x g to remove precipitated material and desalted prior to the Fe and protein content analysis by ferrozine assay and BCA microplate assay, respectively.	TABLE
+109	111	Fe	chemical	Protein-Fe mixtures were centrifuged at 13,000 x g to remove precipitated material and desalted prior to the Fe and protein content analysis by ferrozine assay and BCA microplate assay, respectively.	TABLE
+144	159	ferrozine assay	experimental_method	Protein-Fe mixtures were centrifuged at 13,000 x g to remove precipitated material and desalted prior to the Fe and protein content analysis by ferrozine assay and BCA microplate assay, respectively.	TABLE
+164	184	BCA microplate assay	experimental_method	Protein-Fe mixtures were centrifuged at 13,000 x g to remove precipitated material and desalted prior to the Fe and protein content analysis by ferrozine assay and BCA microplate assay, respectively.	TABLE
+0	2	Fe	chemical	Fe to protein ratio was calculated to indicate the Fe binding capacity of the protein.	TABLE
+51	53	Fe	chemical	Fe to protein ratio was calculated to indicate the Fe binding capacity of the protein.	TABLE
+42	46	iron	chemical	Protein stability was compromised at high iron concentrations; therefore, the highest iron loading with the least protein precipitation was used to derive the maximum iron loading capacity per biological assembly (underlined and highlighted in bold).	TABLE
+86	90	iron	chemical	Protein stability was compromised at high iron concentrations; therefore, the highest iron loading with the least protein precipitation was used to derive the maximum iron loading capacity per biological assembly (underlined and highlighted in bold).	TABLE
+167	171	iron	chemical	Protein stability was compromised at high iron concentrations; therefore, the highest iron loading with the least protein precipitation was used to derive the maximum iron loading capacity per biological assembly (underlined and highlighted in bold).	TABLE
+37	44	decamer	oligomeric_state	The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin.	TABLE
+49	57	EncFtnsH	protein	The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin.	TABLE
+61	66	60mer	oligomeric_state	The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin.	TABLE
+71	81	encapsulin	protein	The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin.	TABLE
+85	90	60mer	oligomeric_state	The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin.	TABLE
+94	104	encapsulin	protein	The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin.	TABLE
+105	116	loaded with	protein_state	The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin.	TABLE
+130	139	decameric	oligomeric_state	The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin.	TABLE
+140	146	EncFtn	protein	The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin.	TABLE
+167	172	24mer	oligomeric_state	The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin.	TABLE
+177	182	horse	taxonomy_domain	The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin.	TABLE
+190	201	apoferritin	protein_state	The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin.	TABLE
+83	118	ferrozine and BCA microplate assays	experimental_method	Errors are quoted as the standard deviation of three technical repeats in both the ferrozine and BCA microplate assays.	TABLE
+21	23	Fe	chemical	The proteins used in Fe loading experiment came from a single preparation.	TABLE
+15	29	Fe(NH4)2(SO4)2	chemical	"Protein sample	Fe(NH4)2(SO4)2 loading (µM)	Fe detected by ferrozine assay (µM)	Protein detected by BCA microplate assay (µM)	Fe / monomeric protein	Maximum Fe loading per biological assembly unit	 	8.46 µM EncFtnsH-10mer	0	4.73 ± 2.32	5.26 ± 0.64	0.90 ± 0.44		 	39.9	9.93 ± 1.20	5.36 ± 0.69	1.85 ± 0.22		 	84	17.99 ± 2.01	4.96 ± 0.04	3.63 ± 0.41		 	147	21.09 ± 1.94	4.44 ± 0.21	4.75 ± 0.44	48 ± 4	 	224	28.68 ± 0.30	3.73 ± 0.53	7.68 ± 0.08		 	301	11.27 ± 1.10	2.50 ± 0.05	4.51 ± 0.44		 	8.50 µM Encapsulin	0	-1.02 ± 0.54	8.63 ± 0.17	-0.12 ± 0.06		 	224	62.24 ± 2.49	10.01 ± 0.58	6.22 ± 0.35		 	301	67.94 ± 3.15	8.69 ± 0.42	7.81 ± 0.36		 	450	107.96 ± 8.88	8.50 ± 0.69	12.71 ± 1.05		 	700	97.51 ± 3.19	7.26 ± 0.20	13.44 ± 0.44		 	1000	308.63 ± 2.06	8.42 ± 0.34	36.66 ± 0.24	2199 ± 15	 	1500	57.09 ± 0.90	1.44 ± 0.21	39.77 ± 0.62		 	2000	9.2 ± 1.16	0.21 ± 0.14	44.73 ± 5.63		 	8.70 µM EncFtn-Enc	0	3.31 ± 1.57	6.85 ± 0.07	0.48 ± 0.23		 	224	116.27 ± 3.74	7.63 ± 0.12	15.25 ± 0.49		 	301	132.86 ± 4.03	6.66 ± 0.31	19.96 ± 0.61		 	450	220.57 ± 27.33	6.12 ± 1.07	36.06 ± 4.47		 	700	344.03 ± 40.38	6.94 ± 0.17	49.58 ± 5.82		 	1000	496.00 ± 38.48	7.19 ± 0.08	68.94 ± 5.35	4137 ± 321	 	1500	569.98 ± 73.63	5.73 ± 0.03	99.44 ± 12.84		 	2000	584.30 ± 28.33	4.88 ± 0.22	119.62 ± 5.80		 	8.50 µM Apoferritin	0	3.95 ± 2.26	9.37 ± 0.24	0.42 ± 0.25		 	42.5	10.27 ± 1.12	8.27 ± 0.30	1.24 ± 0.18		 	212.5	44.48 ± 2.76	7.85 ± 0.77	5.67 ± 0.83		 	637.5	160.93 ± 4.27	6.76 ± 0.81	23.79 ± 3.12	571 ± 75	 	1275	114.92 ± 3.17	3.84 ± 0.30	29.91 ± 2.95		 	1700	91.40 ± 3.37	3.14 ± 0.35	29.13 ± 3.86		 	"	TABLE
+43	45	Fe	chemical	"Protein sample	Fe(NH4)2(SO4)2 loading (µM)	Fe detected by ferrozine assay (µM)	Protein detected by BCA microplate assay (µM)	Fe / monomeric protein	Maximum Fe loading per biological assembly unit	 	8.46 µM EncFtnsH-10mer	0	4.73 ± 2.32	5.26 ± 0.64	0.90 ± 0.44		 	39.9	9.93 ± 1.20	5.36 ± 0.69	1.85 ± 0.22		 	84	17.99 ± 2.01	4.96 ± 0.04	3.63 ± 0.41		 	147	21.09 ± 1.94	4.44 ± 0.21	4.75 ± 0.44	48 ± 4	 	224	28.68 ± 0.30	3.73 ± 0.53	7.68 ± 0.08		 	301	11.27 ± 1.10	2.50 ± 0.05	4.51 ± 0.44		 	8.50 µM Encapsulin	0	-1.02 ± 0.54	8.63 ± 0.17	-0.12 ± 0.06		 	224	62.24 ± 2.49	10.01 ± 0.58	6.22 ± 0.35		 	301	67.94 ± 3.15	8.69 ± 0.42	7.81 ± 0.36		 	450	107.96 ± 8.88	8.50 ± 0.69	12.71 ± 1.05		 	700	97.51 ± 3.19	7.26 ± 0.20	13.44 ± 0.44		 	1000	308.63 ± 2.06	8.42 ± 0.34	36.66 ± 0.24	2199 ± 15	 	1500	57.09 ± 0.90	1.44 ± 0.21	39.77 ± 0.62		 	2000	9.2 ± 1.16	0.21 ± 0.14	44.73 ± 5.63		 	8.70 µM EncFtn-Enc	0	3.31 ± 1.57	6.85 ± 0.07	0.48 ± 0.23		 	224	116.27 ± 3.74	7.63 ± 0.12	15.25 ± 0.49		 	301	132.86 ± 4.03	6.66 ± 0.31	19.96 ± 0.61		 	450	220.57 ± 27.33	6.12 ± 1.07	36.06 ± 4.47		 	700	344.03 ± 40.38	6.94 ± 0.17	49.58 ± 5.82		 	1000	496.00 ± 38.48	7.19 ± 0.08	68.94 ± 5.35	4137 ± 321	 	1500	569.98 ± 73.63	5.73 ± 0.03	99.44 ± 12.84		 	2000	584.30 ± 28.33	4.88 ± 0.22	119.62 ± 5.80		 	8.50 µM Apoferritin	0	3.95 ± 2.26	9.37 ± 0.24	0.42 ± 0.25		 	42.5	10.27 ± 1.12	8.27 ± 0.30	1.24 ± 0.18		 	212.5	44.48 ± 2.76	7.85 ± 0.77	5.67 ± 0.83		 	637.5	160.93 ± 4.27	6.76 ± 0.81	23.79 ± 3.12	571 ± 75	 	1275	114.92 ± 3.17	3.84 ± 0.30	29.91 ± 2.95		 	1700	91.40 ± 3.37	3.14 ± 0.35	29.13 ± 3.86		 	"	TABLE
+58	73	ferrozine assay	experimental_method	"Protein sample	Fe(NH4)2(SO4)2 loading (µM)	Fe detected by ferrozine assay (µM)	Protein detected by BCA microplate assay (µM)	Fe / monomeric protein	Maximum Fe loading per biological assembly unit	 	8.46 µM EncFtnsH-10mer	0	4.73 ± 2.32	5.26 ± 0.64	0.90 ± 0.44		 	39.9	9.93 ± 1.20	5.36 ± 0.69	1.85 ± 0.22		 	84	17.99 ± 2.01	4.96 ± 0.04	3.63 ± 0.41		 	147	21.09 ± 1.94	4.44 ± 0.21	4.75 ± 0.44	48 ± 4	 	224	28.68 ± 0.30	3.73 ± 0.53	7.68 ± 0.08		 	301	11.27 ± 1.10	2.50 ± 0.05	4.51 ± 0.44		 	8.50 µM Encapsulin	0	-1.02 ± 0.54	8.63 ± 0.17	-0.12 ± 0.06		 	224	62.24 ± 2.49	10.01 ± 0.58	6.22 ± 0.35		 	301	67.94 ± 3.15	8.69 ± 0.42	7.81 ± 0.36		 	450	107.96 ± 8.88	8.50 ± 0.69	12.71 ± 1.05		 	700	97.51 ± 3.19	7.26 ± 0.20	13.44 ± 0.44		 	1000	308.63 ± 2.06	8.42 ± 0.34	36.66 ± 0.24	2199 ± 15	 	1500	57.09 ± 0.90	1.44 ± 0.21	39.77 ± 0.62		 	2000	9.2 ± 1.16	0.21 ± 0.14	44.73 ± 5.63		 	8.70 µM EncFtn-Enc	0	3.31 ± 1.57	6.85 ± 0.07	0.48 ± 0.23		 	224	116.27 ± 3.74	7.63 ± 0.12	15.25 ± 0.49		 	301	132.86 ± 4.03	6.66 ± 0.31	19.96 ± 0.61		 	450	220.57 ± 27.33	6.12 ± 1.07	36.06 ± 4.47		 	700	344.03 ± 40.38	6.94 ± 0.17	49.58 ± 5.82		 	1000	496.00 ± 38.48	7.19 ± 0.08	68.94 ± 5.35	4137 ± 321	 	1500	569.98 ± 73.63	5.73 ± 0.03	99.44 ± 12.84		 	2000	584.30 ± 28.33	4.88 ± 0.22	119.62 ± 5.80		 	8.50 µM Apoferritin	0	3.95 ± 2.26	9.37 ± 0.24	0.42 ± 0.25		 	42.5	10.27 ± 1.12	8.27 ± 0.30	1.24 ± 0.18		 	212.5	44.48 ± 2.76	7.85 ± 0.77	5.67 ± 0.83		 	637.5	160.93 ± 4.27	6.76 ± 0.81	23.79 ± 3.12	571 ± 75	 	1275	114.92 ± 3.17	3.84 ± 0.30	29.91 ± 2.95		 	1700	91.40 ± 3.37	3.14 ± 0.35	29.13 ± 3.86		 	"	TABLE
+99	119	BCA microplate assay	experimental_method	"Protein sample	Fe(NH4)2(SO4)2 loading (µM)	Fe detected by ferrozine assay (µM)	Protein detected by BCA microplate assay (µM)	Fe / monomeric protein	Maximum Fe loading per biological assembly unit	 	8.46 µM EncFtnsH-10mer	0	4.73 ± 2.32	5.26 ± 0.64	0.90 ± 0.44		 	39.9	9.93 ± 1.20	5.36 ± 0.69	1.85 ± 0.22		 	84	17.99 ± 2.01	4.96 ± 0.04	3.63 ± 0.41		 	147	21.09 ± 1.94	4.44 ± 0.21	4.75 ± 0.44	48 ± 4	 	224	28.68 ± 0.30	3.73 ± 0.53	7.68 ± 0.08		 	301	11.27 ± 1.10	2.50 ± 0.05	4.51 ± 0.44		 	8.50 µM Encapsulin	0	-1.02 ± 0.54	8.63 ± 0.17	-0.12 ± 0.06		 	224	62.24 ± 2.49	10.01 ± 0.58	6.22 ± 0.35		 	301	67.94 ± 3.15	8.69 ± 0.42	7.81 ± 0.36		 	450	107.96 ± 8.88	8.50 ± 0.69	12.71 ± 1.05		 	700	97.51 ± 3.19	7.26 ± 0.20	13.44 ± 0.44		 	1000	308.63 ± 2.06	8.42 ± 0.34	36.66 ± 0.24	2199 ± 15	 	1500	57.09 ± 0.90	1.44 ± 0.21	39.77 ± 0.62		 	2000	9.2 ± 1.16	0.21 ± 0.14	44.73 ± 5.63		 	8.70 µM EncFtn-Enc	0	3.31 ± 1.57	6.85 ± 0.07	0.48 ± 0.23		 	224	116.27 ± 3.74	7.63 ± 0.12	15.25 ± 0.49		 	301	132.86 ± 4.03	6.66 ± 0.31	19.96 ± 0.61		 	450	220.57 ± 27.33	6.12 ± 1.07	36.06 ± 4.47		 	700	344.03 ± 40.38	6.94 ± 0.17	49.58 ± 5.82		 	1000	496.00 ± 38.48	7.19 ± 0.08	68.94 ± 5.35	4137 ± 321	 	1500	569.98 ± 73.63	5.73 ± 0.03	99.44 ± 12.84		 	2000	584.30 ± 28.33	4.88 ± 0.22	119.62 ± 5.80		 	8.50 µM Apoferritin	0	3.95 ± 2.26	9.37 ± 0.24	0.42 ± 0.25		 	42.5	10.27 ± 1.12	8.27 ± 0.30	1.24 ± 0.18		 	212.5	44.48 ± 2.76	7.85 ± 0.77	5.67 ± 0.83		 	637.5	160.93 ± 4.27	6.76 ± 0.81	23.79 ± 3.12	571 ± 75	 	1275	114.92 ± 3.17	3.84 ± 0.30	29.91 ± 2.95		 	1700	91.40 ± 3.37	3.14 ± 0.35	29.13 ± 3.86		 	"	TABLE
+125	127	Fe	chemical	"Protein sample	Fe(NH4)2(SO4)2 loading (µM)	Fe detected by ferrozine assay (µM)	Protein detected by BCA microplate assay (µM)	Fe / monomeric protein	Maximum Fe loading per biological assembly unit	 	8.46 µM EncFtnsH-10mer	0	4.73 ± 2.32	5.26 ± 0.64	0.90 ± 0.44		 	39.9	9.93 ± 1.20	5.36 ± 0.69	1.85 ± 0.22		 	84	17.99 ± 2.01	4.96 ± 0.04	3.63 ± 0.41		 	147	21.09 ± 1.94	4.44 ± 0.21	4.75 ± 0.44	48 ± 4	 	224	28.68 ± 0.30	3.73 ± 0.53	7.68 ± 0.08		 	301	11.27 ± 1.10	2.50 ± 0.05	4.51 ± 0.44		 	8.50 µM Encapsulin	0	-1.02 ± 0.54	8.63 ± 0.17	-0.12 ± 0.06		 	224	62.24 ± 2.49	10.01 ± 0.58	6.22 ± 0.35		 	301	67.94 ± 3.15	8.69 ± 0.42	7.81 ± 0.36		 	450	107.96 ± 8.88	8.50 ± 0.69	12.71 ± 1.05		 	700	97.51 ± 3.19	7.26 ± 0.20	13.44 ± 0.44		 	1000	308.63 ± 2.06	8.42 ± 0.34	36.66 ± 0.24	2199 ± 15	 	1500	57.09 ± 0.90	1.44 ± 0.21	39.77 ± 0.62		 	2000	9.2 ± 1.16	0.21 ± 0.14	44.73 ± 5.63		 	8.70 µM EncFtn-Enc	0	3.31 ± 1.57	6.85 ± 0.07	0.48 ± 0.23		 	224	116.27 ± 3.74	7.63 ± 0.12	15.25 ± 0.49		 	301	132.86 ± 4.03	6.66 ± 0.31	19.96 ± 0.61		 	450	220.57 ± 27.33	6.12 ± 1.07	36.06 ± 4.47		 	700	344.03 ± 40.38	6.94 ± 0.17	49.58 ± 5.82		 	1000	496.00 ± 38.48	7.19 ± 0.08	68.94 ± 5.35	4137 ± 321	 	1500	569.98 ± 73.63	5.73 ± 0.03	99.44 ± 12.84		 	2000	584.30 ± 28.33	4.88 ± 0.22	119.62 ± 5.80		 	8.50 µM Apoferritin	0	3.95 ± 2.26	9.37 ± 0.24	0.42 ± 0.25		 	42.5	10.27 ± 1.12	8.27 ± 0.30	1.24 ± 0.18		 	212.5	44.48 ± 2.76	7.85 ± 0.77	5.67 ± 0.83		 	637.5	160.93 ± 4.27	6.76 ± 0.81	23.79 ± 3.12	571 ± 75	 	1275	114.92 ± 3.17	3.84 ± 0.30	29.91 ± 2.95		 	1700	91.40 ± 3.37	3.14 ± 0.35	29.13 ± 3.86		 	"	TABLE
+156	158	Fe	chemical	"Protein sample	Fe(NH4)2(SO4)2 loading (µM)	Fe detected by ferrozine assay (µM)	Protein detected by BCA microplate assay (µM)	Fe / monomeric protein	Maximum Fe loading per biological assembly unit	 	8.46 µM EncFtnsH-10mer	0	4.73 ± 2.32	5.26 ± 0.64	0.90 ± 0.44		 	39.9	9.93 ± 1.20	5.36 ± 0.69	1.85 ± 0.22		 	84	17.99 ± 2.01	4.96 ± 0.04	3.63 ± 0.41		 	147	21.09 ± 1.94	4.44 ± 0.21	4.75 ± 0.44	48 ± 4	 	224	28.68 ± 0.30	3.73 ± 0.53	7.68 ± 0.08		 	301	11.27 ± 1.10	2.50 ± 0.05	4.51 ± 0.44		 	8.50 µM Encapsulin	0	-1.02 ± 0.54	8.63 ± 0.17	-0.12 ± 0.06		 	224	62.24 ± 2.49	10.01 ± 0.58	6.22 ± 0.35		 	301	67.94 ± 3.15	8.69 ± 0.42	7.81 ± 0.36		 	450	107.96 ± 8.88	8.50 ± 0.69	12.71 ± 1.05		 	700	97.51 ± 3.19	7.26 ± 0.20	13.44 ± 0.44		 	1000	308.63 ± 2.06	8.42 ± 0.34	36.66 ± 0.24	2199 ± 15	 	1500	57.09 ± 0.90	1.44 ± 0.21	39.77 ± 0.62		 	2000	9.2 ± 1.16	0.21 ± 0.14	44.73 ± 5.63		 	8.70 µM EncFtn-Enc	0	3.31 ± 1.57	6.85 ± 0.07	0.48 ± 0.23		 	224	116.27 ± 3.74	7.63 ± 0.12	15.25 ± 0.49		 	301	132.86 ± 4.03	6.66 ± 0.31	19.96 ± 0.61		 	450	220.57 ± 27.33	6.12 ± 1.07	36.06 ± 4.47		 	700	344.03 ± 40.38	6.94 ± 0.17	49.58 ± 5.82		 	1000	496.00 ± 38.48	7.19 ± 0.08	68.94 ± 5.35	4137 ± 321	 	1500	569.98 ± 73.63	5.73 ± 0.03	99.44 ± 12.84		 	2000	584.30 ± 28.33	4.88 ± 0.22	119.62 ± 5.80		 	8.50 µM Apoferritin	0	3.95 ± 2.26	9.37 ± 0.24	0.42 ± 0.25		 	42.5	10.27 ± 1.12	8.27 ± 0.30	1.24 ± 0.18		 	212.5	44.48 ± 2.76	7.85 ± 0.77	5.67 ± 0.83		 	637.5	160.93 ± 4.27	6.76 ± 0.81	23.79 ± 3.12	571 ± 75	 	1275	114.92 ± 3.17	3.84 ± 0.30	29.91 ± 2.95		 	1700	91.40 ± 3.37	3.14 ± 0.35	29.13 ± 3.86		 	"	TABLE
+206	214	EncFtnsH	protein	"Protein sample	Fe(NH4)2(SO4)2 loading (µM)	Fe detected by ferrozine assay (µM)	Protein detected by BCA microplate assay (µM)	Fe / monomeric protein	Maximum Fe loading per biological assembly unit	 	8.46 µM EncFtnsH-10mer	0	4.73 ± 2.32	5.26 ± 0.64	0.90 ± 0.44		 	39.9	9.93 ± 1.20	5.36 ± 0.69	1.85 ± 0.22		 	84	17.99 ± 2.01	4.96 ± 0.04	3.63 ± 0.41		 	147	21.09 ± 1.94	4.44 ± 0.21	4.75 ± 0.44	48 ± 4	 	224	28.68 ± 0.30	3.73 ± 0.53	7.68 ± 0.08		 	301	11.27 ± 1.10	2.50 ± 0.05	4.51 ± 0.44		 	8.50 µM Encapsulin	0	-1.02 ± 0.54	8.63 ± 0.17	-0.12 ± 0.06		 	224	62.24 ± 2.49	10.01 ± 0.58	6.22 ± 0.35		 	301	67.94 ± 3.15	8.69 ± 0.42	7.81 ± 0.36		 	450	107.96 ± 8.88	8.50 ± 0.69	12.71 ± 1.05		 	700	97.51 ± 3.19	7.26 ± 0.20	13.44 ± 0.44		 	1000	308.63 ± 2.06	8.42 ± 0.34	36.66 ± 0.24	2199 ± 15	 	1500	57.09 ± 0.90	1.44 ± 0.21	39.77 ± 0.62		 	2000	9.2 ± 1.16	0.21 ± 0.14	44.73 ± 5.63		 	8.70 µM EncFtn-Enc	0	3.31 ± 1.57	6.85 ± 0.07	0.48 ± 0.23		 	224	116.27 ± 3.74	7.63 ± 0.12	15.25 ± 0.49		 	301	132.86 ± 4.03	6.66 ± 0.31	19.96 ± 0.61		 	450	220.57 ± 27.33	6.12 ± 1.07	36.06 ± 4.47		 	700	344.03 ± 40.38	6.94 ± 0.17	49.58 ± 5.82		 	1000	496.00 ± 38.48	7.19 ± 0.08	68.94 ± 5.35	4137 ± 321	 	1500	569.98 ± 73.63	5.73 ± 0.03	99.44 ± 12.84		 	2000	584.30 ± 28.33	4.88 ± 0.22	119.62 ± 5.80		 	8.50 µM Apoferritin	0	3.95 ± 2.26	9.37 ± 0.24	0.42 ± 0.25		 	42.5	10.27 ± 1.12	8.27 ± 0.30	1.24 ± 0.18		 	212.5	44.48 ± 2.76	7.85 ± 0.77	5.67 ± 0.83		 	637.5	160.93 ± 4.27	6.76 ± 0.81	23.79 ± 3.12	571 ± 75	 	1275	114.92 ± 3.17	3.84 ± 0.30	29.91 ± 2.95		 	1700	91.40 ± 3.37	3.14 ± 0.35	29.13 ± 3.86		 	"	TABLE
+215	220	10mer	oligomeric_state	"Protein sample	Fe(NH4)2(SO4)2 loading (µM)	Fe detected by ferrozine assay (µM)	Protein detected by BCA microplate assay (µM)	Fe / monomeric protein	Maximum Fe loading per biological assembly unit	 	8.46 µM EncFtnsH-10mer	0	4.73 ± 2.32	5.26 ± 0.64	0.90 ± 0.44		 	39.9	9.93 ± 1.20	5.36 ± 0.69	1.85 ± 0.22		 	84	17.99 ± 2.01	4.96 ± 0.04	3.63 ± 0.41		 	147	21.09 ± 1.94	4.44 ± 0.21	4.75 ± 0.44	48 ± 4	 	224	28.68 ± 0.30	3.73 ± 0.53	7.68 ± 0.08		 	301	11.27 ± 1.10	2.50 ± 0.05	4.51 ± 0.44		 	8.50 µM Encapsulin	0	-1.02 ± 0.54	8.63 ± 0.17	-0.12 ± 0.06		 	224	62.24 ± 2.49	10.01 ± 0.58	6.22 ± 0.35		 	301	67.94 ± 3.15	8.69 ± 0.42	7.81 ± 0.36		 	450	107.96 ± 8.88	8.50 ± 0.69	12.71 ± 1.05		 	700	97.51 ± 3.19	7.26 ± 0.20	13.44 ± 0.44		 	1000	308.63 ± 2.06	8.42 ± 0.34	36.66 ± 0.24	2199 ± 15	 	1500	57.09 ± 0.90	1.44 ± 0.21	39.77 ± 0.62		 	2000	9.2 ± 1.16	0.21 ± 0.14	44.73 ± 5.63		 	8.70 µM EncFtn-Enc	0	3.31 ± 1.57	6.85 ± 0.07	0.48 ± 0.23		 	224	116.27 ± 3.74	7.63 ± 0.12	15.25 ± 0.49		 	301	132.86 ± 4.03	6.66 ± 0.31	19.96 ± 0.61		 	450	220.57 ± 27.33	6.12 ± 1.07	36.06 ± 4.47		 	700	344.03 ± 40.38	6.94 ± 0.17	49.58 ± 5.82		 	1000	496.00 ± 38.48	7.19 ± 0.08	68.94 ± 5.35	4137 ± 321	 	1500	569.98 ± 73.63	5.73 ± 0.03	99.44 ± 12.84		 	2000	584.30 ± 28.33	4.88 ± 0.22	119.62 ± 5.80		 	8.50 µM Apoferritin	0	3.95 ± 2.26	9.37 ± 0.24	0.42 ± 0.25		 	42.5	10.27 ± 1.12	8.27 ± 0.30	1.24 ± 0.18		 	212.5	44.48 ± 2.76	7.85 ± 0.77	5.67 ± 0.83		 	637.5	160.93 ± 4.27	6.76 ± 0.81	23.79 ± 3.12	571 ± 75	 	1275	114.92 ± 3.17	3.84 ± 0.30	29.91 ± 2.95		 	1700	91.40 ± 3.37	3.14 ± 0.35	29.13 ± 3.86		 	"	TABLE
+495	505	Encapsulin	protein	"Protein sample	Fe(NH4)2(SO4)2 loading (µM)	Fe detected by ferrozine assay (µM)	Protein detected by BCA microplate assay (µM)	Fe / monomeric protein	Maximum Fe loading per biological assembly unit	 	8.46 µM EncFtnsH-10mer	0	4.73 ± 2.32	5.26 ± 0.64	0.90 ± 0.44		 	39.9	9.93 ± 1.20	5.36 ± 0.69	1.85 ± 0.22		 	84	17.99 ± 2.01	4.96 ± 0.04	3.63 ± 0.41		 	147	21.09 ± 1.94	4.44 ± 0.21	4.75 ± 0.44	48 ± 4	 	224	28.68 ± 0.30	3.73 ± 0.53	7.68 ± 0.08		 	301	11.27 ± 1.10	2.50 ± 0.05	4.51 ± 0.44		 	8.50 µM Encapsulin	0	-1.02 ± 0.54	8.63 ± 0.17	-0.12 ± 0.06		 	224	62.24 ± 2.49	10.01 ± 0.58	6.22 ± 0.35		 	301	67.94 ± 3.15	8.69 ± 0.42	7.81 ± 0.36		 	450	107.96 ± 8.88	8.50 ± 0.69	12.71 ± 1.05		 	700	97.51 ± 3.19	7.26 ± 0.20	13.44 ± 0.44		 	1000	308.63 ± 2.06	8.42 ± 0.34	36.66 ± 0.24	2199 ± 15	 	1500	57.09 ± 0.90	1.44 ± 0.21	39.77 ± 0.62		 	2000	9.2 ± 1.16	0.21 ± 0.14	44.73 ± 5.63		 	8.70 µM EncFtn-Enc	0	3.31 ± 1.57	6.85 ± 0.07	0.48 ± 0.23		 	224	116.27 ± 3.74	7.63 ± 0.12	15.25 ± 0.49		 	301	132.86 ± 4.03	6.66 ± 0.31	19.96 ± 0.61		 	450	220.57 ± 27.33	6.12 ± 1.07	36.06 ± 4.47		 	700	344.03 ± 40.38	6.94 ± 0.17	49.58 ± 5.82		 	1000	496.00 ± 38.48	7.19 ± 0.08	68.94 ± 5.35	4137 ± 321	 	1500	569.98 ± 73.63	5.73 ± 0.03	99.44 ± 12.84		 	2000	584.30 ± 28.33	4.88 ± 0.22	119.62 ± 5.80		 	8.50 µM Apoferritin	0	3.95 ± 2.26	9.37 ± 0.24	0.42 ± 0.25		 	42.5	10.27 ± 1.12	8.27 ± 0.30	1.24 ± 0.18		 	212.5	44.48 ± 2.76	7.85 ± 0.77	5.67 ± 0.83		 	637.5	160.93 ± 4.27	6.76 ± 0.81	23.79 ± 3.12	571 ± 75	 	1275	114.92 ± 3.17	3.84 ± 0.30	29.91 ± 2.95		 	1700	91.40 ± 3.37	3.14 ± 0.35	29.13 ± 3.86		 	"	TABLE
+883	893	EncFtn-Enc	complex_assembly	"Protein sample	Fe(NH4)2(SO4)2 loading (µM)	Fe detected by ferrozine assay (µM)	Protein detected by BCA microplate assay (µM)	Fe / monomeric protein	Maximum Fe loading per biological assembly unit	 	8.46 µM EncFtnsH-10mer	0	4.73 ± 2.32	5.26 ± 0.64	0.90 ± 0.44		 	39.9	9.93 ± 1.20	5.36 ± 0.69	1.85 ± 0.22		 	84	17.99 ± 2.01	4.96 ± 0.04	3.63 ± 0.41		 	147	21.09 ± 1.94	4.44 ± 0.21	4.75 ± 0.44	48 ± 4	 	224	28.68 ± 0.30	3.73 ± 0.53	7.68 ± 0.08		 	301	11.27 ± 1.10	2.50 ± 0.05	4.51 ± 0.44		 	8.50 µM Encapsulin	0	-1.02 ± 0.54	8.63 ± 0.17	-0.12 ± 0.06		 	224	62.24 ± 2.49	10.01 ± 0.58	6.22 ± 0.35		 	301	67.94 ± 3.15	8.69 ± 0.42	7.81 ± 0.36		 	450	107.96 ± 8.88	8.50 ± 0.69	12.71 ± 1.05		 	700	97.51 ± 3.19	7.26 ± 0.20	13.44 ± 0.44		 	1000	308.63 ± 2.06	8.42 ± 0.34	36.66 ± 0.24	2199 ± 15	 	1500	57.09 ± 0.90	1.44 ± 0.21	39.77 ± 0.62		 	2000	9.2 ± 1.16	0.21 ± 0.14	44.73 ± 5.63		 	8.70 µM EncFtn-Enc	0	3.31 ± 1.57	6.85 ± 0.07	0.48 ± 0.23		 	224	116.27 ± 3.74	7.63 ± 0.12	15.25 ± 0.49		 	301	132.86 ± 4.03	6.66 ± 0.31	19.96 ± 0.61		 	450	220.57 ± 27.33	6.12 ± 1.07	36.06 ± 4.47		 	700	344.03 ± 40.38	6.94 ± 0.17	49.58 ± 5.82		 	1000	496.00 ± 38.48	7.19 ± 0.08	68.94 ± 5.35	4137 ± 321	 	1500	569.98 ± 73.63	5.73 ± 0.03	99.44 ± 12.84		 	2000	584.30 ± 28.33	4.88 ± 0.22	119.62 ± 5.80		 	8.50 µM Apoferritin	0	3.95 ± 2.26	9.37 ± 0.24	0.42 ± 0.25		 	42.5	10.27 ± 1.12	8.27 ± 0.30	1.24 ± 0.18		 	212.5	44.48 ± 2.76	7.85 ± 0.77	5.67 ± 0.83		 	637.5	160.93 ± 4.27	6.76 ± 0.81	23.79 ± 3.12	571 ± 75	 	1275	114.92 ± 3.17	3.84 ± 0.30	29.91 ± 2.95		 	1700	91.40 ± 3.37	3.14 ± 0.35	29.13 ± 3.86		 	"	TABLE
+1285	1296	Apoferritin	protein_state	"Protein sample	Fe(NH4)2(SO4)2 loading (µM)	Fe detected by ferrozine assay (µM)	Protein detected by BCA microplate assay (µM)	Fe / monomeric protein	Maximum Fe loading per biological assembly unit	 	8.46 µM EncFtnsH-10mer	0	4.73 ± 2.32	5.26 ± 0.64	0.90 ± 0.44		 	39.9	9.93 ± 1.20	5.36 ± 0.69	1.85 ± 0.22		 	84	17.99 ± 2.01	4.96 ± 0.04	3.63 ± 0.41		 	147	21.09 ± 1.94	4.44 ± 0.21	4.75 ± 0.44	48 ± 4	 	224	28.68 ± 0.30	3.73 ± 0.53	7.68 ± 0.08		 	301	11.27 ± 1.10	2.50 ± 0.05	4.51 ± 0.44		 	8.50 µM Encapsulin	0	-1.02 ± 0.54	8.63 ± 0.17	-0.12 ± 0.06		 	224	62.24 ± 2.49	10.01 ± 0.58	6.22 ± 0.35		 	301	67.94 ± 3.15	8.69 ± 0.42	7.81 ± 0.36		 	450	107.96 ± 8.88	8.50 ± 0.69	12.71 ± 1.05		 	700	97.51 ± 3.19	7.26 ± 0.20	13.44 ± 0.44		 	1000	308.63 ± 2.06	8.42 ± 0.34	36.66 ± 0.24	2199 ± 15	 	1500	57.09 ± 0.90	1.44 ± 0.21	39.77 ± 0.62		 	2000	9.2 ± 1.16	0.21 ± 0.14	44.73 ± 5.63		 	8.70 µM EncFtn-Enc	0	3.31 ± 1.57	6.85 ± 0.07	0.48 ± 0.23		 	224	116.27 ± 3.74	7.63 ± 0.12	15.25 ± 0.49		 	301	132.86 ± 4.03	6.66 ± 0.31	19.96 ± 0.61		 	450	220.57 ± 27.33	6.12 ± 1.07	36.06 ± 4.47		 	700	344.03 ± 40.38	6.94 ± 0.17	49.58 ± 5.82		 	1000	496.00 ± 38.48	7.19 ± 0.08	68.94 ± 5.35	4137 ± 321	 	1500	569.98 ± 73.63	5.73 ± 0.03	99.44 ± 12.84		 	2000	584.30 ± 28.33	4.88 ± 0.22	119.62 ± 5.80		 	8.50 µM Apoferritin	0	3.95 ± 2.26	9.37 ± 0.24	0.42 ± 0.25		 	42.5	10.27 ± 1.12	8.27 ± 0.30	1.24 ± 0.18		 	212.5	44.48 ± 2.76	7.85 ± 0.77	5.67 ± 0.83		 	637.5	160.93 ± 4.27	6.76 ± 0.81	23.79 ± 3.12	571 ± 75	 	1275	114.92 ± 3.17	3.84 ± 0.30	29.91 ± 2.95		 	1700	91.40 ± 3.37	3.14 ± 0.35	29.13 ± 3.86		 	"	TABLE
+32	39	mutants	protein_state	To understand the impact of the mutants on the organization and metal binding of the FOC, we determined the X-ray crystal structures of each of the EncFtnsH mutants (See Table 4 for data collection and refinement statistics).	RESULTS
+85	88	FOC	site	To understand the impact of the mutants on the organization and metal binding of the FOC, we determined the X-ray crystal structures of each of the EncFtnsH mutants (See Table 4 for data collection and refinement statistics).	RESULTS
+108	132	X-ray crystal structures	evidence	To understand the impact of the mutants on the organization and metal binding of the FOC, we determined the X-ray crystal structures of each of the EncFtnsH mutants (See Table 4 for data collection and refinement statistics).	RESULTS
+148	156	EncFtnsH	protein	To understand the impact of the mutants on the organization and metal binding of the FOC, we determined the X-ray crystal structures of each of the EncFtnsH mutants (See Table 4 for data collection and refinement statistics).	RESULTS
+157	164	mutants	protein_state	To understand the impact of the mutants on the organization and metal binding of the FOC, we determined the X-ray crystal structures of each of the EncFtnsH mutants (See Table 4 for data collection and refinement statistics).	RESULTS
+34	41	mutants	protein_state	The crystal packing of all of the mutants in this study is essentially isomorphous to the EncFtnsH structure.	RESULTS
+90	98	EncFtnsH	protein	The crystal packing of all of the mutants in this study is essentially isomorphous to the EncFtnsH structure.	RESULTS
+99	108	structure	evidence	The crystal packing of all of the mutants in this study is essentially isomorphous to the EncFtnsH structure.	RESULTS
+11	18	mutants	protein_state	All of the mutants display the same decameric arrangement in the crystals as the EncFtnsH structure, and the monomers superimpose with an average RMSDCα of less than 0.2 Å.	RESULTS
+36	45	decameric	oligomeric_state	All of the mutants display the same decameric arrangement in the crystals as the EncFtnsH structure, and the monomers superimpose with an average RMSDCα of less than 0.2 Å.	RESULTS
+65	73	crystals	evidence	All of the mutants display the same decameric arrangement in the crystals as the EncFtnsH structure, and the monomers superimpose with an average RMSDCα of less than 0.2 Å.	RESULTS
+81	89	EncFtnsH	protein	All of the mutants display the same decameric arrangement in the crystals as the EncFtnsH structure, and the monomers superimpose with an average RMSDCα of less than 0.2 Å.	RESULTS
+90	99	structure	evidence	All of the mutants display the same decameric arrangement in the crystals as the EncFtnsH structure, and the monomers superimpose with an average RMSDCα of less than 0.2 Å.	RESULTS
+109	117	monomers	oligomeric_state	All of the mutants display the same decameric arrangement in the crystals as the EncFtnsH structure, and the monomers superimpose with an average RMSDCα of less than 0.2 Å.	RESULTS
+118	129	superimpose	experimental_method	All of the mutants display the same decameric arrangement in the crystals as the EncFtnsH structure, and the monomers superimpose with an average RMSDCα of less than 0.2 Å.	RESULTS
+146	152	RMSDCα	evidence	All of the mutants display the same decameric arrangement in the crystals as the EncFtnsH structure, and the monomers superimpose with an average RMSDCα of less than 0.2 Å.	RESULTS
+0	3	FOC	site	FOC dimer interface of EncFtnsH-E32A mutant.	FIG
+4	19	dimer interface	site	FOC dimer interface of EncFtnsH-E32A mutant.	FIG
+23	36	EncFtnsH-E32A	mutant	FOC dimer interface of EncFtnsH-E32A mutant.	FIG
+37	43	mutant	protein_state	FOC dimer interface of EncFtnsH-E32A mutant.	FIG
+33	69	metal-binding dimerization interface	site	(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-E32A.	FIG
+73	86	EncFtnsH-E32A	mutant	(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-E32A.	FIG
+83	91	subunits	structure_element	Protein residues are shown as sticks with blue and green carbons for the different subunits.	FIG
+83	91	subunits	structure_element	Protein residues are shown as sticks with blue and green carbons for the different subunits.	FIG
+83	91	subunits	structure_element	Protein residues are shown as sticks with blue and green carbons for the different subunits.	FIG
+4	33	2mFo-DFc electron density map	evidence	The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ.	FIG
+4	33	2mFo-DFc electron density map	evidence	The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ.	FIG
+4	33	2mFo-DFc electron density map	evidence	The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ.	FIG
+17	20	FOC	site	(B) Views of the FOC of the EncFtnsH-E32Amutant.	FIG
+28	41	EncFtnsH-E32A	mutant	(B) Views of the FOC of the EncFtnsH-E32Amutant.	FIG
+41	47	mutant	protein_state	(B) Views of the FOC of the EncFtnsH-E32Amutant.	FIG
+0	19	FOC dimer interface	site	FOC dimer interface of EncFtnsH-E62A mutant.	FIG
+23	36	EncFtnsH-E62A	mutant	FOC dimer interface of EncFtnsH-E62A mutant.	FIG
+37	43	mutant	protein_state	FOC dimer interface of EncFtnsH-E62A mutant.	FIG
+33	69	metal-binding dimerization interface	site	(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-E62A.	FIG
+73	86	EncFtnsH-E62A	mutant	(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-E62A.	FIG
+23	30	calcium	chemical	The single coordinated calcium ion is shown as a grey sphere. (B) Views of the FOC of the EncFtnsH-E62A mutant.	FIG
+79	82	FOC	site	The single coordinated calcium ion is shown as a grey sphere. (B) Views of the FOC of the EncFtnsH-E62A mutant.	FIG
+90	103	EncFtnsH-E62A	mutant	The single coordinated calcium ion is shown as a grey sphere. (B) Views of the FOC of the EncFtnsH-E62A mutant.	FIG
+104	110	mutant	protein_state	The single coordinated calcium ion is shown as a grey sphere. (B) Views of the FOC of the EncFtnsH-E62A mutant.	FIG
+0	19	FOC dimer interface	site	FOC dimer interface of EncFtnsH-H65A mutant.	FIG
+23	36	EncFtnsH-H65A	mutant	FOC dimer interface of EncFtnsH-H65A mutant.	FIG
+37	43	mutant	protein_state	FOC dimer interface of EncFtnsH-H65A mutant.	FIG
+33	69	metal-binding dimerization interface	site	(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-H65A.	FIG
+73	86	EncFtnsH-H65A	mutant	(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-H65A.	FIG
+16	23	calcium	chemical	The coordinated calcium ions are shown as a grey spheres with coordination distances in the FOC highlighted with yellow dashed lines.	FIG
+62	74	coordination	bond_interaction	The coordinated calcium ions are shown as a grey spheres with coordination distances in the FOC highlighted with yellow dashed lines.	FIG
+92	95	FOC	site	The coordinated calcium ions are shown as a grey spheres with coordination distances in the FOC highlighted with yellow dashed lines.	FIG
+17	20	FOC	site	(B) Views of the FOC of the EncFtnsH-H65A mutant.	FIG
+28	41	EncFtnsH-H65A	mutant	(B) Views of the FOC of the EncFtnsH-H65A mutant.	FIG
+42	48	mutant	protein_state	(B) Views of the FOC of the EncFtnsH-H65A mutant.	FIG
+18	26	EncFtnsH	protein	Comparison of the EncFtnsH FOC mutants vs wild type.	FIG
+27	30	FOC	site	Comparison of the EncFtnsH FOC mutants vs wild type.	FIG
+31	38	mutants	protein_state	Comparison of the EncFtnsH FOC mutants vs wild type.	FIG
+42	51	wild type	protein_state	Comparison of the EncFtnsH FOC mutants vs wild type.	FIG
+4	14	structures	evidence	The structures of the three EncFtnsH mutants were all determined by X-ray crystallography.	FIG
+28	36	EncFtnsH	protein	The structures of the three EncFtnsH mutants were all determined by X-ray crystallography.	FIG
+37	44	mutants	protein_state	The structures of the three EncFtnsH mutants were all determined by X-ray crystallography.	FIG
+68	89	X-ray crystallography	experimental_method	The structures of the three EncFtnsH mutants were all determined by X-ray crystallography.	FIG
+4	8	E32A	mutant	The E32A, E62A and H65A mutants were crystallized in identical conditions to the wild type.	FIG
+10	14	E62A	mutant	The E32A, E62A and H65A mutants were crystallized in identical conditions to the wild type.	FIG
+19	23	H65A	mutant	The E32A, E62A and H65A mutants were crystallized in identical conditions to the wild type.	FIG
+24	31	mutants	protein_state	The E32A, E62A and H65A mutants were crystallized in identical conditions to the wild type.	FIG
+37	49	crystallized	experimental_method	The E32A, E62A and H65A mutants were crystallized in identical conditions to the wild type.	FIG
+81	90	wild type	protein_state	The E32A, E62A and H65A mutants were crystallized in identical conditions to the wild type.	FIG
+0	8	EncFtnsH	protein	EncFtnsH structure and were essentially isomorphous in terms of their unit cell dimensions.	FIG
+9	18	structure	evidence	EncFtnsH structure and were essentially isomorphous in terms of their unit cell dimensions.	FIG
+4	7	FOC	site	The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue.	FIG
+24	31	mutants	protein_state	The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue.	FIG
+36	42	native	protein_state	The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue.	FIG
+43	51	EncFtnsH	protein	The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue.	FIG
+52	62	structures	evidence	The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue.	FIG
+88	99	coordinated	bond_interaction	The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue.	FIG
+100	104	Fe2+	chemical	The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue.	FIG
+119	123	Ca2+	chemical	The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue.	FIG
+168	177	wild type	protein_state	The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue.	FIG
+185	189	E32A	mutant	The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue.	FIG
+197	201	E62A	mutant	The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue.	FIG
+210	214	H65A	mutant	The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue.	FIG
+7	14	mutants	protein_state	Of the mutants, only H65A has any coordinated metal ions, which appear to be calcium ions from the crystallization condition.	FIG
+21	25	H65A	mutant	Of the mutants, only H65A has any coordinated metal ions, which appear to be calcium ions from the crystallization condition.	FIG
+34	45	coordinated	bond_interaction	Of the mutants, only H65A has any coordinated metal ions, which appear to be calcium ions from the crystallization condition.	FIG
+77	84	calcium	chemical	Of the mutants, only H65A has any coordinated metal ions, which appear to be calcium ions from the crystallization condition.	FIG
+28	31	FOC	site	The overall organization of FOC residues is retained in the mutants, with almost no backbone movements.	FIG
+60	67	mutants	protein_state	The overall organization of FOC residues is retained in the mutants, with almost no backbone movements.	FIG
+38	43	Tyr39	residue_name_number	Significant differences center around Tyr39, which moves to coordinate the bound calcium ions in the H65A mutant; and Glu32, which moves away from the metal ions in this structure.	FIG
+60	70	coordinate	bond_interaction	Significant differences center around Tyr39, which moves to coordinate the bound calcium ions in the H65A mutant; and Glu32, which moves away from the metal ions in this structure.	FIG
+75	80	bound	protein_state	Significant differences center around Tyr39, which moves to coordinate the bound calcium ions in the H65A mutant; and Glu32, which moves away from the metal ions in this structure.	FIG
+81	88	calcium	chemical	Significant differences center around Tyr39, which moves to coordinate the bound calcium ions in the H65A mutant; and Glu32, which moves away from the metal ions in this structure.	FIG
+101	105	H65A	mutant	Significant differences center around Tyr39, which moves to coordinate the bound calcium ions in the H65A mutant; and Glu32, which moves away from the metal ions in this structure.	FIG
+106	112	mutant	protein_state	Significant differences center around Tyr39, which moves to coordinate the bound calcium ions in the H65A mutant; and Glu32, which moves away from the metal ions in this structure.	FIG
+118	123	Glu32	residue_name_number	Significant differences center around Tyr39, which moves to coordinate the bound calcium ions in the H65A mutant; and Glu32, which moves away from the metal ions in this structure.	FIG
+170	179	structure	evidence	Significant differences center around Tyr39, which moves to coordinate the bound calcium ions in the H65A mutant; and Glu32, which moves away from the metal ions in this structure.	FIG
+57	60	FOC	site	Close inspection of the region of the protein around the FOC in each of the mutants highlights their effect on metal binding (Figure 11 and Figure 11—figure supplement 1–3).	RESULTS
+76	83	mutants	protein_state	Close inspection of the region of the protein around the FOC in each of the mutants highlights their effect on metal binding (Figure 11 and Figure 11—figure supplement 1–3).	RESULTS
+7	11	E32A	mutant	In the E32A mutant the position of the side chains of the remaining iron coordinating residues in the FOC is essentially unchanged, but the absence of the axial-metal coordinating ligand provided by the Glu32 side chain abrogates metal binding in this site.	RESULTS
+12	18	mutant	protein_state	In the E32A mutant the position of the side chains of the remaining iron coordinating residues in the FOC is essentially unchanged, but the absence of the axial-metal coordinating ligand provided by the Glu32 side chain abrogates metal binding in this site.	RESULTS
+68	94	iron coordinating residues	site	In the E32A mutant the position of the side chains of the remaining iron coordinating residues in the FOC is essentially unchanged, but the absence of the axial-metal coordinating ligand provided by the Glu32 side chain abrogates metal binding in this site.	RESULTS
+102	105	FOC	site	In the E32A mutant the position of the side chains of the remaining iron coordinating residues in the FOC is essentially unchanged, but the absence of the axial-metal coordinating ligand provided by the Glu32 side chain abrogates metal binding in this site.	RESULTS
+140	150	absence of	protein_state	In the E32A mutant the position of the side chains of the remaining iron coordinating residues in the FOC is essentially unchanged, but the absence of the axial-metal coordinating ligand provided by the Glu32 side chain abrogates metal binding in this site.	RESULTS
+167	179	coordinating	bond_interaction	In the E32A mutant the position of the side chains of the remaining iron coordinating residues in the FOC is essentially unchanged, but the absence of the axial-metal coordinating ligand provided by the Glu32 side chain abrogates metal binding in this site.	RESULTS
+203	208	Glu32	residue_name_number	In the E32A mutant the position of the side chains of the remaining iron coordinating residues in the FOC is essentially unchanged, but the absence of the axial-metal coordinating ligand provided by the Glu32 side chain abrogates metal binding in this site.	RESULTS
+220	243	abrogates metal binding	protein_state	In the E32A mutant the position of the side chains of the remaining iron coordinating residues in the FOC is essentially unchanged, but the absence of the axial-metal coordinating ligand provided by the Glu32 side chain abrogates metal binding in this site.	RESULTS
+4	17	Glu31/34-site	site	The Glu31/34-site also lacks metal, with the side chain of Glu31 rotated by 180° at the Cβ in the absence of metal (Figure 11—figure supplement 1).	RESULTS
+23	28	lacks	protein_state	The Glu31/34-site also lacks metal, with the side chain of Glu31 rotated by 180° at the Cβ in the absence of metal (Figure 11—figure supplement 1).	RESULTS
+29	34	metal	chemical	The Glu31/34-site also lacks metal, with the side chain of Glu31 rotated by 180° at the Cβ in the absence of metal (Figure 11—figure supplement 1).	RESULTS
+59	64	Glu31	residue_name_number	The Glu31/34-site also lacks metal, with the side chain of Glu31 rotated by 180° at the Cβ in the absence of metal (Figure 11—figure supplement 1).	RESULTS
+98	108	absence of	protein_state	The Glu31/34-site also lacks metal, with the side chain of Glu31 rotated by 180° at the Cβ in the absence of metal (Figure 11—figure supplement 1).	RESULTS
+109	114	metal	chemical	The Glu31/34-site also lacks metal, with the side chain of Glu31 rotated by 180° at the Cβ in the absence of metal (Figure 11—figure supplement 1).	RESULTS
+4	8	E62A	mutant	The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11—figure supplement 2).	RESULTS
+9	15	mutant	protein_state	The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11—figure supplement 2).	RESULTS
+44	47	FOC	site	The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11—figure supplement 2).	RESULTS
+55	59	E32A	mutant	The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11—figure supplement 2).	RESULTS
+60	66	mutant	protein_state	The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11—figure supplement 2).	RESULTS
+80	90	entry site	site	The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11—figure supplement 2).	RESULTS
+103	110	calcium	chemical	The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11—figure supplement 2).	RESULTS
+115	126	coordinated	bond_interaction	The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11—figure supplement 2).	RESULTS
+144	149	Glu31	residue_name_number	The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11—figure supplement 2).	RESULTS
+154	159	Glu34	residue_name_number	The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11—figure supplement 2).	RESULTS
+4	8	H65A	mutant	The H65A mutant diverges significantly from the wild type in the position of the residues Glu32 and Tyr39 in the FOC.	RESULTS
+9	15	mutant	protein_state	The H65A mutant diverges significantly from the wild type in the position of the residues Glu32 and Tyr39 in the FOC.	RESULTS
+48	57	wild type	protein_state	The H65A mutant diverges significantly from the wild type in the position of the residues Glu32 and Tyr39 in the FOC.	RESULTS
+90	95	Glu32	residue_name_number	The H65A mutant diverges significantly from the wild type in the position of the residues Glu32 and Tyr39 in the FOC.	RESULTS
+100	105	Tyr39	residue_name_number	The H65A mutant diverges significantly from the wild type in the position of the residues Glu32 and Tyr39 in the FOC.	RESULTS
+113	116	FOC	site	The H65A mutant diverges significantly from the wild type in the position of the residues Glu32 and Tyr39 in the FOC.	RESULTS
+0	3	E32	residue_name_number	E32 appears in either the original orientation as the wild type and coordinates Ca2+ in this position, or it is flipped by 180° at the Cβ, moving away from the coordinated calcium ion in the FOC.	RESULTS
+54	63	wild type	protein_state	E32 appears in either the original orientation as the wild type and coordinates Ca2+ in this position, or it is flipped by 180° at the Cβ, moving away from the coordinated calcium ion in the FOC.	RESULTS
+68	79	coordinates	bond_interaction	E32 appears in either the original orientation as the wild type and coordinates Ca2+ in this position, or it is flipped by 180° at the Cβ, moving away from the coordinated calcium ion in the FOC.	RESULTS
+80	84	Ca2+	chemical	E32 appears in either the original orientation as the wild type and coordinates Ca2+ in this position, or it is flipped by 180° at the Cβ, moving away from the coordinated calcium ion in the FOC.	RESULTS
+160	171	coordinated	bond_interaction	E32 appears in either the original orientation as the wild type and coordinates Ca2+ in this position, or it is flipped by 180° at the Cβ, moving away from the coordinated calcium ion in the FOC.	RESULTS
+172	179	calcium	chemical	E32 appears in either the original orientation as the wild type and coordinates Ca2+ in this position, or it is flipped by 180° at the Cβ, moving away from the coordinated calcium ion in the FOC.	RESULTS
+191	194	FOC	site	E32 appears in either the original orientation as the wild type and coordinates Ca2+ in this position, or it is flipped by 180° at the Cβ, moving away from the coordinated calcium ion in the FOC.	RESULTS
+0	5	Tyr39	residue_name_number	Tyr39 moves closer to Ca2+ compared to the wild-type and coordinates the calcium ion (Figure 11—figure supplement 3).	RESULTS
+22	26	Ca2+	chemical	Tyr39 moves closer to Ca2+ compared to the wild-type and coordinates the calcium ion (Figure 11—figure supplement 3).	RESULTS
+43	52	wild-type	protein_state	Tyr39 moves closer to Ca2+ compared to the wild-type and coordinates the calcium ion (Figure 11—figure supplement 3).	RESULTS
+57	68	coordinates	bond_interaction	Tyr39 moves closer to Ca2+ compared to the wild-type and coordinates the calcium ion (Figure 11—figure supplement 3).	RESULTS
+73	80	calcium	chemical	Tyr39 moves closer to Ca2+ compared to the wild-type and coordinates the calcium ion (Figure 11—figure supplement 3).	RESULTS
+9	16	calcium	chemical	A single calcium ion is present in the entry site of this mutant; however, Glu31 of one chain is rotated away from the metal ion and is not involved in coordination.	RESULTS
+39	49	entry site	site	A single calcium ion is present in the entry site of this mutant; however, Glu31 of one chain is rotated away from the metal ion and is not involved in coordination.	RESULTS
+58	64	mutant	protein_state	A single calcium ion is present in the entry site of this mutant; however, Glu31 of one chain is rotated away from the metal ion and is not involved in coordination.	RESULTS
+75	80	Glu31	residue_name_number	A single calcium ion is present in the entry site of this mutant; however, Glu31 of one chain is rotated away from the metal ion and is not involved in coordination.	RESULTS
+152	164	coordination	bond_interaction	A single calcium ion is present in the entry site of this mutant; however, Glu31 of one chain is rotated away from the metal ion and is not involved in coordination.	RESULTS
+70	73	FOC	site	Taken together the results of our data show that these changes to the FOC of EncFtn still permit the formation of the decameric form of the protein.	RESULTS
+77	83	EncFtn	protein	Taken together the results of our data show that these changes to the FOC of EncFtn still permit the formation of the decameric form of the protein.	RESULTS
+118	127	decameric	oligomeric_state	Taken together the results of our data show that these changes to the FOC of EncFtn still permit the formation of the decameric form of the protein.	RESULTS
+30	39	decameric	oligomeric_state	While the proteins all appear decameric in crystals, their solution and gas-phase behavior differs considerably and the mutants no longer show metal-dependent oligomerization.	RESULTS
+43	51	crystals	evidence	While the proteins all appear decameric in crystals, their solution and gas-phase behavior differs considerably and the mutants no longer show metal-dependent oligomerization.	RESULTS
+120	127	mutants	protein_state	While the proteins all appear decameric in crystals, their solution and gas-phase behavior differs considerably and the mutants no longer show metal-dependent oligomerization.	RESULTS
+42	47	metal	chemical	These results highlight the importance of metal coordination in the FOC for the stability and assembly of the EncFtn protein.	RESULTS
+48	60	coordination	bond_interaction	These results highlight the importance of metal coordination in the FOC for the stability and assembly of the EncFtn protein.	RESULTS
+68	71	FOC	site	These results highlight the importance of metal coordination in the FOC for the stability and assembly of the EncFtn protein.	RESULTS
+110	116	EncFtn	protein	These results highlight the importance of metal coordination in the FOC for the stability and assembly of the EncFtn protein.	RESULTS
+0	15	Progress curves	evidence	Progress curves recording ferroxidase activity of EncFtnsH mutants.	FIG
+26	37	ferroxidase	protein_type	Progress curves recording ferroxidase activity of EncFtnsH mutants.	FIG
+50	58	EncFtnsH	protein	Progress curves recording ferroxidase activity of EncFtnsH mutants.	FIG
+59	66	mutants	protein_state	Progress curves recording ferroxidase activity of EncFtnsH mutants.	FIG
+6	15	wild-type	protein_state	20 µM wild-type EncFtnsH, E32A, E62A and H65A mutants were mixed with 20 µM or 100 µM acidic Fe(NH4)2(SO4)2, respectively.	FIG
+16	24	EncFtnsH	protein	20 µM wild-type EncFtnsH, E32A, E62A and H65A mutants were mixed with 20 µM or 100 µM acidic Fe(NH4)2(SO4)2, respectively.	FIG
+26	30	E32A	mutant	20 µM wild-type EncFtnsH, E32A, E62A and H65A mutants were mixed with 20 µM or 100 µM acidic Fe(NH4)2(SO4)2, respectively.	FIG
+32	36	E62A	mutant	20 µM wild-type EncFtnsH, E32A, E62A and H65A mutants were mixed with 20 µM or 100 µM acidic Fe(NH4)2(SO4)2, respectively.	FIG
+41	45	H65A	mutant	20 µM wild-type EncFtnsH, E32A, E62A and H65A mutants were mixed with 20 µM or 100 µM acidic Fe(NH4)2(SO4)2, respectively.	FIG
+46	53	mutants	protein_state	20 µM wild-type EncFtnsH, E32A, E62A and H65A mutants were mixed with 20 µM or 100 µM acidic Fe(NH4)2(SO4)2, respectively.	FIG
+93	107	Fe(NH4)2(SO4)2	chemical	20 µM wild-type EncFtnsH, E32A, E62A and H65A mutants were mixed with 20 µM or 100 µM acidic Fe(NH4)2(SO4)2, respectively.	FIG
+73	77	Fe3+	chemical	Absorbance at 315 nm was recorded for 1800 s at 25°C as an indication of Fe3+ formation.	FIG
+65	69	Fe2+	chemical	Protein free samples (dashed and dotted lines) were measured for Fe2+ background oxidation as controls.	FIG
+9	20	ferroxidase	protein_type	Relative ferroxidase activity of EncFtnsH mutants.	FIG
+33	41	EncFtnsH	protein	Relative ferroxidase activity of EncFtnsH mutants.	FIG
+42	49	mutants	protein_state	Relative ferroxidase activity of EncFtnsH mutants.	FIG
+0	8	EncFtnsH	protein	EncFtnsH, and the mutant forms E32A, E62A and H65A, each at 20 µM, were mixed with 100 µM acidic Fe(NH4)2(SO4)2.	FIG
+18	24	mutant	protein_state	EncFtnsH, and the mutant forms E32A, E62A and H65A, each at 20 µM, were mixed with 100 µM acidic Fe(NH4)2(SO4)2.	FIG
+31	35	E32A	mutant	EncFtnsH, and the mutant forms E32A, E62A and H65A, each at 20 µM, were mixed with 100 µM acidic Fe(NH4)2(SO4)2.	FIG
+37	41	E62A	mutant	EncFtnsH, and the mutant forms E32A, E62A and H65A, each at 20 µM, were mixed with 100 µM acidic Fe(NH4)2(SO4)2.	FIG
+46	50	H65A	mutant	EncFtnsH, and the mutant forms E32A, E62A and H65A, each at 20 µM, were mixed with 100 µM acidic Fe(NH4)2(SO4)2.	FIG
+97	111	Fe(NH4)2(SO4)2	chemical	EncFtnsH, and the mutant forms E32A, E62A and H65A, each at 20 µM, were mixed with 100 µM acidic Fe(NH4)2(SO4)2.	FIG
+0	11	Ferroxidase	protein_type	Ferroxidase activity of the mutant forms is determined by measuring the absorbance at 315 nm for 1800 s at 25 °C as an indication of Fe3+ formation.	FIG
+28	34	mutant	protein_state	Ferroxidase activity of the mutant forms is determined by measuring the absorbance at 315 nm for 1800 s at 25 °C as an indication of Fe3+ formation.	FIG
+58	92	measuring the absorbance at 315 nm	experimental_method	Ferroxidase activity of the mutant forms is determined by measuring the absorbance at 315 nm for 1800 s at 25 °C as an indication of Fe3+ formation.	FIG
+133	137	Fe3+	chemical	Ferroxidase activity of the mutant forms is determined by measuring the absorbance at 315 nm for 1800 s at 25 °C as an indication of Fe3+ formation.	FIG
+13	24	ferroxidase	protein_type	The relative ferroxidase activity of mutants is plotted as a proportion of the activity of the wild-type protein using the endpoint measurement of A315.	FIG
+37	44	mutants	protein_state	The relative ferroxidase activity of mutants is plotted as a proportion of the activity of the wild-type protein using the endpoint measurement of A315.	FIG
+95	104	wild-type	protein_state	The relative ferroxidase activity of mutants is plotted as a proportion of the activity of the wild-type protein using the endpoint measurement of A315.	FIG
+132	151	measurement of A315	experimental_method	The relative ferroxidase activity of mutants is plotted as a proportion of the activity of the wild-type protein using the endpoint measurement of A315.	FIG
+4	7	FOC	site	The FOC mutants showed reduced ferroxidase activity to varied extents, among which E62A significantly abrogated the ferroxidase activity.	FIG
+8	15	mutants	protein_state	The FOC mutants showed reduced ferroxidase activity to varied extents, among which E62A significantly abrogated the ferroxidase activity.	FIG
+31	42	ferroxidase	protein_type	The FOC mutants showed reduced ferroxidase activity to varied extents, among which E62A significantly abrogated the ferroxidase activity.	FIG
+83	87	E62A	mutant	The FOC mutants showed reduced ferroxidase activity to varied extents, among which E62A significantly abrogated the ferroxidase activity.	FIG
+116	127	ferroxidase	protein_type	The FOC mutants showed reduced ferroxidase activity to varied extents, among which E62A significantly abrogated the ferroxidase activity.	FIG
+31	42	mutagenesis	experimental_method	To address the question of how mutagenesis of the iron coordinating residues affects the enzymatic activity of the EncFtnsH protein we recorded progress curves for the oxidation of Fe2+ to Fe3+ by the different mutants as before.	RESULTS
+50	76	iron coordinating residues	site	To address the question of how mutagenesis of the iron coordinating residues affects the enzymatic activity of the EncFtnsH protein we recorded progress curves for the oxidation of Fe2+ to Fe3+ by the different mutants as before.	RESULTS
+115	123	EncFtnsH	protein	To address the question of how mutagenesis of the iron coordinating residues affects the enzymatic activity of the EncFtnsH protein we recorded progress curves for the oxidation of Fe2+ to Fe3+ by the different mutants as before.	RESULTS
+144	159	progress curves	evidence	To address the question of how mutagenesis of the iron coordinating residues affects the enzymatic activity of the EncFtnsH protein we recorded progress curves for the oxidation of Fe2+ to Fe3+ by the different mutants as before.	RESULTS
+181	185	Fe2+	chemical	To address the question of how mutagenesis of the iron coordinating residues affects the enzymatic activity of the EncFtnsH protein we recorded progress curves for the oxidation of Fe2+ to Fe3+ by the different mutants as before.	RESULTS
+189	193	Fe3+	chemical	To address the question of how mutagenesis of the iron coordinating residues affects the enzymatic activity of the EncFtnsH protein we recorded progress curves for the oxidation of Fe2+ to Fe3+ by the different mutants as before.	RESULTS
+211	218	mutants	protein_state	To address the question of how mutagenesis of the iron coordinating residues affects the enzymatic activity of the EncFtnsH protein we recorded progress curves for the oxidation of Fe2+ to Fe3+ by the different mutants as before.	RESULTS
+0	11	Mutagenesis	experimental_method	Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%; the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12).	RESULTS
+15	19	E32A	mutant	Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%; the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12).	RESULTS
+24	28	H65A	mutant	Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%; the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12).	RESULTS
+53	61	EncFtnsH	protein	Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%; the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12).	RESULTS
+84	88	E62A	mutant	Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%; the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12).	RESULTS
+89	95	mutant	protein_state	Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%; the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12).	RESULTS
+150	157	loss of	protein_state	Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%; the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12).	RESULTS
+171	183	coordination	bond_interaction	Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%; the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12).	RESULTS
+209	231	di-nuclear iron center	site	Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%; the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12).	RESULTS
+239	242	FOC	site	Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%; the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12).	RESULTS
+28	36	mutating	experimental_method	Collectively, the effect of mutating these residues in the FOC confirms the importance of the iron coordinating residues for the ferroxidase activity of the EncFtnsH protein.	RESULTS
+59	62	FOC	site	Collectively, the effect of mutating these residues in the FOC confirms the importance of the iron coordinating residues for the ferroxidase activity of the EncFtnsH protein.	RESULTS
+94	120	iron coordinating residues	site	Collectively, the effect of mutating these residues in the FOC confirms the importance of the iron coordinating residues for the ferroxidase activity of the EncFtnsH protein.	RESULTS
+129	140	ferroxidase	protein_type	Collectively, the effect of mutating these residues in the FOC confirms the importance of the iron coordinating residues for the ferroxidase activity of the EncFtnsH protein.	RESULTS
+157	165	EncFtnsH	protein	Collectively, the effect of mutating these residues in the FOC confirms the importance of the iron coordinating residues for the ferroxidase activity of the EncFtnsH protein.	RESULTS
+0	17	Phylogenetic tree	evidence	Phylogenetic tree of ferritin family proteins.	FIG
+21	29	ferritin	protein_type	Phylogenetic tree of ferritin family proteins.	FIG
+29	52	Neighbor-Joining method	experimental_method	The tree was built using the Neighbor-Joining method based on step-wise amino acid sequence alignment of the four-helical bundle portions of ferritin family proteins (Supplementary file 1).	FIG
+62	101	step-wise amino acid sequence alignment	experimental_method	The tree was built using the Neighbor-Joining method based on step-wise amino acid sequence alignment of the four-helical bundle portions of ferritin family proteins (Supplementary file 1).	FIG
+109	128	four-helical bundle	structure_element	The tree was built using the Neighbor-Joining method based on step-wise amino acid sequence alignment of the four-helical bundle portions of ferritin family proteins (Supplementary file 1).	FIG
+141	149	ferritin	protein_type	The tree was built using the Neighbor-Joining method based on step-wise amino acid sequence alignment of the four-helical bundle portions of ferritin family proteins (Supplementary file 1).	FIG
+4	26	evolutionary distances	evidence	The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site.	FIG
+51	68	p-distance method	experimental_method	The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site.	FIG
+36	44	ferritin	protein_type	Our study reports on a new class of ferritin-like proteins (EncFtn), which are associated with bacterial encapsulin nanocompartments (Enc).	DISCUSS
+60	66	EncFtn	protein	Our study reports on a new class of ferritin-like proteins (EncFtn), which are associated with bacterial encapsulin nanocompartments (Enc).	DISCUSS
+95	104	bacterial	taxonomy_domain	Our study reports on a new class of ferritin-like proteins (EncFtn), which are associated with bacterial encapsulin nanocompartments (Enc).	DISCUSS
+105	115	encapsulin	protein	Our study reports on a new class of ferritin-like proteins (EncFtn), which are associated with bacterial encapsulin nanocompartments (Enc).	DISCUSS
+116	132	nanocompartments	complex_assembly	Our study reports on a new class of ferritin-like proteins (EncFtn), which are associated with bacterial encapsulin nanocompartments (Enc).	DISCUSS
+134	137	Enc	protein	Our study reports on a new class of ferritin-like proteins (EncFtn), which are associated with bacterial encapsulin nanocompartments (Enc).	DISCUSS
+16	22	EncFtn	protein	By studying the EncFtn from R. rubrum we demonstrate that iron binding results in assembly of EncFtn decamers, which display a unique annular architecture.	DISCUSS
+28	37	R. rubrum	species	By studying the EncFtn from R. rubrum we demonstrate that iron binding results in assembly of EncFtn decamers, which display a unique annular architecture.	DISCUSS
+58	62	iron	chemical	By studying the EncFtn from R. rubrum we demonstrate that iron binding results in assembly of EncFtn decamers, which display a unique annular architecture.	DISCUSS
+94	100	EncFtn	protein	By studying the EncFtn from R. rubrum we demonstrate that iron binding results in assembly of EncFtn decamers, which display a unique annular architecture.	DISCUSS
+101	109	decamers	oligomeric_state	By studying the EncFtn from R. rubrum we demonstrate that iron binding results in assembly of EncFtn decamers, which display a unique annular architecture.	DISCUSS
+58	67	classical	protein_state	Despite a radically different quaternary structure to the classical ferritins, the four-helical bundle scaffold and FOC of EncFtnsH are strikingly similar to ferritin (Figure 6A).	DISCUSS
+68	77	ferritins	protein_type	Despite a radically different quaternary structure to the classical ferritins, the four-helical bundle scaffold and FOC of EncFtnsH are strikingly similar to ferritin (Figure 6A).	DISCUSS
+83	111	four-helical bundle scaffold	structure_element	Despite a radically different quaternary structure to the classical ferritins, the four-helical bundle scaffold and FOC of EncFtnsH are strikingly similar to ferritin (Figure 6A).	DISCUSS
+116	119	FOC	site	Despite a radically different quaternary structure to the classical ferritins, the four-helical bundle scaffold and FOC of EncFtnsH are strikingly similar to ferritin (Figure 6A).	DISCUSS
+123	131	EncFtnsH	protein	Despite a radically different quaternary structure to the classical ferritins, the four-helical bundle scaffold and FOC of EncFtnsH are strikingly similar to ferritin (Figure 6A).	DISCUSS
+158	166	ferritin	protein_type	Despite a radically different quaternary structure to the classical ferritins, the four-helical bundle scaffold and FOC of EncFtnsH are strikingly similar to ferritin (Figure 6A).	DISCUSS
+2	34	sequence-based phylogenetic tree	experimental_method	A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn).	DISCUSS
+55	63	ferritin	protein_type	A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn).	DISCUSS
+107	116	classical	protein_state	A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn).	DISCUSS
+117	126	ferritins	protein_type	A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn).	DISCUSS
+128	145	bacterioferritins	protein_type	A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn).	DISCUSS
+150	153	Dps	protein_type	A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn).	DISCUSS
+190	234	encapsulin-associated ferritin-like proteins	protein_type	A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn).	DISCUSS
+236	243	EncFtns	protein_type	A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn).	DISCUSS
+291	301	encapsulin	protein	A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn).	DISCUSS
+312	322	Non-EncFtn	protein_type	A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn).	DISCUSS
+31	37	EncFtn	protein	The analysis revealed that the EncFtn and Non-EncFtn proteins form groups distinct from the other clearly delineated groups of ferritins, and represent outliers in the tree (Figure 13).	DISCUSS
+42	52	Non-EncFtn	protein_type	The analysis revealed that the EncFtn and Non-EncFtn proteins form groups distinct from the other clearly delineated groups of ferritins, and represent outliers in the tree (Figure 13).	DISCUSS
+127	136	ferritins	protein_type	The analysis revealed that the EncFtn and Non-EncFtn proteins form groups distinct from the other clearly delineated groups of ferritins, and represent outliers in the tree (Figure 13).	DISCUSS
+98	118	active site scaffold	site	While it is difficult to infer ancestral lineages in protein families, the similarity seen in the active site scaffold of these proteins highlights a shared evolutionary relationship between EncFtn proteins and other members of the ferritin superfamily that has been noted in previous studies (; ).	DISCUSS
+191	197	EncFtn	protein_type	While it is difficult to infer ancestral lineages in protein families, the similarity seen in the active site scaffold of these proteins highlights a shared evolutionary relationship between EncFtn proteins and other members of the ferritin superfamily that has been noted in previous studies (; ).	DISCUSS
+232	240	ferritin	protein_type	While it is difficult to infer ancestral lineages in protein families, the similarity seen in the active site scaffold of these proteins highlights a shared evolutionary relationship between EncFtn proteins and other members of the ferritin superfamily that has been noted in previous studies (; ).	DISCUSS
+40	57	four-helical fold	structure_element	From this analysis, we propose that the four-helical fold of the classical ferritins may have arisen through gene duplication of an ancestor of EncFtn.	DISCUSS
+65	74	classical	protein_state	From this analysis, we propose that the four-helical fold of the classical ferritins may have arisen through gene duplication of an ancestor of EncFtn.	DISCUSS
+75	84	ferritins	protein_type	From this analysis, we propose that the four-helical fold of the classical ferritins may have arisen through gene duplication of an ancestor of EncFtn.	DISCUSS
+144	150	EncFtn	protein	From this analysis, we propose that the four-helical fold of the classical ferritins may have arisen through gene duplication of an ancestor of EncFtn.	DISCUSS
+42	59	C-terminal region	structure_element	This gene duplication would result in the C-terminal region of one EncFtn monomer being linked to the N-terminus of another and thus stabilizing the four-helix bundle fold within a single polypeptide chain (Figure 6B).	DISCUSS
+67	73	EncFtn	protein	This gene duplication would result in the C-terminal region of one EncFtn monomer being linked to the N-terminus of another and thus stabilizing the four-helix bundle fold within a single polypeptide chain (Figure 6B).	DISCUSS
+74	81	monomer	oligomeric_state	This gene duplication would result in the C-terminal region of one EncFtn monomer being linked to the N-terminus of another and thus stabilizing the four-helix bundle fold within a single polypeptide chain (Figure 6B).	DISCUSS
+149	171	four-helix bundle fold	structure_element	This gene duplication would result in the C-terminal region of one EncFtn monomer being linked to the N-terminus of another and thus stabilizing the four-helix bundle fold within a single polypeptide chain (Figure 6B).	DISCUSS
+102	105	FOC	site	Linking the protein together in this way relaxes the requirement for the maintenance of a symmetrical FOC and thus provides a path to the diversity in active-site residues seen across the ferritin family (Figure 6A, residues Glu95, Gln128 and Glu131 in PmFtn, Supplementary file 1).	DISCUSS
+151	171	active-site residues	site	Linking the protein together in this way relaxes the requirement for the maintenance of a symmetrical FOC and thus provides a path to the diversity in active-site residues seen across the ferritin family (Figure 6A, residues Glu95, Gln128 and Glu131 in PmFtn, Supplementary file 1).	DISCUSS
+188	196	ferritin	protein_type	Linking the protein together in this way relaxes the requirement for the maintenance of a symmetrical FOC and thus provides a path to the diversity in active-site residues seen across the ferritin family (Figure 6A, residues Glu95, Gln128 and Glu131 in PmFtn, Supplementary file 1).	DISCUSS
+225	230	Glu95	residue_name_number	Linking the protein together in this way relaxes the requirement for the maintenance of a symmetrical FOC and thus provides a path to the diversity in active-site residues seen across the ferritin family (Figure 6A, residues Glu95, Gln128 and Glu131 in PmFtn, Supplementary file 1).	DISCUSS
+232	238	Gln128	residue_name_number	Linking the protein together in this way relaxes the requirement for the maintenance of a symmetrical FOC and thus provides a path to the diversity in active-site residues seen across the ferritin family (Figure 6A, residues Glu95, Gln128 and Glu131 in PmFtn, Supplementary file 1).	DISCUSS
+243	249	Glu131	residue_name_number	Linking the protein together in this way relaxes the requirement for the maintenance of a symmetrical FOC and thus provides a path to the diversity in active-site residues seen across the ferritin family (Figure 6A, residues Glu95, Gln128 and Glu131 in PmFtn, Supplementary file 1).	DISCUSS
+253	258	PmFtn	protein	Linking the protein together in this way relaxes the requirement for the maintenance of a symmetrical FOC and thus provides a path to the diversity in active-site residues seen across the ferritin family (Figure 6A, residues Glu95, Gln128 and Glu131 in PmFtn, Supplementary file 1).	DISCUSS
+21	29	ferritin	protein_type	Relationship between ferritin structure and activity	DISCUSS
+30	39	structure	evidence	Relationship between ferritin structure and activity	DISCUSS
+30	39	classical	protein_state	The quaternary arrangement of classical ferritins into an octahedral nanocage and Dps into a dodecamer is absolutely required for their function as iron storage compartments.	DISCUSS
+40	49	ferritins	protein_type	The quaternary arrangement of classical ferritins into an octahedral nanocage and Dps into a dodecamer is absolutely required for their function as iron storage compartments.	DISCUSS
+58	68	octahedral	protein_state	The quaternary arrangement of classical ferritins into an octahedral nanocage and Dps into a dodecamer is absolutely required for their function as iron storage compartments.	DISCUSS
+69	77	nanocage	complex_assembly	The quaternary arrangement of classical ferritins into an octahedral nanocage and Dps into a dodecamer is absolutely required for their function as iron storage compartments.	DISCUSS
+82	85	Dps	protein	The quaternary arrangement of classical ferritins into an octahedral nanocage and Dps into a dodecamer is absolutely required for their function as iron storage compartments.	DISCUSS
+93	102	dodecamer	oligomeric_state	The quaternary arrangement of classical ferritins into an octahedral nanocage and Dps into a dodecamer is absolutely required for their function as iron storage compartments.	DISCUSS
+148	152	iron	chemical	The quaternary arrangement of classical ferritins into an octahedral nanocage and Dps into a dodecamer is absolutely required for their function as iron storage compartments.	DISCUSS
+36	40	iron	chemical	The oxidation and mineralization of iron must be spatially separated from the host cytosol to prevent the formation of damaging hydroxyl radicals in the Fenton and Haber-Weiss reactions.	DISCUSS
+25	34	ferritins	protein_type	 This is achieved in all ferritins by confining the oxidation of iron to the interior of the protein complex, thus achieving sequestration of the Fe3+ mineralization product.	DISCUSS
+65	69	iron	chemical	 This is achieved in all ferritins by confining the oxidation of iron to the interior of the protein complex, thus achieving sequestration of the Fe3+ mineralization product.	DISCUSS
+146	150	Fe3+	chemical	 This is achieved in all ferritins by confining the oxidation of iron to the interior of the protein complex, thus achieving sequestration of the Fe3+ mineralization product.	DISCUSS
+2	22	structural alignment	experimental_method	A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A).	DISCUSS
+30	33	FOC	site	A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A).	DISCUSS
+37	43	EncFtn	protein	A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A).	DISCUSS
+53	62	classical	protein_state	A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A).	DISCUSS
+63	71	ferritin	protein_type	A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A).	DISCUSS
+72	77	PmFtn	protein	A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A).	DISCUSS
+93	105	central ring	structure_element	A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A).	DISCUSS
+109	115	EncFtn	protein	A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A).	DISCUSS
+155	163	ferritin	protein_type	A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A).	DISCUSS
+198	204	EncFtn	protein	A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A).	DISCUSS
+233	255	mineralization surface	site	A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A).	DISCUSS
+259	267	ferritin	protein_type	A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A).	DISCUSS
+5	12	overlay	experimental_method	This overlay highlights the fact that the ferroxidase center of EncFtn faces in the opposite direction relative to the classical ferritins and is essentially inside out regarding iron storage space (Figure 6—figure supplement 1B, boxed region).	DISCUSS
+42	60	ferroxidase center	site	This overlay highlights the fact that the ferroxidase center of EncFtn faces in the opposite direction relative to the classical ferritins and is essentially inside out regarding iron storage space (Figure 6—figure supplement 1B, boxed region).	DISCUSS
+64	70	EncFtn	protein	This overlay highlights the fact that the ferroxidase center of EncFtn faces in the opposite direction relative to the classical ferritins and is essentially inside out regarding iron storage space (Figure 6—figure supplement 1B, boxed region).	DISCUSS
+119	128	classical	protein_state	This overlay highlights the fact that the ferroxidase center of EncFtn faces in the opposite direction relative to the classical ferritins and is essentially inside out regarding iron storage space (Figure 6—figure supplement 1B, boxed region).	DISCUSS
+129	138	ferritins	protein_type	This overlay highlights the fact that the ferroxidase center of EncFtn faces in the opposite direction relative to the classical ferritins and is essentially inside out regarding iron storage space (Figure 6—figure supplement 1B, boxed region).	DISCUSS
+179	183	iron	chemical	This overlay highlights the fact that the ferroxidase center of EncFtn faces in the opposite direction relative to the classical ferritins and is essentially inside out regarding iron storage space (Figure 6—figure supplement 1B, boxed region).	DISCUSS
+31	40	mutations	experimental_method	Analysis of each of the single mutations (E32A, E62A and H65A) made in the FOC highlights the importance of the iron-coordinating residues in the catalytic activity of EncFtn.	DISCUSS
+42	46	E32A	mutant	Analysis of each of the single mutations (E32A, E62A and H65A) made in the FOC highlights the importance of the iron-coordinating residues in the catalytic activity of EncFtn.	DISCUSS
+48	52	E62A	mutant	Analysis of each of the single mutations (E32A, E62A and H65A) made in the FOC highlights the importance of the iron-coordinating residues in the catalytic activity of EncFtn.	DISCUSS
+57	61	H65A	mutant	Analysis of each of the single mutations (E32A, E62A and H65A) made in the FOC highlights the importance of the iron-coordinating residues in the catalytic activity of EncFtn.	DISCUSS
+75	78	FOC	site	Analysis of each of the single mutations (E32A, E62A and H65A) made in the FOC highlights the importance of the iron-coordinating residues in the catalytic activity of EncFtn.	DISCUSS
+112	138	iron-coordinating residues	site	Analysis of each of the single mutations (E32A, E62A and H65A) made in the FOC highlights the importance of the iron-coordinating residues in the catalytic activity of EncFtn.	DISCUSS
+168	174	EncFtn	protein	Analysis of each of the single mutations (E32A, E62A and H65A) made in the FOC highlights the importance of the iron-coordinating residues in the catalytic activity of EncFtn.	DISCUSS
+33	40	calcium	chemical	Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC.	DISCUSS
+45	59	coordinated by	bond_interaction	Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC.	DISCUSS
+60	65	Glu31	residue_name_number	Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC.	DISCUSS
+70	75	Glu34	residue_name_number	Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC.	DISCUSS
+88	96	EncFtnsH	protein	Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC.	DISCUSS
+97	106	structure	evidence	Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC.	DISCUSS
+119	129	entry site	site	Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC.	DISCUSS
+161	164	FOC	site	Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC.	DISCUSS
+207	211	iron	chemical	Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC.	DISCUSS
+275	278	FOC	site	Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC.	DISCUSS
+68	74	EncFtn	protein	The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.	DISCUSS
+76	81	His57	residue_name_number	The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.	DISCUSS
+83	88	Glu61	residue_name_number	The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.	DISCUSS
+93	98	Glu64	residue_name_number	The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.	DISCUSS
+158	180	mineralization surface	site	The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.	DISCUSS
+192	201	classical	protein_state	The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.	DISCUSS
+202	210	ferritin	protein_type	The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.	DISCUSS
+211	219	nanocage	complex_assembly	The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.	DISCUSS
+254	257	FOC	site	The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.	DISCUSS
+281	292	exit portal	site	The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.	DISCUSS
+297	316	mineralization site	site	The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.	DISCUSS
+87	96	ferritins	protein_type	The absolute requirement for the spatial separation of oxidation and mineralization in ferritins suggests that the EncFtn family proteins are not capable of storing iron minerals due to the absence of an enclosed compartment in their structure (Figure 6—figure supplement 1B).	DISCUSS
+115	121	EncFtn	protein_type	The absolute requirement for the spatial separation of oxidation and mineralization in ferritins suggests that the EncFtn family proteins are not capable of storing iron minerals due to the absence of an enclosed compartment in their structure (Figure 6—figure supplement 1B).	DISCUSS
+165	169	iron	chemical	The absolute requirement for the spatial separation of oxidation and mineralization in ferritins suggests that the EncFtn family proteins are not capable of storing iron minerals due to the absence of an enclosed compartment in their structure (Figure 6—figure supplement 1B).	DISCUSS
+190	200	absence of	protein_state	The absolute requirement for the spatial separation of oxidation and mineralization in ferritins suggests that the EncFtn family proteins are not capable of storing iron minerals due to the absence of an enclosed compartment in their structure (Figure 6—figure supplement 1B).	DISCUSS
+4	32	biochemical characterization	experimental_method	Our biochemical characterization of EncFtn supports this hypothesis, indicating that while this protein is capable of oxidizing iron, it does not accrue mineralized iron in an analogous manner to classical ferritins.	DISCUSS
+36	42	EncFtn	protein	Our biochemical characterization of EncFtn supports this hypothesis, indicating that while this protein is capable of oxidizing iron, it does not accrue mineralized iron in an analogous manner to classical ferritins.	DISCUSS
+128	132	iron	chemical	Our biochemical characterization of EncFtn supports this hypothesis, indicating that while this protein is capable of oxidizing iron, it does not accrue mineralized iron in an analogous manner to classical ferritins.	DISCUSS
+165	169	iron	chemical	Our biochemical characterization of EncFtn supports this hypothesis, indicating that while this protein is capable of oxidizing iron, it does not accrue mineralized iron in an analogous manner to classical ferritins.	DISCUSS
+196	205	classical	protein_state	Our biochemical characterization of EncFtn supports this hypothesis, indicating that while this protein is capable of oxidizing iron, it does not accrue mineralized iron in an analogous manner to classical ferritins.	DISCUSS
+206	215	ferritins	protein_type	Our biochemical characterization of EncFtn supports this hypothesis, indicating that while this protein is capable of oxidizing iron, it does not accrue mineralized iron in an analogous manner to classical ferritins.	DISCUSS
+6	12	EncFtn	protein	While EncFtn does not store iron itself, its association with the encapsulin nanocage suggests that mineralization occurs within the cavity of the encapsulin shell.	DISCUSS
+28	32	iron	chemical	While EncFtn does not store iron itself, its association with the encapsulin nanocage suggests that mineralization occurs within the cavity of the encapsulin shell.	DISCUSS
+66	76	encapsulin	protein	While EncFtn does not store iron itself, its association with the encapsulin nanocage suggests that mineralization occurs within the cavity of the encapsulin shell.	DISCUSS
+77	85	nanocage	complex_assembly	While EncFtn does not store iron itself, its association with the encapsulin nanocage suggests that mineralization occurs within the cavity of the encapsulin shell.	DISCUSS
+133	139	cavity	site	While EncFtn does not store iron itself, its association with the encapsulin nanocage suggests that mineralization occurs within the cavity of the encapsulin shell.	DISCUSS
+147	157	encapsulin	protein	While EncFtn does not store iron itself, its association with the encapsulin nanocage suggests that mineralization occurs within the cavity of the encapsulin shell.	DISCUSS
+158	163	shell	structure_element	While EncFtn does not store iron itself, its association with the encapsulin nanocage suggests that mineralization occurs within the cavity of the encapsulin shell.	DISCUSS
+4	21	ferroxidase assay	experimental_method	Our ferroxidase assay data on the recombinant EncFtn-Enc nanocompartments, which accrue over 4100 iron ions per complex and form regular nanoparticles, are consistent with the encapsulin protein acting as the store for iron oxidized by the EncFtn enzyme.	DISCUSS
+46	56	EncFtn-Enc	complex_assembly	Our ferroxidase assay data on the recombinant EncFtn-Enc nanocompartments, which accrue over 4100 iron ions per complex and form regular nanoparticles, are consistent with the encapsulin protein acting as the store for iron oxidized by the EncFtn enzyme.	DISCUSS
+57	73	nanocompartments	complex_assembly	Our ferroxidase assay data on the recombinant EncFtn-Enc nanocompartments, which accrue over 4100 iron ions per complex and form regular nanoparticles, are consistent with the encapsulin protein acting as the store for iron oxidized by the EncFtn enzyme.	DISCUSS
+98	102	iron	chemical	Our ferroxidase assay data on the recombinant EncFtn-Enc nanocompartments, which accrue over 4100 iron ions per complex and form regular nanoparticles, are consistent with the encapsulin protein acting as the store for iron oxidized by the EncFtn enzyme.	DISCUSS
+137	150	nanoparticles	complex_assembly	Our ferroxidase assay data on the recombinant EncFtn-Enc nanocompartments, which accrue over 4100 iron ions per complex and form regular nanoparticles, are consistent with the encapsulin protein acting as the store for iron oxidized by the EncFtn enzyme.	DISCUSS
+176	186	encapsulin	protein	Our ferroxidase assay data on the recombinant EncFtn-Enc nanocompartments, which accrue over 4100 iron ions per complex and form regular nanoparticles, are consistent with the encapsulin protein acting as the store for iron oxidized by the EncFtn enzyme.	DISCUSS
+219	223	iron	chemical	Our ferroxidase assay data on the recombinant EncFtn-Enc nanocompartments, which accrue over 4100 iron ions per complex and form regular nanoparticles, are consistent with the encapsulin protein acting as the store for iron oxidized by the EncFtn enzyme.	DISCUSS
+240	246	EncFtn	protein	Our ferroxidase assay data on the recombinant EncFtn-Enc nanocompartments, which accrue over 4100 iron ions per complex and form regular nanoparticles, are consistent with the encapsulin protein acting as the store for iron oxidized by the EncFtn enzyme.	DISCUSS
+0	3	TEM	experimental_method	TEM analysis of the reaction products shows the production of homogeneous iron nanoparticles only in the EncFtn-Enc nanocompartment (Figure 8—figure supplement 1).	DISCUSS
+74	78	iron	chemical	TEM analysis of the reaction products shows the production of homogeneous iron nanoparticles only in the EncFtn-Enc nanocompartment (Figure 8—figure supplement 1).	DISCUSS
+105	115	EncFtn-Enc	complex_assembly	TEM analysis of the reaction products shows the production of homogeneous iron nanoparticles only in the EncFtn-Enc nanocompartment (Figure 8—figure supplement 1).	DISCUSS
+116	131	nanocompartment	complex_assembly	TEM analysis of the reaction products shows the production of homogeneous iron nanoparticles only in the EncFtn-Enc nanocompartment (Figure 8—figure supplement 1).	DISCUSS
+9	13	iron	chemical	Model of iron oxidation in encapsulin nanocompartments.	FIG
+27	37	encapsulin	protein	Model of iron oxidation in encapsulin nanocompartments.	FIG
+38	54	nanocompartments	complex_assembly	Model of iron oxidation in encapsulin nanocompartments.	FIG
+13	21	EncFtnsH	protein	(A) Model of EncFtnsH docking to the encapsulin shell.	FIG
+22	29	docking	experimental_method	(A) Model of EncFtnsH docking to the encapsulin shell.	FIG
+37	47	encapsulin	protein	(A) Model of EncFtnsH docking to the encapsulin shell.	FIG
+48	53	shell	structure_element	(A) Model of EncFtnsH docking to the encapsulin shell.	FIG
+9	17	pentamer	oligomeric_state	A single pentamer of the icosahedral T. maritima encapsulin structure (PDBID: 3DKT) is shown as a blue surface with the encapsulin localization sequence of EncFtn shown as a purple surface.	FIG
+25	36	icosahedral	protein_state	A single pentamer of the icosahedral T. maritima encapsulin structure (PDBID: 3DKT) is shown as a blue surface with the encapsulin localization sequence of EncFtn shown as a purple surface.	FIG
+37	48	T. maritima	species	A single pentamer of the icosahedral T. maritima encapsulin structure (PDBID: 3DKT) is shown as a blue surface with the encapsulin localization sequence of EncFtn shown as a purple surface.	FIG
+49	59	encapsulin	protein	A single pentamer of the icosahedral T. maritima encapsulin structure (PDBID: 3DKT) is shown as a blue surface with the encapsulin localization sequence of EncFtn shown as a purple surface.	FIG
+60	69	structure	evidence	A single pentamer of the icosahedral T. maritima encapsulin structure (PDBID: 3DKT) is shown as a blue surface with the encapsulin localization sequence of EncFtn shown as a purple surface.	FIG
+120	130	encapsulin	protein	A single pentamer of the icosahedral T. maritima encapsulin structure (PDBID: 3DKT) is shown as a blue surface with the encapsulin localization sequence of EncFtn shown as a purple surface.	FIG
+131	152	localization sequence	structure_element	A single pentamer of the icosahedral T. maritima encapsulin structure (PDBID: 3DKT) is shown as a blue surface with the encapsulin localization sequence of EncFtn shown as a purple surface.	FIG
+156	162	EncFtn	protein	A single pentamer of the icosahedral T. maritima encapsulin structure (PDBID: 3DKT) is shown as a blue surface with the encapsulin localization sequence of EncFtn shown as a purple surface.	FIG
+30	36	EncFtn	protein	The C-terminal regions of the EncFtn subunits correspond to the position of the localization sequences seen in 3DKT.	FIG
+37	45	subunits	structure_element	The C-terminal regions of the EncFtn subunits correspond to the position of the localization sequences seen in 3DKT.	FIG
+80	102	localization sequences	structure_element	The C-terminal regions of the EncFtn subunits correspond to the position of the localization sequences seen in 3DKT.	FIG
+0	9	Alignment	experimental_method	Alignment of EncFtnsH with 3DKT positions the central channel directly above the pore in the 3DKT pentamer axis (shown as a grey pentagon). (B) Surface view of EncFtn within the encapsulin nanocompartment (grey and blue respectively).	FIG
+13	21	EncFtnsH	protein	Alignment of EncFtnsH with 3DKT positions the central channel directly above the pore in the 3DKT pentamer axis (shown as a grey pentagon). (B) Surface view of EncFtn within the encapsulin nanocompartment (grey and blue respectively).	FIG
+46	61	central channel	site	Alignment of EncFtnsH with 3DKT positions the central channel directly above the pore in the 3DKT pentamer axis (shown as a grey pentagon). (B) Surface view of EncFtn within the encapsulin nanocompartment (grey and blue respectively).	FIG
+81	85	pore	site	Alignment of EncFtnsH with 3DKT positions the central channel directly above the pore in the 3DKT pentamer axis (shown as a grey pentagon). (B) Surface view of EncFtn within the encapsulin nanocompartment (grey and blue respectively).	FIG
+98	106	pentamer	oligomeric_state	Alignment of EncFtnsH with 3DKT positions the central channel directly above the pore in the 3DKT pentamer axis (shown as a grey pentagon). (B) Surface view of EncFtn within the encapsulin nanocompartment (grey and blue respectively).	FIG
+160	166	EncFtn	protein	Alignment of EncFtnsH with 3DKT positions the central channel directly above the pore in the 3DKT pentamer axis (shown as a grey pentagon). (B) Surface view of EncFtn within the encapsulin nanocompartment (grey and blue respectively).	FIG
+178	188	encapsulin	protein	Alignment of EncFtnsH with 3DKT positions the central channel directly above the pore in the 3DKT pentamer axis (shown as a grey pentagon). (B) Surface view of EncFtn within the encapsulin nanocompartment (grey and blue respectively).	FIG
+189	204	nanocompartment	complex_assembly	Alignment of EncFtnsH with 3DKT positions the central channel directly above the pore in the 3DKT pentamer axis (shown as a grey pentagon). (B) Surface view of EncFtn within the encapsulin nanocompartment (grey and blue respectively).	FIG
+17	27	encapsulin	protein	The lumen of the encapsulin nanocompartment is considerably larger than the interior of ferritin (shown in orange behind the encapsulin for reference) and thus allows the storage of significantly more iron.	FIG
+28	43	nanocompartment	complex_assembly	The lumen of the encapsulin nanocompartment is considerably larger than the interior of ferritin (shown in orange behind the encapsulin for reference) and thus allows the storage of significantly more iron.	FIG
+88	96	ferritin	protein_type	The lumen of the encapsulin nanocompartment is considerably larger than the interior of ferritin (shown in orange behind the encapsulin for reference) and thus allows the storage of significantly more iron.	FIG
+125	135	encapsulin	protein	The lumen of the encapsulin nanocompartment is considerably larger than the interior of ferritin (shown in orange behind the encapsulin for reference) and thus allows the storage of significantly more iron.	FIG
+201	205	iron	chemical	The lumen of the encapsulin nanocompartment is considerably larger than the interior of ferritin (shown in orange behind the encapsulin for reference) and thus allows the storage of significantly more iron.	FIG
+25	29	iron	chemical	The proposed pathway for iron movement through the encapsulin shell and EncFtn FOC is shown with arrows. (C) Model ofiron oxidation within an encapsulin nanocompartment.	FIG
+51	61	encapsulin	protein	The proposed pathway for iron movement through the encapsulin shell and EncFtn FOC is shown with arrows. (C) Model ofiron oxidation within an encapsulin nanocompartment.	FIG
+62	67	shell	structure_element	The proposed pathway for iron movement through the encapsulin shell and EncFtn FOC is shown with arrows. (C) Model ofiron oxidation within an encapsulin nanocompartment.	FIG
+72	78	EncFtn	protein	The proposed pathway for iron movement through the encapsulin shell and EncFtn FOC is shown with arrows. (C) Model ofiron oxidation within an encapsulin nanocompartment.	FIG
+79	82	FOC	site	The proposed pathway for iron movement through the encapsulin shell and EncFtn FOC is shown with arrows. (C) Model ofiron oxidation within an encapsulin nanocompartment.	FIG
+142	152	encapsulin	protein	The proposed pathway for iron movement through the encapsulin shell and EncFtn FOC is shown with arrows. (C) Model ofiron oxidation within an encapsulin nanocompartment.	FIG
+153	168	nanocompartment	complex_assembly	The proposed pathway for iron movement through the encapsulin shell and EncFtn FOC is shown with arrows. (C) Model ofiron oxidation within an encapsulin nanocompartment.	FIG
+3	9	EncFtn	protein	As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.	FIG
+34	38	iron	chemical	As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.	FIG
+64	68	Fe2+	chemical	As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.	FIG
+91	101	encapsulin	protein	As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.	FIG
+102	107	shell	structure_element	As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.	FIG
+128	146	metal binding site	site	As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.	FIG
+158	173	central channel	site	As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.	FIG
+177	185	EncFtnsH	protein	As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.	FIG
+187	197	entry site	site	As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.	FIG
+229	232	FOC	site	As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.	FIG
+248	252	Fe3+	chemical	As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.	FIG
+341	351	encapsulin	protein	As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.	FIG
+0	7	Docking	experimental_method	Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A).	DISCUSS
+12	19	decamer	oligomeric_state	Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A).	DISCUSS
+20	29	structure	evidence	Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A).	DISCUSS
+33	41	EncFtnsH	protein	Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A).	DISCUSS
+51	59	pentamer	oligomeric_state	Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A).	DISCUSS
+67	78	T. maritima	species	Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A).	DISCUSS
+79	89	encapsulin	protein	Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A).	DISCUSS
+90	100	Tmari_0786	gene	Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A).	DISCUSS
+148	169	C-terminal extensions	structure_element	Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A).	DISCUSS
+177	185	EncFtnsH	protein	Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A).	DISCUSS
+186	195	structure	evidence	Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A).	DISCUSS
+220	242	localization sequences	structure_element	Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A).	DISCUSS
+248	256	bound to	protein_state	Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A).	DISCUSS
+261	271	encapsulin	protein	Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A).	DISCUSS
+26	32	EncFtn	protein	Thus, it appears that the EncFtn decamer is the physiological state of this protein.	DISCUSS
+33	40	decamer	oligomeric_state	Thus, it appears that the EncFtn decamer is the physiological state of this protein.	DISCUSS
+31	43	central ring	structure_element	This arrangement positions the central ring of EncFtn directly above the pore at the five-fold symmetry axis of the encapsulin shell and highlights a potential route for the entry of iron into the encapsulin and towards the active site of EncFtn.	DISCUSS
+47	53	EncFtn	protein	This arrangement positions the central ring of EncFtn directly above the pore at the five-fold symmetry axis of the encapsulin shell and highlights a potential route for the entry of iron into the encapsulin and towards the active site of EncFtn.	DISCUSS
+73	77	pore	site	This arrangement positions the central ring of EncFtn directly above the pore at the five-fold symmetry axis of the encapsulin shell and highlights a potential route for the entry of iron into the encapsulin and towards the active site of EncFtn.	DISCUSS
+116	126	encapsulin	protein	This arrangement positions the central ring of EncFtn directly above the pore at the five-fold symmetry axis of the encapsulin shell and highlights a potential route for the entry of iron into the encapsulin and towards the active site of EncFtn.	DISCUSS
+127	132	shell	structure_element	This arrangement positions the central ring of EncFtn directly above the pore at the five-fold symmetry axis of the encapsulin shell and highlights a potential route for the entry of iron into the encapsulin and towards the active site of EncFtn.	DISCUSS
+183	187	iron	chemical	This arrangement positions the central ring of EncFtn directly above the pore at the five-fold symmetry axis of the encapsulin shell and highlights a potential route for the entry of iron into the encapsulin and towards the active site of EncFtn.	DISCUSS
+197	207	encapsulin	protein	This arrangement positions the central ring of EncFtn directly above the pore at the five-fold symmetry axis of the encapsulin shell and highlights a potential route for the entry of iron into the encapsulin and towards the active site of EncFtn.	DISCUSS
+224	235	active site	site	This arrangement positions the central ring of EncFtn directly above the pore at the five-fold symmetry axis of the encapsulin shell and highlights a potential route for the entry of iron into the encapsulin and towards the active site of EncFtn.	DISCUSS
+239	245	EncFtn	protein	This arrangement positions the central ring of EncFtn directly above the pore at the five-fold symmetry axis of the encapsulin shell and highlights a potential route for the entry of iron into the encapsulin and towards the active site of EncFtn.	DISCUSS
+20	30	encapsulin	protein	A comparison of the encapsulin nanocompartment and the ferritin nanocage highlights the size differential between the two complexes (Figure 14B) that allows the encapsulin to store significantly more iron.	DISCUSS
+31	46	nanocompartment	complex_assembly	A comparison of the encapsulin nanocompartment and the ferritin nanocage highlights the size differential between the two complexes (Figure 14B) that allows the encapsulin to store significantly more iron.	DISCUSS
+55	63	ferritin	protein_type	A comparison of the encapsulin nanocompartment and the ferritin nanocage highlights the size differential between the two complexes (Figure 14B) that allows the encapsulin to store significantly more iron.	DISCUSS
+64	72	nanocage	complex_assembly	A comparison of the encapsulin nanocompartment and the ferritin nanocage highlights the size differential between the two complexes (Figure 14B) that allows the encapsulin to store significantly more iron.	DISCUSS
+161	171	encapsulin	protein	A comparison of the encapsulin nanocompartment and the ferritin nanocage highlights the size differential between the two complexes (Figure 14B) that allows the encapsulin to store significantly more iron.	DISCUSS
+200	204	iron	chemical	A comparison of the encapsulin nanocompartment and the ferritin nanocage highlights the size differential between the two complexes (Figure 14B) that allows the encapsulin to store significantly more iron.	DISCUSS
+4	15	presence of	protein_state	The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment.	DISCUSS
+21	25	FOCs	site	The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment.	DISCUSS
+30	38	EncFtnsH	protein	The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment.	DISCUSS
+39	46	decamer	oligomeric_state	The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment.	DISCUSS
+69	80	icosahedral	protein_state	The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment.	DISCUSS
+81	91	encapsulin	protein	The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment.	DISCUSS
+92	100	nanocage	complex_assembly	The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment.	DISCUSS
+126	135	decameric	oligomeric_state	The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment.	DISCUSS
+136	142	EncFtn	protein	The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment.	DISCUSS
+234	238	iron	chemical	The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment.	DISCUSS
+272	287	nanocompartment	complex_assembly	The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment.	DISCUSS
+93	102	classical	protein_state	This arrangement of multiple reaction centers in a single protein assembly is reminiscent of classical ferritins, which has 24 FOCs distributed around the nanocage.	DISCUSS
+103	112	ferritins	protein_type	This arrangement of multiple reaction centers in a single protein assembly is reminiscent of classical ferritins, which has 24 FOCs distributed around the nanocage.	DISCUSS
+127	131	FOCs	site	This arrangement of multiple reaction centers in a single protein assembly is reminiscent of classical ferritins, which has 24 FOCs distributed around the nanocage.	DISCUSS
+155	163	nanocage	complex_assembly	This arrangement of multiple reaction centers in a single protein assembly is reminiscent of classical ferritins, which has 24 FOCs distributed around the nanocage.	DISCUSS
+4	19	structural data	evidence	Our structural data, coupled with biochemical and ICP-MS analysis, suggest a model for the activity of the encapsulin iron-megastore (Figure 14C).	DISCUSS
+34	56	biochemical and ICP-MS	experimental_method	Our structural data, coupled with biochemical and ICP-MS analysis, suggest a model for the activity of the encapsulin iron-megastore (Figure 14C).	DISCUSS
+107	117	encapsulin	protein	Our structural data, coupled with biochemical and ICP-MS analysis, suggest a model for the activity of the encapsulin iron-megastore (Figure 14C).	DISCUSS
+118	132	iron-megastore	complex_assembly	Our structural data, coupled with biochemical and ICP-MS analysis, suggest a model for the activity of the encapsulin iron-megastore (Figure 14C).	DISCUSS
+4	21	crystal structure	evidence	The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4—figure supplement 1).	DISCUSS
+29	40	T. maritima	species	The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4—figure supplement 1).	DISCUSS
+41	51	encapsulin	protein	The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4—figure supplement 1).	DISCUSS
+52	57	shell	structure_element	The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4—figure supplement 1).	DISCUSS
+72	95	negatively charged pore	site	The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4—figure supplement 1).	DISCUSS
+131	135	Fe2+	chemical	The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4—figure supplement 1).	DISCUSS
+145	155	encapsulin	protein	The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4—figure supplement 1).	DISCUSS
+199	222	negatively charged hole	site	The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4—figure supplement 1).	DISCUSS
+230	236	EncFtn	protein	The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4—figure supplement 1).	DISCUSS
+237	241	ring	structure_element	The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4—figure supplement 1).	DISCUSS
+9	28	metal-binding sites	site	The five metal-binding sites on the interior of the ring (Glu31/34-sites) may select for the Fe2+ ion and direct it towards their cognate FOCs.	DISCUSS
+52	56	ring	structure_element	The five metal-binding sites on the interior of the ring (Glu31/34-sites) may select for the Fe2+ ion and direct it towards their cognate FOCs.	DISCUSS
+58	72	Glu31/34-sites	site	The five metal-binding sites on the interior of the ring (Glu31/34-sites) may select for the Fe2+ ion and direct it towards their cognate FOCs.	DISCUSS
+93	97	Fe2+	chemical	The five metal-binding sites on the interior of the ring (Glu31/34-sites) may select for the Fe2+ ion and direct it towards their cognate FOCs.	DISCUSS
+138	142	FOCs	site	The five metal-binding sites on the interior of the ring (Glu31/34-sites) may select for the Fe2+ ion and direct it towards their cognate FOCs.	DISCUSS
+33	37	Fe2+	chemical	We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by  in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).	DISCUSS
+41	45	Fe3+	chemical	We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by  in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).	DISCUSS
+64	67	FOC	site	We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by  in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).	DISCUSS
+119	122	FOC	site	We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by  in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).	DISCUSS
+133	147	substrate site	site	We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by  in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).	DISCUSS
+162	166	iron	chemical	We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by  in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).	DISCUSS
+196	221	weakly coordinating sites	site	We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by  in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).	DISCUSS
+265	270	His57	residue_name_number	We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by  in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).	DISCUSS
+272	277	Glu61	residue_name_number	We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by  in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).	DISCUSS
+282	287	Glu64	residue_name_number	We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by  in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).	DISCUSS
+315	327	ferrihydrite	chemical	We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by  in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).	DISCUSS
+391	401	encapsulin	protein	We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by  in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).	DISCUSS
+402	417	nanocompartment	complex_assembly	We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by  in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).	DISCUSS
+40	49	structure	evidence	Here we describe for the first time the structure and biochemistry of a new class of encapsulin-associated ferritin-like protein and demonstrate that it has an absolute requirement for compartmentalization within an encapsulin nanocage to act as an iron store.	DISCUSS
+85	128	encapsulin-associated ferritin-like protein	protein_type	Here we describe for the first time the structure and biochemistry of a new class of encapsulin-associated ferritin-like protein and demonstrate that it has an absolute requirement for compartmentalization within an encapsulin nanocage to act as an iron store.	DISCUSS
+216	226	encapsulin	protein	Here we describe for the first time the structure and biochemistry of a new class of encapsulin-associated ferritin-like protein and demonstrate that it has an absolute requirement for compartmentalization within an encapsulin nanocage to act as an iron store.	DISCUSS
+227	235	nanocage	complex_assembly	Here we describe for the first time the structure and biochemistry of a new class of encapsulin-associated ferritin-like protein and demonstrate that it has an absolute requirement for compartmentalization within an encapsulin nanocage to act as an iron store.	DISCUSS
+249	253	iron	chemical	Here we describe for the first time the structure and biochemistry of a new class of encapsulin-associated ferritin-like protein and demonstrate that it has an absolute requirement for compartmentalization within an encapsulin nanocage to act as an iron store.	DISCUSS
+20	30	EncFtn-Enc	complex_assembly	Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment.	DISCUSS
+31	46	nanocompartment	complex_assembly	Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment.	DISCUSS
+103	107	iron	chemical	Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment.	DISCUSS
+120	130	encapsulin	protein	Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment.	DISCUSS
+131	136	shell	structure_element	Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment.	DISCUSS
+155	159	iron	chemical	Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment.	DISCUSS
+177	183	EncFtn	protein	Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment.	DISCUSS
+184	187	FOC	site	Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment.	DISCUSS
+235	245	encapsulin	protein	Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment.	DISCUSS
+246	261	nanocompartment	complex_assembly	Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment.	DISCUSS
+77	88	apoferritin	protein_state	TEM imaging was performed on purified encapsulin, EncFtn, and EncFtn-Enc and apoferritin.	METHODS
+129	140	apoferritin	protein_state	To observe iron mineral formation by TEM, protein samples at 8.5 µM concentration including EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were supplemented with acidic Fe(NH4)2(SO4)2 at their maximum iron loading ratio in room temperature for 1 hr.	METHODS
+13	24	apoferritin	protein_state	Horse spleen apoferritin preparation	METHODS
+13	24	apoferritin	protein_state	Horse spleen apoferritin purchased from Sigma Aldrich (UK) was dissolved in deaerated MOPS buffer (100 mM MOPS, 100 mM NaCl, 3 g/100 ml Na2S2O4 and 0.5 M EDTA, pH 6.5).	METHODS
+14	25	apoferritin	protein_state	Fe content of apoferritin was detected using ferrozine assay.	METHODS
+0	11	Apoferritin	protein_state	Apoferritin containing less than 0.5 Fe per 24-mer was used in the ferroxidase assay.	METHODS
+0	11	Apoferritin	protein_state	Apoferritin used in the Fe loading capacity experiment was prepared in the same way with 5–15 Fe per 24-mer.	METHODS
+137	148	apoferritin	protein_state	In order to determine the maximum iron loading capacity, around 8.5 µM proteins including decameric EncFtnsH, Encapsulin, EncFtn-Enc and apoferritin were loaded with various amount of acidic Fe(NH4)2(SO4)2 ranging from 0 to 1700 µM. Protein mixtures were incubated in room temperature for 3 hrs before desalting in Zebra spin desalting columns (7 kDa cut-off, Thermo Fisher Scientific, UK) to remove free iron ions.	METHODS
+180	190	absence of	protein_state	Both monomer and decamer fractions of EncFtnsH left at room temperature for 2 hrs, or overnight, were also analysed as controls to show the stability of the protein samples in the absence of additional metal ions.	METHODS
+49	64	nanocompartment	complex_assembly	Characterization of a Mycobacterium tuberculosis nanocompartment and its potential cargo proteins	REF
+20	35	nanocompartment	complex_assembly	A virus capsid-like nanocompartment that stores iron and protects bacteria from oxidative stress	REF
+48	63	nanocompartment	complex_assembly	Self-sorting of foreign proteins in a bacterial nanocompartment	REF
+58	73	nanocompartment	complex_assembly	Structural basis of enzyme encapsulation into a bacterial nanocompartment	REF
+256	262	oxygen	chemical	1) Methods: What procedures and analyses did the author use to assess whether the iron added to the various ferritin derivatives was protein coated or was simply balls of rust attached to protein fragments? If the latter, it could easily generate reactive oxygen species in air under physiological conditions.	REVIEW_INFO
+109	120	apoferritin	protein_state	Even an experimental situation: 24 subunit (monomer) ferritin with a biomineral prepared experimentally from apoferritin and containing, on average, only 1000 iron atoms/24 subunit cage, the equivalent parameter appears to be 1000/24 = 42.	REVIEW_INFO
+109	120	apoferritin	protein_state	Even an experimental situation: 24 subunit (monomer) ferritin with a biomineral prepared experimentally from apoferritin and containing, on average, only 1000 iron atoms/24 subunit cage, the equivalent parameter appears to be 1000/24 = 42.	REVIEW_INFO
+67	78	apoferritin	protein_state	Missing are data for the starting material, 24 subunit ferritin or apoferritin (ferritin with the iron removed, by reduction and chelation, as a control.)	REVIEW_INFO
+67	78	apoferritin	protein_state	Missing are data for the starting material, 24 subunit ferritin or apoferritin (ferritin with the iron removed, by reduction and chelation, as a control.)	REVIEW_INFO
+34	45	mutagenesis	experimental_method	4) I would have liked to see some mutagenesis experiments to test the models of assembly, iron binding and ferroxidase activity.	REVIEW_INFO
+107	118	ferroxidase	protein_type	4) I would have liked to see some mutagenesis experiments to test the models of assembly, iron binding and ferroxidase activity.	REVIEW_INFO
+44	55	apoferritin	protein_state	These results show the production of ROS by apoferritin, which is consistent with the published data on the reaction mechanism of certain ferritins; however, no significant ROS were detected for the EncFtn or encapsulin proteins.	REVIEW_INFO
+103	118	nanocompartment	complex_assembly	We have clarified this key difference in the discussion of the iron storage function of the encapsulin nanocompartment (subsection “Iron storage in encapsulin nanocompartments”, second paragraph).	REVIEW_INFO
+160	175	nanocompartment	complex_assembly	The key conclusion of the paper is that the iron storage and iron oxidation functions that are combined in classical ferritins are split between the encapsulin nanocompartment and the EncFtn protein.	REVIEW_INFO
+17	28	apoferritin	protein_state	Control data for apoferritin have been added to this table and are illustrated in Figure 8.	REVIEW_INFO
+75	86	apoferritin	protein_state	We note that we do not reach the experimental maximum loading capacity for apoferritin; however, we also note that the EncFtn-encapsulin nanocompartment sequesters five times more iron than the ferritin under the same reaction conditions, supporting the published observations that these nanocompartments can store more iron than classical ferritin nanocages.	REVIEW_INFO
+137	152	nanocompartment	complex_assembly	We note that we do not reach the experimental maximum loading capacity for apoferritin; however, we also note that the EncFtn-encapsulin nanocompartment sequesters five times more iron than the ferritin under the same reaction conditions, supporting the published observations that these nanocompartments can store more iron than classical ferritin nanocages.	REVIEW_INFO
diff --git a/annotation_CSV/PMC5014086.csv b/annotation_CSV/PMC5014086.csv
new file mode 100644
index 0000000000000000000000000000000000000000..0d8f4b191f7a5455fac5587e1c07504dc1af5a7d
--- /dev/null
+++ b/annotation_CSV/PMC5014086.csv
@@ -0,0 +1,677 @@
+anno_start	anno_end	anno_text	entity_type	sentence	section
+0	9	Structure	evidence	Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf	TITLE
+27	30	Wnt	protein_type	Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf	TITLE
+41	48	Kremen1	protein	Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf	TITLE
+97	101	LRP6	protein	Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf	TITLE
+106	114	Dickkopf	protein_type	Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf	TITLE
+0	14	Kremen 1 and 2	protein_type	Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling.	ABSTRACT
+39	51	co-receptors	protein_type	Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling.	ABSTRACT
+56	64	Dickkopf	protein_type	Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling.	ABSTRACT
+66	69	Dkk	protein_type	Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling.	ABSTRACT
+124	127	Wnt	protein_type	Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling.	ABSTRACT
+22	40	crystal structures	evidence	We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains.	ABSTRACT
+48	58	ectodomain	structure_element	We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains.	ABSTRACT
+62	67	human	species	We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains.	ABSTRACT
+68	75	Kremen1	protein	We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains.	ABSTRACT
+77	81	KRM1	protein	We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains.	ABSTRACT
+81	84	ECD	structure_element	We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains.	ABSTRACT
+124	128	KRM1	protein	We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains.	ABSTRACT
+128	131	ECD	structure_element	We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains.	ABSTRACT
+198	220	triangular arrangement	protein_state	We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains.	ABSTRACT
+228	235	Kringle	structure_element	We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains.	ABSTRACT
+237	240	WSC	structure_element	We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains.	ABSTRACT
+246	249	CUB	structure_element	We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains.	ABSTRACT
+4	14	structures	evidence	The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor.	ABSTRACT
+53	56	WSC	structure_element	The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor.	ABSTRACT
+67	91	hepatocyte growth factor	protein_type	The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor.	ABSTRACT
+80	83	Wnt	protein_type	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+84	95	co-receptor	protein_type	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+96	102	Lrp5/6	protein_type	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+104	107	Dkk	protein_type	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+113	116	Krm	protein_type	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+159	176	crystal structure	evidence	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+185	221	β-propeller/EGF repeats (PE) 3 and 4	structure_element	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+229	232	Wnt	protein_type	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+233	244	co-receptor	protein_type	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+245	249	LRP6	protein	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+251	255	LRP6	protein	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+255	261	PE3PE4	structure_element	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+268	290	cysteine-rich domain 2	structure_element	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+292	296	CRD2	structure_element	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+301	305	DKK1	protein	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+311	315	KRM1	protein	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+315	318	ECD	structure_element	We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.	ABSTRACT
+0	4	DKK1	protein	DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC.	ABSTRACT
+4	8	CRD2	structure_element	DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC.	ABSTRACT
+31	35	LRP6	protein	DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC.	ABSTRACT
+35	38	PE3	structure_element	DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC.	ABSTRACT
+43	47	KRM1	protein	DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC.	ABSTRACT
+47	58	Kringle-WSC	structure_element	DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC.	ABSTRACT
+0	8	Modeling	experimental_method	Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.	ABSTRACT
+30	55	surface plasmon resonance	experimental_method	Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.	ABSTRACT
+73	89	interaction site	site	Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.	ABSTRACT
+98	102	Krm1	protein	Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.	ABSTRACT
+102	105	CUB	structure_element	Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.	ABSTRACT
+110	114	Lrp6	protein	Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.	ABSTRACT
+114	117	PE2	structure_element	Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.	ABSTRACT
+4	13	structure	evidence	The structure of the KREMEN 1 ectodomain is solved from three crystal forms	ABSTRACT
+21	29	KREMEN 1	protein	The structure of the KREMEN 1 ectodomain is solved from three crystal forms	ABSTRACT
+30	40	ectodomain	structure_element	The structure of the KREMEN 1 ectodomain is solved from three crystal forms	ABSTRACT
+44	50	solved	experimental_method	The structure of the KREMEN 1 ectodomain is solved from three crystal forms	ABSTRACT
+62	75	crystal forms	evidence	The structure of the KREMEN 1 ectodomain is solved from three crystal forms	ABSTRACT
+0	7	Kringle	structure_element	Kringle, WSC, and CUB subdomains interact tightly to form a single structural unit	ABSTRACT
+9	12	WSC	structure_element	Kringle, WSC, and CUB subdomains interact tightly to form a single structural unit	ABSTRACT
+18	21	CUB	structure_element	Kringle, WSC, and CUB subdomains interact tightly to form a single structural unit	ABSTRACT
+4	13	interface	site	The interface to DKKs is formed from the Kringle and WSC domains	ABSTRACT
+17	21	DKKs	protein_type	The interface to DKKs is formed from the Kringle and WSC domains	ABSTRACT
+41	48	Kringle	structure_element	The interface to DKKs is formed from the Kringle and WSC domains	ABSTRACT
+53	56	WSC	structure_element	The interface to DKKs is formed from the Kringle and WSC domains	ABSTRACT
+4	7	CUB	structure_element	The CUB domain is found to interact directly with LRP6PE1PE2	ABSTRACT
+50	54	LRP6	protein	The CUB domain is found to interact directly with LRP6PE1PE2	ABSTRACT
+54	60	PE1PE2	structure_element	The CUB domain is found to interact directly with LRP6PE1PE2	ABSTRACT
+28	38	ectodomain	structure_element	Zebisch et al. describe the ectodomain structure of KREMEN 1, a receptor for Wnt antagonists of the DKK family.	ABSTRACT
+39	48	structure	evidence	Zebisch et al. describe the ectodomain structure of KREMEN 1, a receptor for Wnt antagonists of the DKK family.	ABSTRACT
+52	60	KREMEN 1	protein	Zebisch et al. describe the ectodomain structure of KREMEN 1, a receptor for Wnt antagonists of the DKK family.	ABSTRACT
+64	72	receptor	protein_type	Zebisch et al. describe the ectodomain structure of KREMEN 1, a receptor for Wnt antagonists of the DKK family.	ABSTRACT
+77	80	Wnt	protein_type	Zebisch et al. describe the ectodomain structure of KREMEN 1, a receptor for Wnt antagonists of the DKK family.	ABSTRACT
+100	103	DKK	protein_type	Zebisch et al. describe the ectodomain structure of KREMEN 1, a receptor for Wnt antagonists of the DKK family.	ABSTRACT
+0	3	Apo	protein_state	Apo structures and a complex with functional fragments of DKK1 and LRP6 shed light on the function of this dual-mode regulator of Wnt signaling.	ABSTRACT
+4	14	structures	evidence	Apo structures and a complex with functional fragments of DKK1 and LRP6 shed light on the function of this dual-mode regulator of Wnt signaling.	ABSTRACT
+21	33	complex with	protein_state	Apo structures and a complex with functional fragments of DKK1 and LRP6 shed light on the function of this dual-mode regulator of Wnt signaling.	ABSTRACT
+34	54	functional fragments	protein_state	Apo structures and a complex with functional fragments of DKK1 and LRP6 shed light on the function of this dual-mode regulator of Wnt signaling.	ABSTRACT
+58	62	DKK1	protein	Apo structures and a complex with functional fragments of DKK1 and LRP6 shed light on the function of this dual-mode regulator of Wnt signaling.	ABSTRACT
+67	71	LRP6	protein	Apo structures and a complex with functional fragments of DKK1 and LRP6 shed light on the function of this dual-mode regulator of Wnt signaling.	ABSTRACT
+130	133	Wnt	protein_type	Apo structures and a complex with functional fragments of DKK1 and LRP6 shed light on the function of this dual-mode regulator of Wnt signaling.	ABSTRACT
+13	16	Wnt	protein_type	Signaling by Wnt morphogens is renowned for its fundamental roles in embryonic development, tissue homeostasis, and stem cell maintenance.	INTRO
+68	71	Wnt	protein_type	Due to these functions, generation, delivery, and interpretation of Wnt signals are all heavily regulated in the animal body.	INTRO
+0	10	Vertebrate	taxonomy_domain	Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6.	INTRO
+11	19	Dickkopf	protein_type	Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6.	INTRO
+30	34	Dkk1	protein_type	Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6.	INTRO
+36	37	2	protein_type	Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6.	INTRO
+43	44	4	protein_type	Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6.	INTRO
+86	89	Wnt	protein_type	Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6.	INTRO
+129	132	Wnt	protein_type	Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6.	INTRO
+133	144	co-receptor	protein_type	Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6.	INTRO
+145	151	LRP5/6	protein	Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6.	INTRO
+0	6	Kremen	protein_type	Kremen proteins (Krm1 and Krm2) have been identified as additional high-affinity transmembrane receptors for Dkk.	INTRO
+17	21	Krm1	protein_type	Kremen proteins (Krm1 and Krm2) have been identified as additional high-affinity transmembrane receptors for Dkk.	INTRO
+26	30	Krm2	protein_type	Kremen proteins (Krm1 and Krm2) have been identified as additional high-affinity transmembrane receptors for Dkk.	INTRO
+81	104	transmembrane receptors	protein_type	Kremen proteins (Krm1 and Krm2) have been identified as additional high-affinity transmembrane receptors for Dkk.	INTRO
+109	112	Dkk	protein_type	Kremen proteins (Krm1 and Krm2) have been identified as additional high-affinity transmembrane receptors for Dkk.	INTRO
+0	3	Krm	protein_type	Krm and Dkk synergize in Wnt inhibition during Xenopus embryogenesis to regulate anterior-posterior patterning.	INTRO
+8	11	Dkk	protein_type	Krm and Dkk synergize in Wnt inhibition during Xenopus embryogenesis to regulate anterior-posterior patterning.	INTRO
+25	28	Wnt	protein_type	Krm and Dkk synergize in Wnt inhibition during Xenopus embryogenesis to regulate anterior-posterior patterning.	INTRO
+47	54	Xenopus	taxonomy_domain	Krm and Dkk synergize in Wnt inhibition during Xenopus embryogenesis to regulate anterior-posterior patterning.	INTRO
+43	54	presence of	protein_state	Mechanistically it is thought that, in the presence of Dkk, Krm forms a ternary complex with Lrp6, which is then rapidly endocytosed.	INTRO
+55	58	Dkk	protein_type	Mechanistically it is thought that, in the presence of Dkk, Krm forms a ternary complex with Lrp6, which is then rapidly endocytosed.	INTRO
+60	63	Krm	protein_type	Mechanistically it is thought that, in the presence of Dkk, Krm forms a ternary complex with Lrp6, which is then rapidly endocytosed.	INTRO
+80	92	complex with	protein_state	Mechanistically it is thought that, in the presence of Dkk, Krm forms a ternary complex with Lrp6, which is then rapidly endocytosed.	INTRO
+93	97	Lrp6	protein_type	Mechanistically it is thought that, in the presence of Dkk, Krm forms a ternary complex with Lrp6, which is then rapidly endocytosed.	INTRO
+29	32	Wnt	protein_type	This amplifies the intrinsic Wnt antagonistic activity of Dkk by efficiently depleting the cell surface of the Wnt co-receptor.	INTRO
+58	61	Dkk	protein_type	This amplifies the intrinsic Wnt antagonistic activity of Dkk by efficiently depleting the cell surface of the Wnt co-receptor.	INTRO
+111	114	Wnt	protein_type	This amplifies the intrinsic Wnt antagonistic activity of Dkk by efficiently depleting the cell surface of the Wnt co-receptor.	INTRO
+115	126	co-receptor	protein_type	This amplifies the intrinsic Wnt antagonistic activity of Dkk by efficiently depleting the cell surface of the Wnt co-receptor.	INTRO
+25	29	Krm1	protein_type	In accordance with this, Krm1−/− and Krm2−/− double knockout mice show a high bone mass phenotype typical of increased Wnt signaling, as well as growth of ectopic forelimb digits.	INTRO
+37	41	Krm2	protein_type	In accordance with this, Krm1−/− and Krm2−/− double knockout mice show a high bone mass phenotype typical of increased Wnt signaling, as well as growth of ectopic forelimb digits.	INTRO
+45	60	double knockout	experimental_method	In accordance with this, Krm1−/− and Krm2−/− double knockout mice show a high bone mass phenotype typical of increased Wnt signaling, as well as growth of ectopic forelimb digits.	INTRO
+61	65	mice	taxonomy_domain	In accordance with this, Krm1−/− and Krm2−/− double knockout mice show a high bone mass phenotype typical of increased Wnt signaling, as well as growth of ectopic forelimb digits.	INTRO
+119	122	Wnt	protein_type	In accordance with this, Krm1−/− and Krm2−/− double knockout mice show a high bone mass phenotype typical of increased Wnt signaling, as well as growth of ectopic forelimb digits.	INTRO
+69	72	dkk	protein_type	Growth of ectopic digits is further enhanced upon additional loss of dkk expression.	INTRO
+4	7	Wnt	protein_type	The Wnt antagonistic activity of Krm1 is also linked to its importance for correct thymus epithelium formation in mice.	INTRO
+33	37	Krm1	protein_type	The Wnt antagonistic activity of Krm1 is also linked to its importance for correct thymus epithelium formation in mice.	INTRO
+114	118	mice	taxonomy_domain	The Wnt antagonistic activity of Krm1 is also linked to its importance for correct thymus epithelium formation in mice.	INTRO
+18	24	intact	protein_state	The importance of intact KRM1 for normal human development and health is highlighted by the recent finding that a homozygous mutation in the ectodomain of KRM1 leads to severe ectodermal dysplasia including oligodontia.	INTRO
+25	29	KRM1	protein	The importance of intact KRM1 for normal human development and health is highlighted by the recent finding that a homozygous mutation in the ectodomain of KRM1 leads to severe ectodermal dysplasia including oligodontia.	INTRO
+41	46	human	species	The importance of intact KRM1 for normal human development and health is highlighted by the recent finding that a homozygous mutation in the ectodomain of KRM1 leads to severe ectodermal dysplasia including oligodontia.	INTRO
+141	151	ectodomain	structure_element	The importance of intact KRM1 for normal human development and health is highlighted by the recent finding that a homozygous mutation in the ectodomain of KRM1 leads to severe ectodermal dysplasia including oligodontia.	INTRO
+155	159	KRM1	protein	The importance of intact KRM1 for normal human development and health is highlighted by the recent finding that a homozygous mutation in the ectodomain of KRM1 leads to severe ectodermal dysplasia including oligodontia.	INTRO
+19	22	Wnt	protein_type	Interestingly, the Wnt antagonistic activity of Krm is context dependent, and Krm proteins are actually dual-mode Wnt regulators.	INTRO
+48	51	Krm	protein_type	Interestingly, the Wnt antagonistic activity of Krm is context dependent, and Krm proteins are actually dual-mode Wnt regulators.	INTRO
+78	81	Krm	protein_type	Interestingly, the Wnt antagonistic activity of Krm is context dependent, and Krm proteins are actually dual-mode Wnt regulators.	INTRO
+114	117	Wnt	protein_type	Interestingly, the Wnt antagonistic activity of Krm is context dependent, and Krm proteins are actually dual-mode Wnt regulators.	INTRO
+7	17	absence of	protein_state	In the absence of Dkk, Krm1 and 2 change their function from inhibition to enhancement of Lrp6-mediated signaling.	INTRO
+18	21	Dkk	protein_type	In the absence of Dkk, Krm1 and 2 change their function from inhibition to enhancement of Lrp6-mediated signaling.	INTRO
+23	27	Krm1	protein_type	In the absence of Dkk, Krm1 and 2 change their function from inhibition to enhancement of Lrp6-mediated signaling.	INTRO
+32	33	2	protein_type	In the absence of Dkk, Krm1 and 2 change their function from inhibition to enhancement of Lrp6-mediated signaling.	INTRO
+90	94	Lrp6	protein_type	In the absence of Dkk, Krm1 and 2 change their function from inhibition to enhancement of Lrp6-mediated signaling.	INTRO
+21	25	Lrp6	protein_type	By direct binding to Lrp6 via the ectodomains, Krm proteins promote Lrp6 cell-surface localization and hence increase receptor availability.	INTRO
+34	45	ectodomains	structure_element	By direct binding to Lrp6 via the ectodomains, Krm proteins promote Lrp6 cell-surface localization and hence increase receptor availability.	INTRO
+47	50	Krm	protein_type	By direct binding to Lrp6 via the ectodomains, Krm proteins promote Lrp6 cell-surface localization and hence increase receptor availability.	INTRO
+68	72	Lrp6	protein_type	By direct binding to Lrp6 via the ectodomains, Krm proteins promote Lrp6 cell-surface localization and hence increase receptor availability.	INTRO
+37	40	Krm	protein_type	Further increasing the complexity of Krm functionality, it was recently found that Krm1 (but not Krm2) can also act independently of LRP5/6 and Wnt as a dependence receptor, triggering apoptosis unless bound to Dkk.	INTRO
+83	87	Krm1	protein_type	Further increasing the complexity of Krm functionality, it was recently found that Krm1 (but not Krm2) can also act independently of LRP5/6 and Wnt as a dependence receptor, triggering apoptosis unless bound to Dkk.	INTRO
+97	101	Krm2	protein_type	Further increasing the complexity of Krm functionality, it was recently found that Krm1 (but not Krm2) can also act independently of LRP5/6 and Wnt as a dependence receptor, triggering apoptosis unless bound to Dkk.	INTRO
+133	139	LRP5/6	protein	Further increasing the complexity of Krm functionality, it was recently found that Krm1 (but not Krm2) can also act independently of LRP5/6 and Wnt as a dependence receptor, triggering apoptosis unless bound to Dkk.	INTRO
+144	147	Wnt	protein_type	Further increasing the complexity of Krm functionality, it was recently found that Krm1 (but not Krm2) can also act independently of LRP5/6 and Wnt as a dependence receptor, triggering apoptosis unless bound to Dkk.	INTRO
+202	210	bound to	protein_state	Further increasing the complexity of Krm functionality, it was recently found that Krm1 (but not Krm2) can also act independently of LRP5/6 and Wnt as a dependence receptor, triggering apoptosis unless bound to Dkk.	INTRO
+211	214	Dkk	protein_type	Further increasing the complexity of Krm functionality, it was recently found that Krm1 (but not Krm2) can also act independently of LRP5/6 and Wnt as a dependence receptor, triggering apoptosis unless bound to Dkk.	INTRO
+14	18	Krm1	protein_type	Structurally, Krm1 and 2 are type I transmembrane proteins with a 40 kDa ectodomain and a flexible cytoplasmic tail consisting of 60–75 residues.	INTRO
+23	24	2	protein_type	Structurally, Krm1 and 2 are type I transmembrane proteins with a 40 kDa ectodomain and a flexible cytoplasmic tail consisting of 60–75 residues.	INTRO
+29	58	type I transmembrane proteins	protein_type	Structurally, Krm1 and 2 are type I transmembrane proteins with a 40 kDa ectodomain and a flexible cytoplasmic tail consisting of 60–75 residues.	INTRO
+73	83	ectodomain	structure_element	Structurally, Krm1 and 2 are type I transmembrane proteins with a 40 kDa ectodomain and a flexible cytoplasmic tail consisting of 60–75 residues.	INTRO
+90	98	flexible	protein_state	Structurally, Krm1 and 2 are type I transmembrane proteins with a 40 kDa ectodomain and a flexible cytoplasmic tail consisting of 60–75 residues.	INTRO
+99	115	cytoplasmic tail	structure_element	Structurally, Krm1 and 2 are type I transmembrane proteins with a 40 kDa ectodomain and a flexible cytoplasmic tail consisting of 60–75 residues.	INTRO
+130	132	60	residue_range	Structurally, Krm1 and 2 are type I transmembrane proteins with a 40 kDa ectodomain and a flexible cytoplasmic tail consisting of 60–75 residues.	INTRO
+133	135	75	residue_range	Structurally, Krm1 and 2 are type I transmembrane proteins with a 40 kDa ectodomain and a flexible cytoplasmic tail consisting of 60–75 residues.	INTRO
+4	14	ectodomain	structure_element	The ectodomain consists of three similarly sized structural domains of around 10 kDa each: the N-terminal Kringle domain (KR) is followed by a WSC domain of unknown fold.	INTRO
+106	113	Kringle	structure_element	The ectodomain consists of three similarly sized structural domains of around 10 kDa each: the N-terminal Kringle domain (KR) is followed by a WSC domain of unknown fold.	INTRO
+122	124	KR	structure_element	The ectodomain consists of three similarly sized structural domains of around 10 kDa each: the N-terminal Kringle domain (KR) is followed by a WSC domain of unknown fold.	INTRO
+143	146	WSC	structure_element	The ectodomain consists of three similarly sized structural domains of around 10 kDa each: the N-terminal Kringle domain (KR) is followed by a WSC domain of unknown fold.	INTRO
+33	36	CUB	structure_element	The third structural domain is a CUB domain.	INTRO
+3	27	approximately 70-residue	residue_range	An approximately 70-residue linker connects the CUB domain to the transmembrane span.	INTRO
+28	34	linker	structure_element	An approximately 70-residue linker connects the CUB domain to the transmembrane span.	INTRO
+48	51	CUB	structure_element	An approximately 70-residue linker connects the CUB domain to the transmembrane span.	INTRO
+66	84	transmembrane span	structure_element	An approximately 70-residue linker connects the CUB domain to the transmembrane span.	INTRO
+3	9	intact	protein_state	An intact KR-WSC-CUB domain triplet and membrane attachment is required for Wnt antagonism.	INTRO
+10	20	KR-WSC-CUB	structure_element	An intact KR-WSC-CUB domain triplet and membrane attachment is required for Wnt antagonism.	INTRO
+76	79	Wnt	protein_type	An intact KR-WSC-CUB domain triplet and membrane attachment is required for Wnt antagonism.	INTRO
+4	22	transmembrane span	structure_element	The transmembrane span and cytoplasmic tail can be replaced with a GPI linker without impact on Wnt antagonism.	INTRO
+27	43	cytoplasmic tail	structure_element	The transmembrane span and cytoplasmic tail can be replaced with a GPI linker without impact on Wnt antagonism.	INTRO
+67	70	GPI	structure_element	The transmembrane span and cytoplasmic tail can be replaced with a GPI linker without impact on Wnt antagonism.	INTRO
+71	77	linker	structure_element	The transmembrane span and cytoplasmic tail can be replaced with a GPI linker without impact on Wnt antagonism.	INTRO
+96	99	Wnt	protein_type	The transmembrane span and cytoplasmic tail can be replaced with a GPI linker without impact on Wnt antagonism.	INTRO
+4	14	structures	evidence	The structures presented here reveal the unknown fold of the WSC domain and the tight interactions of all three domains.	INTRO
+61	64	WSC	structure_element	The structures presented here reveal the unknown fold of the WSC domain and the tight interactions of all three domains.	INTRO
+58	85	LRP6PE3PE4-DKK1CRD2-KRM1ECD	complex_assembly	We further succeeded in determination of a low-resolution LRP6PE3PE4-DKK1CRD2-KRM1ECD complex, defining the architecture of the Wnt inhibitory complex that leads to Lrp6 cell-surface depletion.	INTRO
+128	131	Wnt	protein_type	We further succeeded in determination of a low-resolution LRP6PE3PE4-DKK1CRD2-KRM1ECD complex, defining the architecture of the Wnt inhibitory complex that leads to Lrp6 cell-surface depletion.	INTRO
+132	150	inhibitory complex	complex_assembly	We further succeeded in determination of a low-resolution LRP6PE3PE4-DKK1CRD2-KRM1ECD complex, defining the architecture of the Wnt inhibitory complex that leads to Lrp6 cell-surface depletion.	INTRO
+165	169	Lrp6	protein	We further succeeded in determination of a low-resolution LRP6PE3PE4-DKK1CRD2-KRM1ECD complex, defining the architecture of the Wnt inhibitory complex that leads to Lrp6 cell-surface depletion.	INTRO
+34	54	extracellular domain	structure_element	The recombinant production of the extracellular domain of Krm for structural studies proved challenging (see Experimental Procedures).	RESULTS
+58	61	Krm	protein_type	The recombinant production of the extracellular domain of Krm for structural studies proved challenging (see Experimental Procedures).	RESULTS
+66	84	structural studies	experimental_method	The recombinant production of the extracellular domain of Krm for structural studies proved challenging (see Experimental Procedures).	RESULTS
+26	30	KRM1	protein	We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown).	RESULTS
+30	33	ECD	structure_element	We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown).	RESULTS
+34	48	complexes with	protein_state	We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown).	RESULTS
+49	55	DKK1fl	protein	We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown).	RESULTS
+57	61	DKK1	protein	We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown).	RESULTS
+61	72	Linker-CRD2	structure_element	We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown).	RESULTS
+78	82	DKK1	protein	We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown).	RESULTS
+82	86	CRD2	structure_element	We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown).	RESULTS
+124	138	gel filtration	experimental_method	We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown).	RESULTS
+8	21	crystal forms	evidence	Several crystal forms were obtained from these complexes, however, crystals always contained only KRM1 protein.	RESULTS
+67	75	crystals	evidence	Several crystal forms were obtained from these complexes, however, crystals always contained only KRM1 protein.	RESULTS
+98	102	KRM1	protein	Several crystal forms were obtained from these complexes, however, crystals always contained only KRM1 protein.	RESULTS
+3	9	solved	experimental_method	We solved the structure of KRM1ECD in three crystal forms at 1.9, 2.8, and 3.2 Å resolution (Table 1).	RESULTS
+14	23	structure	evidence	We solved the structure of KRM1ECD in three crystal forms at 1.9, 2.8, and 3.2 Å resolution (Table 1).	RESULTS
+27	31	KRM1	protein	We solved the structure of KRM1ECD in three crystal forms at 1.9, 2.8, and 3.2 Å resolution (Table 1).	RESULTS
+31	34	ECD	structure_element	We solved the structure of KRM1ECD in three crystal forms at 1.9, 2.8, and 3.2 Å resolution (Table 1).	RESULTS
+20	29	structure	evidence	The high-resolution structure is a near full-length model (Figure 1).	RESULTS
+40	51	full-length	protein_state	The high-resolution structure is a near full-length model (Figure 1).	RESULTS
+4	9	small	protein_state	The small, flexible, and charged 98AEHED102 loop could only be modeled in a slightly lower resolution structure and in crystal form III.	RESULTS
+11	19	flexible	protein_state	The small, flexible, and charged 98AEHED102 loop could only be modeled in a slightly lower resolution structure and in crystal form III.	RESULTS
+25	32	charged	protein_state	The small, flexible, and charged 98AEHED102 loop could only be modeled in a slightly lower resolution structure and in crystal form III.	RESULTS
+33	48	98AEHED102 loop	structure_element	The small, flexible, and charged 98AEHED102 loop could only be modeled in a slightly lower resolution structure and in crystal form III.	RESULTS
+102	111	structure	evidence	The small, flexible, and charged 98AEHED102 loop could only be modeled in a slightly lower resolution structure and in crystal form III.	RESULTS
+4	6	KR	structure_element	The KR, WSC, and CUB are arranged in a roughly triangular fashion with tight interactions between all three domains.	RESULTS
+8	11	WSC	structure_element	The KR, WSC, and CUB are arranged in a roughly triangular fashion with tight interactions between all three domains.	RESULTS
+17	20	CUB	structure_element	The KR, WSC, and CUB are arranged in a roughly triangular fashion with tight interactions between all three domains.	RESULTS
+4	6	KR	structure_element	The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements.	RESULTS
+43	62	glycosylation sites	site	The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements.	RESULTS
+93	110	disulfide bridges	ptm	The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements.	RESULTS
+112	115	C32	residue_name_number	The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements.	RESULTS
+116	120	C114	residue_name_number	The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements.	RESULTS
+122	125	C55	residue_name_number	The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements.	RESULTS
+126	129	C95	residue_name_number	The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements.	RESULTS
+131	134	C84	residue_name_number	The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements.	RESULTS
+135	139	C109	residue_name_number	The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements.	RESULTS
+157	164	Kringle	structure_element	The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements.	RESULTS
+30	37	Kringle	structure_element	The structurally most similar Kringle domain is that of human plasminogen (PDB: 1PKR) with an root-mean-square deviation (RMSD) of 1.7 Å for 73 aligned Cα (Figure 1B).	RESULTS
+56	61	human	species	The structurally most similar Kringle domain is that of human plasminogen (PDB: 1PKR) with an root-mean-square deviation (RMSD) of 1.7 Å for 73 aligned Cα (Figure 1B).	RESULTS
+62	73	plasminogen	protein	The structurally most similar Kringle domain is that of human plasminogen (PDB: 1PKR) with an root-mean-square deviation (RMSD) of 1.7 Å for 73 aligned Cα (Figure 1B).	RESULTS
+94	120	root-mean-square deviation	evidence	The structurally most similar Kringle domain is that of human plasminogen (PDB: 1PKR) with an root-mean-square deviation (RMSD) of 1.7 Å for 73 aligned Cα (Figure 1B).	RESULTS
+122	126	RMSD	evidence	The structurally most similar Kringle domain is that of human plasminogen (PDB: 1PKR) with an root-mean-square deviation (RMSD) of 1.7 Å for 73 aligned Cα (Figure 1B).	RESULTS
+4	8	KRM1	protein	The KRM1 structure reveals the fold of the WSC domain for the first time.	RESULTS
+9	18	structure	evidence	The KRM1 structure reveals the fold of the WSC domain for the first time.	RESULTS
+43	46	WSC	structure_element	The KRM1 structure reveals the fold of the WSC domain for the first time.	RESULTS
+4	13	structure	evidence	The structure is best described as a sandwich of a β1-β5-β3-β4-β2 antiparallel β sheet and a single α helix.	RESULTS
+37	45	sandwich	structure_element	The structure is best described as a sandwich of a β1-β5-β3-β4-β2 antiparallel β sheet and a single α helix.	RESULTS
+51	86	β1-β5-β3-β4-β2 antiparallel β sheet	structure_element	The structure is best described as a sandwich of a β1-β5-β3-β4-β2 antiparallel β sheet and a single α helix.	RESULTS
+100	107	α helix	structure_element	The structure is best described as a sandwich of a β1-β5-β3-β4-β2 antiparallel β sheet and a single α helix.	RESULTS
+4	13	structure	evidence	The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198).	RESULTS
+30	35	loops	structure_element	The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198).	RESULTS
+62	79	disulfide bridges	ptm	The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198).	RESULTS
+81	85	C122	residue_name_number	The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198).	RESULTS
+86	90	C186	residue_name_number	The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198).	RESULTS
+92	96	C147	residue_name_number	The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198).	RESULTS
+97	101	C167	residue_name_number	The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198).	RESULTS
+103	107	C151	residue_name_number	The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198).	RESULTS
+108	112	C169	residue_name_number	The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198).	RESULTS
+114	118	C190	residue_name_number	The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198).	RESULTS
+119	123	C198	residue_name_number	The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198).	RESULTS
+10	25	PDBeFold server	experimental_method	Using the PDBeFold server, we detected a surprising yet significant homology to PAN module domains.	RESULTS
+80	98	PAN module domains	structure_element	Using the PDBeFold server, we detected a surprising yet significant homology to PAN module domains.	RESULTS
+35	59	hepatocyte growth factor	protein_type	The closest structural relative is hepatocyte growth factor (HGF, PDB: 1GP9), which superposes with an RMSD of 2.3 Å for 58 aligned Cα (Figure 1B).	RESULTS
+61	64	HGF	protein_type	The closest structural relative is hepatocyte growth factor (HGF, PDB: 1GP9), which superposes with an RMSD of 2.3 Å for 58 aligned Cα (Figure 1B).	RESULTS
+84	94	superposes	experimental_method	The closest structural relative is hepatocyte growth factor (HGF, PDB: 1GP9), which superposes with an RMSD of 2.3 Å for 58 aligned Cα (Figure 1B).	RESULTS
+103	107	RMSD	evidence	The closest structural relative is hepatocyte growth factor (HGF, PDB: 1GP9), which superposes with an RMSD of 2.3 Å for 58 aligned Cα (Figure 1B).	RESULTS
+4	7	CUB	structure_element	The CUB domain bears two glycosylation sites.	RESULTS
+25	44	glycosylation sites	site	The CUB domain bears two glycosylation sites.	RESULTS
+37	53	electron density	evidence	Although present, the quality of the electron density around N217 did not allow modeling of the sugar moiety.	RESULTS
+61	65	N217	residue_name_number	Although present, the quality of the electron density around N217 did not allow modeling of the sugar moiety.	RESULTS
+3	17	crystal form I	evidence	In crystal form I, a calcium ion is present at the canonical position coordinated by the carboxylates of D263, D266 (bidentate), and D306, as well as the carbonyl of N309 and a water molecule.	RESULTS
+21	28	calcium	chemical	In crystal form I, a calcium ion is present at the canonical position coordinated by the carboxylates of D263, D266 (bidentate), and D306, as well as the carbonyl of N309 and a water molecule.	RESULTS
+70	84	coordinated by	bond_interaction	In crystal form I, a calcium ion is present at the canonical position coordinated by the carboxylates of D263, D266 (bidentate), and D306, as well as the carbonyl of N309 and a water molecule.	RESULTS
+105	109	D263	residue_name_number	In crystal form I, a calcium ion is present at the canonical position coordinated by the carboxylates of D263, D266 (bidentate), and D306, as well as the carbonyl of N309 and a water molecule.	RESULTS
+111	115	D266	residue_name_number	In crystal form I, a calcium ion is present at the canonical position coordinated by the carboxylates of D263, D266 (bidentate), and D306, as well as the carbonyl of N309 and a water molecule.	RESULTS
+133	137	D306	residue_name_number	In crystal form I, a calcium ion is present at the canonical position coordinated by the carboxylates of D263, D266 (bidentate), and D306, as well as the carbonyl of N309 and a water molecule.	RESULTS
+166	170	N309	residue_name_number	In crystal form I, a calcium ion is present at the canonical position coordinated by the carboxylates of D263, D266 (bidentate), and D306, as well as the carbonyl of N309 and a water molecule.	RESULTS
+177	182	water	chemical	In crystal form I, a calcium ion is present at the canonical position coordinated by the carboxylates of D263, D266 (bidentate), and D306, as well as the carbonyl of N309 and a water molecule.	RESULTS
+4	23	coordination sphere	site	The coordination sphere deviates significantly from perfectly octahedral (not shown).	RESULTS
+72	79	calcium	chemical	This might result in the site having a low affinity and may explain why calcium is not present in the two low-resolution crystal forms.	RESULTS
+121	134	crystal forms	evidence	This might result in the site having a low affinity and may explain why calcium is not present in the two low-resolution crystal forms.	RESULTS
+0	7	Loss of	protein_state	Loss of calcium has led to loop rearrangements and partial disorder in these crystal forms.	RESULTS
+8	15	calcium	chemical	Loss of calcium has led to loop rearrangements and partial disorder in these crystal forms.	RESULTS
+27	31	loop	structure_element	Loss of calcium has led to loop rearrangements and partial disorder in these crystal forms.	RESULTS
+77	90	crystal forms	evidence	Loss of calcium has led to loop rearrangements and partial disorder in these crystal forms.	RESULTS
+39	44	CUB_C	structure_element	The closest structural relative is the CUB_C domain of Tsg-6 (PDB: 2WNO), which superposes with KRMCUB with an RMSD of 1.6 Å for 104 Cα (Figure 1B).	RESULTS
+55	60	Tsg-6	protein	The closest structural relative is the CUB_C domain of Tsg-6 (PDB: 2WNO), which superposes with KRMCUB with an RMSD of 1.6 Å for 104 Cα (Figure 1B).	RESULTS
+80	90	superposes	experimental_method	The closest structural relative is the CUB_C domain of Tsg-6 (PDB: 2WNO), which superposes with KRMCUB with an RMSD of 1.6 Å for 104 Cα (Figure 1B).	RESULTS
+96	99	KRM	protein	The closest structural relative is the CUB_C domain of Tsg-6 (PDB: 2WNO), which superposes with KRMCUB with an RMSD of 1.6 Å for 104 Cα (Figure 1B).	RESULTS
+99	102	CUB	structure_element	The closest structural relative is the CUB_C domain of Tsg-6 (PDB: 2WNO), which superposes with KRMCUB with an RMSD of 1.6 Å for 104 Cα (Figure 1B).	RESULTS
+111	115	RMSD	evidence	The closest structural relative is the CUB_C domain of Tsg-6 (PDB: 2WNO), which superposes with KRMCUB with an RMSD of 1.6 Å for 104 Cα (Figure 1B).	RESULTS
+2	15	superposition	experimental_method	A superposition of the three KRM1 structures reveals no major structural differences (Figure 1C) as anticipated from the plethora of interactions between the three domains.	RESULTS
+29	33	KRM1	protein	A superposition of the three KRM1 structures reveals no major structural differences (Figure 1C) as anticipated from the plethora of interactions between the three domains.	RESULTS
+34	44	structures	evidence	A superposition of the three KRM1 structures reveals no major structural differences (Figure 1C) as anticipated from the plethora of interactions between the three domains.	RESULTS
+52	69	Ca2+ binding site	site	Minor differences are caused by the collapse of the Ca2+ binding site in crystal forms II and III and loop flexibility in the KR domain.	RESULTS
+73	97	crystal forms II and III	evidence	Minor differences are caused by the collapse of the Ca2+ binding site in crystal forms II and III and loop flexibility in the KR domain.	RESULTS
+102	106	loop	structure_element	Minor differences are caused by the collapse of the Ca2+ binding site in crystal forms II and III and loop flexibility in the KR domain.	RESULTS
+126	128	KR	structure_element	Minor differences are caused by the collapse of the Ca2+ binding site in crystal forms II and III and loop flexibility in the KR domain.	RESULTS
+4	9	F207S	mutant	The F207S mutation recently found to cause ectodermal dysplasia in Palestinian families maps to the hydrophobic core of the protein at the interface of the three subdomains (Figure 1A).	RESULTS
+100	116	hydrophobic core	site	The F207S mutation recently found to cause ectodermal dysplasia in Palestinian families maps to the hydrophobic core of the protein at the interface of the three subdomains (Figure 1A).	RESULTS
+139	148	interface	site	The F207S mutation recently found to cause ectodermal dysplasia in Palestinian families maps to the hydrophobic core of the protein at the interface of the three subdomains (Figure 1A).	RESULTS
+19	27	bound to	protein_state	Such a mutation is bound to severely destabilize the protein structure of KRM1, leading to disturbance of its Wnt antagonistic, Wnt stimulatory, and Wnt independent activity.	RESULTS
+74	78	KRM1	protein	Such a mutation is bound to severely destabilize the protein structure of KRM1, leading to disturbance of its Wnt antagonistic, Wnt stimulatory, and Wnt independent activity.	RESULTS
+110	113	Wnt	protein_type	Such a mutation is bound to severely destabilize the protein structure of KRM1, leading to disturbance of its Wnt antagonistic, Wnt stimulatory, and Wnt independent activity.	RESULTS
+128	131	Wnt	protein_type	Such a mutation is bound to severely destabilize the protein structure of KRM1, leading to disturbance of its Wnt antagonistic, Wnt stimulatory, and Wnt independent activity.	RESULTS
+149	152	Wnt	protein_type	Such a mutation is bound to severely destabilize the protein structure of KRM1, leading to disturbance of its Wnt antagonistic, Wnt stimulatory, and Wnt independent activity.	RESULTS
+0	18	Co-crystallization	experimental_method	Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands.	RESULTS
+22	26	LRP6	protein	Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands.	RESULTS
+26	32	PE3PE4	structure_element	Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands.	RESULTS
+38	42	DKK1	protein	Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands.	RESULTS
+42	46	CRD2	structure_element	Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands.	RESULTS
+52	56	LRP6	protein	Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands.	RESULTS
+56	59	PE1	structure_element	Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands.	RESULTS
+90	94	DKK1	protein	Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands.	RESULTS
+148	151	Wnt	protein_type	Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands.	RESULTS
+166	169	Dkk	protein_type	Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands.	RESULTS
+23	27	flat	protein_state	One face of the rather flat DKK1CRD2 fragment binds to the third β propeller of LRP6.	RESULTS
+28	32	DKK1	protein	One face of the rather flat DKK1CRD2 fragment binds to the third β propeller of LRP6.	RESULTS
+32	36	CRD2	structure_element	One face of the rather flat DKK1CRD2 fragment binds to the third β propeller of LRP6.	RESULTS
+46	54	binds to	protein_state	One face of the rather flat DKK1CRD2 fragment binds to the third β propeller of LRP6.	RESULTS
+59	76	third β propeller	structure_element	One face of the rather flat DKK1CRD2 fragment binds to the third β propeller of LRP6.	RESULTS
+80	84	LRP6	protein	One face of the rather flat DKK1CRD2 fragment binds to the third β propeller of LRP6.	RESULTS
+0	19	Mutational analyses	experimental_method	Mutational analyses further implied that the LRP6PE3-averted face of DKK1CRD2 bears the Krm binding site, hence suggesting how Dkk can recruit both receptors into a ternary complex.	RESULTS
+45	49	LRP6	protein	Mutational analyses further implied that the LRP6PE3-averted face of DKK1CRD2 bears the Krm binding site, hence suggesting how Dkk can recruit both receptors into a ternary complex.	RESULTS
+49	52	PE3	structure_element	Mutational analyses further implied that the LRP6PE3-averted face of DKK1CRD2 bears the Krm binding site, hence suggesting how Dkk can recruit both receptors into a ternary complex.	RESULTS
+69	73	DKK1	protein	Mutational analyses further implied that the LRP6PE3-averted face of DKK1CRD2 bears the Krm binding site, hence suggesting how Dkk can recruit both receptors into a ternary complex.	RESULTS
+73	77	CRD2	structure_element	Mutational analyses further implied that the LRP6PE3-averted face of DKK1CRD2 bears the Krm binding site, hence suggesting how Dkk can recruit both receptors into a ternary complex.	RESULTS
+88	104	Krm binding site	site	Mutational analyses further implied that the LRP6PE3-averted face of DKK1CRD2 bears the Krm binding site, hence suggesting how Dkk can recruit both receptors into a ternary complex.	RESULTS
+127	130	Dkk	protein_type	Mutational analyses further implied that the LRP6PE3-averted face of DKK1CRD2 bears the Krm binding site, hence suggesting how Dkk can recruit both receptors into a ternary complex.	RESULTS
+148	157	receptors	protein_type	Mutational analyses further implied that the LRP6PE3-averted face of DKK1CRD2 bears the Krm binding site, hence suggesting how Dkk can recruit both receptors into a ternary complex.	RESULTS
+59	65	Lrp5/6	protein_type	To obtain direct insight into ternary complex formation by Lrp5/6, Dkk, and Krm, we subjected an LRP6PE3PE4-DKK1fl-KRM1ECD complex to crystallization trials.	RESULTS
+67	70	Dkk	protein_type	To obtain direct insight into ternary complex formation by Lrp5/6, Dkk, and Krm, we subjected an LRP6PE3PE4-DKK1fl-KRM1ECD complex to crystallization trials.	RESULTS
+76	79	Krm	protein_type	To obtain direct insight into ternary complex formation by Lrp5/6, Dkk, and Krm, we subjected an LRP6PE3PE4-DKK1fl-KRM1ECD complex to crystallization trials.	RESULTS
+97	122	LRP6PE3PE4-DKK1fl-KRM1ECD	complex_assembly	To obtain direct insight into ternary complex formation by Lrp5/6, Dkk, and Krm, we subjected an LRP6PE3PE4-DKK1fl-KRM1ECD complex to crystallization trials.	RESULTS
+134	156	crystallization trials	experimental_method	To obtain direct insight into ternary complex formation by Lrp5/6, Dkk, and Krm, we subjected an LRP6PE3PE4-DKK1fl-KRM1ECD complex to crystallization trials.	RESULTS
+0	16	Diffraction data	evidence	Diffraction data collected from the resulting crystals were highly anisotropic with diffraction extending in the best directions to 3.5 Å and 3.7 Å but only to 6.4 Å in the third direction.	RESULTS
+46	54	crystals	evidence	Diffraction data collected from the resulting crystals were highly anisotropic with diffraction extending in the best directions to 3.5 Å and 3.7 Å but only to 6.4 Å in the third direction.	RESULTS
+36	47	diffraction	evidence	Despite the lack of high-resolution diffraction, the general architecture of the ternary complex is revealed (Figure 2A).	RESULTS
+0	4	DKK1	protein	DKK1CRD2 binds to the top face of the LRP6 PE3 β propeller as described earlier for the binary complex.	RESULTS
+4	8	CRD2	structure_element	DKK1CRD2 binds to the top face of the LRP6 PE3 β propeller as described earlier for the binary complex.	RESULTS
+9	17	binds to	protein_state	DKK1CRD2 binds to the top face of the LRP6 PE3 β propeller as described earlier for the binary complex.	RESULTS
+38	42	LRP6	protein	DKK1CRD2 binds to the top face of the LRP6 PE3 β propeller as described earlier for the binary complex.	RESULTS
+43	46	PE3	structure_element	DKK1CRD2 binds to the top face of the LRP6 PE3 β propeller as described earlier for the binary complex.	RESULTS
+47	58	β propeller	structure_element	DKK1CRD2 binds to the top face of the LRP6 PE3 β propeller as described earlier for the binary complex.	RESULTS
+0	4	KRM1	protein	KRM1ECD does indeed bind on the opposite side of DKK1CRD2 with only its KR and WSC domains engaged in binding (Figure 2A).	RESULTS
+4	7	ECD	structure_element	KRM1ECD does indeed bind on the opposite side of DKK1CRD2 with only its KR and WSC domains engaged in binding (Figure 2A).	RESULTS
+20	27	bind on	protein_state	KRM1ECD does indeed bind on the opposite side of DKK1CRD2 with only its KR and WSC domains engaged in binding (Figure 2A).	RESULTS
+49	53	DKK1	protein	KRM1ECD does indeed bind on the opposite side of DKK1CRD2 with only its KR and WSC domains engaged in binding (Figure 2A).	RESULTS
+53	57	CRD2	structure_element	KRM1ECD does indeed bind on the opposite side of DKK1CRD2 with only its KR and WSC domains engaged in binding (Figure 2A).	RESULTS
+72	74	KR	structure_element	KRM1ECD does indeed bind on the opposite side of DKK1CRD2 with only its KR and WSC domains engaged in binding (Figure 2A).	RESULTS
+79	82	WSC	structure_element	KRM1ECD does indeed bind on the opposite side of DKK1CRD2 with only its KR and WSC domains engaged in binding (Figure 2A).	RESULTS
+45	60	crystallization	experimental_method	Although present in the complex subjected to crystallization, we observe no density that could correspond to CRD1 or the domain linker (L).	RESULTS
+76	83	density	evidence	Although present in the complex subjected to crystallization, we observe no density that could correspond to CRD1 or the domain linker (L).	RESULTS
+109	113	CRD1	structure_element	Although present in the complex subjected to crystallization, we observe no density that could correspond to CRD1 or the domain linker (L).	RESULTS
+121	134	domain linker	structure_element	Although present in the complex subjected to crystallization, we observe no density that could correspond to CRD1 or the domain linker (L).	RESULTS
+136	137	L	structure_element	Although present in the complex subjected to crystallization, we observe no density that could correspond to CRD1 or the domain linker (L).	RESULTS
+20	24	CRD2	structure_element	We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure 2B).	RESULTS
+28	32	DKK1	protein	We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure 2B).	RESULTS
+75	79	KRM1	protein	We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure 2B).	RESULTS
+84	109	surface plasmon resonance	experimental_method	We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure 2B).	RESULTS
+111	114	SPR	experimental_method	We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure 2B).	RESULTS
+144	152	affinity	evidence	We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure 2B).	RESULTS
+161	172	full-length	protein_state	We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure 2B).	RESULTS
+173	177	DKK1	protein	We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure 2B).	RESULTS
+194	198	KRM1	protein	We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure 2B).	RESULTS
+198	201	ECD	structure_element	We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure 2B).	RESULTS
+2	13	SUMO fusion	experimental_method	A SUMO fusion of DKK1L-CRD2 displayed a similar (slightly higher) affinity.	RESULTS
+17	27	DKK1L-CRD2	structure_element	A SUMO fusion of DKK1L-CRD2 displayed a similar (slightly higher) affinity.	RESULTS
+66	74	affinity	evidence	A SUMO fusion of DKK1L-CRD2 displayed a similar (slightly higher) affinity.	RESULTS
+15	26	SUMO fusion	experimental_method	In contrast, a SUMO fusion of DKK1CRD1-L did not display binding for concentrations tested up to 325 μM (Figure 2B).	RESULTS
+30	40	DKK1CRD1-L	structure_element	In contrast, a SUMO fusion of DKK1CRD1-L did not display binding for concentrations tested up to 325 μM (Figure 2B).	RESULTS
+13	32	DKK1-KRM1 interface	site	Overall, the DKK1-KRM1 interface is characterized by a large number of polar interactions but only few hydrophobic contacts (Figure 2C).	RESULTS
+71	89	polar interactions	bond_interaction	Overall, the DKK1-KRM1 interface is characterized by a large number of polar interactions but only few hydrophobic contacts (Figure 2C).	RESULTS
+103	123	hydrophobic contacts	bond_interaction	Overall, the DKK1-KRM1 interface is characterized by a large number of polar interactions but only few hydrophobic contacts (Figure 2C).	RESULTS
+4	21	crystal structure	evidence	The crystal structure gives an explanation for DKK1 loss-of-binding mutations identified previously: R191 of DKK1 forms a double salt bridge to D125 and E162 of KRM1 (Figure 2C).	RESULTS
+47	51	DKK1	protein	The crystal structure gives an explanation for DKK1 loss-of-binding mutations identified previously: R191 of DKK1 forms a double salt bridge to D125 and E162 of KRM1 (Figure 2C).	RESULTS
+101	105	R191	residue_name_number	The crystal structure gives an explanation for DKK1 loss-of-binding mutations identified previously: R191 of DKK1 forms a double salt bridge to D125 and E162 of KRM1 (Figure 2C).	RESULTS
+109	113	DKK1	protein	The crystal structure gives an explanation for DKK1 loss-of-binding mutations identified previously: R191 of DKK1 forms a double salt bridge to D125 and E162 of KRM1 (Figure 2C).	RESULTS
+129	140	salt bridge	bond_interaction	The crystal structure gives an explanation for DKK1 loss-of-binding mutations identified previously: R191 of DKK1 forms a double salt bridge to D125 and E162 of KRM1 (Figure 2C).	RESULTS
+144	148	D125	residue_name_number	The crystal structure gives an explanation for DKK1 loss-of-binding mutations identified previously: R191 of DKK1 forms a double salt bridge to D125 and E162 of KRM1 (Figure 2C).	RESULTS
+153	157	E162	residue_name_number	The crystal structure gives an explanation for DKK1 loss-of-binding mutations identified previously: R191 of DKK1 forms a double salt bridge to D125 and E162 of KRM1 (Figure 2C).	RESULTS
+161	165	KRM1	protein	The crystal structure gives an explanation for DKK1 loss-of-binding mutations identified previously: R191 of DKK1 forms a double salt bridge to D125 and E162 of KRM1 (Figure 2C).	RESULTS
+2	17	charge reversal	experimental_method	A charge reversal as in the mouse Dkk1 (mDkk1) R197E variant would severely disrupt the binding.	RESULTS
+28	33	mouse	taxonomy_domain	A charge reversal as in the mouse Dkk1 (mDkk1) R197E variant would severely disrupt the binding.	RESULTS
+34	38	Dkk1	protein	A charge reversal as in the mouse Dkk1 (mDkk1) R197E variant would severely disrupt the binding.	RESULTS
+40	45	mDkk1	protein	A charge reversal as in the mouse Dkk1 (mDkk1) R197E variant would severely disrupt the binding.	RESULTS
+47	52	R197E	mutant	A charge reversal as in the mouse Dkk1 (mDkk1) R197E variant would severely disrupt the binding.	RESULTS
+15	19	K226	residue_name_number	Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90.	RESULTS
+34	38	DKK1	protein	Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90.	RESULTS
+64	82	hydrophobic pocket	site	Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90.	RESULTS
+101	105	KRM1	protein	Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90.	RESULTS
+116	120	Y108	residue_name_number	Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90.	RESULTS
+122	125	W94	residue_name_number	Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90.	RESULTS
+131	135	W106	residue_name_number	Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90.	RESULTS
+143	155	salt bridges	bond_interaction	Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90.	RESULTS
+180	184	KRM1	protein	Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90.	RESULTS
+185	188	D88	residue_name_number	Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90.	RESULTS
+193	196	D90	residue_name_number	Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90.	RESULTS
+9	24	charge reversal	experimental_method	Again, a charge reversal as shown before for mDkk1 K232E would be incompatible with binding.	RESULTS
+45	50	mDkk1	protein	Again, a charge reversal as shown before for mDkk1 K232E would be incompatible with binding.	RESULTS
+51	56	K232E	mutant	Again, a charge reversal as shown before for mDkk1 K232E would be incompatible with binding.	RESULTS
+18	22	DKK1	protein	The side chain of DKK1 S192 was also predicted to be involved in Krm binding.	RESULTS
+23	27	S192	residue_name_number	The side chain of DKK1 S192 was also predicted to be involved in Krm binding.	RESULTS
+65	68	Krm	protein_type	The side chain of DKK1 S192 was also predicted to be involved in Krm binding.	RESULTS
+52	56	D201	residue_name_number	Indeed, we found (Figure 2C) that the side chain of D201 of KRM1 forms two hydrogen bonds to the side-chain hydroxyl and the backbone amide of S192 (mouse, S198).	RESULTS
+60	64	KRM1	protein	Indeed, we found (Figure 2C) that the side chain of D201 of KRM1 forms two hydrogen bonds to the side-chain hydroxyl and the backbone amide of S192 (mouse, S198).	RESULTS
+75	89	hydrogen bonds	bond_interaction	Indeed, we found (Figure 2C) that the side chain of D201 of KRM1 forms two hydrogen bonds to the side-chain hydroxyl and the backbone amide of S192 (mouse, S198).	RESULTS
+143	147	S192	residue_name_number	Indeed, we found (Figure 2C) that the side chain of D201 of KRM1 forms two hydrogen bonds to the side-chain hydroxyl and the backbone amide of S192 (mouse, S198).	RESULTS
+149	154	mouse	taxonomy_domain	Indeed, we found (Figure 2C) that the side chain of D201 of KRM1 forms two hydrogen bonds to the side-chain hydroxyl and the backbone amide of S192 (mouse, S198).	RESULTS
+156	160	S198	residue_name_number	Indeed, we found (Figure 2C) that the side chain of D201 of KRM1 forms two hydrogen bonds to the side-chain hydroxyl and the backbone amide of S192 (mouse, S198).	RESULTS
+11	29	polar interactions	bond_interaction	Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively.	RESULTS
+53	57	N140	residue_name_number	Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively.	RESULTS
+59	63	S163	residue_name_number	Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively.	RESULTS
+69	73	Y165	residue_name_number	Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively.	RESULTS
+89	93	KRM1	protein	Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively.	RESULTS
+98	102	DKK1	protein	Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively.	RESULTS
+125	129	W206	residue_name_number	Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively.	RESULTS
+131	135	L190	residue_name_number	Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively.	RESULTS
+141	145	C189	residue_name_number	Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively.	RESULTS
+16	20	DKK1	protein	The carbonyl of DKK1 R224 is hydrogen bonded to Y105 and W106 of KRM1.	RESULTS
+21	25	R224	residue_name_number	The carbonyl of DKK1 R224 is hydrogen bonded to Y105 and W106 of KRM1.	RESULTS
+29	44	hydrogen bonded	bond_interaction	The carbonyl of DKK1 R224 is hydrogen bonded to Y105 and W106 of KRM1.	RESULTS
+48	52	Y105	residue_name_number	The carbonyl of DKK1 R224 is hydrogen bonded to Y105 and W106 of KRM1.	RESULTS
+57	61	W106	residue_name_number	The carbonyl of DKK1 R224 is hydrogen bonded to Y105 and W106 of KRM1.	RESULTS
+65	69	KRM1	protein	The carbonyl of DKK1 R224 is hydrogen bonded to Y105 and W106 of KRM1.	RESULTS
+20	23	Dkk	protein_type	We suspect that the Dkk charge reversal mutations performed in the murine background and shown to diminish Krm binding K211E and R203E (mouse K217E and R209E) do so likely indirectly by disruption of the Dkk fold.	RESULTS
+24	49	charge reversal mutations	experimental_method	We suspect that the Dkk charge reversal mutations performed in the murine background and shown to diminish Krm binding K211E and R203E (mouse K217E and R209E) do so likely indirectly by disruption of the Dkk fold.	RESULTS
+67	73	murine	taxonomy_domain	We suspect that the Dkk charge reversal mutations performed in the murine background and shown to diminish Krm binding K211E and R203E (mouse K217E and R209E) do so likely indirectly by disruption of the Dkk fold.	RESULTS
+107	110	Krm	protein_type	We suspect that the Dkk charge reversal mutations performed in the murine background and shown to diminish Krm binding K211E and R203E (mouse K217E and R209E) do so likely indirectly by disruption of the Dkk fold.	RESULTS
+119	124	K211E	mutant	We suspect that the Dkk charge reversal mutations performed in the murine background and shown to diminish Krm binding K211E and R203E (mouse K217E and R209E) do so likely indirectly by disruption of the Dkk fold.	RESULTS
+129	134	R203E	mutant	We suspect that the Dkk charge reversal mutations performed in the murine background and shown to diminish Krm binding K211E and R203E (mouse K217E and R209E) do so likely indirectly by disruption of the Dkk fold.	RESULTS
+136	141	mouse	taxonomy_domain	We suspect that the Dkk charge reversal mutations performed in the murine background and shown to diminish Krm binding K211E and R203E (mouse K217E and R209E) do so likely indirectly by disruption of the Dkk fold.	RESULTS
+142	147	K217E	mutant	We suspect that the Dkk charge reversal mutations performed in the murine background and shown to diminish Krm binding K211E and R203E (mouse K217E and R209E) do so likely indirectly by disruption of the Dkk fold.	RESULTS
+152	157	R209E	mutant	We suspect that the Dkk charge reversal mutations performed in the murine background and shown to diminish Krm binding K211E and R203E (mouse K217E and R209E) do so likely indirectly by disruption of the Dkk fold.	RESULTS
+204	207	Dkk	protein_type	We suspect that the Dkk charge reversal mutations performed in the murine background and shown to diminish Krm binding K211E and R203E (mouse K217E and R209E) do so likely indirectly by disruption of the Dkk fold.	RESULTS
+25	42	DKK1 binding site	site	We further validated the DKK1 binding site on KRM1 by introducing glycosylation sites at the KR (90DVS92→NVS) and WSC (189VCF191→NCS) domains pointing toward DKK (Figures 2A and 2D).	RESULTS
+46	50	KRM1	protein	We further validated the DKK1 binding site on KRM1 by introducing glycosylation sites at the KR (90DVS92→NVS) and WSC (189VCF191→NCS) domains pointing toward DKK (Figures 2A and 2D).	RESULTS
+54	65	introducing	experimental_method	We further validated the DKK1 binding site on KRM1 by introducing glycosylation sites at the KR (90DVS92→NVS) and WSC (189VCF191→NCS) domains pointing toward DKK (Figures 2A and 2D).	RESULTS
+66	85	glycosylation sites	site	We further validated the DKK1 binding site on KRM1 by introducing glycosylation sites at the KR (90DVS92→NVS) and WSC (189VCF191→NCS) domains pointing toward DKK (Figures 2A and 2D).	RESULTS
+93	95	KR	structure_element	We further validated the DKK1 binding site on KRM1 by introducing glycosylation sites at the KR (90DVS92→NVS) and WSC (189VCF191→NCS) domains pointing toward DKK (Figures 2A and 2D).	RESULTS
+97	108	90DVS92→NVS	mutant	We further validated the DKK1 binding site on KRM1 by introducing glycosylation sites at the KR (90DVS92→NVS) and WSC (189VCF191→NCS) domains pointing toward DKK (Figures 2A and 2D).	RESULTS
+114	117	WSC	structure_element	We further validated the DKK1 binding site on KRM1 by introducing glycosylation sites at the KR (90DVS92→NVS) and WSC (189VCF191→NCS) domains pointing toward DKK (Figures 2A and 2D).	RESULTS
+119	132	189VCF191→NCS	mutant	We further validated the DKK1 binding site on KRM1 by introducing glycosylation sites at the KR (90DVS92→NVS) and WSC (189VCF191→NCS) domains pointing toward DKK (Figures 2A and 2D).	RESULTS
+158	161	DKK	protein	We further validated the DKK1 binding site on KRM1 by introducing glycosylation sites at the KR (90DVS92→NVS) and WSC (189VCF191→NCS) domains pointing toward DKK (Figures 2A and 2D).	RESULTS
+16	32	N-linked glycans	ptm	Introduction of N-linked glycans in protein-protein-binding sites is an established way of disrupting protein-binding interfaces.	RESULTS
+36	65	protein-protein-binding sites	site	Introduction of N-linked glycans in protein-protein-binding sites is an established way of disrupting protein-binding interfaces.	RESULTS
+102	128	protein-binding interfaces	site	Introduction of N-linked glycans in protein-protein-binding sites is an established way of disrupting protein-binding interfaces.	RESULTS
+5	15	ectodomain	structure_element	Both ectodomain mutants were secreted comparably with the wild-type, indicating correct folding, but failed to achieve any detectable binding in SPR using full-length DKK1 as analyte.	RESULTS
+16	23	mutants	protein_state	Both ectodomain mutants were secreted comparably with the wild-type, indicating correct folding, but failed to achieve any detectable binding in SPR using full-length DKK1 as analyte.	RESULTS
+58	67	wild-type	protein_state	Both ectodomain mutants were secreted comparably with the wild-type, indicating correct folding, but failed to achieve any detectable binding in SPR using full-length DKK1 as analyte.	RESULTS
+145	148	SPR	experimental_method	Both ectodomain mutants were secreted comparably with the wild-type, indicating correct folding, but failed to achieve any detectable binding in SPR using full-length DKK1 as analyte.	RESULTS
+155	166	full-length	protein_state	Both ectodomain mutants were secreted comparably with the wild-type, indicating correct folding, but failed to achieve any detectable binding in SPR using full-length DKK1 as analyte.	RESULTS
+167	171	DKK1	protein	Both ectodomain mutants were secreted comparably with the wild-type, indicating correct folding, but failed to achieve any detectable binding in SPR using full-length DKK1 as analyte.	RESULTS
+15	21	mutant	protein_state	In contrast, a mutant carrying an additional N-glycan outside the interface at the CUB domain (309NQA311→NQS), was wild-type-like in DKK1 binding (Figure 2D).	RESULTS
+45	53	N-glycan	ptm	In contrast, a mutant carrying an additional N-glycan outside the interface at the CUB domain (309NQA311→NQS), was wild-type-like in DKK1 binding (Figure 2D).	RESULTS
+66	75	interface	site	In contrast, a mutant carrying an additional N-glycan outside the interface at the CUB domain (309NQA311→NQS), was wild-type-like in DKK1 binding (Figure 2D).	RESULTS
+83	86	CUB	structure_element	In contrast, a mutant carrying an additional N-glycan outside the interface at the CUB domain (309NQA311→NQS), was wild-type-like in DKK1 binding (Figure 2D).	RESULTS
+95	108	309NQA311→NQS	mutant	In contrast, a mutant carrying an additional N-glycan outside the interface at the CUB domain (309NQA311→NQS), was wild-type-like in DKK1 binding (Figure 2D).	RESULTS
+115	124	wild-type	protein_state	In contrast, a mutant carrying an additional N-glycan outside the interface at the CUB domain (309NQA311→NQS), was wild-type-like in DKK1 binding (Figure 2D).	RESULTS
+133	137	DKK1	protein	In contrast, a mutant carrying an additional N-glycan outside the interface at the CUB domain (309NQA311→NQS), was wild-type-like in DKK1 binding (Figure 2D).	RESULTS
+27	49	LRP6-KRM1 Binding Site	site	Identification of a Direct LRP6-KRM1 Binding Site	RESULTS
+4	31	LRP6PE3PE4-DKK1CRD2-KRM1ECD	complex_assembly	The LRP6PE3PE4-DKK1CRD2-KRM1ECD complex structure reveals no direct interactions between KRM1 and LRP6.	RESULTS
+40	49	structure	evidence	The LRP6PE3PE4-DKK1CRD2-KRM1ECD complex structure reveals no direct interactions between KRM1 and LRP6.	RESULTS
+89	93	KRM1	protein	The LRP6PE3PE4-DKK1CRD2-KRM1ECD complex structure reveals no direct interactions between KRM1 and LRP6.	RESULTS
+98	102	LRP6	protein	The LRP6PE3PE4-DKK1CRD2-KRM1ECD complex structure reveals no direct interactions between KRM1 and LRP6.	RESULTS
+35	47	complex with	protein_state	We constructed in silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data.	RESULTS
+59	70	full-length	protein_state	We constructed in silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data.	RESULTS
+71	75	LRP6	protein	We constructed in silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data.	RESULTS
+76	86	ectodomain	structure_element	We constructed in silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data.	RESULTS
+88	100	PE1PE2PE3PE4	structure_element	We constructed in silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data.	RESULTS
+101	111	horse shoe	structure_element	We constructed in silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data.	RESULTS
+155	174	electron microscopy	experimental_method	We constructed in silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data.	RESULTS
+176	178	EM	experimental_method	We constructed in silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data.	RESULTS
+183	211	small-angle X-ray scattering	experimental_method	We constructed in silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data.	RESULTS
+13	19	PE3PE4	structure_element	An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A).	RESULTS
+33	45	superimposed	experimental_method	An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A).	RESULTS
+50	53	PE4	structure_element	An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A).	RESULTS
+59	62	PE3	structure_element	An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A).	RESULTS
+70	87	crystal structure	evidence	An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A).	RESULTS
+97	101	LRP6	protein	An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A).	RESULTS
+101	107	PE1PE2	structure_element	An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A).	RESULTS
+108	117	structure	evidence	An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A).	RESULTS
+122	134	superimposed	experimental_method	An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A).	RESULTS
+139	142	PE2	structure_element	An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A).	RESULTS
+148	151	PE3	structure_element	An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A).	RESULTS
+98	117	Ca2+-binding region	site	For this crude approximation of a true ternary complex, we noted very close proximity between the Ca2+-binding region of KRM1 and the top face of the PE2 β propeller of LRP6.	RESULTS
+121	125	KRM1	protein	For this crude approximation of a true ternary complex, we noted very close proximity between the Ca2+-binding region of KRM1 and the top face of the PE2 β propeller of LRP6.	RESULTS
+150	153	PE2	structure_element	For this crude approximation of a true ternary complex, we noted very close proximity between the Ca2+-binding region of KRM1 and the top face of the PE2 β propeller of LRP6.	RESULTS
+154	165	β propeller	structure_element	For this crude approximation of a true ternary complex, we noted very close proximity between the Ca2+-binding region of KRM1 and the top face of the PE2 β propeller of LRP6.	RESULTS
+169	173	LRP6	protein	For this crude approximation of a true ternary complex, we noted very close proximity between the Ca2+-binding region of KRM1 and the top face of the PE2 β propeller of LRP6.	RESULTS
+4	19	solvent-exposed	protein_state	The solvent-exposed residues R307, I308, and N309 of the central Ca2+-binding β connection loop of KRM1 would be almost ideally positioned for binding to this face, which is commonly used as a binding site on β propellers.	RESULTS
+29	33	R307	residue_name_number	The solvent-exposed residues R307, I308, and N309 of the central Ca2+-binding β connection loop of KRM1 would be almost ideally positioned for binding to this face, which is commonly used as a binding site on β propellers.	RESULTS
+35	39	I308	residue_name_number	The solvent-exposed residues R307, I308, and N309 of the central Ca2+-binding β connection loop of KRM1 would be almost ideally positioned for binding to this face, which is commonly used as a binding site on β propellers.	RESULTS
+45	49	N309	residue_name_number	The solvent-exposed residues R307, I308, and N309 of the central Ca2+-binding β connection loop of KRM1 would be almost ideally positioned for binding to this face, which is commonly used as a binding site on β propellers.	RESULTS
+65	95	Ca2+-binding β connection loop	structure_element	The solvent-exposed residues R307, I308, and N309 of the central Ca2+-binding β connection loop of KRM1 would be almost ideally positioned for binding to this face, which is commonly used as a binding site on β propellers.	RESULTS
+99	103	KRM1	protein	The solvent-exposed residues R307, I308, and N309 of the central Ca2+-binding β connection loop of KRM1 would be almost ideally positioned for binding to this face, which is commonly used as a binding site on β propellers.	RESULTS
+193	205	binding site	site	The solvent-exposed residues R307, I308, and N309 of the central Ca2+-binding β connection loop of KRM1 would be almost ideally positioned for binding to this face, which is commonly used as a binding site on β propellers.	RESULTS
+209	221	β propellers	structure_element	The solvent-exposed residues R307, I308, and N309 of the central Ca2+-binding β connection loop of KRM1 would be almost ideally positioned for binding to this face, which is commonly used as a binding site on β propellers.	RESULTS
+20	28	arginine	residue_name	Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure 3B).	RESULTS
+29	35	lysine	residue_name	Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure 3B).	RESULTS
+37	47	isoleucine	residue_name	Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure 3B).	RESULTS
+53	63	asparagine	residue_name	Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure 3B).	RESULTS
+84	97	N-X-I-(G)-R/K	structure_element	Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure 3B).	RESULTS
+120	124	DKK1	protein	Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure 3B).	RESULTS
+129	133	SOST	protein	Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure 3B).	RESULTS
+145	149	LRP6	protein	Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure 3B).	RESULTS
+161	172	propeller 1	structure_element	Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure 3B).	RESULTS
+31	35	KRM1	protein	To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment.	RESULTS
+35	38	CUB	structure_element	To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment.	RESULTS
+39	47	binds to	protein_state	To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment.	RESULTS
+48	52	LRP6	protein	To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment.	RESULTS
+52	55	PE2	structure_element	To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment.	RESULTS
+65	68	SPR	experimental_method	To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment.	RESULTS
+97	106	wild-type	protein_state	To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment.	RESULTS
+115	130	GlycoCUB mutant	protein_state	To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment.	RESULTS
+134	138	KRM1	protein	To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment.	RESULTS
+138	141	ECD	structure_element	To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment.	RESULTS
+154	174	N-glycosylation site	site	To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment.	RESULTS
+178	182	N309	residue_name_number	To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment.	RESULTS
+200	204	LRP6	protein	To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment.	RESULTS
+204	210	PE1PE2	structure_element	To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment.	RESULTS
+29	39	absence of	protein_state	Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2 (Figure 3C).	RESULTS
+40	43	Dkk	protein_type	Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2 (Figure 3C).	RESULTS
+45	49	KRM1	protein	Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2 (Figure 3C).	RESULTS
+49	52	ECD	structure_element	Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2 (Figure 3C).	RESULTS
+53	58	bound	protein_state	Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2 (Figure 3C).	RESULTS
+86	88	to	protein_state	Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2 (Figure 3C).	RESULTS
+89	93	LRP6	protein	Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2 (Figure 3C).	RESULTS
+93	99	PE1PE2	structure_element	Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2 (Figure 3C).	RESULTS
+55	59	KRM1	protein	In contrast, no saturable binding was observed between KRM1 and LRP6PE3PE4.	RESULTS
+64	68	LRP6	protein	In contrast, no saturable binding was observed between KRM1 and LRP6PE3PE4.	RESULTS
+68	74	PE3PE4	structure_element	In contrast, no saturable binding was observed between KRM1 and LRP6PE3PE4.	RESULTS
+0	15	Introduction of	experimental_method	Introduction of an N-glycosylation site at N309 in KRM1ECD abolished LRP6PE1PE2 binding (Figure 3C), while binding to DKK1 was unaffected (Figure 2D).	RESULTS
+19	39	N-glycosylation site	site	Introduction of an N-glycosylation site at N309 in KRM1ECD abolished LRP6PE1PE2 binding (Figure 3C), while binding to DKK1 was unaffected (Figure 2D).	RESULTS
+43	47	N309	residue_name_number	Introduction of an N-glycosylation site at N309 in KRM1ECD abolished LRP6PE1PE2 binding (Figure 3C), while binding to DKK1 was unaffected (Figure 2D).	RESULTS
+51	55	KRM1	protein	Introduction of an N-glycosylation site at N309 in KRM1ECD abolished LRP6PE1PE2 binding (Figure 3C), while binding to DKK1 was unaffected (Figure 2D).	RESULTS
+55	58	ECD	structure_element	Introduction of an N-glycosylation site at N309 in KRM1ECD abolished LRP6PE1PE2 binding (Figure 3C), while binding to DKK1 was unaffected (Figure 2D).	RESULTS
+69	73	LRP6	protein	Introduction of an N-glycosylation site at N309 in KRM1ECD abolished LRP6PE1PE2 binding (Figure 3C), while binding to DKK1 was unaffected (Figure 2D).	RESULTS
+73	79	PE1PE2	structure_element	Introduction of an N-glycosylation site at N309 in KRM1ECD abolished LRP6PE1PE2 binding (Figure 3C), while binding to DKK1 was unaffected (Figure 2D).	RESULTS
+118	122	DKK1	protein	Introduction of an N-glycosylation site at N309 in KRM1ECD abolished LRP6PE1PE2 binding (Figure 3C), while binding to DKK1 was unaffected (Figure 2D).	RESULTS
+31	43	binding site	site	We conclude that the predicted binding site between KRM1CUB and LRP6PE2 is a strong candidate for mediating the direct Lrp6-Krm interaction, which is thought to increase Wnt responsiveness by stabilizing Lrp6 at the cell surface.	RESULTS
+52	56	KRM1	protein	We conclude that the predicted binding site between KRM1CUB and LRP6PE2 is a strong candidate for mediating the direct Lrp6-Krm interaction, which is thought to increase Wnt responsiveness by stabilizing Lrp6 at the cell surface.	RESULTS
+56	59	CUB	structure_element	We conclude that the predicted binding site between KRM1CUB and LRP6PE2 is a strong candidate for mediating the direct Lrp6-Krm interaction, which is thought to increase Wnt responsiveness by stabilizing Lrp6 at the cell surface.	RESULTS
+64	68	LRP6	protein	We conclude that the predicted binding site between KRM1CUB and LRP6PE2 is a strong candidate for mediating the direct Lrp6-Krm interaction, which is thought to increase Wnt responsiveness by stabilizing Lrp6 at the cell surface.	RESULTS
+68	71	PE2	structure_element	We conclude that the predicted binding site between KRM1CUB and LRP6PE2 is a strong candidate for mediating the direct Lrp6-Krm interaction, which is thought to increase Wnt responsiveness by stabilizing Lrp6 at the cell surface.	RESULTS
+119	127	Lrp6-Krm	complex_assembly	We conclude that the predicted binding site between KRM1CUB and LRP6PE2 is a strong candidate for mediating the direct Lrp6-Krm interaction, which is thought to increase Wnt responsiveness by stabilizing Lrp6 at the cell surface.	RESULTS
+170	173	Wnt	protein_type	We conclude that the predicted binding site between KRM1CUB and LRP6PE2 is a strong candidate for mediating the direct Lrp6-Krm interaction, which is thought to increase Wnt responsiveness by stabilizing Lrp6 at the cell surface.	RESULTS
+204	208	Lrp6	protein	We conclude that the predicted binding site between KRM1CUB and LRP6PE2 is a strong candidate for mediating the direct Lrp6-Krm interaction, which is thought to increase Wnt responsiveness by stabilizing Lrp6 at the cell surface.	RESULTS
+55	67	binding site	site	Further experiments are required to pinpoint the exact binding site.	RESULTS
+9	13	LRP6	protein	Although LRP6PE1 appears somewhat out of reach in the modeled ternary complex, it cannot be excluded as the Krm binding site in the ternary complex and LRP6-Krm binary complex.	RESULTS
+13	16	PE1	structure_element	Although LRP6PE1 appears somewhat out of reach in the modeled ternary complex, it cannot be excluded as the Krm binding site in the ternary complex and LRP6-Krm binary complex.	RESULTS
+108	124	Krm binding site	site	Although LRP6PE1 appears somewhat out of reach in the modeled ternary complex, it cannot be excluded as the Krm binding site in the ternary complex and LRP6-Krm binary complex.	RESULTS
+152	160	LRP6-Krm	complex_assembly	Although LRP6PE1 appears somewhat out of reach in the modeled ternary complex, it cannot be excluded as the Krm binding site in the ternary complex and LRP6-Krm binary complex.	RESULTS
+4	15	presence of	protein_state	The presence of DKK may govern which propeller (PE1 versus PE2) of LRP6 is available for Krm binding.	RESULTS
+16	19	DKK	protein	The presence of DKK may govern which propeller (PE1 versus PE2) of LRP6 is available for Krm binding.	RESULTS
+37	46	propeller	structure_element	The presence of DKK may govern which propeller (PE1 versus PE2) of LRP6 is available for Krm binding.	RESULTS
+48	51	PE1	structure_element	The presence of DKK may govern which propeller (PE1 versus PE2) of LRP6 is available for Krm binding.	RESULTS
+59	62	PE2	structure_element	The presence of DKK may govern which propeller (PE1 versus PE2) of LRP6 is available for Krm binding.	RESULTS
+67	71	LRP6	protein	The presence of DKK may govern which propeller (PE1 versus PE2) of LRP6 is available for Krm binding.	RESULTS
+89	92	Krm	protein_type	The presence of DKK may govern which propeller (PE1 versus PE2) of LRP6 is available for Krm binding.	RESULTS
+37	62	KRM1CUB-LRP6PE2 interface	site	Apparent binding across the proposed KRM1CUB-LRP6PE2 interface is expected to be higher once Krm is also cross-linked to LRP6PE3 via DKK1CRD2 (Figure 3D).	RESULTS
+93	96	Krm	protein_type	Apparent binding across the proposed KRM1CUB-LRP6PE2 interface is expected to be higher once Krm is also cross-linked to LRP6PE3 via DKK1CRD2 (Figure 3D).	RESULTS
+121	125	LRP6	protein	Apparent binding across the proposed KRM1CUB-LRP6PE2 interface is expected to be higher once Krm is also cross-linked to LRP6PE3 via DKK1CRD2 (Figure 3D).	RESULTS
+125	128	PE3	structure_element	Apparent binding across the proposed KRM1CUB-LRP6PE2 interface is expected to be higher once Krm is also cross-linked to LRP6PE3 via DKK1CRD2 (Figure 3D).	RESULTS
+133	137	DKK1	protein	Apparent binding across the proposed KRM1CUB-LRP6PE2 interface is expected to be higher once Krm is also cross-linked to LRP6PE3 via DKK1CRD2 (Figure 3D).	RESULTS
+137	141	CRD2	structure_element	Apparent binding across the proposed KRM1CUB-LRP6PE2 interface is expected to be higher once Krm is also cross-linked to LRP6PE3 via DKK1CRD2 (Figure 3D).	RESULTS
+15	32	negative-stain EM	experimental_method	Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.	RESULTS
+37	65	small-angle X-ray scattering	experimental_method	Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.	RESULTS
+77	81	LRP6	protein	Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.	RESULTS
+81	93	PE1PE2PE3PE4	structure_element	Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.	RESULTS
+95	107	in isolation	protein_state	Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.	RESULTS
+112	127	in complex with	protein_state	Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.	RESULTS
+128	132	Dkk1	protein_type	Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.	RESULTS
+139	156	negative-stain EM	experimental_method	Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.	RESULTS
+160	171	full-length	protein_state	Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.	RESULTS
+172	176	LRP6	protein	Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.	RESULTS
+177	187	ectodomain	structure_element	Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.	RESULTS
+204	210	curved	protein_state	Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.	RESULTS
+212	225	platform-like	protein_state	Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.	RESULTS
+279	282	PE2	structure_element	Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.	RESULTS
+287	290	PE3	structure_element	Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.	RESULTS
+47	50	Krm	protein_type	It is therefore possible that the interplay of Krm and Dkk binding can promote changes in LRP6 ectodomain conformation with functional consequences; however, such ideas await investigation.	RESULTS
+55	58	Dkk	protein_type	It is therefore possible that the interplay of Krm and Dkk binding can promote changes in LRP6 ectodomain conformation with functional consequences; however, such ideas await investigation.	RESULTS
+90	94	LRP6	protein	It is therefore possible that the interplay of Krm and Dkk binding can promote changes in LRP6 ectodomain conformation with functional consequences; however, such ideas await investigation.	RESULTS
+95	105	ectodomain	structure_element	It is therefore possible that the interplay of Krm and Dkk binding can promote changes in LRP6 ectodomain conformation with functional consequences; however, such ideas await investigation.	RESULTS
+20	54	structural and biophysical studies	experimental_method	Taken together, the structural and biophysical studies we report here extend our mechanistic understanding of Wnt signal regulation.	RESULTS
+110	113	Wnt	protein_type	Taken together, the structural and biophysical studies we report here extend our mechanistic understanding of Wnt signal regulation.	RESULTS
+16	26	ectodomain	structure_element	We describe the ectodomain structure of the dual Wnt regulator Krm1, providing an explanation for the detrimental effect on health and development of a homozygous KRM1 mutation.	RESULTS
+27	36	structure	evidence	We describe the ectodomain structure of the dual Wnt regulator Krm1, providing an explanation for the detrimental effect on health and development of a homozygous KRM1 mutation.	RESULTS
+49	52	Wnt	protein_type	We describe the ectodomain structure of the dual Wnt regulator Krm1, providing an explanation for the detrimental effect on health and development of a homozygous KRM1 mutation.	RESULTS
+63	67	Krm1	protein_type	We describe the ectodomain structure of the dual Wnt regulator Krm1, providing an explanation for the detrimental effect on health and development of a homozygous KRM1 mutation.	RESULTS
+163	167	KRM1	protein	We describe the ectodomain structure of the dual Wnt regulator Krm1, providing an explanation for the detrimental effect on health and development of a homozygous KRM1 mutation.	RESULTS
+39	46	Krm-Dkk	complex_assembly	We also reveal the interaction mode of Krm-Dkk and the architecture of the ternary complex formed by Lrp5/6, Dkk, and Krm.	RESULTS
+101	107	Lrp5/6	protein_type	We also reveal the interaction mode of Krm-Dkk and the architecture of the ternary complex formed by Lrp5/6, Dkk, and Krm.	RESULTS
+109	112	Dkk	protein_type	We also reveal the interaction mode of Krm-Dkk and the architecture of the ternary complex formed by Lrp5/6, Dkk, and Krm.	RESULTS
+118	121	Krm	protein_type	We also reveal the interaction mode of Krm-Dkk and the architecture of the ternary complex formed by Lrp5/6, Dkk, and Krm.	RESULTS
+25	42	crystal structure	evidence	Furthermore, the ternary crystal structure has guided in silico and biophysical analyses to suggest a direct LRP6-KRM1 interaction site.	RESULTS
+54	88	in silico and biophysical analyses	experimental_method	Furthermore, the ternary crystal structure has guided in silico and biophysical analyses to suggest a direct LRP6-KRM1 interaction site.	RESULTS
+109	135	LRP6-KRM1 interaction site	site	Furthermore, the ternary crystal structure has guided in silico and biophysical analyses to suggest a direct LRP6-KRM1 interaction site.	RESULTS
+136	139	Krm	protein_type	Our findings provide a solid foundation for additional studies to probe how ternary complex formation triggers internalization, whereas Krm binding in the absence of Dkk stabilizes the Wnt co-receptor at the cell surface.	RESULTS
+155	165	absence of	protein_state	Our findings provide a solid foundation for additional studies to probe how ternary complex formation triggers internalization, whereas Krm binding in the absence of Dkk stabilizes the Wnt co-receptor at the cell surface.	RESULTS
+166	169	Dkk	protein_type	Our findings provide a solid foundation for additional studies to probe how ternary complex formation triggers internalization, whereas Krm binding in the absence of Dkk stabilizes the Wnt co-receptor at the cell surface.	RESULTS
+185	188	Wnt	protein_type	Our findings provide a solid foundation for additional studies to probe how ternary complex formation triggers internalization, whereas Krm binding in the absence of Dkk stabilizes the Wnt co-receptor at the cell surface.	RESULTS
+189	200	co-receptor	protein_type	Our findings provide a solid foundation for additional studies to probe how ternary complex formation triggers internalization, whereas Krm binding in the absence of Dkk stabilizes the Wnt co-receptor at the cell surface.	RESULTS
+0	9	Structure	evidence	Structure of Unliganded KRM1ECD	FIG
+13	23	Unliganded	protein_state	Structure of Unliganded KRM1ECD	FIG
+24	28	KRM1	protein	Structure of Unliganded KRM1ECD	FIG
+28	31	ECD	structure_element	Structure of Unliganded KRM1ECD	FIG
+8	12	KRM1	protein	(A) The KRM1ECD fold (crystal form I) colored blue to red from the N to C terminus.	FIG
+12	15	ECD	structure_element	(A) The KRM1ECD fold (crystal form I) colored blue to red from the N to C terminus.	FIG
+22	36	crystal form I	evidence	(A) The KRM1ECD fold (crystal form I) colored blue to red from the N to C terminus.	FIG
+0	9	Cysteines	residue_name	Cysteines as ball and sticks, glycosylation sites as sticks.	FIG
+30	49	glycosylation sites	site	Cysteines as ball and sticks, glycosylation sites as sticks.	FIG
+10	17	calcium	chemical	The bound calcium is shown as a gray sphere.	FIG
+16	21	F207S	mutant	The site of the F207S mutation associated with ectodermal dysplasia in humans is shown as mesh.	FIG
+71	77	humans	species	The site of the F207S mutation associated with ectodermal dysplasia in humans is shown as mesh.	FIG
+4	17	Superposition	experimental_method	(B) Superposition of the three KRM1ECD subdomains (solid) with their next structurally characterized homologs (half transparent).	FIG
+31	35	KRM1	protein	(B) Superposition of the three KRM1ECD subdomains (solid) with their next structurally characterized homologs (half transparent).	FIG
+35	38	ECD	structure_element	(B) Superposition of the three KRM1ECD subdomains (solid) with their next structurally characterized homologs (half transparent).	FIG
+4	17	Superposition	experimental_method	(C) Superposition of KRM1ECD from the three crystal forms.	FIG
+21	25	KRM1	protein	(C) Superposition of KRM1ECD from the three crystal forms.	FIG
+25	28	ECD	structure_element	(C) Superposition of KRM1ECD from the three crystal forms.	FIG
+44	57	crystal forms	evidence	(C) Superposition of KRM1ECD from the three crystal forms.	FIG
+0	16	Alignment scores	evidence	Alignment scores for each pairing are indicated on the dashed triangle.	FIG
+8	17	structure	evidence	(A) The structure of the ternary LRP6PE3PE4-DKK1CRD2-KRM1ECD complex.	FIG
+33	60	LRP6PE3PE4-DKK1CRD2-KRM1ECD	complex_assembly	(A) The structure of the ternary LRP6PE3PE4-DKK1CRD2-KRM1ECD complex.	FIG
+0	4	DKK1	protein	DKK1 (orange) is sandwiched between the PE3 module of LRP6 (blue) and the KR-WSC domain pair of KRM1 (green).	FIG
+40	43	PE3	structure_element	DKK1 (orange) is sandwiched between the PE3 module of LRP6 (blue) and the KR-WSC domain pair of KRM1 (green).	FIG
+54	58	LRP6	protein	DKK1 (orange) is sandwiched between the PE3 module of LRP6 (blue) and the KR-WSC domain pair of KRM1 (green).	FIG
+74	80	KR-WSC	structure_element	DKK1 (orange) is sandwiched between the PE3 module of LRP6 (blue) and the KR-WSC domain pair of KRM1 (green).	FIG
+96	100	KRM1	protein	DKK1 (orange) is sandwiched between the PE3 module of LRP6 (blue) and the KR-WSC domain pair of KRM1 (green).	FIG
+36	61	N-glycan attachment sites	site	Colored symbols indicate introduced N-glycan attachment sites (see D).	FIG
+4	7	SPR	experimental_method	(B) SPR data comparing binding of full-length DKK1 and SUMO fusions of DKK1 truncations for binding to immobilized wild-type KRM1ECD.	FIG
+34	45	full-length	protein_state	(B) SPR data comparing binding of full-length DKK1 and SUMO fusions of DKK1 truncations for binding to immobilized wild-type KRM1ECD.	FIG
+46	50	DKK1	protein	(B) SPR data comparing binding of full-length DKK1 and SUMO fusions of DKK1 truncations for binding to immobilized wild-type KRM1ECD.	FIG
+55	67	SUMO fusions	experimental_method	(B) SPR data comparing binding of full-length DKK1 and SUMO fusions of DKK1 truncations for binding to immobilized wild-type KRM1ECD.	FIG
+71	75	DKK1	protein	(B) SPR data comparing binding of full-length DKK1 and SUMO fusions of DKK1 truncations for binding to immobilized wild-type KRM1ECD.	FIG
+115	124	wild-type	protein_state	(B) SPR data comparing binding of full-length DKK1 and SUMO fusions of DKK1 truncations for binding to immobilized wild-type KRM1ECD.	FIG
+125	129	KRM1	protein	(B) SPR data comparing binding of full-length DKK1 and SUMO fusions of DKK1 truncations for binding to immobilized wild-type KRM1ECD.	FIG
+129	132	ECD	structure_element	(B) SPR data comparing binding of full-length DKK1 and SUMO fusions of DKK1 truncations for binding to immobilized wild-type KRM1ECD.	FIG
+25	51	DKK1CRD2-KRM1ECD interface	site	(C) Close-up view of the DKK1CRD2-KRM1ECD interface.	FIG
+21	30	interface	site	Residues involved in interface formation are shown as sticks; those mentioned in the text are labeled.	FIG
+0	12	Salt bridges	bond_interaction	Salt bridges are in pink and hydrogen bonds in black.	FIG
+29	43	hydrogen bonds	bond_interaction	Salt bridges are in pink and hydrogen bonds in black.	FIG
+4	7	SPR	experimental_method	(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange).	FIG
+8	20	binding data	evidence	(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange).	FIG
+31	35	DKK1	protein	(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange).	FIG
+57	66	wild-type	protein_state	(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange).	FIG
+67	71	KRM1	protein	(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange).	FIG
+71	74	ECD	structure_element	(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange).	FIG
+102	112	engineered	protein_state	(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange).	FIG
+113	132	glycosylation sites	site	(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange).	FIG
+140	142	KR	structure_element	(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange).	FIG
+147	150	WSC	structure_element	(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange).	FIG
+187	191	DKK1	protein	(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange).	FIG
+204	207	CUB	structure_element	(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange).	FIG
+0	9	LRP6-KRM1	complex_assembly	LRP6-KRM1 Direct Interaction and Summary	FIG
+35	47	complex with	protein_state	(A) In a construction of a ternary complex with all four β propellers of LRP6 intact, the CUB domain points via its Ca2+-binding region toward the top face of the second β propeller.	FIG
+57	69	β propellers	structure_element	(A) In a construction of a ternary complex with all four β propellers of LRP6 intact, the CUB domain points via its Ca2+-binding region toward the top face of the second β propeller.	FIG
+73	77	LRP6	protein	(A) In a construction of a ternary complex with all four β propellers of LRP6 intact, the CUB domain points via its Ca2+-binding region toward the top face of the second β propeller.	FIG
+78	84	intact	protein_state	(A) In a construction of a ternary complex with all four β propellers of LRP6 intact, the CUB domain points via its Ca2+-binding region toward the top face of the second β propeller.	FIG
+90	93	CUB	structure_element	(A) In a construction of a ternary complex with all four β propellers of LRP6 intact, the CUB domain points via its Ca2+-binding region toward the top face of the second β propeller.	FIG
+116	135	Ca2+-binding region	site	(A) In a construction of a ternary complex with all four β propellers of LRP6 intact, the CUB domain points via its Ca2+-binding region toward the top face of the second β propeller.	FIG
+163	181	second β propeller	structure_element	(A) In a construction of a ternary complex with all four β propellers of LRP6 intact, the CUB domain points via its Ca2+-binding region toward the top face of the second β propeller.	FIG
+35	51	interaction site	site	(B) Close-up view of the potential interaction site.	FIG
+13	17	LRP6	protein	In addition, LRP6PE2 has been superimposed with DKK1 (yellow) and SOST (pink) peptide complexes of LRP6PE1.	FIG
+17	20	PE2	structure_element	In addition, LRP6PE2 has been superimposed with DKK1 (yellow) and SOST (pink) peptide complexes of LRP6PE1.	FIG
+30	42	superimposed	experimental_method	In addition, LRP6PE2 has been superimposed with DKK1 (yellow) and SOST (pink) peptide complexes of LRP6PE1.	FIG
+48	52	DKK1	protein	In addition, LRP6PE2 has been superimposed with DKK1 (yellow) and SOST (pink) peptide complexes of LRP6PE1.	FIG
+66	70	SOST	protein	In addition, LRP6PE2 has been superimposed with DKK1 (yellow) and SOST (pink) peptide complexes of LRP6PE1.	FIG
+99	103	LRP6	protein	In addition, LRP6PE2 has been superimposed with DKK1 (yellow) and SOST (pink) peptide complexes of LRP6PE1.	FIG
+103	106	PE1	structure_element	In addition, LRP6PE2 has been superimposed with DKK1 (yellow) and SOST (pink) peptide complexes of LRP6PE1.	FIG
+4	20	SPR measurements	experimental_method	(C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309.	FIG
+31	35	LRP6	protein	(C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309.	FIG
+35	41	PE1PE2	structure_element	(C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309.	FIG
+55	64	wild-type	protein_state	(C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309.	FIG
+65	69	KRM1	protein	(C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309.	FIG
+69	72	ECD	structure_element	(C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309.	FIG
+81	96	GlycoCUB mutant	protein_state	(C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309.	FIG
+108	116	N-glycan	ptm	(C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309.	FIG
+120	124	N309	residue_name_number	(C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309.	FIG
+95	98	Wnt	protein_type	(D) Schematic representation of structural and biophysical findings and their implications for Wnt-dependent (left, middle) and independent (right) signaling.	FIG
+48	52	LRP6	protein	Conformational differences in the depictions of LRP6 are included purely for ease of representation.	FIG
+0	37	Diffraction and Refinement Statistics	evidence	Diffraction and Refinement Statistics	TABLE
+1	5	KRM1	protein	"	KRM1ECD	KRM1ECD	KRM1ECD	KRM1ECD	LRP6PE3PE4-DKKCRD2-KRM1ECD	 	Crystal form	I	I	II	III	I	 	X-ray source	Diamond i04	Diamond i03	Diamond i03	Diamond i04	Diamond i04	 	Wavelength (Å)	0.9793	0.9700	0.9700	0.9795	0.9795	 	Space group	P3121	P3121	P43	P41212	C2221	 	Unit cell a/α (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	86.9/90	 	b/β (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	100.1/90	 	c/γ (Å/°)	188.4/120	187.4/120	75.0/90	198.2/90	270.7/90	 	Wilson B factor (Å2)	31	41	76	77	NA	 	Resolution range (Å)	47.10–1.90 (1.95–1.90)	62.47–2.10 (2.16–2.10)	75.00–2.80 (2.99–2.80)	67.80–3.20 (3.42–3.20)	67.68–3.50 (7.16–6.40, 3.92–3.50)	 	Unique reflections	23,300 (1,524)	17,089 (1,428)	7,964 (1,448)	8,171 (1,343)	8,070 (723, 645)	 	Average multiplicity	9.1 (9.2)	5.2 (5.3)	3.7 (3.7)	22.7 (12.6)	3.8 (3.5, 4.4)	 	Completeness (%)	99.8 (98.5)	100 (100)	99.8 (100)	98.8 (93.4)	51.6 (98.5, 14.1)	 	<I/σI>	11.4 (1.7)	12.0 (1.7)	14.9 (1.5)	13.1 (1.9)	4.6 (4.1, 2.2)	 	Rmerge (%)	14.8 (158.3)	9.3 (98.0)	6.2 (98.9)	29.8 (142.2)	44.9 (40.5, 114.2)	 	Rpim (%)	15.7 (55.3)	10.3 (109.0)	3.7 (53.8)	6.3 (40.0)	24.7 (23.9, 59.9)	 		 	Refinement	 		 	Rwork (%)	17.9	18.4	21.6	20.2	32.1	 	Rfree (%)	22.7	23.2	30.7	27.1	35.5	 		 	No. of Non-Hydrogen Atoms	 		 	Protein	2,260	2,301	2,102	2,305	7,730	 	N-glycans	42	42	28	28	0	 	Water	79	54	0	2	0	 	Ligands	6	6	2	5	0	 		 	Average B factor (Å2)	 		 	Protein	63	65	108	84	–	 	N-glycans	35	46	102	18	–	 	Water	68	85	–	75	–	 	Ligands	36	47	91	75	66	 		 	RMSD from Ideality	 		 	Bond lengths (Å)	0.020	0.016	0.019	0.016	0.004	 	Bond angles (°)	2.050	1.748	1.952	1.796	0.770	 		 	Ramachandran Plot	 		 	Favored (%)	96.8	95.5	96.9	94.9	92.3	 	Allowed (%)	99.7	100.0	100.0	99.7	99.8	 	Number of outliers	1	0	0	1	2	 	PDB code	5FWS	5FWT	5FWU	5FWV	5FWW	 	"	TABLE
+5	8	ECD	structure_element	"	KRM1ECD	KRM1ECD	KRM1ECD	KRM1ECD	LRP6PE3PE4-DKKCRD2-KRM1ECD	 	Crystal form	I	I	II	III	I	 	X-ray source	Diamond i04	Diamond i03	Diamond i03	Diamond i04	Diamond i04	 	Wavelength (Å)	0.9793	0.9700	0.9700	0.9795	0.9795	 	Space group	P3121	P3121	P43	P41212	C2221	 	Unit cell a/α (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	86.9/90	 	b/β (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	100.1/90	 	c/γ (Å/°)	188.4/120	187.4/120	75.0/90	198.2/90	270.7/90	 	Wilson B factor (Å2)	31	41	76	77	NA	 	Resolution range (Å)	47.10–1.90 (1.95–1.90)	62.47–2.10 (2.16–2.10)	75.00–2.80 (2.99–2.80)	67.80–3.20 (3.42–3.20)	67.68–3.50 (7.16–6.40, 3.92–3.50)	 	Unique reflections	23,300 (1,524)	17,089 (1,428)	7,964 (1,448)	8,171 (1,343)	8,070 (723, 645)	 	Average multiplicity	9.1 (9.2)	5.2 (5.3)	3.7 (3.7)	22.7 (12.6)	3.8 (3.5, 4.4)	 	Completeness (%)	99.8 (98.5)	100 (100)	99.8 (100)	98.8 (93.4)	51.6 (98.5, 14.1)	 	<I/σI>	11.4 (1.7)	12.0 (1.7)	14.9 (1.5)	13.1 (1.9)	4.6 (4.1, 2.2)	 	Rmerge (%)	14.8 (158.3)	9.3 (98.0)	6.2 (98.9)	29.8 (142.2)	44.9 (40.5, 114.2)	 	Rpim (%)	15.7 (55.3)	10.3 (109.0)	3.7 (53.8)	6.3 (40.0)	24.7 (23.9, 59.9)	 		 	Refinement	 		 	Rwork (%)	17.9	18.4	21.6	20.2	32.1	 	Rfree (%)	22.7	23.2	30.7	27.1	35.5	 		 	No. of Non-Hydrogen Atoms	 		 	Protein	2,260	2,301	2,102	2,305	7,730	 	N-glycans	42	42	28	28	0	 	Water	79	54	0	2	0	 	Ligands	6	6	2	5	0	 		 	Average B factor (Å2)	 		 	Protein	63	65	108	84	–	 	N-glycans	35	46	102	18	–	 	Water	68	85	–	75	–	 	Ligands	36	47	91	75	66	 		 	RMSD from Ideality	 		 	Bond lengths (Å)	0.020	0.016	0.019	0.016	0.004	 	Bond angles (°)	2.050	1.748	1.952	1.796	0.770	 		 	Ramachandran Plot	 		 	Favored (%)	96.8	95.5	96.9	94.9	92.3	 	Allowed (%)	99.7	100.0	100.0	99.7	99.8	 	Number of outliers	1	0	0	1	2	 	PDB code	5FWS	5FWT	5FWU	5FWV	5FWW	 	"	TABLE
+9	13	KRM1	protein	"	KRM1ECD	KRM1ECD	KRM1ECD	KRM1ECD	LRP6PE3PE4-DKKCRD2-KRM1ECD	 	Crystal form	I	I	II	III	I	 	X-ray source	Diamond i04	Diamond i03	Diamond i03	Diamond i04	Diamond i04	 	Wavelength (Å)	0.9793	0.9700	0.9700	0.9795	0.9795	 	Space group	P3121	P3121	P43	P41212	C2221	 	Unit cell a/α (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	86.9/90	 	b/β (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	100.1/90	 	c/γ (Å/°)	188.4/120	187.4/120	75.0/90	198.2/90	270.7/90	 	Wilson B factor (Å2)	31	41	76	77	NA	 	Resolution range (Å)	47.10–1.90 (1.95–1.90)	62.47–2.10 (2.16–2.10)	75.00–2.80 (2.99–2.80)	67.80–3.20 (3.42–3.20)	67.68–3.50 (7.16–6.40, 3.92–3.50)	 	Unique reflections	23,300 (1,524)	17,089 (1,428)	7,964 (1,448)	8,171 (1,343)	8,070 (723, 645)	 	Average multiplicity	9.1 (9.2)	5.2 (5.3)	3.7 (3.7)	22.7 (12.6)	3.8 (3.5, 4.4)	 	Completeness (%)	99.8 (98.5)	100 (100)	99.8 (100)	98.8 (93.4)	51.6 (98.5, 14.1)	 	<I/σI>	11.4 (1.7)	12.0 (1.7)	14.9 (1.5)	13.1 (1.9)	4.6 (4.1, 2.2)	 	Rmerge (%)	14.8 (158.3)	9.3 (98.0)	6.2 (98.9)	29.8 (142.2)	44.9 (40.5, 114.2)	 	Rpim (%)	15.7 (55.3)	10.3 (109.0)	3.7 (53.8)	6.3 (40.0)	24.7 (23.9, 59.9)	 		 	Refinement	 		 	Rwork (%)	17.9	18.4	21.6	20.2	32.1	 	Rfree (%)	22.7	23.2	30.7	27.1	35.5	 		 	No. of Non-Hydrogen Atoms	 		 	Protein	2,260	2,301	2,102	2,305	7,730	 	N-glycans	42	42	28	28	0	 	Water	79	54	0	2	0	 	Ligands	6	6	2	5	0	 		 	Average B factor (Å2)	 		 	Protein	63	65	108	84	–	 	N-glycans	35	46	102	18	–	 	Water	68	85	–	75	–	 	Ligands	36	47	91	75	66	 		 	RMSD from Ideality	 		 	Bond lengths (Å)	0.020	0.016	0.019	0.016	0.004	 	Bond angles (°)	2.050	1.748	1.952	1.796	0.770	 		 	Ramachandran Plot	 		 	Favored (%)	96.8	95.5	96.9	94.9	92.3	 	Allowed (%)	99.7	100.0	100.0	99.7	99.8	 	Number of outliers	1	0	0	1	2	 	PDB code	5FWS	5FWT	5FWU	5FWV	5FWW	 	"	TABLE
+13	16	ECD	structure_element	"	KRM1ECD	KRM1ECD	KRM1ECD	KRM1ECD	LRP6PE3PE4-DKKCRD2-KRM1ECD	 	Crystal form	I	I	II	III	I	 	X-ray source	Diamond i04	Diamond i03	Diamond i03	Diamond i04	Diamond i04	 	Wavelength (Å)	0.9793	0.9700	0.9700	0.9795	0.9795	 	Space group	P3121	P3121	P43	P41212	C2221	 	Unit cell a/α (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	86.9/90	 	b/β (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	100.1/90	 	c/γ (Å/°)	188.4/120	187.4/120	75.0/90	198.2/90	270.7/90	 	Wilson B factor (Å2)	31	41	76	77	NA	 	Resolution range (Å)	47.10–1.90 (1.95–1.90)	62.47–2.10 (2.16–2.10)	75.00–2.80 (2.99–2.80)	67.80–3.20 (3.42–3.20)	67.68–3.50 (7.16–6.40, 3.92–3.50)	 	Unique reflections	23,300 (1,524)	17,089 (1,428)	7,964 (1,448)	8,171 (1,343)	8,070 (723, 645)	 	Average multiplicity	9.1 (9.2)	5.2 (5.3)	3.7 (3.7)	22.7 (12.6)	3.8 (3.5, 4.4)	 	Completeness (%)	99.8 (98.5)	100 (100)	99.8 (100)	98.8 (93.4)	51.6 (98.5, 14.1)	 	<I/σI>	11.4 (1.7)	12.0 (1.7)	14.9 (1.5)	13.1 (1.9)	4.6 (4.1, 2.2)	 	Rmerge (%)	14.8 (158.3)	9.3 (98.0)	6.2 (98.9)	29.8 (142.2)	44.9 (40.5, 114.2)	 	Rpim (%)	15.7 (55.3)	10.3 (109.0)	3.7 (53.8)	6.3 (40.0)	24.7 (23.9, 59.9)	 		 	Refinement	 		 	Rwork (%)	17.9	18.4	21.6	20.2	32.1	 	Rfree (%)	22.7	23.2	30.7	27.1	35.5	 		 	No. of Non-Hydrogen Atoms	 		 	Protein	2,260	2,301	2,102	2,305	7,730	 	N-glycans	42	42	28	28	0	 	Water	79	54	0	2	0	 	Ligands	6	6	2	5	0	 		 	Average B factor (Å2)	 		 	Protein	63	65	108	84	–	 	N-glycans	35	46	102	18	–	 	Water	68	85	–	75	–	 	Ligands	36	47	91	75	66	 		 	RMSD from Ideality	 		 	Bond lengths (Å)	0.020	0.016	0.019	0.016	0.004	 	Bond angles (°)	2.050	1.748	1.952	1.796	0.770	 		 	Ramachandran Plot	 		 	Favored (%)	96.8	95.5	96.9	94.9	92.3	 	Allowed (%)	99.7	100.0	100.0	99.7	99.8	 	Number of outliers	1	0	0	1	2	 	PDB code	5FWS	5FWT	5FWU	5FWV	5FWW	 	"	TABLE
+17	21	KRM1	protein	"	KRM1ECD	KRM1ECD	KRM1ECD	KRM1ECD	LRP6PE3PE4-DKKCRD2-KRM1ECD	 	Crystal form	I	I	II	III	I	 	X-ray source	Diamond i04	Diamond i03	Diamond i03	Diamond i04	Diamond i04	 	Wavelength (Å)	0.9793	0.9700	0.9700	0.9795	0.9795	 	Space group	P3121	P3121	P43	P41212	C2221	 	Unit cell a/α (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	86.9/90	 	b/β (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	100.1/90	 	c/γ (Å/°)	188.4/120	187.4/120	75.0/90	198.2/90	270.7/90	 	Wilson B factor (Å2)	31	41	76	77	NA	 	Resolution range (Å)	47.10–1.90 (1.95–1.90)	62.47–2.10 (2.16–2.10)	75.00–2.80 (2.99–2.80)	67.80–3.20 (3.42–3.20)	67.68–3.50 (7.16–6.40, 3.92–3.50)	 	Unique reflections	23,300 (1,524)	17,089 (1,428)	7,964 (1,448)	8,171 (1,343)	8,070 (723, 645)	 	Average multiplicity	9.1 (9.2)	5.2 (5.3)	3.7 (3.7)	22.7 (12.6)	3.8 (3.5, 4.4)	 	Completeness (%)	99.8 (98.5)	100 (100)	99.8 (100)	98.8 (93.4)	51.6 (98.5, 14.1)	 	<I/σI>	11.4 (1.7)	12.0 (1.7)	14.9 (1.5)	13.1 (1.9)	4.6 (4.1, 2.2)	 	Rmerge (%)	14.8 (158.3)	9.3 (98.0)	6.2 (98.9)	29.8 (142.2)	44.9 (40.5, 114.2)	 	Rpim (%)	15.7 (55.3)	10.3 (109.0)	3.7 (53.8)	6.3 (40.0)	24.7 (23.9, 59.9)	 		 	Refinement	 		 	Rwork (%)	17.9	18.4	21.6	20.2	32.1	 	Rfree (%)	22.7	23.2	30.7	27.1	35.5	 		 	No. of Non-Hydrogen Atoms	 		 	Protein	2,260	2,301	2,102	2,305	7,730	 	N-glycans	42	42	28	28	0	 	Water	79	54	0	2	0	 	Ligands	6	6	2	5	0	 		 	Average B factor (Å2)	 		 	Protein	63	65	108	84	–	 	N-glycans	35	46	102	18	–	 	Water	68	85	–	75	–	 	Ligands	36	47	91	75	66	 		 	RMSD from Ideality	 		 	Bond lengths (Å)	0.020	0.016	0.019	0.016	0.004	 	Bond angles (°)	2.050	1.748	1.952	1.796	0.770	 		 	Ramachandran Plot	 		 	Favored (%)	96.8	95.5	96.9	94.9	92.3	 	Allowed (%)	99.7	100.0	100.0	99.7	99.8	 	Number of outliers	1	0	0	1	2	 	PDB code	5FWS	5FWT	5FWU	5FWV	5FWW	 	"	TABLE
+21	24	ECD	structure_element	"	KRM1ECD	KRM1ECD	KRM1ECD	KRM1ECD	LRP6PE3PE4-DKKCRD2-KRM1ECD	 	Crystal form	I	I	II	III	I	 	X-ray source	Diamond i04	Diamond i03	Diamond i03	Diamond i04	Diamond i04	 	Wavelength (Å)	0.9793	0.9700	0.9700	0.9795	0.9795	 	Space group	P3121	P3121	P43	P41212	C2221	 	Unit cell a/α (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	86.9/90	 	b/β (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	100.1/90	 	c/γ (Å/°)	188.4/120	187.4/120	75.0/90	198.2/90	270.7/90	 	Wilson B factor (Å2)	31	41	76	77	NA	 	Resolution range (Å)	47.10–1.90 (1.95–1.90)	62.47–2.10 (2.16–2.10)	75.00–2.80 (2.99–2.80)	67.80–3.20 (3.42–3.20)	67.68–3.50 (7.16–6.40, 3.92–3.50)	 	Unique reflections	23,300 (1,524)	17,089 (1,428)	7,964 (1,448)	8,171 (1,343)	8,070 (723, 645)	 	Average multiplicity	9.1 (9.2)	5.2 (5.3)	3.7 (3.7)	22.7 (12.6)	3.8 (3.5, 4.4)	 	Completeness (%)	99.8 (98.5)	100 (100)	99.8 (100)	98.8 (93.4)	51.6 (98.5, 14.1)	 	<I/σI>	11.4 (1.7)	12.0 (1.7)	14.9 (1.5)	13.1 (1.9)	4.6 (4.1, 2.2)	 	Rmerge (%)	14.8 (158.3)	9.3 (98.0)	6.2 (98.9)	29.8 (142.2)	44.9 (40.5, 114.2)	 	Rpim (%)	15.7 (55.3)	10.3 (109.0)	3.7 (53.8)	6.3 (40.0)	24.7 (23.9, 59.9)	 		 	Refinement	 		 	Rwork (%)	17.9	18.4	21.6	20.2	32.1	 	Rfree (%)	22.7	23.2	30.7	27.1	35.5	 		 	No. of Non-Hydrogen Atoms	 		 	Protein	2,260	2,301	2,102	2,305	7,730	 	N-glycans	42	42	28	28	0	 	Water	79	54	0	2	0	 	Ligands	6	6	2	5	0	 		 	Average B factor (Å2)	 		 	Protein	63	65	108	84	–	 	N-glycans	35	46	102	18	–	 	Water	68	85	–	75	–	 	Ligands	36	47	91	75	66	 		 	RMSD from Ideality	 		 	Bond lengths (Å)	0.020	0.016	0.019	0.016	0.004	 	Bond angles (°)	2.050	1.748	1.952	1.796	0.770	 		 	Ramachandran Plot	 		 	Favored (%)	96.8	95.5	96.9	94.9	92.3	 	Allowed (%)	99.7	100.0	100.0	99.7	99.8	 	Number of outliers	1	0	0	1	2	 	PDB code	5FWS	5FWT	5FWU	5FWV	5FWW	 	"	TABLE
+25	29	KRM1	protein	"	KRM1ECD	KRM1ECD	KRM1ECD	KRM1ECD	LRP6PE3PE4-DKKCRD2-KRM1ECD	 	Crystal form	I	I	II	III	I	 	X-ray source	Diamond i04	Diamond i03	Diamond i03	Diamond i04	Diamond i04	 	Wavelength (Å)	0.9793	0.9700	0.9700	0.9795	0.9795	 	Space group	P3121	P3121	P43	P41212	C2221	 	Unit cell a/α (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	86.9/90	 	b/β (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	100.1/90	 	c/γ (Å/°)	188.4/120	187.4/120	75.0/90	198.2/90	270.7/90	 	Wilson B factor (Å2)	31	41	76	77	NA	 	Resolution range (Å)	47.10–1.90 (1.95–1.90)	62.47–2.10 (2.16–2.10)	75.00–2.80 (2.99–2.80)	67.80–3.20 (3.42–3.20)	67.68–3.50 (7.16–6.40, 3.92–3.50)	 	Unique reflections	23,300 (1,524)	17,089 (1,428)	7,964 (1,448)	8,171 (1,343)	8,070 (723, 645)	 	Average multiplicity	9.1 (9.2)	5.2 (5.3)	3.7 (3.7)	22.7 (12.6)	3.8 (3.5, 4.4)	 	Completeness (%)	99.8 (98.5)	100 (100)	99.8 (100)	98.8 (93.4)	51.6 (98.5, 14.1)	 	<I/σI>	11.4 (1.7)	12.0 (1.7)	14.9 (1.5)	13.1 (1.9)	4.6 (4.1, 2.2)	 	Rmerge (%)	14.8 (158.3)	9.3 (98.0)	6.2 (98.9)	29.8 (142.2)	44.9 (40.5, 114.2)	 	Rpim (%)	15.7 (55.3)	10.3 (109.0)	3.7 (53.8)	6.3 (40.0)	24.7 (23.9, 59.9)	 		 	Refinement	 		 	Rwork (%)	17.9	18.4	21.6	20.2	32.1	 	Rfree (%)	22.7	23.2	30.7	27.1	35.5	 		 	No. of Non-Hydrogen Atoms	 		 	Protein	2,260	2,301	2,102	2,305	7,730	 	N-glycans	42	42	28	28	0	 	Water	79	54	0	2	0	 	Ligands	6	6	2	5	0	 		 	Average B factor (Å2)	 		 	Protein	63	65	108	84	–	 	N-glycans	35	46	102	18	–	 	Water	68	85	–	75	–	 	Ligands	36	47	91	75	66	 		 	RMSD from Ideality	 		 	Bond lengths (Å)	0.020	0.016	0.019	0.016	0.004	 	Bond angles (°)	2.050	1.748	1.952	1.796	0.770	 		 	Ramachandran Plot	 		 	Favored (%)	96.8	95.5	96.9	94.9	92.3	 	Allowed (%)	99.7	100.0	100.0	99.7	99.8	 	Number of outliers	1	0	0	1	2	 	PDB code	5FWS	5FWT	5FWU	5FWV	5FWW	 	"	TABLE
+29	32	ECD	structure_element	"	KRM1ECD	KRM1ECD	KRM1ECD	KRM1ECD	LRP6PE3PE4-DKKCRD2-KRM1ECD	 	Crystal form	I	I	II	III	I	 	X-ray source	Diamond i04	Diamond i03	Diamond i03	Diamond i04	Diamond i04	 	Wavelength (Å)	0.9793	0.9700	0.9700	0.9795	0.9795	 	Space group	P3121	P3121	P43	P41212	C2221	 	Unit cell a/α (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	86.9/90	 	b/β (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	100.1/90	 	c/γ (Å/°)	188.4/120	187.4/120	75.0/90	198.2/90	270.7/90	 	Wilson B factor (Å2)	31	41	76	77	NA	 	Resolution range (Å)	47.10–1.90 (1.95–1.90)	62.47–2.10 (2.16–2.10)	75.00–2.80 (2.99–2.80)	67.80–3.20 (3.42–3.20)	67.68–3.50 (7.16–6.40, 3.92–3.50)	 	Unique reflections	23,300 (1,524)	17,089 (1,428)	7,964 (1,448)	8,171 (1,343)	8,070 (723, 645)	 	Average multiplicity	9.1 (9.2)	5.2 (5.3)	3.7 (3.7)	22.7 (12.6)	3.8 (3.5, 4.4)	 	Completeness (%)	99.8 (98.5)	100 (100)	99.8 (100)	98.8 (93.4)	51.6 (98.5, 14.1)	 	<I/σI>	11.4 (1.7)	12.0 (1.7)	14.9 (1.5)	13.1 (1.9)	4.6 (4.1, 2.2)	 	Rmerge (%)	14.8 (158.3)	9.3 (98.0)	6.2 (98.9)	29.8 (142.2)	44.9 (40.5, 114.2)	 	Rpim (%)	15.7 (55.3)	10.3 (109.0)	3.7 (53.8)	6.3 (40.0)	24.7 (23.9, 59.9)	 		 	Refinement	 		 	Rwork (%)	17.9	18.4	21.6	20.2	32.1	 	Rfree (%)	22.7	23.2	30.7	27.1	35.5	 		 	No. of Non-Hydrogen Atoms	 		 	Protein	2,260	2,301	2,102	2,305	7,730	 	N-glycans	42	42	28	28	0	 	Water	79	54	0	2	0	 	Ligands	6	6	2	5	0	 		 	Average B factor (Å2)	 		 	Protein	63	65	108	84	–	 	N-glycans	35	46	102	18	–	 	Water	68	85	–	75	–	 	Ligands	36	47	91	75	66	 		 	RMSD from Ideality	 		 	Bond lengths (Å)	0.020	0.016	0.019	0.016	0.004	 	Bond angles (°)	2.050	1.748	1.952	1.796	0.770	 		 	Ramachandran Plot	 		 	Favored (%)	96.8	95.5	96.9	94.9	92.3	 	Allowed (%)	99.7	100.0	100.0	99.7	99.8	 	Number of outliers	1	0	0	1	2	 	PDB code	5FWS	5FWT	5FWU	5FWV	5FWW	 	"	TABLE
+33	59	LRP6PE3PE4-DKKCRD2-KRM1ECD	complex_assembly	"	KRM1ECD	KRM1ECD	KRM1ECD	KRM1ECD	LRP6PE3PE4-DKKCRD2-KRM1ECD	 	Crystal form	I	I	II	III	I	 	X-ray source	Diamond i04	Diamond i03	Diamond i03	Diamond i04	Diamond i04	 	Wavelength (Å)	0.9793	0.9700	0.9700	0.9795	0.9795	 	Space group	P3121	P3121	P43	P41212	C2221	 	Unit cell a/α (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	86.9/90	 	b/β (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	100.1/90	 	c/γ (Å/°)	188.4/120	187.4/120	75.0/90	198.2/90	270.7/90	 	Wilson B factor (Å2)	31	41	76	77	NA	 	Resolution range (Å)	47.10–1.90 (1.95–1.90)	62.47–2.10 (2.16–2.10)	75.00–2.80 (2.99–2.80)	67.80–3.20 (3.42–3.20)	67.68–3.50 (7.16–6.40, 3.92–3.50)	 	Unique reflections	23,300 (1,524)	17,089 (1,428)	7,964 (1,448)	8,171 (1,343)	8,070 (723, 645)	 	Average multiplicity	9.1 (9.2)	5.2 (5.3)	3.7 (3.7)	22.7 (12.6)	3.8 (3.5, 4.4)	 	Completeness (%)	99.8 (98.5)	100 (100)	99.8 (100)	98.8 (93.4)	51.6 (98.5, 14.1)	 	<I/σI>	11.4 (1.7)	12.0 (1.7)	14.9 (1.5)	13.1 (1.9)	4.6 (4.1, 2.2)	 	Rmerge (%)	14.8 (158.3)	9.3 (98.0)	6.2 (98.9)	29.8 (142.2)	44.9 (40.5, 114.2)	 	Rpim (%)	15.7 (55.3)	10.3 (109.0)	3.7 (53.8)	6.3 (40.0)	24.7 (23.9, 59.9)	 		 	Refinement	 		 	Rwork (%)	17.9	18.4	21.6	20.2	32.1	 	Rfree (%)	22.7	23.2	30.7	27.1	35.5	 		 	No. of Non-Hydrogen Atoms	 		 	Protein	2,260	2,301	2,102	2,305	7,730	 	N-glycans	42	42	28	28	0	 	Water	79	54	0	2	0	 	Ligands	6	6	2	5	0	 		 	Average B factor (Å2)	 		 	Protein	63	65	108	84	–	 	N-glycans	35	46	102	18	–	 	Water	68	85	–	75	–	 	Ligands	36	47	91	75	66	 		 	RMSD from Ideality	 		 	Bond lengths (Å)	0.020	0.016	0.019	0.016	0.004	 	Bond angles (°)	2.050	1.748	1.952	1.796	0.770	 		 	Ramachandran Plot	 		 	Favored (%)	96.8	95.5	96.9	94.9	92.3	 	Allowed (%)	99.7	100.0	100.0	99.7	99.8	 	Number of outliers	1	0	0	1	2	 	PDB code	5FWS	5FWT	5FWU	5FWV	5FWW	 	"	TABLE
+1295	1300	Water	chemical	"	KRM1ECD	KRM1ECD	KRM1ECD	KRM1ECD	LRP6PE3PE4-DKKCRD2-KRM1ECD	 	Crystal form	I	I	II	III	I	 	X-ray source	Diamond i04	Diamond i03	Diamond i03	Diamond i04	Diamond i04	 	Wavelength (Å)	0.9793	0.9700	0.9700	0.9795	0.9795	 	Space group	P3121	P3121	P43	P41212	C2221	 	Unit cell a/α (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	86.9/90	 	b/β (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	100.1/90	 	c/γ (Å/°)	188.4/120	187.4/120	75.0/90	198.2/90	270.7/90	 	Wilson B factor (Å2)	31	41	76	77	NA	 	Resolution range (Å)	47.10–1.90 (1.95–1.90)	62.47–2.10 (2.16–2.10)	75.00–2.80 (2.99–2.80)	67.80–3.20 (3.42–3.20)	67.68–3.50 (7.16–6.40, 3.92–3.50)	 	Unique reflections	23,300 (1,524)	17,089 (1,428)	7,964 (1,448)	8,171 (1,343)	8,070 (723, 645)	 	Average multiplicity	9.1 (9.2)	5.2 (5.3)	3.7 (3.7)	22.7 (12.6)	3.8 (3.5, 4.4)	 	Completeness (%)	99.8 (98.5)	100 (100)	99.8 (100)	98.8 (93.4)	51.6 (98.5, 14.1)	 	<I/σI>	11.4 (1.7)	12.0 (1.7)	14.9 (1.5)	13.1 (1.9)	4.6 (4.1, 2.2)	 	Rmerge (%)	14.8 (158.3)	9.3 (98.0)	6.2 (98.9)	29.8 (142.2)	44.9 (40.5, 114.2)	 	Rpim (%)	15.7 (55.3)	10.3 (109.0)	3.7 (53.8)	6.3 (40.0)	24.7 (23.9, 59.9)	 		 	Refinement	 		 	Rwork (%)	17.9	18.4	21.6	20.2	32.1	 	Rfree (%)	22.7	23.2	30.7	27.1	35.5	 		 	No. of Non-Hydrogen Atoms	 		 	Protein	2,260	2,301	2,102	2,305	7,730	 	N-glycans	42	42	28	28	0	 	Water	79	54	0	2	0	 	Ligands	6	6	2	5	0	 		 	Average B factor (Å2)	 		 	Protein	63	65	108	84	–	 	N-glycans	35	46	102	18	–	 	Water	68	85	–	75	–	 	Ligands	36	47	91	75	66	 		 	RMSD from Ideality	 		 	Bond lengths (Å)	0.020	0.016	0.019	0.016	0.004	 	Bond angles (°)	2.050	1.748	1.952	1.796	0.770	 		 	Ramachandran Plot	 		 	Favored (%)	96.8	95.5	96.9	94.9	92.3	 	Allowed (%)	99.7	100.0	100.0	99.7	99.8	 	Number of outliers	1	0	0	1	2	 	PDB code	5FWS	5FWT	5FWU	5FWV	5FWW	 	"	TABLE
+1417	1422	Water	chemical	"	KRM1ECD	KRM1ECD	KRM1ECD	KRM1ECD	LRP6PE3PE4-DKKCRD2-KRM1ECD	 	Crystal form	I	I	II	III	I	 	X-ray source	Diamond i04	Diamond i03	Diamond i03	Diamond i04	Diamond i04	 	Wavelength (Å)	0.9793	0.9700	0.9700	0.9795	0.9795	 	Space group	P3121	P3121	P43	P41212	C2221	 	Unit cell a/α (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	86.9/90	 	b/β (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	100.1/90	 	c/γ (Å/°)	188.4/120	187.4/120	75.0/90	198.2/90	270.7/90	 	Wilson B factor (Å2)	31	41	76	77	NA	 	Resolution range (Å)	47.10–1.90 (1.95–1.90)	62.47–2.10 (2.16–2.10)	75.00–2.80 (2.99–2.80)	67.80–3.20 (3.42–3.20)	67.68–3.50 (7.16–6.40, 3.92–3.50)	 	Unique reflections	23,300 (1,524)	17,089 (1,428)	7,964 (1,448)	8,171 (1,343)	8,070 (723, 645)	 	Average multiplicity	9.1 (9.2)	5.2 (5.3)	3.7 (3.7)	22.7 (12.6)	3.8 (3.5, 4.4)	 	Completeness (%)	99.8 (98.5)	100 (100)	99.8 (100)	98.8 (93.4)	51.6 (98.5, 14.1)	 	<I/σI>	11.4 (1.7)	12.0 (1.7)	14.9 (1.5)	13.1 (1.9)	4.6 (4.1, 2.2)	 	Rmerge (%)	14.8 (158.3)	9.3 (98.0)	6.2 (98.9)	29.8 (142.2)	44.9 (40.5, 114.2)	 	Rpim (%)	15.7 (55.3)	10.3 (109.0)	3.7 (53.8)	6.3 (40.0)	24.7 (23.9, 59.9)	 		 	Refinement	 		 	Rwork (%)	17.9	18.4	21.6	20.2	32.1	 	Rfree (%)	22.7	23.2	30.7	27.1	35.5	 		 	No. of Non-Hydrogen Atoms	 		 	Protein	2,260	2,301	2,102	2,305	7,730	 	N-glycans	42	42	28	28	0	 	Water	79	54	0	2	0	 	Ligands	6	6	2	5	0	 		 	Average B factor (Å2)	 		 	Protein	63	65	108	84	–	 	N-glycans	35	46	102	18	–	 	Water	68	85	–	75	–	 	Ligands	36	47	91	75	66	 		 	RMSD from Ideality	 		 	Bond lengths (Å)	0.020	0.016	0.019	0.016	0.004	 	Bond angles (°)	2.050	1.748	1.952	1.796	0.770	 		 	Ramachandran Plot	 		 	Favored (%)	96.8	95.5	96.9	94.9	92.3	 	Allowed (%)	99.7	100.0	100.0	99.7	99.8	 	Number of outliers	1	0	0	1	2	 	PDB code	5FWS	5FWT	5FWU	5FWV	5FWW	 	"	TABLE
+1466	1470	RMSD	evidence	"	KRM1ECD	KRM1ECD	KRM1ECD	KRM1ECD	LRP6PE3PE4-DKKCRD2-KRM1ECD	 	Crystal form	I	I	II	III	I	 	X-ray source	Diamond i04	Diamond i03	Diamond i03	Diamond i04	Diamond i04	 	Wavelength (Å)	0.9793	0.9700	0.9700	0.9795	0.9795	 	Space group	P3121	P3121	P43	P41212	C2221	 	Unit cell a/α (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	86.9/90	 	b/β (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	100.1/90	 	c/γ (Å/°)	188.4/120	187.4/120	75.0/90	198.2/90	270.7/90	 	Wilson B factor (Å2)	31	41	76	77	NA	 	Resolution range (Å)	47.10–1.90 (1.95–1.90)	62.47–2.10 (2.16–2.10)	75.00–2.80 (2.99–2.80)	67.80–3.20 (3.42–3.20)	67.68–3.50 (7.16–6.40, 3.92–3.50)	 	Unique reflections	23,300 (1,524)	17,089 (1,428)	7,964 (1,448)	8,171 (1,343)	8,070 (723, 645)	 	Average multiplicity	9.1 (9.2)	5.2 (5.3)	3.7 (3.7)	22.7 (12.6)	3.8 (3.5, 4.4)	 	Completeness (%)	99.8 (98.5)	100 (100)	99.8 (100)	98.8 (93.4)	51.6 (98.5, 14.1)	 	<I/σI>	11.4 (1.7)	12.0 (1.7)	14.9 (1.5)	13.1 (1.9)	4.6 (4.1, 2.2)	 	Rmerge (%)	14.8 (158.3)	9.3 (98.0)	6.2 (98.9)	29.8 (142.2)	44.9 (40.5, 114.2)	 	Rpim (%)	15.7 (55.3)	10.3 (109.0)	3.7 (53.8)	6.3 (40.0)	24.7 (23.9, 59.9)	 		 	Refinement	 		 	Rwork (%)	17.9	18.4	21.6	20.2	32.1	 	Rfree (%)	22.7	23.2	30.7	27.1	35.5	 		 	No. of Non-Hydrogen Atoms	 		 	Protein	2,260	2,301	2,102	2,305	7,730	 	N-glycans	42	42	28	28	0	 	Water	79	54	0	2	0	 	Ligands	6	6	2	5	0	 		 	Average B factor (Å2)	 		 	Protein	63	65	108	84	–	 	N-glycans	35	46	102	18	–	 	Water	68	85	–	75	–	 	Ligands	36	47	91	75	66	 		 	RMSD from Ideality	 		 	Bond lengths (Å)	0.020	0.016	0.019	0.016	0.004	 	Bond angles (°)	2.050	1.748	1.952	1.796	0.770	 		 	Ramachandran Plot	 		 	Favored (%)	96.8	95.5	96.9	94.9	92.3	 	Allowed (%)	99.7	100.0	100.0	99.7	99.8	 	Number of outliers	1	0	0	1	2	 	PDB code	5FWS	5FWT	5FWU	5FWV	5FWW	 	"	TABLE
+99	115	diffraction data	evidence	An additional shell given for the ternary complex corresponds to the last shell with near-complete diffraction data.	TABLE
diff --git a/annotation_CSV/PMC5063996.csv b/annotation_CSV/PMC5063996.csv
new file mode 100644
index 0000000000000000000000000000000000000000..6cd57fef48735a42cf4c86f3f171597621ba6f2d
--- /dev/null
+++ b/annotation_CSV/PMC5063996.csv
@@ -0,0 +1,1047 @@
+anno_start	anno_end	anno_text	entity_type	sentence	section
+23	39	Arabinoxylanases	protein_type	The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans*	TITLE
+54	70	Highly Decorated	protein_state	The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans*	TITLE
+71	77	Xylans	chemical	The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans*	TITLE
+29	34	plant	taxonomy_domain	The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance.	ABSTRACT
+0	5	Xylan	chemical	Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains.	ABSTRACT
+32	37	plant	taxonomy_domain	Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains.	ABSTRACT
+75	87	β-1,4-xylose	chemical	Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains.	ABSTRACT
+89	93	Xylp	chemical	Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains.	ABSTRACT
+131	146	arabinofuranose	chemical	Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains.	ABSTRACT
+148	152	Araf	chemical	Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains.	ABSTRACT
+8	28	penta-modular enzyme	protein_type	A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans.	ABSTRACT
+30	37	CtXyl5A	protein	A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans.	ABSTRACT
+83	96	arabinoxylans	chemical	A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans.	ABSTRACT
+19	36	crystal structure	evidence	Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands.	ABSTRACT
+44	59	arabinoxylanase	protein_type	Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands.	ABSTRACT
+75	90	in complex with	protein_state	Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands.	ABSTRACT
+91	98	ligands	chemical	Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands.	ABSTRACT
+95	111	catalytic domain	structure_element	The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain.	ABSTRACT
+15	56	non-catalytic carbohydrate binding module	structure_element	The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility.	ABSTRACT
+86	103	crystal structure	evidence	The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility.	ABSTRACT
+4	13	structure	evidence	The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center.	ABSTRACT
+28	43	in complex with	protein_state	The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center.	ABSTRACT
+44	94	Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp	chemical	The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center.	ABSTRACT
+111	115	Araf	chemical	The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center.	ABSTRACT
+144	150	xylose	chemical	The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center.	ABSTRACT
+158	169	active site	site	The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center.	ABSTRACT
+188	194	pocket	site	The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center.	ABSTRACT
+196	207	−2* subsite	site	The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center.	ABSTRACT
+229	245	catalytic center	site	The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center.	ABSTRACT
+4	15	−2* subsite	site	The −2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants.	ABSTRACT
+33	37	Xylp	chemical	The −2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants.	ABSTRACT
+42	46	Arap	chemical	The −2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants.	ABSTRACT
+86	92	xylose	chemical	The −2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants.	ABSTRACT
+97	106	arabinose	chemical	The −2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants.	ABSTRACT
+0	20	Alanine substitution	experimental_method	Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity.	ABSTRACT
+24	29	Glu68	residue_name_number	Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity.	ABSTRACT
+31	36	Tyr92	residue_name_number	Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity.	ABSTRACT
+41	47	Asn139	residue_name_number	Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity.	ABSTRACT
+69	78	arabinose	chemical	Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity.	ABSTRACT
+83	89	xylose	chemical	Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity.	ABSTRACT
+109	120	−2* subsite	site	Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity.	ABSTRACT
+14	25	active site	site	Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed.	ABSTRACT
+31	36	xylan	chemical	Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed.	ABSTRACT
+115	130	solvent-exposed	protein_state	Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed.	ABSTRACT
+18	25	CtXyl5A	protein	This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack.	ABSTRACT
+52	58	xylans	chemical	This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack.	ABSTRACT
+127	140	endo-xylanase	protein_type	This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack.	ABSTRACT
+4	9	plant	taxonomy_domain	The plant cell wall is an important biological substrate.	INTRO
+53	67	microorganisms	taxonomy_domain	This complex composite structure is depolymerized by microorganisms that occupy important highly competitive ecological niches, whereas the process makes an important contribution to the carbon cycle.	INTRO
+15	20	plant	taxonomy_domain	Given that the plant cell wall is the most abundant source of renewable organic carbon on the planet, this macromolecular substrate has substantial industrial potential.	INTRO
+45	50	plant	taxonomy_domain	An example of the chemical complexity of the plant cell wall is provided by xylan, which is the major hemicellulosic component.	INTRO
+76	81	xylan	chemical	An example of the chemical complexity of the plant cell wall is provided by xylan, which is the major hemicellulosic component.	INTRO
+5	19	polysaccharide	chemical	This polysaccharide comprises a backbone of β-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with α-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated.	INTRO
+44	58	β-1,4-d-xylose	chemical	This polysaccharide comprises a backbone of β-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with α-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated.	INTRO
+77	85	pyranose	chemical	This polysaccharide comprises a backbone of β-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with α-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated.	INTRO
+101	105	Xylp	chemical	This polysaccharide comprises a backbone of β-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with α-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated.	INTRO
+137	165	4-O-methyl-d-glucuronic acid	chemical	This polysaccharide comprises a backbone of β-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with α-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated.	INTRO
+167	171	GlcA	chemical	This polysaccharide comprises a backbone of β-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with α-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated.	INTRO
+198	217	α-l-arabinofuranose	chemical	This polysaccharide comprises a backbone of β-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with α-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated.	INTRO
+219	223	Araf	chemical	This polysaccharide comprises a backbone of β-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with α-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated.	INTRO
+247	261	polysaccharide	chemical	This polysaccharide comprises a backbone of β-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with α-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated.	INTRO
+17	21	Araf	chemical	In addition, the Araf side chain decorations can also be esterified to ferulic acid that, in some species, provide a chemical link between hemicellulose and lignin.	INTRO
+71	83	ferulic acid	chemical	In addition, the Araf side chain decorations can also be esterified to ferulic acid that, in some species, provide a chemical link between hemicellulose and lignin.	INTRO
+139	152	hemicellulose	chemical	In addition, the Araf side chain decorations can also be esterified to ferulic acid that, in some species, provide a chemical link between hemicellulose and lignin.	INTRO
+157	163	lignin	chemical	In addition, the Araf side chain decorations can also be esterified to ferulic acid that, in some species, provide a chemical link between hemicellulose and lignin.	INTRO
+25	31	xylans	chemical	The precise structure of xylans varies between plant species, in particular in different tissues and during cellular differentiation.	INTRO
+47	52	plant	taxonomy_domain	The precise structure of xylans varies between plant species, in particular in different tissues and during cellular differentiation.	INTRO
+15	20	plant	taxonomy_domain	In specialized plant tissues, such as the outer layer of cereal grains, xylans are extremely complex, and side chains may comprise a range of other sugars including l- and d-galactose and β- and α-Xylp units.	INTRO
+57	63	cereal	taxonomy_domain	In specialized plant tissues, such as the outer layer of cereal grains, xylans are extremely complex, and side chains may comprise a range of other sugars including l- and d-galactose and β- and α-Xylp units.	INTRO
+72	78	xylans	chemical	In specialized plant tissues, such as the outer layer of cereal grains, xylans are extremely complex, and side chains may comprise a range of other sugars including l- and d-galactose and β- and α-Xylp units.	INTRO
+148	154	sugars	chemical	In specialized plant tissues, such as the outer layer of cereal grains, xylans are extremely complex, and side chains may comprise a range of other sugars including l- and d-galactose and β- and α-Xylp units.	INTRO
+165	183	l- and d-galactose	chemical	In specialized plant tissues, such as the outer layer of cereal grains, xylans are extremely complex, and side chains may comprise a range of other sugars including l- and d-galactose and β- and α-Xylp units.	INTRO
+188	201	β- and α-Xylp	chemical	In specialized plant tissues, such as the outer layer of cereal grains, xylans are extremely complex, and side chains may comprise a range of other sugars including l- and d-galactose and β- and α-Xylp units.	INTRO
+17	23	cereal	taxonomy_domain	Indeed, in these cereal brans, xylans have very few backbone Xylp units that are undecorated, and the side chains can contain up to six sugars.	INTRO
+31	37	xylans	chemical	Indeed, in these cereal brans, xylans have very few backbone Xylp units that are undecorated, and the side chains can contain up to six sugars.	INTRO
+61	65	Xylp	chemical	Indeed, in these cereal brans, xylans have very few backbone Xylp units that are undecorated, and the side chains can contain up to six sugars.	INTRO
+136	142	sugars	chemical	Indeed, in these cereal brans, xylans have very few backbone Xylp units that are undecorated, and the side chains can contain up to six sugars.	INTRO
+55	60	plant	taxonomy_domain	Reflecting the chemical and physical complexity of the plant cell wall, microorganisms that utilize these composite structures express a large number of polysaccharide-degrading enzymes, primarily glycoside hydrolases, but also polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases.	INTRO
+72	86	microorganisms	taxonomy_domain	Reflecting the chemical and physical complexity of the plant cell wall, microorganisms that utilize these composite structures express a large number of polysaccharide-degrading enzymes, primarily glycoside hydrolases, but also polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases.	INTRO
+153	185	polysaccharide-degrading enzymes	protein_type	Reflecting the chemical and physical complexity of the plant cell wall, microorganisms that utilize these composite structures express a large number of polysaccharide-degrading enzymes, primarily glycoside hydrolases, but also polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases.	INTRO
+197	217	glycoside hydrolases	protein_type	Reflecting the chemical and physical complexity of the plant cell wall, microorganisms that utilize these composite structures express a large number of polysaccharide-degrading enzymes, primarily glycoside hydrolases, but also polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases.	INTRO
+228	249	polysaccharide lyases	protein_type	Reflecting the chemical and physical complexity of the plant cell wall, microorganisms that utilize these composite structures express a large number of polysaccharide-degrading enzymes, primarily glycoside hydrolases, but also polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases.	INTRO
+251	273	carbohydrate esterases	protein_type	Reflecting the chemical and physical complexity of the plant cell wall, microorganisms that utilize these composite structures express a large number of polysaccharide-degrading enzymes, primarily glycoside hydrolases, but also polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases.	INTRO
+279	314	lytic polysaccharide monooxygenases	protein_type	Reflecting the chemical and physical complexity of the plant cell wall, microorganisms that utilize these composite structures express a large number of polysaccharide-degrading enzymes, primarily glycoside hydrolases, but also polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases.	INTRO
+6	33	carbohydrate active enzymes	protein_type	These carbohydrate active enzymes are grouped into sequence-based families in the CAZy database.	INTRO
+16	21	xylan	chemical	With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8.	INTRO
+58	64	xylans	chemical	With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8.	INTRO
+82	103	endo-acting xylanases	protein_type	With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8.	INTRO
+142	161	glycoside hydrolase	protein_type	With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8.	INTRO
+163	165	GH	protein_type	With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8.	INTRO
+166	167	5	protein_type	With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8.	INTRO
+177	181	GH10	protein_type	With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8.	INTRO
+186	190	GH11	protein_type	With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8.	INTRO
+226	229	GH8	protein_type	With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8.	INTRO
+32	37	xylan	chemical	The extensive decoration of the xylan backbone generally restricts the capacity of these enzymes to attack the polysaccharide prior to removal of the side chains by a range of α-glucuronidases, α-arabinofuranosidases, and esterases.	INTRO
+111	125	polysaccharide	chemical	The extensive decoration of the xylan backbone generally restricts the capacity of these enzymes to attack the polysaccharide prior to removal of the side chains by a range of α-glucuronidases, α-arabinofuranosidases, and esterases.	INTRO
+176	192	α-glucuronidases	protein_type	The extensive decoration of the xylan backbone generally restricts the capacity of these enzymes to attack the polysaccharide prior to removal of the side chains by a range of α-glucuronidases, α-arabinofuranosidases, and esterases.	INTRO
+194	216	α-arabinofuranosidases	protein_type	The extensive decoration of the xylan backbone generally restricts the capacity of these enzymes to attack the polysaccharide prior to removal of the side chains by a range of α-glucuronidases, α-arabinofuranosidases, and esterases.	INTRO
+222	231	esterases	protein_type	The extensive decoration of the xylan backbone generally restricts the capacity of these enzymes to attack the polysaccharide prior to removal of the side chains by a range of α-glucuronidases, α-arabinofuranosidases, and esterases.	INTRO
+4	13	xylanases	protein_type	Two xylanases, however, utilize the side chains as essential specificity determinants and thus target decorated forms of the hemicellulose.	INTRO
+125	138	hemicellulose	chemical	Two xylanases, however, utilize the side chains as essential specificity determinants and thus target decorated forms of the hemicellulose.	INTRO
+4	8	GH30	protein_type	The GH30 glucuronoxylanases require the Xylp bound at the −2 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites −1 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively).	INTRO
+9	27	glucuronoxylanases	protein_type	The GH30 glucuronoxylanases require the Xylp bound at the −2 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites −1 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively).	INTRO
+40	44	Xylp	chemical	The GH30 glucuronoxylanases require the Xylp bound at the −2 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites −1 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively).	INTRO
+45	53	bound at	protein_state	The GH30 glucuronoxylanases require the Xylp bound at the −2 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites −1 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively).	INTRO
+58	60	−2	site	The GH30 glucuronoxylanases require the Xylp bound at the −2 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites −1 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively).	INTRO
+74	78	GlcA	chemical	The GH30 glucuronoxylanases require the Xylp bound at the −2 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites −1 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively).	INTRO
+121	141	glycoside hydrolases	protein_type	The GH30 glucuronoxylanases require the Xylp bound at the −2 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites −1 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively).	INTRO
+153	171	subsites −1 and +1	site	The GH30 glucuronoxylanases require the Xylp bound at the −2 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites −1 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively).	INTRO
+177	185	subsites	site	The GH30 glucuronoxylanases require the Xylp bound at the −2 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites −1 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively).	INTRO
+4	7	GH5	protein_type	The GH5 arabinoxylanase (CtXyl5A) derived from Clostridium thermocellum displays an absolute requirement for xylans that contain Araf side chains.	INTRO
+8	23	arabinoxylanase	protein_type	The GH5 arabinoxylanase (CtXyl5A) derived from Clostridium thermocellum displays an absolute requirement for xylans that contain Araf side chains.	INTRO
+25	32	CtXyl5A	protein	The GH5 arabinoxylanase (CtXyl5A) derived from Clostridium thermocellum displays an absolute requirement for xylans that contain Araf side chains.	INTRO
+47	71	Clostridium thermocellum	species	The GH5 arabinoxylanase (CtXyl5A) derived from Clostridium thermocellum displays an absolute requirement for xylans that contain Araf side chains.	INTRO
+109	115	xylans	chemical	The GH5 arabinoxylanase (CtXyl5A) derived from Clostridium thermocellum displays an absolute requirement for xylans that contain Araf side chains.	INTRO
+129	133	Araf	chemical	The GH5 arabinoxylanase (CtXyl5A) derived from Clostridium thermocellum displays an absolute requirement for xylans that contain Araf side chains.	INTRO
+55	59	Araf	chemical	In this enzyme, the key specificity determinant is the Araf appended to O3 of the Xylp bound in the active site (−1 subsite).	INTRO
+82	86	Xylp	chemical	In this enzyme, the key specificity determinant is the Araf appended to O3 of the Xylp bound in the active site (−1 subsite).	INTRO
+87	95	bound in	protein_state	In this enzyme, the key specificity determinant is the Araf appended to O3 of the Xylp bound in the active site (−1 subsite).	INTRO
+100	111	active site	site	In this enzyme, the key specificity determinant is the Araf appended to O3 of the Xylp bound in the active site (−1 subsite).	INTRO
+113	123	−1 subsite	site	In this enzyme, the key specificity determinant is the Araf appended to O3 of the Xylp bound in the active site (−1 subsite).	INTRO
+37	50	arabinoxylans	chemical	The reaction products generated from arabinoxylans, however, suggest that Araf can be accommodated at subsites distal to the active site.	INTRO
+74	78	Araf	chemical	The reaction products generated from arabinoxylans, however, suggest that Araf can be accommodated at subsites distal to the active site.	INTRO
+102	110	subsites	site	The reaction products generated from arabinoxylans, however, suggest that Araf can be accommodated at subsites distal to the active site.	INTRO
+125	136	active site	site	The reaction products generated from arabinoxylans, however, suggest that Araf can be accommodated at subsites distal to the active site.	INTRO
+0	7	CtXyl5A	protein	CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.	INTRO
+64	67	GH5	protein_type	CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.	INTRO
+68	84	catalytic module	structure_element	CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.	INTRO
+86	91	CtGH5	structure_element	CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.	INTRO
+100	142	non-catalytic carbohydrate binding modules	structure_element	CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.	INTRO
+144	148	CBMs	structure_element	CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.	INTRO
+172	173	6	protein_type	CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.	INTRO
+175	181	CtCBM6	structure_element	CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.	INTRO
+184	186	13	protein_type	CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.	INTRO
+188	195	CtCBM13	structure_element	CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.	INTRO
+202	204	62	protein_type	CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.	INTRO
+206	213	CtCBM62	structure_element	CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.	INTRO
+216	234	fibronectin type 3	protein_type	CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.	INTRO
+236	239	Fn3	structure_element	CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.	INTRO
+266	274	dockerin	structure_element	CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.	INTRO
+20	23	Fn3	structure_element	Previous studies of Fn3 domains have indicated that they might function as ligand-binding modules, as a compact form of peptide linkers or spacers between other domains, as cellulose-disrupting modules, or as proteins that help large enzyme complexes remain soluble.	INTRO
+75	97	ligand-binding modules	structure_element	Previous studies of Fn3 domains have indicated that they might function as ligand-binding modules, as a compact form of peptide linkers or spacers between other domains, as cellulose-disrupting modules, or as proteins that help large enzyme complexes remain soluble.	INTRO
+173	201	cellulose-disrupting modules	structure_element	Previous studies of Fn3 domains have indicated that they might function as ligand-binding modules, as a compact form of peptide linkers or spacers between other domains, as cellulose-disrupting modules, or as proteins that help large enzyme complexes remain soluble.	INTRO
+4	12	dockerin	structure_element	The dockerin domain recruits the enzyme into the cellulosome, a multienzyme plant cell wall degrading complex presented on the surface of C. thermocellum.	INTRO
+49	60	cellulosome	complex_assembly	The dockerin domain recruits the enzyme into the cellulosome, a multienzyme plant cell wall degrading complex presented on the surface of C. thermocellum.	INTRO
+76	81	plant	taxonomy_domain	The dockerin domain recruits the enzyme into the cellulosome, a multienzyme plant cell wall degrading complex presented on the surface of C. thermocellum.	INTRO
+138	153	C. thermocellum	species	The dockerin domain recruits the enzyme into the cellulosome, a multienzyme plant cell wall degrading complex presented on the surface of C. thermocellum.	INTRO
+0	6	CtCBM6	structure_element	CtCBM6 stabilizes CtGH5, and CtCBM62 binds to d-galactopyranose and l-arabinopyranose.	INTRO
+18	23	CtGH5	structure_element	CtCBM6 stabilizes CtGH5, and CtCBM62 binds to d-galactopyranose and l-arabinopyranose.	INTRO
+29	36	CtCBM62	structure_element	CtCBM6 stabilizes CtGH5, and CtCBM62 binds to d-galactopyranose and l-arabinopyranose.	INTRO
+46	63	d-galactopyranose	chemical	CtCBM6 stabilizes CtGH5, and CtCBM62 binds to d-galactopyranose and l-arabinopyranose.	INTRO
+68	85	l-arabinopyranose	chemical	CtCBM6 stabilizes CtGH5, and CtCBM62 binds to d-galactopyranose and l-arabinopyranose.	INTRO
+20	27	CtCBM13	structure_element	The function of the CtCBM13 and Fn3 modules remains unclear.	INTRO
+32	35	Fn3	structure_element	The function of the CtCBM13 and Fn3 modules remains unclear.	INTRO
+25	42	crystal structure	evidence	This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity.	INTRO
+46	52	mature	protein_state	This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity.	INTRO
+53	60	CtXyl5A	protein	This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity.	INTRO
+61	68	lacking	protein_state	This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity.	INTRO
+84	92	dockerin	structure_element	This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity.	INTRO
+101	112	CtXyl5A-Doc	mutant	This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity.	INTRO
+130	145	in complex with	protein_state	This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity.	INTRO
+146	153	ligands	chemical	This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity.	INTRO
+106	112	xylans	chemical	The data show that the plasticity in substrate recognition enables the enzyme to hydrolyze highly complex xylans that are not accessible to classical GH10 and GH11 endo-xylanases.	INTRO
+150	154	GH10	protein_type	The data show that the plasticity in substrate recognition enables the enzyme to hydrolyze highly complex xylans that are not accessible to classical GH10 and GH11 endo-xylanases.	INTRO
+159	163	GH11	protein_type	The data show that the plasticity in substrate recognition enables the enzyme to hydrolyze highly complex xylans that are not accessible to classical GH10 and GH11 endo-xylanases.	INTRO
+164	178	endo-xylanases	protein_type	The data show that the plasticity in substrate recognition enables the enzyme to hydrolyze highly complex xylans that are not accessible to classical GH10 and GH11 endo-xylanases.	INTRO
+26	32	GH5_34	protein_type	Molecular architecture of GH5_34 enzymes.	FIG
+20	22	GH	structure_element	Modules prefaced by GH, CBM, or CE are modules in the indicated glycoside hydrolase, carbohydrate binding module, or carbohydrate esterase families, respectively.	FIG
+24	27	CBM	structure_element	Modules prefaced by GH, CBM, or CE are modules in the indicated glycoside hydrolase, carbohydrate binding module, or carbohydrate esterase families, respectively.	FIG
+32	34	CE	structure_element	Modules prefaced by GH, CBM, or CE are modules in the indicated glycoside hydrolase, carbohydrate binding module, or carbohydrate esterase families, respectively.	FIG
+64	83	glycoside hydrolase	protein_type	Modules prefaced by GH, CBM, or CE are modules in the indicated glycoside hydrolase, carbohydrate binding module, or carbohydrate esterase families, respectively.	FIG
+85	112	carbohydrate binding module	structure_element	Modules prefaced by GH, CBM, or CE are modules in the indicated glycoside hydrolase, carbohydrate binding module, or carbohydrate esterase families, respectively.	FIG
+117	138	carbohydrate esterase	protein_type	Modules prefaced by GH, CBM, or CE are modules in the indicated glycoside hydrolase, carbohydrate binding module, or carbohydrate esterase families, respectively.	FIG
+0	11	Laminin_3_G	structure_element	Laminin_3_G domain belongs to the concanavalin A lectin superfamily, and FN3 denotes a fibronectin type 3 domain.	FIG
+34	67	concanavalin A lectin superfamily	protein_type	Laminin_3_G domain belongs to the concanavalin A lectin superfamily, and FN3 denotes a fibronectin type 3 domain.	FIG
+73	76	FN3	structure_element	Laminin_3_G domain belongs to the concanavalin A lectin superfamily, and FN3 denotes a fibronectin type 3 domain.	FIG
+87	112	fibronectin type 3 domain	structure_element	Laminin_3_G domain belongs to the concanavalin A lectin superfamily, and FN3 denotes a fibronectin type 3 domain.	FIG
+23	31	dockerin	structure_element	Segments labeled D are dockerin domains.	FIG
+25	32	CtXyl5A	protein	Substrate Specificity of CtXyl5A	RESULTS
+29	36	CtXyl5A	protein	Previous studies showed that CtXyl5A is an arabinoxylan-specific xylanase that generates xylooligosaccharides with an arabinose linked O3 to the reducing end xylose.	RESULTS
+43	73	arabinoxylan-specific xylanase	protein_type	Previous studies showed that CtXyl5A is an arabinoxylan-specific xylanase that generates xylooligosaccharides with an arabinose linked O3 to the reducing end xylose.	RESULTS
+89	109	xylooligosaccharides	chemical	Previous studies showed that CtXyl5A is an arabinoxylan-specific xylanase that generates xylooligosaccharides with an arabinose linked O3 to the reducing end xylose.	RESULTS
+118	127	arabinose	chemical	Previous studies showed that CtXyl5A is an arabinoxylan-specific xylanase that generates xylooligosaccharides with an arabinose linked O3 to the reducing end xylose.	RESULTS
+158	164	xylose	chemical	Previous studies showed that CtXyl5A is an arabinoxylan-specific xylanase that generates xylooligosaccharides with an arabinose linked O3 to the reducing end xylose.	RESULTS
+34	39	wheat	taxonomy_domain	The enzyme is active against both wheat and rye arabinoxylans (abbreviated as WAX and RAX, respectively).	RESULTS
+44	47	rye	taxonomy_domain	The enzyme is active against both wheat and rye arabinoxylans (abbreviated as WAX and RAX, respectively).	RESULTS
+48	61	arabinoxylans	chemical	The enzyme is active against both wheat and rye arabinoxylans (abbreviated as WAX and RAX, respectively).	RESULTS
+78	81	WAX	chemical	The enzyme is active against both wheat and rye arabinoxylans (abbreviated as WAX and RAX, respectively).	RESULTS
+86	89	RAX	chemical	The enzyme is active against both wheat and rye arabinoxylans (abbreviated as WAX and RAX, respectively).	RESULTS
+21	30	arabinose	chemical	It was proposed that arabinose decorations make productive interactions with a pocket (−2*) that is abutted onto the active site or −1 subsite.	RESULTS
+79	85	pocket	site	It was proposed that arabinose decorations make productive interactions with a pocket (−2*) that is abutted onto the active site or −1 subsite.	RESULTS
+87	90	−2*	site	It was proposed that arabinose decorations make productive interactions with a pocket (−2*) that is abutted onto the active site or −1 subsite.	RESULTS
+117	128	active site	site	It was proposed that arabinose decorations make productive interactions with a pocket (−2*) that is abutted onto the active site or −1 subsite.	RESULTS
+132	142	−1 subsite	site	It was proposed that arabinose decorations make productive interactions with a pocket (−2*) that is abutted onto the active site or −1 subsite.	RESULTS
+0	9	Arabinose	chemical	Arabinose side chains of the other backbone xylose units in the oligosaccharides generated by CtXyl5A were essentially random.	RESULTS
+44	50	xylose	chemical	Arabinose side chains of the other backbone xylose units in the oligosaccharides generated by CtXyl5A were essentially random.	RESULTS
+64	80	oligosaccharides	chemical	Arabinose side chains of the other backbone xylose units in the oligosaccharides generated by CtXyl5A were essentially random.	RESULTS
+94	101	CtXyl5A	protein	Arabinose side chains of the other backbone xylose units in the oligosaccharides generated by CtXyl5A were essentially random.	RESULTS
+52	58	xylose	chemical	These data suggest that O3, and possibly O2, on the xylose residues at subsites distal to the active site and −2* pocket are solvent-exposed, implying that the enzyme can access highly decorated xylans.	RESULTS
+71	79	subsites	site	These data suggest that O3, and possibly O2, on the xylose residues at subsites distal to the active site and −2* pocket are solvent-exposed, implying that the enzyme can access highly decorated xylans.	RESULTS
+94	105	active site	site	These data suggest that O3, and possibly O2, on the xylose residues at subsites distal to the active site and −2* pocket are solvent-exposed, implying that the enzyme can access highly decorated xylans.	RESULTS
+110	120	−2* pocket	site	These data suggest that O3, and possibly O2, on the xylose residues at subsites distal to the active site and −2* pocket are solvent-exposed, implying that the enzyme can access highly decorated xylans.	RESULTS
+125	140	solvent-exposed	protein_state	These data suggest that O3, and possibly O2, on the xylose residues at subsites distal to the active site and −2* pocket are solvent-exposed, implying that the enzyme can access highly decorated xylans.	RESULTS
+195	201	xylans	chemical	These data suggest that O3, and possibly O2, on the xylose residues at subsites distal to the active site and −2* pocket are solvent-exposed, implying that the enzyme can access highly decorated xylans.	RESULTS
+41	48	CtXyl5A	protein	To test this hypothesis, the activity of CtXyl5A against xylans from cereal brans was assessed.	RESULTS
+57	63	xylans	chemical	To test this hypothesis, the activity of CtXyl5A against xylans from cereal brans was assessed.	RESULTS
+69	75	cereal	taxonomy_domain	To test this hypothesis, the activity of CtXyl5A against xylans from cereal brans was assessed.	RESULTS
+0	7	CtXyl5a	protein	CtXyl5a was incubated with a range of xylans for 16 h at 60 °C, and the limit products were visualized by TLC.	RESULTS
+12	21	incubated	experimental_method	CtXyl5a was incubated with a range of xylans for 16 h at 60 °C, and the limit products were visualized by TLC.	RESULTS
+38	44	xylans	chemical	CtXyl5a was incubated with a range of xylans for 16 h at 60 °C, and the limit products were visualized by TLC.	RESULTS
+106	109	TLC	experimental_method	CtXyl5a was incubated with a range of xylans for 16 h at 60 °C, and the limit products were visualized by TLC.	RESULTS
+6	12	xylans	chemical	These xylans are highly decorated not only with Araf and GlcA units but also with l-Gal, d-Gal, and d-Xyl.	RESULTS
+48	52	Araf	chemical	These xylans are highly decorated not only with Araf and GlcA units but also with l-Gal, d-Gal, and d-Xyl.	RESULTS
+57	61	GlcA	chemical	These xylans are highly decorated not only with Araf and GlcA units but also with l-Gal, d-Gal, and d-Xyl.	RESULTS
+82	87	l-Gal	chemical	These xylans are highly decorated not only with Araf and GlcA units but also with l-Gal, d-Gal, and d-Xyl.	RESULTS
+89	94	d-Gal	chemical	These xylans are highly decorated not only with Araf and GlcA units but also with l-Gal, d-Gal, and d-Xyl.	RESULTS
+100	105	d-Xyl	chemical	These xylans are highly decorated not only with Araf and GlcA units but also with l-Gal, d-Gal, and d-Xyl.	RESULTS
+17	23	xylose	chemical	Indeed, very few xylose units in the backbone of bran xylans lack side chains.	RESULTS
+54	60	xylans	chemical	Indeed, very few xylose units in the backbone of bran xylans lack side chains.	RESULTS
+42	49	CtXyl5A	protein	The data presented in Table 1 showed that CtXyl5A was active against corn bran xylan (CX).	RESULTS
+69	73	corn	taxonomy_domain	The data presented in Table 1 showed that CtXyl5A was active against corn bran xylan (CX).	RESULTS
+79	84	xylan	chemical	The data presented in Table 1 showed that CtXyl5A was active against corn bran xylan (CX).	RESULTS
+86	88	CX	chemical	The data presented in Table 1 showed that CtXyl5A was active against corn bran xylan (CX).	RESULTS
+20	34	endo-xylanases	protein_type	In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues).	RESULTS
+40	44	GH10	protein_type	In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues).	RESULTS
+49	53	GH11	protein_type	In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues).	RESULTS
+76	78	CX	chemical	In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues).	RESULTS
+95	102	lack of	protein_state	In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues).	RESULTS
+115	121	xylose	chemical	In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues).	RESULTS
+149	160	active site	site	In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues).	RESULTS
+187	194	bind to	protein_state	In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues).	RESULTS
+211	217	xylose	chemical	In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues).	RESULTS
+32	39	CtXyl5A	protein	The limit products generated by CtXyl5A from CX consisted of an extensive range of oligosaccharides.	RESULTS
+45	47	CX	chemical	The limit products generated by CtXyl5A from CX consisted of an extensive range of oligosaccharides.	RESULTS
+83	99	oligosaccharides	chemical	The limit products generated by CtXyl5A from CX consisted of an extensive range of oligosaccharides.	RESULTS
+36	44	subsites	site	These data support the view that in subsites out with the active site the O2 and O3 groups of the bound xylose units are solvent-exposed and will thus tolerate decoration.	RESULTS
+58	69	active site	site	These data support the view that in subsites out with the active site the O2 and O3 groups of the bound xylose units are solvent-exposed and will thus tolerate decoration.	RESULTS
+104	110	xylose	chemical	These data support the view that in subsites out with the active site the O2 and O3 groups of the bound xylose units are solvent-exposed and will thus tolerate decoration.	RESULTS
+121	136	solvent-exposed	protein_state	These data support the view that in subsites out with the active site the O2 and O3 groups of the bound xylose units are solvent-exposed and will thus tolerate decoration.	RESULTS
+0	8	Kinetics	evidence	Kinetics of GH5_34 arabinoxylanases	TABLE
+12	18	GH5_34	protein_type	Kinetics of GH5_34 arabinoxylanases	TABLE
+19	35	arabinoxylanases	protein_type	Kinetics of GH5_34 arabinoxylanases	TABLE
+15	19	kcat	evidence	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+20	22	Km	evidence	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+25	28	WAX	chemical	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+29	32	RAX	chemical	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+33	35	CX	chemical	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+54	61	CtXyl5A	protein	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+62	88	CtGH5-CBM6-CBM13-Fn3-CBM62	structure_element	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+102	109	CtXyl5A	protein	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+110	130	CtGH5-CBM6-CBM13-Fn3	structure_element	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+146	153	CtXyl5A	protein	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+154	170	CtGH5-CBM6-CBM13	structure_element	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+186	193	CtXyl5A	protein	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+194	204	CtGH5-CBM6	structure_element	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+218	225	CtXyl5A	protein	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+226	236	CtGH5-CBM6	structure_element	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+238	242	E68A	mutant	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+254	261	CtXyl5A	protein	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+262	272	CtGH5-CBM6	structure_element	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+274	278	Y92A	mutant	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+290	297	CtXyl5A	protein	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+298	308	CtGH5-CBM6	structure_element	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+310	315	N135A	mutant	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+328	335	CtXyl5A	protein	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+336	346	CtGH5-CBM6	structure_element	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+348	353	N139A	mutant	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+365	370	AcGH5	protein	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+371	380	Wild type	protein_state	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+397	402	GpGH5	protein	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+403	412	Wild type	protein_state	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+431	436	VbGH5	protein	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+437	446	Wild type	protein_state	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+458	463	VbGH5	protein	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+464	468	D45A	mutant	"Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	"	TABLE
+29	42	bound only at	protein_state	To explore whether substrate bound only at −2* and −1 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study.	RESULTS
+43	46	−2*	site	To explore whether substrate bound only at −2* and −1 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study.	RESULTS
+51	53	−1	site	To explore whether substrate bound only at −2* and −1 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study.	RESULTS
+61	78	negative subsites	site	To explore whether substrate bound only at −2* and −1 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study.	RESULTS
+97	104	CtXyl5A	protein	To explore whether substrate bound only at −2* and −1 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study.	RESULTS
+128	130	CX	chemical	To explore whether substrate bound only at −2* and −1 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study.	RESULTS
+147	162	arabinoxylanase	protein_type	To explore whether substrate bound only at −2* and −1 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study.	RESULTS
+181	210	size exclusion chromatography	experimental_method	To explore whether substrate bound only at −2* and −1 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study.	RESULTS
+249	265	oligosaccharides	chemical	To explore whether substrate bound only at −2* and −1 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study.	RESULTS
+0	5	HPAEC	experimental_method	HPAEC analysis of the smallest oligosaccharide fraction (pool 4) contained two species with retention times of 14.0 min (oligosaccharide 1) and 20.8 min (oligosaccharide 2) (Fig. 2).	RESULTS
+31	46	oligosaccharide	chemical	HPAEC analysis of the smallest oligosaccharide fraction (pool 4) contained two species with retention times of 14.0 min (oligosaccharide 1) and 20.8 min (oligosaccharide 2) (Fig. 2).	RESULTS
+121	136	oligosaccharide	chemical	HPAEC analysis of the smallest oligosaccharide fraction (pool 4) contained two species with retention times of 14.0 min (oligosaccharide 1) and 20.8 min (oligosaccharide 2) (Fig. 2).	RESULTS
+154	169	oligosaccharide	chemical	HPAEC analysis of the smallest oligosaccharide fraction (pool 4) contained two species with retention times of 14.0 min (oligosaccharide 1) and 20.8 min (oligosaccharide 2) (Fig. 2).	RESULTS
+0	44	Positive mode electrospray mass spectrometry	experimental_method	Positive mode electrospray mass spectrometry showed that pool 4 contained exclusively molecular ions with a m/z = 305 [M + Na]+, which corresponds to a pentose-pentose disaccharide (molecular mass = 282 Da) as a sodium ion adduct, whereas a dimer of the disaccharide with a sodium adduct (m/z = 587 [2M+Na]+) was also evident.	RESULTS
+152	159	pentose	chemical	Positive mode electrospray mass spectrometry showed that pool 4 contained exclusively molecular ions with a m/z = 305 [M + Na]+, which corresponds to a pentose-pentose disaccharide (molecular mass = 282 Da) as a sodium ion adduct, whereas a dimer of the disaccharide with a sodium adduct (m/z = 587 [2M+Na]+) was also evident.	RESULTS
+160	167	pentose	chemical	Positive mode electrospray mass spectrometry showed that pool 4 contained exclusively molecular ions with a m/z = 305 [M + Na]+, which corresponds to a pentose-pentose disaccharide (molecular mass = 282 Da) as a sodium ion adduct, whereas a dimer of the disaccharide with a sodium adduct (m/z = 587 [2M+Na]+) was also evident.	RESULTS
+168	180	disaccharide	chemical	Positive mode electrospray mass spectrometry showed that pool 4 contained exclusively molecular ions with a m/z = 305 [M + Na]+, which corresponds to a pentose-pentose disaccharide (molecular mass = 282 Da) as a sodium ion adduct, whereas a dimer of the disaccharide with a sodium adduct (m/z = 587 [2M+Na]+) was also evident.	RESULTS
+254	266	disaccharide	chemical	Positive mode electrospray mass spectrometry showed that pool 4 contained exclusively molecular ions with a m/z = 305 [M + Na]+, which corresponds to a pentose-pentose disaccharide (molecular mass = 282 Da) as a sodium ion adduct, whereas a dimer of the disaccharide with a sodium adduct (m/z = 587 [2M+Na]+) was also evident.	RESULTS
+55	69	TFA hydrolysis	experimental_method	The monosaccharide composition of pool 4 determined by TFA hydrolysis contained xylose and arabinose in a 3:1 ratio.	RESULTS
+80	86	xylose	chemical	The monosaccharide composition of pool 4 determined by TFA hydrolysis contained xylose and arabinose in a 3:1 ratio.	RESULTS
+91	100	arabinose	chemical	The monosaccharide composition of pool 4 determined by TFA hydrolysis contained xylose and arabinose in a 3:1 ratio.	RESULTS
+27	43	oligosaccharides	chemical	This suggests that the two oligosaccharides consist of two disaccharides: one consisting of two xylose residues and the other consisting of an arabinose linked to a xylose.	RESULTS
+59	72	disaccharides	chemical	This suggests that the two oligosaccharides consist of two disaccharides: one consisting of two xylose residues and the other consisting of an arabinose linked to a xylose.	RESULTS
+96	102	xylose	chemical	This suggests that the two oligosaccharides consist of two disaccharides: one consisting of two xylose residues and the other consisting of an arabinose linked to a xylose.	RESULTS
+143	152	arabinose	chemical	This suggests that the two oligosaccharides consist of two disaccharides: one consisting of two xylose residues and the other consisting of an arabinose linked to a xylose.	RESULTS
+165	171	xylose	chemical	This suggests that the two oligosaccharides consist of two disaccharides: one consisting of two xylose residues and the other consisting of an arabinose linked to a xylose.	RESULTS
+29	60	nonspecific arabinofuranosidase	protein_type	Treatment of pool 4 with the nonspecific arabinofuranosidase, CjAbf51A, resulted in the loss of oligosaccharide 2 and the production of both xylose and arabinose, indicative of a disaccharide of xylose and arabinose.	RESULTS
+62	70	CjAbf51A	protein	Treatment of pool 4 with the nonspecific arabinofuranosidase, CjAbf51A, resulted in the loss of oligosaccharide 2 and the production of both xylose and arabinose, indicative of a disaccharide of xylose and arabinose.	RESULTS
+96	111	oligosaccharide	chemical	Treatment of pool 4 with the nonspecific arabinofuranosidase, CjAbf51A, resulted in the loss of oligosaccharide 2 and the production of both xylose and arabinose, indicative of a disaccharide of xylose and arabinose.	RESULTS
+141	147	xylose	chemical	Treatment of pool 4 with the nonspecific arabinofuranosidase, CjAbf51A, resulted in the loss of oligosaccharide 2 and the production of both xylose and arabinose, indicative of a disaccharide of xylose and arabinose.	RESULTS
+152	161	arabinose	chemical	Treatment of pool 4 with the nonspecific arabinofuranosidase, CjAbf51A, resulted in the loss of oligosaccharide 2 and the production of both xylose and arabinose, indicative of a disaccharide of xylose and arabinose.	RESULTS
+179	191	disaccharide	chemical	Treatment of pool 4 with the nonspecific arabinofuranosidase, CjAbf51A, resulted in the loss of oligosaccharide 2 and the production of both xylose and arabinose, indicative of a disaccharide of xylose and arabinose.	RESULTS
+195	201	xylose	chemical	Treatment of pool 4 with the nonspecific arabinofuranosidase, CjAbf51A, resulted in the loss of oligosaccharide 2 and the production of both xylose and arabinose, indicative of a disaccharide of xylose and arabinose.	RESULTS
+206	215	arabinose	chemical	Treatment of pool 4 with the nonspecific arabinofuranosidase, CjAbf51A, resulted in the loss of oligosaccharide 2 and the production of both xylose and arabinose, indicative of a disaccharide of xylose and arabinose.	RESULTS
+28	44	β-1,3-xylosidase	protein_type	Incubation of pool 4 with a β-1,3-xylosidase (XynB) converted oligosaccharide 1 into xylose, demonstrating that this molecule is the disaccharide β-1,3-xylobiose.	RESULTS
+46	50	XynB	protein	Incubation of pool 4 with a β-1,3-xylosidase (XynB) converted oligosaccharide 1 into xylose, demonstrating that this molecule is the disaccharide β-1,3-xylobiose.	RESULTS
+62	77	oligosaccharide	chemical	Incubation of pool 4 with a β-1,3-xylosidase (XynB) converted oligosaccharide 1 into xylose, demonstrating that this molecule is the disaccharide β-1,3-xylobiose.	RESULTS
+85	91	xylose	chemical	Incubation of pool 4 with a β-1,3-xylosidase (XynB) converted oligosaccharide 1 into xylose, demonstrating that this molecule is the disaccharide β-1,3-xylobiose.	RESULTS
+133	145	disaccharide	chemical	Incubation of pool 4 with a β-1,3-xylosidase (XynB) converted oligosaccharide 1 into xylose, demonstrating that this molecule is the disaccharide β-1,3-xylobiose.	RESULTS
+146	161	β-1,3-xylobiose	chemical	Incubation of pool 4 with a β-1,3-xylosidase (XynB) converted oligosaccharide 1 into xylose, demonstrating that this molecule is the disaccharide β-1,3-xylobiose.	RESULTS
+45	70	β-1,4-specific xylosidase	protein_type	This view is supported by the inability of a β-1,4-specific xylosidase to hydrolyze oligosaccharide 1 or oligosaccharide 2 (data not shown).	RESULTS
+84	99	oligosaccharide	chemical	This view is supported by the inability of a β-1,4-specific xylosidase to hydrolyze oligosaccharide 1 or oligosaccharide 2 (data not shown).	RESULTS
+105	120	oligosaccharide	chemical	This view is supported by the inability of a β-1,4-specific xylosidase to hydrolyze oligosaccharide 1 or oligosaccharide 2 (data not shown).	RESULTS
+43	53	−2* pocket	site	The crucial importance of occupancy of the −2* pocket for catalytic competence is illustrated by the inability of the enzyme to hydrolyze linear β-1,4-xylooligosaccharides.	RESULTS
+145	171	β-1,4-xylooligosaccharides	chemical	The crucial importance of occupancy of the −2* pocket for catalytic competence is illustrated by the inability of the enzyme to hydrolyze linear β-1,4-xylooligosaccharides.	RESULTS
+18	27	Araf-Xylp	chemical	The generation of Araf-Xylp and Xyl-β-1,3-Xyl as reaction products demonstrates that occupancy of the −2 subsite is not essential for catalytic activity, which is in contrast to all endo-acting xylanases where this subsite plays a critical role in enzyme activity.	RESULTS
+32	45	Xyl-β-1,3-Xyl	chemical	The generation of Araf-Xylp and Xyl-β-1,3-Xyl as reaction products demonstrates that occupancy of the −2 subsite is not essential for catalytic activity, which is in contrast to all endo-acting xylanases where this subsite plays a critical role in enzyme activity.	RESULTS
+102	112	−2 subsite	site	The generation of Araf-Xylp and Xyl-β-1,3-Xyl as reaction products demonstrates that occupancy of the −2 subsite is not essential for catalytic activity, which is in contrast to all endo-acting xylanases where this subsite plays a critical role in enzyme activity.	RESULTS
+182	203	endo-acting xylanases	protein_type	The generation of Araf-Xylp and Xyl-β-1,3-Xyl as reaction products demonstrates that occupancy of the −2 subsite is not essential for catalytic activity, which is in contrast to all endo-acting xylanases where this subsite plays a critical role in enzyme activity.	RESULTS
+215	222	subsite	site	The generation of Araf-Xylp and Xyl-β-1,3-Xyl as reaction products demonstrates that occupancy of the −2 subsite is not essential for catalytic activity, which is in contrast to all endo-acting xylanases where this subsite plays a critical role in enzyme activity.	RESULTS
+34	37	−2*	site	Indeed, the data demonstrate that −2* plays a more important role in productive substrate binding than the −2 subsite.	RESULTS
+107	117	−2 subsite	site	Indeed, the data demonstrate that −2* plays a more important role in productive substrate binding than the −2 subsite.	RESULTS
+57	89	(Xyl-β-1,4)n-[β-1,3-Xyl/Ara]-Xyl	chemical	Unfortunately, the inability to generate highly purified (Xyl-β-1,4)n-[β-1,3-Xyl/Ara]-Xyl oligosaccharides from arabinoxylans prevented the precise binding energies at the negative subsites to be determined.	RESULTS
+90	106	oligosaccharides	chemical	Unfortunately, the inability to generate highly purified (Xyl-β-1,4)n-[β-1,3-Xyl/Ara]-Xyl oligosaccharides from arabinoxylans prevented the precise binding energies at the negative subsites to be determined.	RESULTS
+112	125	arabinoxylans	chemical	Unfortunately, the inability to generate highly purified (Xyl-β-1,4)n-[β-1,3-Xyl/Ara]-Xyl oligosaccharides from arabinoxylans prevented the precise binding energies at the negative subsites to be determined.	RESULTS
+22	34	disaccharide	chemical	Identification of the disaccharide reaction products generated from CX.	FIG
+68	70	CX	chemical	Identification of the disaccharide reaction products generated from CX.	FIG
+48	77	size exclusion chromatography	experimental_method	The smallest reaction products were purified by size exclusion chromatography and analyzed by HPAEC (A) and positive mode ESI-MS (B), respectively.	FIG
+94	99	HPAEC	experimental_method	The smallest reaction products were purified by size exclusion chromatography and analyzed by HPAEC (A) and positive mode ESI-MS (B), respectively.	FIG
+122	128	ESI-MS	experimental_method	The smallest reaction products were purified by size exclusion chromatography and analyzed by HPAEC (A) and positive mode ESI-MS (B), respectively.	FIG
+32	63	nonspecific arabinofuranosidase	protein_type	The samples were treated with a nonspecific arabinofuranosidase (CjAbf51A) and a GH3 xylosidase (XynB) that targeted β-1,3-xylosidic bonds.	FIG
+65	73	CjAbf51A	protein	The samples were treated with a nonspecific arabinofuranosidase (CjAbf51A) and a GH3 xylosidase (XynB) that targeted β-1,3-xylosidic bonds.	FIG
+81	95	GH3 xylosidase	protein_type	The samples were treated with a nonspecific arabinofuranosidase (CjAbf51A) and a GH3 xylosidase (XynB) that targeted β-1,3-xylosidic bonds.	FIG
+97	101	XynB	protein	The samples were treated with a nonspecific arabinofuranosidase (CjAbf51A) and a GH3 xylosidase (XynB) that targeted β-1,3-xylosidic bonds.	FIG
+3	9	xylose	chemical	X, xylose; A, arabinose.	FIG
+14	23	arabinose	chemical	X, xylose; A, arabinose.	FIG
+32	39	pentose	chemical	The m/z = 305 species denotes a pentose disaccharide as a sodium adduct [M + Na]+, whereas the m/z = 587 signal corresponds to an ESI-MS dimer of the pentose disaccharide also as a sodium adduct [2M + Na]+.	FIG
+40	52	disaccharide	chemical	The m/z = 305 species denotes a pentose disaccharide as a sodium adduct [M + Na]+, whereas the m/z = 587 signal corresponds to an ESI-MS dimer of the pentose disaccharide also as a sodium adduct [2M + Na]+.	FIG
+130	136	ESI-MS	experimental_method	The m/z = 305 species denotes a pentose disaccharide as a sodium adduct [M + Na]+, whereas the m/z = 587 signal corresponds to an ESI-MS dimer of the pentose disaccharide also as a sodium adduct [2M + Na]+.	FIG
+150	157	pentose	chemical	The m/z = 305 species denotes a pentose disaccharide as a sodium adduct [M + Na]+, whereas the m/z = 587 signal corresponds to an ESI-MS dimer of the pentose disaccharide also as a sodium adduct [2M + Na]+.	FIG
+158	170	disaccharide	chemical	The m/z = 305 species denotes a pentose disaccharide as a sodium adduct [M + Na]+, whereas the m/z = 587 signal corresponds to an ESI-MS dimer of the pentose disaccharide also as a sodium adduct [2M + Na]+.	FIG
+0	17	Crystal Structure	evidence	Crystal Structure of the Catalytic Module of CtXyl5A in Complex with Ligands	RESULTS
+25	41	Catalytic Module	structure_element	Crystal Structure of the Catalytic Module of CtXyl5A in Complex with Ligands	RESULTS
+45	52	CtXyl5A	protein	Crystal Structure of the Catalytic Module of CtXyl5A in Complex with Ligands	RESULTS
+53	68	in Complex with	protein_state	Crystal Structure of the Catalytic Module of CtXyl5A in Complex with Ligands	RESULTS
+69	76	Ligands	chemical	Crystal Structure of the Catalytic Module of CtXyl5A in Complex with Ligands	RESULTS
+69	76	CtXyl5A	protein	To understand the structural basis for the biochemical properties of CtXyl5A, the crystal structure of the enzyme with ligands that occupy the substrate binding cleft and the critical −2* subsite were sought.	RESULTS
+82	99	crystal structure	evidence	To understand the structural basis for the biochemical properties of CtXyl5A, the crystal structure of the enzyme with ligands that occupy the substrate binding cleft and the critical −2* subsite were sought.	RESULTS
+143	166	substrate binding cleft	site	To understand the structural basis for the biochemical properties of CtXyl5A, the crystal structure of the enzyme with ligands that occupy the substrate binding cleft and the critical −2* subsite were sought.	RESULTS
+184	195	−2* subsite	site	To understand the structural basis for the biochemical properties of CtXyl5A, the crystal structure of the enzyme with ligands that occupy the substrate binding cleft and the critical −2* subsite were sought.	RESULTS
+39	48	structure	evidence	The data presented in Fig. 3A show the structure of the CtXyl5A derivative CtGH5-CtCBM6 in complex with arabinose bound in the −2* pocket.	RESULTS
+56	63	CtXyl5A	protein	The data presented in Fig. 3A show the structure of the CtXyl5A derivative CtGH5-CtCBM6 in complex with arabinose bound in the −2* pocket.	RESULTS
+75	87	CtGH5-CtCBM6	structure_element	The data presented in Fig. 3A show the structure of the CtXyl5A derivative CtGH5-CtCBM6 in complex with arabinose bound in the −2* pocket.	RESULTS
+88	103	in complex with	protein_state	The data presented in Fig. 3A show the structure of the CtXyl5A derivative CtGH5-CtCBM6 in complex with arabinose bound in the −2* pocket.	RESULTS
+104	113	arabinose	chemical	The data presented in Fig. 3A show the structure of the CtXyl5A derivative CtGH5-CtCBM6 in complex with arabinose bound in the −2* pocket.	RESULTS
+114	122	bound in	protein_state	The data presented in Fig. 3A show the structure of the CtXyl5A derivative CtGH5-CtCBM6 in complex with arabinose bound in the −2* pocket.	RESULTS
+127	137	−2* pocket	site	The data presented in Fig. 3A show the structure of the CtXyl5A derivative CtGH5-CtCBM6 in complex with arabinose bound in the −2* pocket.	RESULTS
+19	24	bound	protein_state	Interestingly, the bound arabinose was in the pyranose conformation rather than in its furanose form found in arabinoxylans.	RESULTS
+25	34	arabinose	chemical	Interestingly, the bound arabinose was in the pyranose conformation rather than in its furanose form found in arabinoxylans.	RESULTS
+46	54	pyranose	chemical	Interestingly, the bound arabinose was in the pyranose conformation rather than in its furanose form found in arabinoxylans.	RESULTS
+87	95	furanose	chemical	Interestingly, the bound arabinose was in the pyranose conformation rather than in its furanose form found in arabinoxylans.	RESULTS
+110	123	arabinoxylans	chemical	Interestingly, the bound arabinose was in the pyranose conformation rather than in its furanose form found in arabinoxylans.	RESULTS
+25	36	active site	site	O1 was facing toward the active site −1 subsite, indicative of the bound arabinose being in the right orientation to be linked to the xylan backbone via an α-1,3 linkage.	RESULTS
+37	47	−1 subsite	site	O1 was facing toward the active site −1 subsite, indicative of the bound arabinose being in the right orientation to be linked to the xylan backbone via an α-1,3 linkage.	RESULTS
+67	72	bound	protein_state	O1 was facing toward the active site −1 subsite, indicative of the bound arabinose being in the right orientation to be linked to the xylan backbone via an α-1,3 linkage.	RESULTS
+73	82	arabinose	chemical	O1 was facing toward the active site −1 subsite, indicative of the bound arabinose being in the right orientation to be linked to the xylan backbone via an α-1,3 linkage.	RESULTS
+134	139	xylan	chemical	O1 was facing toward the active site −1 subsite, indicative of the bound arabinose being in the right orientation to be linked to the xylan backbone via an α-1,3 linkage.	RESULTS
+43	47	Arap	chemical	As discussed on below, the axial O4 of the Arap did not interact with the −2* subsite, suggesting that the pocket might be capable of binding a xylose molecule.	RESULTS
+74	85	−2* subsite	site	As discussed on below, the axial O4 of the Arap did not interact with the −2* subsite, suggesting that the pocket might be capable of binding a xylose molecule.	RESULTS
+107	113	pocket	site	As discussed on below, the axial O4 of the Arap did not interact with the −2* subsite, suggesting that the pocket might be capable of binding a xylose molecule.	RESULTS
+144	150	xylose	chemical	As discussed on below, the axial O4 of the Arap did not interact with the −2* subsite, suggesting that the pocket might be capable of binding a xylose molecule.	RESULTS
+8	15	soaking	experimental_method	Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the −2* subsite in its pyranose conformation (Fig. 3B).	RESULTS
+16	19	apo	protein_state	Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the −2* subsite in its pyranose conformation (Fig. 3B).	RESULTS
+20	28	crystals	evidence	Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the −2* subsite in its pyranose conformation (Fig. 3B).	RESULTS
+34	40	xylose	chemical	Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the −2* subsite in its pyranose conformation (Fig. 3B).	RESULTS
+57	64	pentose	chemical	Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the −2* subsite in its pyranose conformation (Fig. 3B).	RESULTS
+65	70	sugar	chemical	Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the −2* subsite in its pyranose conformation (Fig. 3B).	RESULTS
+76	84	bound in	protein_state	Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the −2* subsite in its pyranose conformation (Fig. 3B).	RESULTS
+89	100	−2* subsite	site	Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the −2* subsite in its pyranose conformation (Fig. 3B).	RESULTS
+108	116	pyranose	chemical	Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the −2* subsite in its pyranose conformation (Fig. 3B).	RESULTS
+6	24	crystal structures	evidence	These crystal structures support the biochemical data presented above showing that the enzyme generated β-1,3-xylobiose from CX, which would require the disaccharide to bind at the −1 and −2* subsites.	RESULTS
+104	119	β-1,3-xylobiose	chemical	These crystal structures support the biochemical data presented above showing that the enzyme generated β-1,3-xylobiose from CX, which would require the disaccharide to bind at the −1 and −2* subsites.	RESULTS
+125	127	CX	chemical	These crystal structures support the biochemical data presented above showing that the enzyme generated β-1,3-xylobiose from CX, which would require the disaccharide to bind at the −1 and −2* subsites.	RESULTS
+153	165	disaccharide	chemical	These crystal structures support the biochemical data presented above showing that the enzyme generated β-1,3-xylobiose from CX, which would require the disaccharide to bind at the −1 and −2* subsites.	RESULTS
+181	200	−1 and −2* subsites	site	These crystal structures support the biochemical data presented above showing that the enzyme generated β-1,3-xylobiose from CX, which would require the disaccharide to bind at the −1 and −2* subsites.	RESULTS
+41	57	co-crystallizing	experimental_method	A third product complex was generated by co-crystallizing the nucleophile inactive mutant CtGH5E279S-CtCBM6 with a WAX-derived oligosaccharide (Fig. 3C).	RESULTS
+62	82	nucleophile inactive	protein_state	A third product complex was generated by co-crystallizing the nucleophile inactive mutant CtGH5E279S-CtCBM6 with a WAX-derived oligosaccharide (Fig. 3C).	RESULTS
+83	89	mutant	protein_state	A third product complex was generated by co-crystallizing the nucleophile inactive mutant CtGH5E279S-CtCBM6 with a WAX-derived oligosaccharide (Fig. 3C).	RESULTS
+90	100	CtGH5E279S	mutant	A third product complex was generated by co-crystallizing the nucleophile inactive mutant CtGH5E279S-CtCBM6 with a WAX-derived oligosaccharide (Fig. 3C).	RESULTS
+101	107	CtCBM6	structure_element	A third product complex was generated by co-crystallizing the nucleophile inactive mutant CtGH5E279S-CtCBM6 with a WAX-derived oligosaccharide (Fig. 3C).	RESULTS
+115	118	WAX	chemical	A third product complex was generated by co-crystallizing the nucleophile inactive mutant CtGH5E279S-CtCBM6 with a WAX-derived oligosaccharide (Fig. 3C).	RESULTS
+127	142	oligosaccharide	chemical	A third product complex was generated by co-crystallizing the nucleophile inactive mutant CtGH5E279S-CtCBM6 with a WAX-derived oligosaccharide (Fig. 3C).	RESULTS
+20	35	pentasaccharide	chemical	The data revealed a pentasaccharide bound to the enzyme, comprising β-1,4-xylotetraose with an Araf linked α-1,3 to the reducing end xylose.	RESULTS
+36	44	bound to	protein_state	The data revealed a pentasaccharide bound to the enzyme, comprising β-1,4-xylotetraose with an Araf linked α-1,3 to the reducing end xylose.	RESULTS
+68	86	β-1,4-xylotetraose	chemical	The data revealed a pentasaccharide bound to the enzyme, comprising β-1,4-xylotetraose with an Araf linked α-1,3 to the reducing end xylose.	RESULTS
+95	99	Araf	chemical	The data revealed a pentasaccharide bound to the enzyme, comprising β-1,4-xylotetraose with an Araf linked α-1,3 to the reducing end xylose.	RESULTS
+133	139	xylose	chemical	The data revealed a pentasaccharide bound to the enzyme, comprising β-1,4-xylotetraose with an Araf linked α-1,3 to the reducing end xylose.	RESULTS
+4	16	xylotetraose	chemical	The xylotetraose was positioned in subsites −1 to −4 and the Araf in the −2* pocket.	RESULTS
+35	52	subsites −1 to −4	site	The xylotetraose was positioned in subsites −1 to −4 and the Araf in the −2* pocket.	RESULTS
+61	65	Araf	chemical	The xylotetraose was positioned in subsites −1 to −4 and the Araf in the −2* pocket.	RESULTS
+73	83	−2* pocket	site	The xylotetraose was positioned in subsites −1 to −4 and the Araf in the −2* pocket.	RESULTS
+22	32	structures	evidence	Analysis of the three structures showed that O1, O2, O3, and the endocyclic oxygen occupied identical positions in the Arap, Araf, and Xylp ligands bound in the −2* subsite and thus made identical interactions with the pocket.	RESULTS
+119	123	Arap	chemical	Analysis of the three structures showed that O1, O2, O3, and the endocyclic oxygen occupied identical positions in the Arap, Araf, and Xylp ligands bound in the −2* subsite and thus made identical interactions with the pocket.	RESULTS
+125	129	Araf	chemical	Analysis of the three structures showed that O1, O2, O3, and the endocyclic oxygen occupied identical positions in the Arap, Araf, and Xylp ligands bound in the −2* subsite and thus made identical interactions with the pocket.	RESULTS
+135	139	Xylp	chemical	Analysis of the three structures showed that O1, O2, O3, and the endocyclic oxygen occupied identical positions in the Arap, Araf, and Xylp ligands bound in the −2* subsite and thus made identical interactions with the pocket.	RESULTS
+148	156	bound in	protein_state	Analysis of the three structures showed that O1, O2, O3, and the endocyclic oxygen occupied identical positions in the Arap, Araf, and Xylp ligands bound in the −2* subsite and thus made identical interactions with the pocket.	RESULTS
+161	172	−2* subsite	site	Analysis of the three structures showed that O1, O2, O3, and the endocyclic oxygen occupied identical positions in the Arap, Araf, and Xylp ligands bound in the −2* subsite and thus made identical interactions with the pocket.	RESULTS
+219	225	pocket	site	Analysis of the three structures showed that O1, O2, O3, and the endocyclic oxygen occupied identical positions in the Arap, Araf, and Xylp ligands bound in the −2* subsite and thus made identical interactions with the pocket.	RESULTS
+11	24	polar contact	bond_interaction	O1 makes a polar contact with Nδ2 of Asn139, O2 is within hydrogen bonding distance with Oδ1 of Asn139 and the backbone N of Asn135, and O3 interacts with the N of Gly136 and Oϵ2 of Glu68.	RESULTS
+37	43	Asn139	residue_name_number	O1 makes a polar contact with Nδ2 of Asn139, O2 is within hydrogen bonding distance with Oδ1 of Asn139 and the backbone N of Asn135, and O3 interacts with the N of Gly136 and Oϵ2 of Glu68.	RESULTS
+58	74	hydrogen bonding	bond_interaction	O1 makes a polar contact with Nδ2 of Asn139, O2 is within hydrogen bonding distance with Oδ1 of Asn139 and the backbone N of Asn135, and O3 interacts with the N of Gly136 and Oϵ2 of Glu68.	RESULTS
+96	102	Asn139	residue_name_number	O1 makes a polar contact with Nδ2 of Asn139, O2 is within hydrogen bonding distance with Oδ1 of Asn139 and the backbone N of Asn135, and O3 interacts with the N of Gly136 and Oϵ2 of Glu68.	RESULTS
+125	131	Asn135	residue_name_number	O1 makes a polar contact with Nδ2 of Asn139, O2 is within hydrogen bonding distance with Oδ1 of Asn139 and the backbone N of Asn135, and O3 interacts with the N of Gly136 and Oϵ2 of Glu68.	RESULTS
+164	170	Gly136	residue_name_number	O1 makes a polar contact with Nδ2 of Asn139, O2 is within hydrogen bonding distance with Oδ1 of Asn139 and the backbone N of Asn135, and O3 interacts with the N of Gly136 and Oϵ2 of Glu68.	RESULTS
+182	187	Glu68	residue_name_number	O1 makes a polar contact with Nδ2 of Asn139, O2 is within hydrogen bonding distance with Oδ1 of Asn139 and the backbone N of Asn135, and O3 interacts with the N of Gly136 and Oϵ2 of Glu68.	RESULTS
+15	19	Arap	chemical	Although O4 of Arap does not make a direct interaction with the enzyme, O4 and O5 of Xylp and Araf, respectively, form hydrogen bonds with Oϵ1 of Glu68.	RESULTS
+85	89	Xylp	chemical	Although O4 of Arap does not make a direct interaction with the enzyme, O4 and O5 of Xylp and Araf, respectively, form hydrogen bonds with Oϵ1 of Glu68.	RESULTS
+94	98	Araf	chemical	Although O4 of Arap does not make a direct interaction with the enzyme, O4 and O5 of Xylp and Araf, respectively, form hydrogen bonds with Oϵ1 of Glu68.	RESULTS
+119	133	hydrogen bonds	bond_interaction	Although O4 of Arap does not make a direct interaction with the enzyme, O4 and O5 of Xylp and Araf, respectively, form hydrogen bonds with Oϵ1 of Glu68.	RESULTS
+146	151	Glu68	residue_name_number	Although O4 of Arap does not make a direct interaction with the enzyme, O4 and O5 of Xylp and Araf, respectively, form hydrogen bonds with Oϵ1 of Glu68.	RESULTS
+8	13	Tyr92	residue_name_number	Finally Tyr92 makes apolar parallel interactions with the pyranose or furanose rings of the three sugars.	RESULTS
+27	48	parallel interactions	bond_interaction	Finally Tyr92 makes apolar parallel interactions with the pyranose or furanose rings of the three sugars.	RESULTS
+58	66	pyranose	chemical	Finally Tyr92 makes apolar parallel interactions with the pyranose or furanose rings of the three sugars.	RESULTS
+70	78	furanose	chemical	Finally Tyr92 makes apolar parallel interactions with the pyranose or furanose rings of the three sugars.	RESULTS
+74	85	−2* subsite	site	Representation of the residues involved in the ligands recognition at the −2* subsite.	FIG
+63	81	catalytic residues	site	Interacting residues are represented as stick in blue, and the catalytic residues and the mutated glutamate (into a serine) are in magenta.	FIG
+90	97	mutated	experimental_method	Interacting residues are represented as stick in blue, and the catalytic residues and the mutated glutamate (into a serine) are in magenta.	FIG
+98	107	glutamate	residue_name	Interacting residues are represented as stick in blue, and the catalytic residues and the mutated glutamate (into a serine) are in magenta.	FIG
+116	122	serine	residue_name	Interacting residues are represented as stick in blue, and the catalytic residues and the mutated glutamate (into a serine) are in magenta.	FIG
+3	13	CtGH5-CBM6	structure_element	A, CtGH5-CBM6 in complex with an arabinopyranose.	FIG
+14	29	in complex with	protein_state	A, CtGH5-CBM6 in complex with an arabinopyranose.	FIG
+33	48	arabinopyranose	chemical	A, CtGH5-CBM6 in complex with an arabinopyranose.	FIG
+3	13	CtGH5-CBM6	structure_element	B, CtGH5-CBM6 in complex with a xylopyranose.	FIG
+14	29	in complex with	protein_state	B, CtGH5-CBM6 in complex with a xylopyranose.	FIG
+32	44	xylopyranose	chemical	B, CtGH5-CBM6 in complex with a xylopyranose.	FIG
+3	13	CtGH5E279S	mutant	C, CtGH5E279S-CBM6 in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose).	FIG
+14	18	CBM6	structure_element	C, CtGH5E279S-CBM6 in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose).	FIG
+19	34	in complex with	protein_state	C, CtGH5E279S-CBM6 in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose).	FIG
+37	52	pentasaccharide	chemical	C, CtGH5E279S-CBM6 in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose).	FIG
+54	71	β1,4-xylotetraose	chemical	C, CtGH5E279S-CBM6 in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose).	FIG
+80	86	l-Araf	chemical	C, CtGH5E279S-CBM6 in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose).	FIG
+119	125	xylose	chemical	C, CtGH5E279S-CBM6 in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose).	FIG
+4	9	xylan	chemical	The xylan backbone is shown transparently for more clarity.	FIG
+0	9	Densities	evidence	Densities shown in blue are RefMac maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ.	FIG
+35	83	maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ	evidence	Densities shown in blue are RefMac maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ.	FIG
+98	108	−2* pocket	site	The importance of the interactions between the ligands and the side chains of the residues in the −2* pocket were evaluated by alanine substitution of these amino acids.	RESULTS
+127	147	alanine substitution	experimental_method	The importance of the interactions between the ligands and the side chains of the residues in the −2* pocket were evaluated by alanine substitution of these amino acids.	RESULTS
+4	11	mutants	protein_state	The mutants E68A, Y92A, and N139A were all inactive (Table 1), demonstrating the importance of the interactions of these residues with the substrate and reinforcing the critical role the −2* subsite plays in the activity of the enzyme.	RESULTS
+12	16	E68A	mutant	The mutants E68A, Y92A, and N139A were all inactive (Table 1), demonstrating the importance of the interactions of these residues with the substrate and reinforcing the critical role the −2* subsite plays in the activity of the enzyme.	RESULTS
+18	22	Y92A	mutant	The mutants E68A, Y92A, and N139A were all inactive (Table 1), demonstrating the importance of the interactions of these residues with the substrate and reinforcing the critical role the −2* subsite plays in the activity of the enzyme.	RESULTS
+28	33	N139A	mutant	The mutants E68A, Y92A, and N139A were all inactive (Table 1), demonstrating the importance of the interactions of these residues with the substrate and reinforcing the critical role the −2* subsite plays in the activity of the enzyme.	RESULTS
+43	51	inactive	protein_state	The mutants E68A, Y92A, and N139A were all inactive (Table 1), demonstrating the importance of the interactions of these residues with the substrate and reinforcing the critical role the −2* subsite plays in the activity of the enzyme.	RESULTS
+187	198	−2* subsite	site	The mutants E68A, Y92A, and N139A were all inactive (Table 1), demonstrating the importance of the interactions of these residues with the substrate and reinforcing the critical role the −2* subsite plays in the activity of the enzyme.	RESULTS
+0	5	N135A	mutant	N135A retained wild type activity because the O2 of the sugars interacts with the backbone N of Asn135 and not with the side chain.	RESULTS
+15	24	wild type	protein_state	N135A retained wild type activity because the O2 of the sugars interacts with the backbone N of Asn135 and not with the side chain.	RESULTS
+96	102	Asn135	residue_name_number	N135A retained wild type activity because the O2 of the sugars interacts with the backbone N of Asn135 and not with the side chain.	RESULTS
+25	29	Xylp	chemical	Because the hydroxyls of Xylp or Araf in the −2* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration.	RESULTS
+33	37	Araf	chemical	Because the hydroxyls of Xylp or Araf in the −2* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration.	RESULTS
+45	55	−2* pocket	site	Because the hydroxyls of Xylp or Araf in the −2* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration.	RESULTS
+64	79	solvent-exposed	protein_state	Because the hydroxyls of Xylp or Araf in the −2* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration.	RESULTS
+85	96	active site	site	Because the hydroxyls of Xylp or Araf in the −2* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration.	RESULTS
+104	119	arabinoxylanase	protein_type	Because the hydroxyls of Xylp or Araf in the −2* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration.	RESULTS
+137	143	xylose	chemical	Because the hydroxyls of Xylp or Araf in the −2* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration.	RESULTS
+175	181	xylose	chemical	Because the hydroxyls of Xylp or Araf in the −2* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration.	RESULTS
+185	194	arabinose	chemical	Because the hydroxyls of Xylp or Araf in the −2* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration.	RESULTS
+25	29	kcat	evidence	This may explain why the kcat/Km for CtXyl5A against WAX was 2-fold higher than against CX (Table 1).	RESULTS
+30	32	Km	evidence	This may explain why the kcat/Km for CtXyl5A against WAX was 2-fold higher than against CX (Table 1).	RESULTS
+37	44	CtXyl5A	protein	This may explain why the kcat/Km for CtXyl5A against WAX was 2-fold higher than against CX (Table 1).	RESULTS
+53	56	WAX	chemical	This may explain why the kcat/Km for CtXyl5A against WAX was 2-fold higher than against CX (Table 1).	RESULTS
+88	90	CX	chemical	This may explain why the kcat/Km for CtXyl5A against WAX was 2-fold higher than against CX (Table 1).	RESULTS
+0	3	WAX	chemical	WAX is likely to have a higher concentration of single Araf decorations compared with CX and thus contain more substrate available to the arabinoxylanase.	RESULTS
+55	59	Araf	chemical	WAX is likely to have a higher concentration of single Araf decorations compared with CX and thus contain more substrate available to the arabinoxylanase.	RESULTS
+86	88	CX	chemical	WAX is likely to have a higher concentration of single Araf decorations compared with CX and thus contain more substrate available to the arabinoxylanase.	RESULTS
+138	153	arabinoxylanase	protein_type	WAX is likely to have a higher concentration of single Araf decorations compared with CX and thus contain more substrate available to the arabinoxylanase.	RESULTS
+7	18	active site	site	In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92.	RESULTS
+22	29	CtXyl5A	protein	In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92.	RESULTS
+34	42	α-d-Xylp	chemical	In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92.	RESULTS
+153	167	hydrogen bonds	bond_interaction	In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92.	RESULTS
+184	190	His253	residue_name_number	In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92.	RESULTS
+202	208	Glu171	residue_name_number	In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92.	RESULTS
+257	270	polar contact	bond_interaction	In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92.	RESULTS
+286	292	Tyr255	residue_name_number	In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92.	RESULTS
+303	309	Ser279	residue_name_number	In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92.	RESULTS
+354	368	hydrogen bonds	bond_interaction	In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92.	RESULTS
+381	387	Asn170	residue_name_number	In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92.	RESULTS
+398	403	Tyr92	residue_name_number	In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92.	RESULTS
+14	18	Araf	chemical	O3 (O1 of the Araf at the −2* subsite) makes a polar contact with Nδ2 of Asn139; the endocyclic oxygen hydrogens bonds with the OH of Tyr255.	RESULTS
+26	37	−2* subsite	site	O3 (O1 of the Araf at the −2* subsite) makes a polar contact with Nδ2 of Asn139; the endocyclic oxygen hydrogens bonds with the OH of Tyr255.	RESULTS
+47	60	polar contact	bond_interaction	O3 (O1 of the Araf at the −2* subsite) makes a polar contact with Nδ2 of Asn139; the endocyclic oxygen hydrogens bonds with the OH of Tyr255.	RESULTS
+73	79	Asn139	residue_name_number	O3 (O1 of the Araf at the −2* subsite) makes a polar contact with Nδ2 of Asn139; the endocyclic oxygen hydrogens bonds with the OH of Tyr255.	RESULTS
+103	118	hydrogens bonds	bond_interaction	O3 (O1 of the Araf at the −2* subsite) makes a polar contact with Nδ2 of Asn139; the endocyclic oxygen hydrogens bonds with the OH of Tyr255.	RESULTS
+134	140	Tyr255	residue_name_number	O3 (O1 of the Araf at the −2* subsite) makes a polar contact with Nδ2 of Asn139; the endocyclic oxygen hydrogens bonds with the OH of Tyr255.	RESULTS
+4	8	Xylp	chemical	The Xylp in the active site makes strong parallel apolar interactions with Phe310.	RESULTS
+16	27	active site	site	The Xylp in the active site makes strong parallel apolar interactions with Phe310.	RESULTS
+41	69	parallel apolar interactions	bond_interaction	The Xylp in the active site makes strong parallel apolar interactions with Phe310.	RESULTS
+75	81	Phe310	residue_name_number	The Xylp in the active site makes strong parallel apolar interactions with Phe310.	RESULTS
+29	40	active site	site	Substrate recognition in the active site is conserved between CtXyl5A and the closest GH5 structural homolog, the endoglucanase BaCel5A (PDB code 1qi2) as noted previously.	RESULTS
+44	53	conserved	protein_state	Substrate recognition in the active site is conserved between CtXyl5A and the closest GH5 structural homolog, the endoglucanase BaCel5A (PDB code 1qi2) as noted previously.	RESULTS
+62	69	CtXyl5A	protein	Substrate recognition in the active site is conserved between CtXyl5A and the closest GH5 structural homolog, the endoglucanase BaCel5A (PDB code 1qi2) as noted previously.	RESULTS
+86	89	GH5	protein_type	Substrate recognition in the active site is conserved between CtXyl5A and the closest GH5 structural homolog, the endoglucanase BaCel5A (PDB code 1qi2) as noted previously.	RESULTS
+114	127	endoglucanase	protein_type	Substrate recognition in the active site is conserved between CtXyl5A and the closest GH5 structural homolog, the endoglucanase BaCel5A (PDB code 1qi2) as noted previously.	RESULTS
+128	135	BaCel5A	protein	Substrate recognition in the active site is conserved between CtXyl5A and the closest GH5 structural homolog, the endoglucanase BaCel5A (PDB code 1qi2) as noted previously.	RESULTS
+51	68	negative subsites	site	Comparison of the ligand recognition at the distal negative subsites between CtGH5E279S-CBM6, the cellulase BaCel5A, and the xylanase GH10.	FIG
+77	87	CtGH5E279S	mutant	Comparison of the ligand recognition at the distal negative subsites between CtGH5E279S-CBM6, the cellulase BaCel5A, and the xylanase GH10.	FIG
+88	92	CBM6	structure_element	Comparison of the ligand recognition at the distal negative subsites between CtGH5E279S-CBM6, the cellulase BaCel5A, and the xylanase GH10.	FIG
+98	107	cellulase	protein_type	Comparison of the ligand recognition at the distal negative subsites between CtGH5E279S-CBM6, the cellulase BaCel5A, and the xylanase GH10.	FIG
+108	115	BaCel5A	protein	Comparison of the ligand recognition at the distal negative subsites between CtGH5E279S-CBM6, the cellulase BaCel5A, and the xylanase GH10.	FIG
+125	133	xylanase	protein_type	Comparison of the ligand recognition at the distal negative subsites between CtGH5E279S-CBM6, the cellulase BaCel5A, and the xylanase GH10.	FIG
+134	138	GH10	protein_type	Comparison of the ligand recognition at the distal negative subsites between CtGH5E279S-CBM6, the cellulase BaCel5A, and the xylanase GH10.	FIG
+10	20	CtGH5E279S	mutant	 A–C show CtGH5E279S-CBM6 is in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose).	FIG
+29	44	in complex with	protein_state	 A–C show CtGH5E279S-CBM6 is in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose).	FIG
+47	62	pentasaccharide	chemical	 A–C show CtGH5E279S-CBM6 is in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose).	FIG
+64	81	β1,4-xylotetraose	chemical	 A–C show CtGH5E279S-CBM6 is in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose).	FIG
+90	96	l-Araf	chemical	 A–C show CtGH5E279S-CBM6 is in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose).	FIG
+129	135	xylose	chemical	 A–C show CtGH5E279S-CBM6 is in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose).	FIG
+44	60	hydrogen bonding	bond_interaction	A, Poseview representation highlighting the hydrogen bonding and the hydrophobic interactions that occur in the negative subsites.	FIG
+69	93	hydrophobic interactions	bond_interaction	A, Poseview representation highlighting the hydrogen bonding and the hydrophobic interactions that occur in the negative subsites.	FIG
+112	129	negative subsites	site	A, Poseview representation highlighting the hydrogen bonding and the hydrophobic interactions that occur in the negative subsites.	FIG
+3	10	density	evidence	C, density of the ligand shown in blue is RefMac maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ.	FIG
+49	97	maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ	evidence	C, density of the ligand shown in blue is RefMac maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ.	FIG
+16	23	BaCel5A	protein	D and E display BaCel5A in complex with deoxy-2-fluoro-β-d-cellotrioside (PDB code 1qi2), and F and G show CmXyn10B in complex with a xylotriose (PDB code 1uqy).	FIG
+24	39	in complex with	protein_state	D and E display BaCel5A in complex with deoxy-2-fluoro-β-d-cellotrioside (PDB code 1qi2), and F and G show CmXyn10B in complex with a xylotriose (PDB code 1uqy).	FIG
+40	72	deoxy-2-fluoro-β-d-cellotrioside	chemical	D and E display BaCel5A in complex with deoxy-2-fluoro-β-d-cellotrioside (PDB code 1qi2), and F and G show CmXyn10B in complex with a xylotriose (PDB code 1uqy).	FIG
+107	115	CmXyn10B	protein	D and E display BaCel5A in complex with deoxy-2-fluoro-β-d-cellotrioside (PDB code 1qi2), and F and G show CmXyn10B in complex with a xylotriose (PDB code 1uqy).	FIG
+116	131	in complex with	protein_state	D and E display BaCel5A in complex with deoxy-2-fluoro-β-d-cellotrioside (PDB code 1qi2), and F and G show CmXyn10B in complex with a xylotriose (PDB code 1uqy).	FIG
+134	144	xylotriose	chemical	D and E display BaCel5A in complex with deoxy-2-fluoro-β-d-cellotrioside (PDB code 1qi2), and F and G show CmXyn10B in complex with a xylotriose (PDB code 1uqy).	FIG
+41	51	CtGH5E279S	mutant	B, D, and F are surface representations (CtGH5E279S-CBM6 in gray, BaCel5A in cyan, and the xylanase GH10 in light brown).	FIG
+66	73	BaCel5A	protein	B, D, and F are surface representations (CtGH5E279S-CBM6 in gray, BaCel5A in cyan, and the xylanase GH10 in light brown).	FIG
+91	99	xylanase	protein_type	B, D, and F are surface representations (CtGH5E279S-CBM6 in gray, BaCel5A in cyan, and the xylanase GH10 in light brown).	FIG
+100	104	GH10	protein_type	B, D, and F are surface representations (CtGH5E279S-CBM6 in gray, BaCel5A in cyan, and the xylanase GH10 in light brown).	FIG
+31	45	hydrogen bonds	bond_interaction	The black dashes represent the hydrogen bonds.	FIG
+31	45	hydrogen bonds	bond_interaction	The black dashes represent the hydrogen bonds.	FIG
+16	23	CtXyl5A	protein	The capacity of CtXyl5A to act on the highly decorated xylan CX indicates that O3 and possibly O2 of the backbone Xylp units are solvent-exposed.	RESULTS
+55	60	xylan	chemical	The capacity of CtXyl5A to act on the highly decorated xylan CX indicates that O3 and possibly O2 of the backbone Xylp units are solvent-exposed.	RESULTS
+61	63	CX	chemical	The capacity of CtXyl5A to act on the highly decorated xylan CX indicates that O3 and possibly O2 of the backbone Xylp units are solvent-exposed.	RESULTS
+114	118	Xylp	chemical	The capacity of CtXyl5A to act on the highly decorated xylan CX indicates that O3 and possibly O2 of the backbone Xylp units are solvent-exposed.	RESULTS
+129	144	solvent-exposed	protein_state	The capacity of CtXyl5A to act on the highly decorated xylan CX indicates that O3 and possibly O2 of the backbone Xylp units are solvent-exposed.	RESULTS
+47	59	xylotetraose	chemical	This is consistent with the interaction of the xylotetraose backbone with the enzyme distal to the active site.	RESULTS
+99	110	active site	site	This is consistent with the interaction of the xylotetraose backbone with the enzyme distal to the active site.	RESULTS
+73	79	xylose	chemical	A surface representation of the enzyme (Fig. 4B) shows that O3 and O2 of xylose units at subsites −2 to −4 are solvent-exposed and are thus available for decoration.	RESULTS
+89	106	subsites −2 to −4	site	A surface representation of the enzyme (Fig. 4B) shows that O3 and O2 of xylose units at subsites −2 to −4 are solvent-exposed and are thus available for decoration.	RESULTS
+111	126	solvent-exposed	protein_state	A surface representation of the enzyme (Fig. 4B) shows that O3 and O2 of xylose units at subsites −2 to −4 are solvent-exposed and are thus available for decoration.	RESULTS
+14	22	pyranose	chemical	Indeed, these pyranose sugars make very weak apolar interactions with the arabinoxylanase.	RESULTS
+23	29	sugars	chemical	Indeed, these pyranose sugars make very weak apolar interactions with the arabinoxylanase.	RESULTS
+45	64	apolar interactions	bond_interaction	Indeed, these pyranose sugars make very weak apolar interactions with the arabinoxylanase.	RESULTS
+74	89	arabinoxylanase	protein_type	Indeed, these pyranose sugars make very weak apolar interactions with the arabinoxylanase.	RESULTS
+3	5	−2	site	At −2, Xylp makes planar apolar interactions with the Araf bound to the −2* subsite (Fig. 4C).	RESULTS
+7	11	Xylp	chemical	At −2, Xylp makes planar apolar interactions with the Araf bound to the −2* subsite (Fig. 4C).	RESULTS
+18	44	planar apolar interactions	bond_interaction	At −2, Xylp makes planar apolar interactions with the Araf bound to the −2* subsite (Fig. 4C).	RESULTS
+54	58	Araf	chemical	At −2, Xylp makes planar apolar interactions with the Araf bound to the −2* subsite (Fig. 4C).	RESULTS
+59	67	bound to	protein_state	At −2, Xylp makes planar apolar interactions with the Araf bound to the −2* subsite (Fig. 4C).	RESULTS
+72	83	−2* subsite	site	At −2, Xylp makes planar apolar interactions with the Araf bound to the −2* subsite (Fig. 4C).	RESULTS
+0	4	Xylp	chemical	Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69.	RESULTS
+8	26	subsites −2 and −3	site	Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69.	RESULTS
+52	71	hydrophobic contact	bond_interaction	Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69.	RESULTS
+77	83	Val318	residue_name_number	Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69.	RESULTS
+89	91	−3	site	Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69.	RESULTS
+92	96	Xylp	chemical	Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69.	RESULTS
+103	129	planar apolar interactions	bond_interaction	Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69.	RESULTS
+135	141	Ala137	residue_name_number	Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69.	RESULTS
+155	161	xylose	chemical	Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69.	RESULTS
+165	167	−4	site	Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69.	RESULTS
+174	198	parallel apolar contacts	bond_interaction	Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69.	RESULTS
+204	209	Trp69	residue_name_number	Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69.	RESULTS
+25	42	negative subsites	site	Comparison of the distal negative subsites of CtXyl5A with BaCel5A and a typical GH10 xylanase (CmXyn10B, PDB code 1uqy) highlights the paucity of interactions between the arabinoxylanase and its substrate out with the active site (Fig. 4).	RESULTS
+46	53	CtXyl5A	protein	Comparison of the distal negative subsites of CtXyl5A with BaCel5A and a typical GH10 xylanase (CmXyn10B, PDB code 1uqy) highlights the paucity of interactions between the arabinoxylanase and its substrate out with the active site (Fig. 4).	RESULTS
+59	66	BaCel5A	protein	Comparison of the distal negative subsites of CtXyl5A with BaCel5A and a typical GH10 xylanase (CmXyn10B, PDB code 1uqy) highlights the paucity of interactions between the arabinoxylanase and its substrate out with the active site (Fig. 4).	RESULTS
+81	85	GH10	protein_type	Comparison of the distal negative subsites of CtXyl5A with BaCel5A and a typical GH10 xylanase (CmXyn10B, PDB code 1uqy) highlights the paucity of interactions between the arabinoxylanase and its substrate out with the active site (Fig. 4).	RESULTS
+86	94	xylanase	protein_type	Comparison of the distal negative subsites of CtXyl5A with BaCel5A and a typical GH10 xylanase (CmXyn10B, PDB code 1uqy) highlights the paucity of interactions between the arabinoxylanase and its substrate out with the active site (Fig. 4).	RESULTS
+96	104	CmXyn10B	protein	Comparison of the distal negative subsites of CtXyl5A with BaCel5A and a typical GH10 xylanase (CmXyn10B, PDB code 1uqy) highlights the paucity of interactions between the arabinoxylanase and its substrate out with the active site (Fig. 4).	RESULTS
+172	187	arabinoxylanase	protein_type	Comparison of the distal negative subsites of CtXyl5A with BaCel5A and a typical GH10 xylanase (CmXyn10B, PDB code 1uqy) highlights the paucity of interactions between the arabinoxylanase and its substrate out with the active site (Fig. 4).	RESULTS
+219	230	active site	site	Comparison of the distal negative subsites of CtXyl5A with BaCel5A and a typical GH10 xylanase (CmXyn10B, PDB code 1uqy) highlights the paucity of interactions between the arabinoxylanase and its substrate out with the active site (Fig. 4).	RESULTS
+10	19	cellulase	protein_type	Thus, the cellulase contains three negative subsites and the sugars bound in the −2 and −3 subsites make a total of 9 polar interactions with the enzyme (Fig. 4, D and E).	RESULTS
+35	52	negative subsites	site	Thus, the cellulase contains three negative subsites and the sugars bound in the −2 and −3 subsites make a total of 9 polar interactions with the enzyme (Fig. 4, D and E).	RESULTS
+61	67	sugars	chemical	Thus, the cellulase contains three negative subsites and the sugars bound in the −2 and −3 subsites make a total of 9 polar interactions with the enzyme (Fig. 4, D and E).	RESULTS
+68	76	bound in	protein_state	Thus, the cellulase contains three negative subsites and the sugars bound in the −2 and −3 subsites make a total of 9 polar interactions with the enzyme (Fig. 4, D and E).	RESULTS
+81	99	−2 and −3 subsites	site	Thus, the cellulase contains three negative subsites and the sugars bound in the −2 and −3 subsites make a total of 9 polar interactions with the enzyme (Fig. 4, D and E).	RESULTS
+118	136	polar interactions	bond_interaction	Thus, the cellulase contains three negative subsites and the sugars bound in the −2 and −3 subsites make a total of 9 polar interactions with the enzyme (Fig. 4, D and E).	RESULTS
+4	8	GH10	protein_type	The GH10 xylanase also contains a −2 subsite that, similar to the cellulase, makes numerous interactions with the substrate (Fig. 4, F and G).	RESULTS
+9	17	xylanase	protein_type	The GH10 xylanase also contains a −2 subsite that, similar to the cellulase, makes numerous interactions with the substrate (Fig. 4, F and G).	RESULTS
+34	44	−2 subsite	site	The GH10 xylanase also contains a −2 subsite that, similar to the cellulase, makes numerous interactions with the substrate (Fig. 4, F and G).	RESULTS
+66	75	cellulase	protein_type	The GH10 xylanase also contains a −2 subsite that, similar to the cellulase, makes numerous interactions with the substrate (Fig. 4, F and G).	RESULTS
+45	52	CtXyl5A	protein	The Influence of the Modular Architecture of CtXyl5A on Catalytic Activity	RESULTS
+0	7	CtXyl5A	protein	CtXyl5A, in addition to its catalytic module, contains three CBMs (CtCBM6, CtCBM13, and CtCBM62) and a fibronectin domain (CtFn3).	RESULTS
+28	44	catalytic module	structure_element	CtXyl5A, in addition to its catalytic module, contains three CBMs (CtCBM6, CtCBM13, and CtCBM62) and a fibronectin domain (CtFn3).	RESULTS
+61	65	CBMs	structure_element	CtXyl5A, in addition to its catalytic module, contains three CBMs (CtCBM6, CtCBM13, and CtCBM62) and a fibronectin domain (CtFn3).	RESULTS
+67	73	CtCBM6	structure_element	CtXyl5A, in addition to its catalytic module, contains three CBMs (CtCBM6, CtCBM13, and CtCBM62) and a fibronectin domain (CtFn3).	RESULTS
+75	82	CtCBM13	structure_element	CtXyl5A, in addition to its catalytic module, contains three CBMs (CtCBM6, CtCBM13, and CtCBM62) and a fibronectin domain (CtFn3).	RESULTS
+88	95	CtCBM62	structure_element	CtXyl5A, in addition to its catalytic module, contains three CBMs (CtCBM6, CtCBM13, and CtCBM62) and a fibronectin domain (CtFn3).	RESULTS
+103	121	fibronectin domain	structure_element	CtXyl5A, in addition to its catalytic module, contains three CBMs (CtCBM6, CtCBM13, and CtCBM62) and a fibronectin domain (CtFn3).	RESULTS
+123	128	CtFn3	structure_element	CtXyl5A, in addition to its catalytic module, contains three CBMs (CtCBM6, CtCBM13, and CtCBM62) and a fibronectin domain (CtFn3).	RESULTS
+42	46	CBM6	structure_element	A previous study showed that although the CBM6 bound in an exo-mode to xylo- and cellulooligosaccharides, the primary role of this module was to stabilize the structure of the GH5 catalytic module.	RESULTS
+47	55	bound in	protein_state	A previous study showed that although the CBM6 bound in an exo-mode to xylo- and cellulooligosaccharides, the primary role of this module was to stabilize the structure of the GH5 catalytic module.	RESULTS
+59	67	exo-mode	protein_state	A previous study showed that although the CBM6 bound in an exo-mode to xylo- and cellulooligosaccharides, the primary role of this module was to stabilize the structure of the GH5 catalytic module.	RESULTS
+71	104	xylo- and cellulooligosaccharides	chemical	A previous study showed that although the CBM6 bound in an exo-mode to xylo- and cellulooligosaccharides, the primary role of this module was to stabilize the structure of the GH5 catalytic module.	RESULTS
+176	179	GH5	protein_type	A previous study showed that although the CBM6 bound in an exo-mode to xylo- and cellulooligosaccharides, the primary role of this module was to stabilize the structure of the GH5 catalytic module.	RESULTS
+180	196	catalytic module	structure_element	A previous study showed that although the CBM6 bound in an exo-mode to xylo- and cellulooligosaccharides, the primary role of this module was to stabilize the structure of the GH5 catalytic module.	RESULTS
+41	62	non-catalytic modules	structure_element	To explore the contribution of the other non-catalytic modules to CtXyl5A function, the activity of a series of truncated derivatives of the arabinoxylanase were assessed.	RESULTS
+66	73	CtXyl5A	protein	To explore the contribution of the other non-catalytic modules to CtXyl5A function, the activity of a series of truncated derivatives of the arabinoxylanase were assessed.	RESULTS
+112	121	truncated	protein_state	To explore the contribution of the other non-catalytic modules to CtXyl5A function, the activity of a series of truncated derivatives of the arabinoxylanase were assessed.	RESULTS
+141	156	arabinoxylanase	protein_type	To explore the contribution of the other non-catalytic modules to CtXyl5A function, the activity of a series of truncated derivatives of the arabinoxylanase were assessed.	RESULTS
+30	40	removal of	experimental_method	The data in Table 1 show that removal of CtCBM62 caused a modest increase in activity against both WAX and CX, whereas deletion of the Fn3 domain had no further impact on catalytic performance.	RESULTS
+41	48	CtCBM62	structure_element	The data in Table 1 show that removal of CtCBM62 caused a modest increase in activity against both WAX and CX, whereas deletion of the Fn3 domain had no further impact on catalytic performance.	RESULTS
+99	102	WAX	chemical	The data in Table 1 show that removal of CtCBM62 caused a modest increase in activity against both WAX and CX, whereas deletion of the Fn3 domain had no further impact on catalytic performance.	RESULTS
+107	109	CX	chemical	The data in Table 1 show that removal of CtCBM62 caused a modest increase in activity against both WAX and CX, whereas deletion of the Fn3 domain had no further impact on catalytic performance.	RESULTS
+119	130	deletion of	experimental_method	The data in Table 1 show that removal of CtCBM62 caused a modest increase in activity against both WAX and CX, whereas deletion of the Fn3 domain had no further impact on catalytic performance.	RESULTS
+135	138	Fn3	structure_element	The data in Table 1 show that removal of CtCBM62 caused a modest increase in activity against both WAX and CX, whereas deletion of the Fn3 domain had no further impact on catalytic performance.	RESULTS
+0	10	Truncation	experimental_method	Truncation of CtCBM13, however, caused a 4–5-fold reduction in activity against both substrates.	RESULTS
+14	21	CtCBM13	structure_element	Truncation of CtCBM13, however, caused a 4–5-fold reduction in activity against both substrates.	RESULTS
+11	16	CBM13	structure_element	Members of CBM13 have been shown to bind to xylans, mannose, and galactose residues in complex glycans, hinting that the function of CtCBM13 is to increase the proximity of substrate to the catalytic module of CtXyl5A.	RESULTS
+44	50	xylans	chemical	Members of CBM13 have been shown to bind to xylans, mannose, and galactose residues in complex glycans, hinting that the function of CtCBM13 is to increase the proximity of substrate to the catalytic module of CtXyl5A.	RESULTS
+52	59	mannose	chemical	Members of CBM13 have been shown to bind to xylans, mannose, and galactose residues in complex glycans, hinting that the function of CtCBM13 is to increase the proximity of substrate to the catalytic module of CtXyl5A.	RESULTS
+65	74	galactose	chemical	Members of CBM13 have been shown to bind to xylans, mannose, and galactose residues in complex glycans, hinting that the function of CtCBM13 is to increase the proximity of substrate to the catalytic module of CtXyl5A.	RESULTS
+87	102	complex glycans	chemical	Members of CBM13 have been shown to bind to xylans, mannose, and galactose residues in complex glycans, hinting that the function of CtCBM13 is to increase the proximity of substrate to the catalytic module of CtXyl5A.	RESULTS
+133	140	CtCBM13	structure_element	Members of CBM13 have been shown to bind to xylans, mannose, and galactose residues in complex glycans, hinting that the function of CtCBM13 is to increase the proximity of substrate to the catalytic module of CtXyl5A.	RESULTS
+190	206	catalytic module	structure_element	Members of CBM13 have been shown to bind to xylans, mannose, and galactose residues in complex glycans, hinting that the function of CtCBM13 is to increase the proximity of substrate to the catalytic module of CtXyl5A.	RESULTS
+210	217	CtXyl5A	protein	Members of CBM13 have been shown to bind to xylans, mannose, and galactose residues in complex glycans, hinting that the function of CtCBM13 is to increase the proximity of substrate to the catalytic module of CtXyl5A.	RESULTS
+0	15	Binding studies	experimental_method	Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown).	RESULTS
+38	45	CtCBM13	structure_element	Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown).	RESULTS
+92	99	glycans	chemical	Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown).	RESULTS
+110	113	WAX	chemical	Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown).	RESULTS
+115	117	CX	chemical	Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown).	RESULTS
+119	125	xylose	chemical	Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown).	RESULTS
+127	134	mannose	chemical	Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown).	RESULTS
+136	145	galactose	chemical	Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown).	RESULTS
+151	166	birchwood xylan	chemical	Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown).	RESULTS
+168	170	BX	chemical	Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown).	RESULTS
+33	40	CtCBM13	structure_element	It would appear, therefore, that CtCBM13 makes a structural contribution to the function of CtXyl5A.	RESULTS
+92	99	CtXyl5A	protein	It would appear, therefore, that CtCBM13 makes a structural contribution to the function of CtXyl5A.	RESULTS
+0	17	Crystal Structure	evidence	Crystal Structure of CtXyl5A-D	RESULTS
+21	30	CtXyl5A-D	mutant	Crystal Structure of CtXyl5A-D	RESULTS
+35	56	non-catalytic modules	structure_element	To explore further the role of the non-catalytic modules in CtXyl5A the crystal structure of CtXyl5A extending from CtGH5 to CtCBM62 was sought.	RESULTS
+60	67	CtXyl5A	protein	To explore further the role of the non-catalytic modules in CtXyl5A the crystal structure of CtXyl5A extending from CtGH5 to CtCBM62 was sought.	RESULTS
+72	89	crystal structure	evidence	To explore further the role of the non-catalytic modules in CtXyl5A the crystal structure of CtXyl5A extending from CtGH5 to CtCBM62 was sought.	RESULTS
+93	100	CtXyl5A	protein	To explore further the role of the non-catalytic modules in CtXyl5A the crystal structure of CtXyl5A extending from CtGH5 to CtCBM62 was sought.	RESULTS
+116	121	CtGH5	structure_element	To explore further the role of the non-catalytic modules in CtXyl5A the crystal structure of CtXyl5A extending from CtGH5 to CtCBM62 was sought.	RESULTS
+125	132	CtCBM62	structure_element	To explore further the role of the non-catalytic modules in CtXyl5A the crystal structure of CtXyl5A extending from CtGH5 to CtCBM62 was sought.	RESULTS
+48	60	crystallized	experimental_method	To obtain a construct that could potentially be crystallized, the protein was generated without the C-terminal dockerin domain because it is known to be unstable and prone to cleavage.	RESULTS
+88	95	without	protein_state	To obtain a construct that could potentially be crystallized, the protein was generated without the C-terminal dockerin domain because it is known to be unstable and prone to cleavage.	RESULTS
+111	119	dockerin	structure_element	To obtain a construct that could potentially be crystallized, the protein was generated without the C-terminal dockerin domain because it is known to be unstable and prone to cleavage.	RESULTS
+22	31	CtXyl5A-D	mutant	Using this construct (CtXyl5A-D) the crystal structure of the arabinoxylanase was determined by molecular replacement to a resolution of 2.64 Å with Rwork and Rfree at 23.7% and 27.8%, respectively.	RESULTS
+37	54	crystal structure	evidence	Using this construct (CtXyl5A-D) the crystal structure of the arabinoxylanase was determined by molecular replacement to a resolution of 2.64 Å with Rwork and Rfree at 23.7% and 27.8%, respectively.	RESULTS
+62	77	arabinoxylanase	protein_type	Using this construct (CtXyl5A-D) the crystal structure of the arabinoxylanase was determined by molecular replacement to a resolution of 2.64 Å with Rwork and Rfree at 23.7% and 27.8%, respectively.	RESULTS
+96	117	molecular replacement	experimental_method	Using this construct (CtXyl5A-D) the crystal structure of the arabinoxylanase was determined by molecular replacement to a resolution of 2.64 Å with Rwork and Rfree at 23.7% and 27.8%, respectively.	RESULTS
+149	154	Rwork	evidence	Using this construct (CtXyl5A-D) the crystal structure of the arabinoxylanase was determined by molecular replacement to a resolution of 2.64 Å with Rwork and Rfree at 23.7% and 27.8%, respectively.	RESULTS
+159	164	Rfree	evidence	Using this construct (CtXyl5A-D) the crystal structure of the arabinoxylanase was determined by molecular replacement to a resolution of 2.64 Å with Rwork and Rfree at 23.7% and 27.8%, respectively.	RESULTS
+4	13	structure	evidence	The structure comprises a continuous polypeptide extending from Ala36 to Trp742 displaying four modules GH5-CBM6-CBM13-Fn3.	RESULTS
+64	79	Ala36 to Trp742	residue_range	The structure comprises a continuous polypeptide extending from Ala36 to Trp742 displaying four modules GH5-CBM6-CBM13-Fn3.	RESULTS
+104	122	GH5-CBM6-CBM13-Fn3	structure_element	The structure comprises a continuous polypeptide extending from Ala36 to Trp742 displaying four modules GH5-CBM6-CBM13-Fn3.	RESULTS
+24	40	electron density	evidence	Although there was some electron density for CtCBM62, it was not sufficient to confidently build the module (Fig. 5).	RESULTS
+45	52	CtCBM62	structure_element	Although there was some electron density for CtCBM62, it was not sufficient to confidently build the module (Fig. 5).	RESULTS
+29	44	crystal packing	evidence	Further investigation of the crystal packing revealed a large solvent channel adjacent to the area the CBM62 occupies.	RESULTS
+62	77	solvent channel	site	Further investigation of the crystal packing revealed a large solvent channel adjacent to the area the CBM62 occupies.	RESULTS
+103	108	CBM62	structure_element	Further investigation of the crystal packing revealed a large solvent channel adjacent to the area the CBM62 occupies.	RESULTS
+42	58	electron density	evidence	We postulate that the reason for the poor electron density is due to the CtCBM62 being mobile compared with the rest of the protein.	RESULTS
+73	80	CtCBM62	structure_element	We postulate that the reason for the poor electron density is due to the CtCBM62 being mobile compared with the rest of the protein.	RESULTS
+87	93	mobile	protein_state	We postulate that the reason for the poor electron density is due to the CtCBM62 being mobile compared with the rest of the protein.	RESULTS
+4	14	structures	evidence	The structures of CtGH5 and CtCBM6 have been described previously.	RESULTS
+18	23	CtGH5	structure_element	The structures of CtGH5 and CtCBM6 have been described previously.	RESULTS
+28	34	CtCBM6	structure_element	The structures of CtGH5 and CtCBM6 have been described previously.	RESULTS
+44	59	arabinoxylanase	protein_type	Surface representation of the tetra-modular arabinoxylanase and zoom view on the CtGH5 loop.	FIG
+81	86	CtGH5	structure_element	Surface representation of the tetra-modular arabinoxylanase and zoom view on the CtGH5 loop.	FIG
+87	91	loop	structure_element	Surface representation of the tetra-modular arabinoxylanase and zoom view on the CtGH5 loop.	FIG
+23	28	CtGH5	structure_element	The blue module is the CtGH5 catalytic domain, the green module corresponds to the CtCBM6, the yellow module is the CtCBM13, and the salmon module is the fibronectin domain.	FIG
+29	45	catalytic domain	structure_element	The blue module is the CtGH5 catalytic domain, the green module corresponds to the CtCBM6, the yellow module is the CtCBM13, and the salmon module is the fibronectin domain.	FIG
+83	89	CtCBM6	structure_element	The blue module is the CtGH5 catalytic domain, the green module corresponds to the CtCBM6, the yellow module is the CtCBM13, and the salmon module is the fibronectin domain.	FIG
+116	123	CtCBM13	structure_element	The blue module is the CtGH5 catalytic domain, the green module corresponds to the CtCBM6, the yellow module is the CtCBM13, and the salmon module is the fibronectin domain.	FIG
+154	172	fibronectin domain	structure_element	The blue module is the CtGH5 catalytic domain, the green module corresponds to the CtCBM6, the yellow module is the CtCBM13, and the salmon module is the fibronectin domain.	FIG
+4	9	CtGH5	structure_element	The CtGH5 loop is stabilized between the CtCBM6 and the CtCBM13 modules.	FIG
+10	14	loop	structure_element	The CtGH5 loop is stabilized between the CtCBM6 and the CtCBM13 modules.	FIG
+41	47	CtCBM6	structure_element	The CtGH5 loop is stabilized between the CtCBM6 and the CtCBM13 modules.	FIG
+56	63	CtCBM13	structure_element	The CtGH5 loop is stabilized between the CtCBM6 and the CtCBM13 modules.	FIG
+0	7	CtCBM13	structure_element	CtCBM13 extends from Gly567 to Pro648.	RESULTS
+21	37	Gly567 to Pro648	residue_range	CtCBM13 extends from Gly567 to Pro648.	RESULTS
+11	16	CBM13	protein_type	Typical of CBM13 proteins CtCBM13 displays a β-trefoil fold comprising the canonical pseudo 3-fold symmetry with a 3-fold repeating unit of 40–50 amino acid residues characteristic of the Ricin superfamily.	RESULTS
+26	33	CtCBM13	structure_element	Typical of CBM13 proteins CtCBM13 displays a β-trefoil fold comprising the canonical pseudo 3-fold symmetry with a 3-fold repeating unit of 40–50 amino acid residues characteristic of the Ricin superfamily.	RESULTS
+45	59	β-trefoil fold	structure_element	Typical of CBM13 proteins CtCBM13 displays a β-trefoil fold comprising the canonical pseudo 3-fold symmetry with a 3-fold repeating unit of 40–50 amino acid residues characteristic of the Ricin superfamily.	RESULTS
+115	136	3-fold repeating unit	structure_element	Typical of CBM13 proteins CtCBM13 displays a β-trefoil fold comprising the canonical pseudo 3-fold symmetry with a 3-fold repeating unit of 40–50 amino acid residues characteristic of the Ricin superfamily.	RESULTS
+140	156	40–50 amino acid	residue_range	Typical of CBM13 proteins CtCBM13 displays a β-trefoil fold comprising the canonical pseudo 3-fold symmetry with a 3-fold repeating unit of 40–50 amino acid residues characteristic of the Ricin superfamily.	RESULTS
+188	205	Ricin superfamily	protein_type	Typical of CBM13 proteins CtCBM13 displays a β-trefoil fold comprising the canonical pseudo 3-fold symmetry with a 3-fold repeating unit of 40–50 amino acid residues characteristic of the Ricin superfamily.	RESULTS
+5	11	repeat	structure_element	Each repeat contains two pairs of antiparallel β-strands.	RESULTS
+34	56	antiparallel β-strands	structure_element	Each repeat contains two pairs of antiparallel β-strands.	RESULTS
+2	13	Dali search	experimental_method	A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).	RESULTS
+52	57	CBM13	protein_type	A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).	RESULTS
+73	99	root mean square deviation	evidence	A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).	RESULTS
+205	220	C. thermocellum	species	A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).	RESULTS
+221	242	exo-β-1,3-galactanase	protein_type	A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).	RESULTS
+260	284	Streptomyces avermitilis	species	A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).	RESULTS
+285	308	β-l-arabinopyranosidase	protein_type	A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).	RESULTS
+326	347	Streptomyces lividans	species	A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).	RESULTS
+348	360	xylanase 10A	protein	A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).	RESULTS
+383	416	Streptomyces olivaceoviridis E-86	species	A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).	RESULTS
+417	429	xylanase 10A	protein	A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).	RESULTS
+4	7	Fn3	structure_element	The Fn3 module displays a typical β-sandwich fold with the two sheets comprising, primarily, three antiparallel strands in the order β1-β2-β5 in β-sheet 1 and β4-β3-β6 in β-sheet 2.	RESULTS
+34	49	β-sandwich fold	structure_element	The Fn3 module displays a typical β-sandwich fold with the two sheets comprising, primarily, three antiparallel strands in the order β1-β2-β5 in β-sheet 1 and β4-β3-β6 in β-sheet 2.	RESULTS
+63	69	sheets	structure_element	The Fn3 module displays a typical β-sandwich fold with the two sheets comprising, primarily, three antiparallel strands in the order β1-β2-β5 in β-sheet 1 and β4-β3-β6 in β-sheet 2.	RESULTS
+99	119	antiparallel strands	structure_element	The Fn3 module displays a typical β-sandwich fold with the two sheets comprising, primarily, three antiparallel strands in the order β1-β2-β5 in β-sheet 1 and β4-β3-β6 in β-sheet 2.	RESULTS
+133	141	β1-β2-β5	structure_element	The Fn3 module displays a typical β-sandwich fold with the two sheets comprising, primarily, three antiparallel strands in the order β1-β2-β5 in β-sheet 1 and β4-β3-β6 in β-sheet 2.	RESULTS
+145	154	β-sheet 1	structure_element	The Fn3 module displays a typical β-sandwich fold with the two sheets comprising, primarily, three antiparallel strands in the order β1-β2-β5 in β-sheet 1 and β4-β3-β6 in β-sheet 2.	RESULTS
+159	167	β4-β3-β6	structure_element	The Fn3 module displays a typical β-sandwich fold with the two sheets comprising, primarily, three antiparallel strands in the order β1-β2-β5 in β-sheet 1 and β4-β3-β6 in β-sheet 2.	RESULTS
+171	180	β-sheet 2	structure_element	The Fn3 module displays a typical β-sandwich fold with the two sheets comprising, primarily, three antiparallel strands in the order β1-β2-β5 in β-sheet 1 and β4-β3-β6 in β-sheet 2.	RESULTS
+9	18	β-sheet 2	structure_element	Although β-sheet 2 presents a cleft-like topology, typical of endo-binding CBMs, the surface lacks aromatic residues that play a key role in ligand recognition, and in the context of the full-length enzyme, the cleft abuts into CtCBM13 and thus would not be able to accommodate an extended polysaccharide chain (see below).	RESULTS
+30	35	cleft	site	Although β-sheet 2 presents a cleft-like topology, typical of endo-binding CBMs, the surface lacks aromatic residues that play a key role in ligand recognition, and in the context of the full-length enzyme, the cleft abuts into CtCBM13 and thus would not be able to accommodate an extended polysaccharide chain (see below).	RESULTS
+62	79	endo-binding CBMs	protein_type	Although β-sheet 2 presents a cleft-like topology, typical of endo-binding CBMs, the surface lacks aromatic residues that play a key role in ligand recognition, and in the context of the full-length enzyme, the cleft abuts into CtCBM13 and thus would not be able to accommodate an extended polysaccharide chain (see below).	RESULTS
+187	198	full-length	protein_state	Although β-sheet 2 presents a cleft-like topology, typical of endo-binding CBMs, the surface lacks aromatic residues that play a key role in ligand recognition, and in the context of the full-length enzyme, the cleft abuts into CtCBM13 and thus would not be able to accommodate an extended polysaccharide chain (see below).	RESULTS
+199	205	enzyme	protein	Although β-sheet 2 presents a cleft-like topology, typical of endo-binding CBMs, the surface lacks aromatic residues that play a key role in ligand recognition, and in the context of the full-length enzyme, the cleft abuts into CtCBM13 and thus would not be able to accommodate an extended polysaccharide chain (see below).	RESULTS
+211	216	cleft	site	Although β-sheet 2 presents a cleft-like topology, typical of endo-binding CBMs, the surface lacks aromatic residues that play a key role in ligand recognition, and in the context of the full-length enzyme, the cleft abuts into CtCBM13 and thus would not be able to accommodate an extended polysaccharide chain (see below).	RESULTS
+228	235	CtCBM13	structure_element	Although β-sheet 2 presents a cleft-like topology, typical of endo-binding CBMs, the surface lacks aromatic residues that play a key role in ligand recognition, and in the context of the full-length enzyme, the cleft abuts into CtCBM13 and thus would not be able to accommodate an extended polysaccharide chain (see below).	RESULTS
+290	304	polysaccharide	chemical	Although β-sheet 2 presents a cleft-like topology, typical of endo-binding CBMs, the surface lacks aromatic residues that play a key role in ligand recognition, and in the context of the full-length enzyme, the cleft abuts into CtCBM13 and thus would not be able to accommodate an extended polysaccharide chain (see below).	RESULTS
+7	16	structure	evidence	In the structure of CtXyl5A-D, the four modules form a three-leaf clover-like structure (Fig. 5).	RESULTS
+20	29	CtXyl5A-D	mutant	In the structure of CtXyl5A-D, the four modules form a three-leaf clover-like structure (Fig. 5).	RESULTS
+40	47	modules	structure_element	In the structure of CtXyl5A-D, the four modules form a three-leaf clover-like structure (Fig. 5).	RESULTS
+12	22	interfaces	site	Between the interfaces of CtGH5-CBM6-CBM13 there are a number of interactions that maintain the modules in a fixed position relative to each other.	RESULTS
+26	42	CtGH5-CBM6-CBM13	structure_element	Between the interfaces of CtGH5-CBM6-CBM13 there are a number of interactions that maintain the modules in a fixed position relative to each other.	RESULTS
+19	24	CtGH5	structure_element	The interaction of CtGH5 and CtCBM6, which buries a substantial apolar solvent-exposed surface of the two modules, has been described previously.	RESULTS
+29	35	CtCBM6	structure_element	The interaction of CtGH5 and CtCBM6, which buries a substantial apolar solvent-exposed surface of the two modules, has been described previously.	RESULTS
+64	94	apolar solvent-exposed surface	site	The interaction of CtGH5 and CtCBM6, which buries a substantial apolar solvent-exposed surface of the two modules, has been described previously.	RESULTS
+4	22	polar interactions	bond_interaction	The polar interactions between these two modules comprise 14 hydrogen bonds and 5 salt bridges.	RESULTS
+61	75	hydrogen bonds	bond_interaction	The polar interactions between these two modules comprise 14 hydrogen bonds and 5 salt bridges.	RESULTS
+82	94	salt bridges	bond_interaction	The polar interactions between these two modules comprise 14 hydrogen bonds and 5 salt bridges.	RESULTS
+4	33	apolar and polar interactions	bond_interaction	The apolar and polar interactions between these two modules likely explaining why they do not fold independently compared with other glycoside hydrolases that contain CBMs.	RESULTS
+133	153	glycoside hydrolases	protein_type	The apolar and polar interactions between these two modules likely explaining why they do not fold independently compared with other glycoside hydrolases that contain CBMs.	RESULTS
+167	171	CBMs	structure_element	The apolar and polar interactions between these two modules likely explaining why they do not fold independently compared with other glycoside hydrolases that contain CBMs.	RESULTS
+0	7	CtCBM13	structure_element	CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of ∼450, 350, and 500 Å2, respectively, to form a compact heterotetramer.	RESULTS
+20	34	central domain	structure_element	CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of ∼450, 350, and 500 Å2, respectively, to form a compact heterotetramer.	RESULTS
+42	56	interacts with	protein_state	CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of ∼450, 350, and 500 Å2, respectively, to form a compact heterotetramer.	RESULTS
+57	62	CtGH5	structure_element	CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of ∼450, 350, and 500 Å2, respectively, to form a compact heterotetramer.	RESULTS
+64	70	CtCBM6	structure_element	CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of ∼450, 350, and 500 Å2, respectively, to form a compact heterotetramer.	RESULTS
+76	81	CtFn3	structure_element	CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of ∼450, 350, and 500 Å2, respectively, to form a compact heterotetramer.	RESULTS
+98	112	hydrogen bonds	bond_interaction	CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of ∼450, 350, and 500 Å2, respectively, to form a compact heterotetramer.	RESULTS
+201	208	compact	protein_state	CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of ∼450, 350, and 500 Å2, respectively, to form a compact heterotetramer.	RESULTS
+209	223	heterotetramer	oligomeric_state	CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of ∼450, 350, and 500 Å2, respectively, to form a compact heterotetramer.	RESULTS
+20	42	CtCBM6-CBM13 interface	site	With respect to the CtCBM6-CBM13 interface, the linker (SPISTGTIP) between the two modules, extending from Ser514 to Pro522, adopts a fixed conformation.	RESULTS
+48	54	linker	structure_element	With respect to the CtCBM6-CBM13 interface, the linker (SPISTGTIP) between the two modules, extending from Ser514 to Pro522, adopts a fixed conformation.	RESULTS
+56	65	SPISTGTIP	structure_element	With respect to the CtCBM6-CBM13 interface, the linker (SPISTGTIP) between the two modules, extending from Ser514 to Pro522, adopts a fixed conformation.	RESULTS
+83	90	modules	structure_element	With respect to the CtCBM6-CBM13 interface, the linker (SPISTGTIP) between the two modules, extending from Ser514 to Pro522, adopts a fixed conformation.	RESULTS
+107	113	Ser514	residue_name_number	With respect to the CtCBM6-CBM13 interface, the linker (SPISTGTIP) between the two modules, extending from Ser514 to Pro522, adopts a fixed conformation.	RESULTS
+117	123	Pro522	residue_name_number	With respect to the CtCBM6-CBM13 interface, the linker (SPISTGTIP) between the two modules, extending from Ser514 to Pro522, adopts a fixed conformation.	RESULTS
+134	152	fixed conformation	protein_state	With respect to the CtCBM6-CBM13 interface, the linker (SPISTGTIP) between the two modules, extending from Ser514 to Pro522, adopts a fixed conformation.	RESULTS
+65	68	Ile	residue_name	Such sequences are normally extremely flexible; however, the two Ile residues make extensive apolar contacts within the linker and with the two CBMs, leading to conformational stabilization.	RESULTS
+93	108	apolar contacts	bond_interaction	Such sequences are normally extremely flexible; however, the two Ile residues make extensive apolar contacts within the linker and with the two CBMs, leading to conformational stabilization.	RESULTS
+120	126	linker	structure_element	Such sequences are normally extremely flexible; however, the two Ile residues make extensive apolar contacts within the linker and with the two CBMs, leading to conformational stabilization.	RESULTS
+144	148	CBMs	structure_element	Such sequences are normally extremely flexible; however, the two Ile residues make extensive apolar contacts within the linker and with the two CBMs, leading to conformational stabilization.	RESULTS
+25	30	CtGH5	structure_element	The interactions between CtGH5 and the two CBMs, which are mediated by the tip of the loop between β-7 and α-7 (loop 7) of CtGH5, not only stabilize the trimodular clover-like structure but also make a contribution to catalytic function.	RESULTS
+43	47	CBMs	structure_element	The interactions between CtGH5 and the two CBMs, which are mediated by the tip of the loop between β-7 and α-7 (loop 7) of CtGH5, not only stabilize the trimodular clover-like structure but also make a contribution to catalytic function.	RESULTS
+86	90	loop	structure_element	The interactions between CtGH5 and the two CBMs, which are mediated by the tip of the loop between β-7 and α-7 (loop 7) of CtGH5, not only stabilize the trimodular clover-like structure but also make a contribution to catalytic function.	RESULTS
+99	102	β-7	structure_element	The interactions between CtGH5 and the two CBMs, which are mediated by the tip of the loop between β-7 and α-7 (loop 7) of CtGH5, not only stabilize the trimodular clover-like structure but also make a contribution to catalytic function.	RESULTS
+107	110	α-7	structure_element	The interactions between CtGH5 and the two CBMs, which are mediated by the tip of the loop between β-7 and α-7 (loop 7) of CtGH5, not only stabilize the trimodular clover-like structure but also make a contribution to catalytic function.	RESULTS
+112	118	loop 7	structure_element	The interactions between CtGH5 and the two CBMs, which are mediated by the tip of the loop between β-7 and α-7 (loop 7) of CtGH5, not only stabilize the trimodular clover-like structure but also make a contribution to catalytic function.	RESULTS
+123	128	CtGH5	structure_element	The interactions between CtGH5 and the two CBMs, which are mediated by the tip of the loop between β-7 and α-7 (loop 7) of CtGH5, not only stabilize the trimodular clover-like structure but also make a contribution to catalytic function.	RESULTS
+153	170	trimodular clover	structure_element	The interactions between CtGH5 and the two CBMs, which are mediated by the tip of the loop between β-7 and α-7 (loop 7) of CtGH5, not only stabilize the trimodular clover-like structure but also make a contribution to catalytic function.	RESULTS
+46	53	modules	structure_element	Central to the interactions between the three modules is Trp285, which is intercalated between the two CBMs.	RESULTS
+57	63	Trp285	residue_name_number	Central to the interactions between the three modules is Trp285, which is intercalated between the two CBMs.	RESULTS
+74	94	intercalated between	bond_interaction	Central to the interactions between the three modules is Trp285, which is intercalated between the two CBMs.	RESULTS
+103	107	CBMs	structure_element	Central to the interactions between the three modules is Trp285, which is intercalated between the two CBMs.	RESULTS
+38	52	hydrogen bonds	bond_interaction	The Nϵ of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5).	RESULTS
+83	89	Val615	residue_name_number	The Nϵ of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5).	RESULTS
+94	100	Gly616	residue_name_number	The Nϵ of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5).	RESULTS
+104	111	CtCBM13	structure_element	The Nϵ of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5).	RESULTS
+147	162	apolar contacts	bond_interaction	The Nϵ of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5).	RESULTS
+168	174	CtCBM6	structure_element	The Nϵ of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5).	RESULTS
+176	182	Pro440	residue_name_number	The Nϵ of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5).	RESULTS
+184	190	Phe489	residue_name_number	The Nϵ of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5).	RESULTS
+192	198	Gly491	residue_name_number	The Nϵ of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5).	RESULTS
+204	210	Ala492	residue_name_number	The Nϵ of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5).	RESULTS
+8	14	loop 7	structure_element	Indeed, loop 7 is completely disordered in the truncated derivative of CtXyl5A comprising CtGH5 and CtCBM6, demonstrating that the interactions with CtCBM13 stabilize the conformation of this loop.	RESULTS
+18	39	completely disordered	protein_state	Indeed, loop 7 is completely disordered in the truncated derivative of CtXyl5A comprising CtGH5 and CtCBM6, demonstrating that the interactions with CtCBM13 stabilize the conformation of this loop.	RESULTS
+47	56	truncated	protein_state	Indeed, loop 7 is completely disordered in the truncated derivative of CtXyl5A comprising CtGH5 and CtCBM6, demonstrating that the interactions with CtCBM13 stabilize the conformation of this loop.	RESULTS
+71	78	CtXyl5A	protein	Indeed, loop 7 is completely disordered in the truncated derivative of CtXyl5A comprising CtGH5 and CtCBM6, demonstrating that the interactions with CtCBM13 stabilize the conformation of this loop.	RESULTS
+90	95	CtGH5	structure_element	Indeed, loop 7 is completely disordered in the truncated derivative of CtXyl5A comprising CtGH5 and CtCBM6, demonstrating that the interactions with CtCBM13 stabilize the conformation of this loop.	RESULTS
+100	106	CtCBM6	structure_element	Indeed, loop 7 is completely disordered in the truncated derivative of CtXyl5A comprising CtGH5 and CtCBM6, demonstrating that the interactions with CtCBM13 stabilize the conformation of this loop.	RESULTS
+149	156	CtCBM13	structure_element	Indeed, loop 7 is completely disordered in the truncated derivative of CtXyl5A comprising CtGH5 and CtCBM6, demonstrating that the interactions with CtCBM13 stabilize the conformation of this loop.	RESULTS
+192	196	loop	structure_element	Indeed, loop 7 is completely disordered in the truncated derivative of CtXyl5A comprising CtGH5 and CtCBM6, demonstrating that the interactions with CtCBM13 stabilize the conformation of this loop.	RESULTS
+20	26	loop 7	structure_element	Although the tip of loop 7 does not directly contribute to the topology of the active site, it is only ∼12 Å from the catalytic nucleophile Glu279.	RESULTS
+79	90	active site	site	Although the tip of loop 7 does not directly contribute to the topology of the active site, it is only ∼12 Å from the catalytic nucleophile Glu279.	RESULTS
+140	146	Glu279	residue_name_number	Although the tip of loop 7 does not directly contribute to the topology of the active site, it is only ∼12 Å from the catalytic nucleophile Glu279.	RESULTS
+30	34	loop	structure_element	Thus, any perturbation of the loop (through the removal of CtCBM13) is likely to influence the electrostatic and apolar environment of the catalytic apparatus, which could explain the reduction in activity associated with the deletion of CtCBM13.	RESULTS
+48	55	removal	experimental_method	Thus, any perturbation of the loop (through the removal of CtCBM13) is likely to influence the electrostatic and apolar environment of the catalytic apparatus, which could explain the reduction in activity associated with the deletion of CtCBM13.	RESULTS
+59	66	CtCBM13	structure_element	Thus, any perturbation of the loop (through the removal of CtCBM13) is likely to influence the electrostatic and apolar environment of the catalytic apparatus, which could explain the reduction in activity associated with the deletion of CtCBM13.	RESULTS
+226	234	deletion	experimental_method	Thus, any perturbation of the loop (through the removal of CtCBM13) is likely to influence the electrostatic and apolar environment of the catalytic apparatus, which could explain the reduction in activity associated with the deletion of CtCBM13.	RESULTS
+238	245	CtCBM13	structure_element	Thus, any perturbation of the loop (through the removal of CtCBM13) is likely to influence the electrostatic and apolar environment of the catalytic apparatus, which could explain the reduction in activity associated with the deletion of CtCBM13.	RESULTS
+36	42	CtCBM6	structure_element	Similar to the interactions between CtCBM6 and CtCBM13, there are extensive hydrophobic interactions between CtCBM13 and CtFn3, resulting in very little flexibility between these modules.	RESULTS
+47	54	CtCBM13	structure_element	Similar to the interactions between CtCBM6 and CtCBM13, there are extensive hydrophobic interactions between CtCBM13 and CtFn3, resulting in very little flexibility between these modules.	RESULTS
+76	100	hydrophobic interactions	bond_interaction	Similar to the interactions between CtCBM6 and CtCBM13, there are extensive hydrophobic interactions between CtCBM13 and CtFn3, resulting in very little flexibility between these modules.	RESULTS
+109	116	CtCBM13	structure_element	Similar to the interactions between CtCBM6 and CtCBM13, there are extensive hydrophobic interactions between CtCBM13 and CtFn3, resulting in very little flexibility between these modules.	RESULTS
+121	126	CtFn3	structure_element	Similar to the interactions between CtCBM6 and CtCBM13, there are extensive hydrophobic interactions between CtCBM13 and CtFn3, resulting in very little flexibility between these modules.	RESULTS
+179	186	modules	structure_element	Similar to the interactions between CtCBM6 and CtCBM13, there are extensive hydrophobic interactions between CtCBM13 and CtFn3, resulting in very little flexibility between these modules.	RESULTS
+21	31	absence of	protein_state	As stated above, the absence of CtCBM62 in the structure suggests that the module can adopt multiple positions with respect to the rest of the protein.	RESULTS
+32	39	CtCBM62	structure_element	As stated above, the absence of CtCBM62 in the structure suggests that the module can adopt multiple positions with respect to the rest of the protein.	RESULTS
+47	56	structure	evidence	As stated above, the absence of CtCBM62 in the structure suggests that the module can adopt multiple positions with respect to the rest of the protein.	RESULTS
+75	81	module	structure_element	As stated above, the absence of CtCBM62 in the structure suggests that the module can adopt multiple positions with respect to the rest of the protein.	RESULTS
+4	11	CtCBM62	structure_element	The CtCBM62, by binding to its ligands (d-Galp and l-Arap) in plant cell walls, may be able to recruit the enzyme onto its target substrate.	RESULTS
+16	26	binding to	protein_state	The CtCBM62, by binding to its ligands (d-Galp and l-Arap) in plant cell walls, may be able to recruit the enzyme onto its target substrate.	RESULTS
+40	46	d-Galp	chemical	The CtCBM62, by binding to its ligands (d-Galp and l-Arap) in plant cell walls, may be able to recruit the enzyme onto its target substrate.	RESULTS
+51	57	l-Arap	chemical	The CtCBM62, by binding to its ligands (d-Galp and l-Arap) in plant cell walls, may be able to recruit the enzyme onto its target substrate.	RESULTS
+62	67	plant	taxonomy_domain	The CtCBM62, by binding to its ligands (d-Galp and l-Arap) in plant cell walls, may be able to recruit the enzyme onto its target substrate.	RESULTS
+0	6	Xylans	chemical	Xylans are not generally thought to contain such sugars.	RESULTS
+49	55	sugars	chemical	Xylans are not generally thought to contain such sugars.	RESULTS
+0	6	d-Galp	chemical	d-Galp, however, has been detected in xylans in the outer layer of cereal grains and in eucalyptus trees, which are substrates used by CtXyl5A.	RESULTS
+38	44	xylans	chemical	d-Galp, however, has been detected in xylans in the outer layer of cereal grains and in eucalyptus trees, which are substrates used by CtXyl5A.	RESULTS
+67	73	cereal	taxonomy_domain	d-Galp, however, has been detected in xylans in the outer layer of cereal grains and in eucalyptus trees, which are substrates used by CtXyl5A.	RESULTS
+88	104	eucalyptus trees	taxonomy_domain	d-Galp, however, has been detected in xylans in the outer layer of cereal grains and in eucalyptus trees, which are substrates used by CtXyl5A.	RESULTS
+135	142	CtXyl5A	protein	d-Galp, however, has been detected in xylans in the outer layer of cereal grains and in eucalyptus trees, which are substrates used by CtXyl5A.	RESULTS
+6	13	CtCBM62	structure_element	Thus, CtCBM62 may direct the enzyme to particularly complex xylans containing d-Galp at the non-reducing termini of the side chains, consistent with the open substrate binding cleft of the arabinoxylanase that is optimized to bind highly decorated forms of the hemicellulose.	RESULTS
+60	66	xylans	chemical	Thus, CtCBM62 may direct the enzyme to particularly complex xylans containing d-Galp at the non-reducing termini of the side chains, consistent with the open substrate binding cleft of the arabinoxylanase that is optimized to bind highly decorated forms of the hemicellulose.	RESULTS
+78	84	d-Galp	chemical	Thus, CtCBM62 may direct the enzyme to particularly complex xylans containing d-Galp at the non-reducing termini of the side chains, consistent with the open substrate binding cleft of the arabinoxylanase that is optimized to bind highly decorated forms of the hemicellulose.	RESULTS
+153	157	open	protein_state	Thus, CtCBM62 may direct the enzyme to particularly complex xylans containing d-Galp at the non-reducing termini of the side chains, consistent with the open substrate binding cleft of the arabinoxylanase that is optimized to bind highly decorated forms of the hemicellulose.	RESULTS
+158	181	substrate binding cleft	site	Thus, CtCBM62 may direct the enzyme to particularly complex xylans containing d-Galp at the non-reducing termini of the side chains, consistent with the open substrate binding cleft of the arabinoxylanase that is optimized to bind highly decorated forms of the hemicellulose.	RESULTS
+189	204	arabinoxylanase	protein_type	Thus, CtCBM62 may direct the enzyme to particularly complex xylans containing d-Galp at the non-reducing termini of the side chains, consistent with the open substrate binding cleft of the arabinoxylanase that is optimized to bind highly decorated forms of the hemicellulose.	RESULTS
+261	274	hemicellulose	chemical	Thus, CtCBM62 may direct the enzyme to particularly complex xylans containing d-Galp at the non-reducing termini of the side chains, consistent with the open substrate binding cleft of the arabinoxylanase that is optimized to bind highly decorated forms of the hemicellulose.	RESULTS
+11	15	CBMs	structure_element	In general CBMs have little influence on enzyme activity against soluble substrates but have a significant impact on glycans within plant cell walls.	RESULTS
+117	124	glycans	chemical	In general CBMs have little influence on enzyme activity against soluble substrates but have a significant impact on glycans within plant cell walls.	RESULTS
+132	137	plant	taxonomy_domain	In general CBMs have little influence on enzyme activity against soluble substrates but have a significant impact on glycans within plant cell walls.	RESULTS
+18	23	CBM62	structure_element	Thus, the role of CBM62 will likely only be evident against insoluble composite substrates.	RESULTS
+10	26	GH5 Subfamily 34	protein_type	Exploring GH5 Subfamily 34	RESULTS
+0	7	CtXyl5A	protein	CtXyl5A is a member of a seven-protein subfamily of GH5, GH5_34.	RESULTS
+52	55	GH5	protein_type	CtXyl5A is a member of a seven-protein subfamily of GH5, GH5_34.	RESULTS
+57	63	GH5_34	protein_type	CtXyl5A is a member of a seven-protein subfamily of GH5, GH5_34.	RESULTS
+130	145	C. thermocellum	species	Four of these proteins are distinct, whereas the other three members are essentially identical (derived from different strains of C. thermocellum).	RESULTS
+110	116	GH5_34	protein_type	To investigate further the substrate specificity within this subfamily, recombinant forms of three members of GH5_34 that were distinct from CtXyl5A were generated.	RESULTS
+141	148	CtXyl5A	protein	To investigate further the substrate specificity within this subfamily, recombinant forms of three members of GH5_34 that were distinct from CtXyl5A were generated.	RESULTS
+0	5	AcGH5	protein	AcGH5 has a similar molecular architecture to CtXyl5A with the exception of an additional carbohydrate esterase family 6 module at the C terminus (Fig. 1).	RESULTS
+46	53	CtXyl5A	protein	AcGH5 has a similar molecular architecture to CtXyl5A with the exception of an additional carbohydrate esterase family 6 module at the C terminus (Fig. 1).	RESULTS
+90	127	carbohydrate esterase family 6 module	structure_element	AcGH5 has a similar molecular architecture to CtXyl5A with the exception of an additional carbohydrate esterase family 6 module at the C terminus (Fig. 1).	RESULTS
+4	10	GH5_34	protein_type	The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function.	RESULTS
+16	32	Verrucomicrobiae	taxonomy_domain	The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function.	RESULTS
+33	42	bacterium	taxonomy_domain	The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function.	RESULTS
+44	49	VbGH5	protein	The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function.	RESULTS
+64	78	GH5-CBM6-CBM13	structure_element	The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function.	RESULTS
+114	132	Fn3-CBM62-dockerin	structure_element	The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function.	RESULTS
+153	160	CtXyl5A	protein	The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function.	RESULTS
+182	200	Laminin_3_G domain	structure_element	The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function.	RESULTS
+283	296	carbohydrates	chemical	The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function.	RESULTS
+312	318	glycan	chemical	The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function.	RESULTS
+4	19	Verrucomicobiae	taxonomy_domain	The Verrucomicobiae enzyme also has an N-terminal GH43 subfamily 10 (GH43_10) catalytic module.	RESULTS
+50	67	GH43 subfamily 10	protein_type	The Verrucomicobiae enzyme also has an N-terminal GH43 subfamily 10 (GH43_10) catalytic module.	RESULTS
+69	76	GH43_10	protein_type	The Verrucomicobiae enzyme also has an N-terminal GH43 subfamily 10 (GH43_10) catalytic module.	RESULTS
+78	94	catalytic module	structure_element	The Verrucomicobiae enzyme also has an N-terminal GH43 subfamily 10 (GH43_10) catalytic module.	RESULTS
+4	10	fungal	taxonomy_domain	The fungal GH5_34, GpGH5, unlike the two bacterial homologs, comprises a single GH5 catalytic module lacking all of the other accessory modules (Fig. 1).	RESULTS
+11	17	GH5_34	protein_type	The fungal GH5_34, GpGH5, unlike the two bacterial homologs, comprises a single GH5 catalytic module lacking all of the other accessory modules (Fig. 1).	RESULTS
+19	24	GpGH5	protein	The fungal GH5_34, GpGH5, unlike the two bacterial homologs, comprises a single GH5 catalytic module lacking all of the other accessory modules (Fig. 1).	RESULTS
+41	50	bacterial	taxonomy_domain	The fungal GH5_34, GpGH5, unlike the two bacterial homologs, comprises a single GH5 catalytic module lacking all of the other accessory modules (Fig. 1).	RESULTS
+80	83	GH5	protein_type	The fungal GH5_34, GpGH5, unlike the two bacterial homologs, comprises a single GH5 catalytic module lacking all of the other accessory modules (Fig. 1).	RESULTS
+84	100	catalytic module	structure_element	The fungal GH5_34, GpGH5, unlike the two bacterial homologs, comprises a single GH5 catalytic module lacking all of the other accessory modules (Fig. 1).	RESULTS
+0	5	GpGh5	protein	GpGh5 is particularly interesting as Gonapodya prolifera is the only fungus of the several hundred fungal genomes that encodes a GH5_34 enzyme.	RESULTS
+37	56	Gonapodya prolifera	species	GpGh5 is particularly interesting as Gonapodya prolifera is the only fungus of the several hundred fungal genomes that encodes a GH5_34 enzyme.	RESULTS
+69	75	fungus	taxonomy_domain	GpGh5 is particularly interesting as Gonapodya prolifera is the only fungus of the several hundred fungal genomes that encodes a GH5_34 enzyme.	RESULTS
+99	105	fungal	taxonomy_domain	GpGh5 is particularly interesting as Gonapodya prolifera is the only fungus of the several hundred fungal genomes that encodes a GH5_34 enzyme.	RESULTS
+129	135	GH5_34	protein_type	GpGh5 is particularly interesting as Gonapodya prolifera is the only fungus of the several hundred fungal genomes that encodes a GH5_34 enzyme.	RESULTS
+33	39	GH5_34	protein_type	In fact there are four potential GH5_34 sequences in the G. prolifera genome, all of which show high sequence homology to Clostridium GH5_34 sequences.	RESULTS
+57	69	G. prolifera	species	In fact there are four potential GH5_34 sequences in the G. prolifera genome, all of which show high sequence homology to Clostridium GH5_34 sequences.	RESULTS
+122	133	Clostridium	taxonomy_domain	In fact there are four potential GH5_34 sequences in the G. prolifera genome, all of which show high sequence homology to Clostridium GH5_34 sequences.	RESULTS
+134	140	GH5_34	protein_type	In fact there are four potential GH5_34 sequences in the G. prolifera genome, all of which show high sequence homology to Clostridium GH5_34 sequences.	RESULTS
+0	12	G. prolifera	species	G. prolifera and Clostridium occupy similar environments, suggesting that the GpGH5_34 gene was acquired from a Clostridium species, which was followed by duplication of the gene in the fungal genome.	RESULTS
+17	28	Clostridium	taxonomy_domain	G. prolifera and Clostridium occupy similar environments, suggesting that the GpGH5_34 gene was acquired from a Clostridium species, which was followed by duplication of the gene in the fungal genome.	RESULTS
+78	86	GpGH5_34	protein	G. prolifera and Clostridium occupy similar environments, suggesting that the GpGH5_34 gene was acquired from a Clostridium species, which was followed by duplication of the gene in the fungal genome.	RESULTS
+112	123	Clostridium	taxonomy_domain	G. prolifera and Clostridium occupy similar environments, suggesting that the GpGH5_34 gene was acquired from a Clostridium species, which was followed by duplication of the gene in the fungal genome.	RESULTS
+186	192	fungal	taxonomy_domain	G. prolifera and Clostridium occupy similar environments, suggesting that the GpGH5_34 gene was acquired from a Clostridium species, which was followed by duplication of the gene in the fungal genome.	RESULTS
+29	35	GH5_34	protein_type	The sequence identity of the GH5_34 catalytic modules with CtXyl5A ranged from 55 to 80% (supplemental Fig. S1).	RESULTS
+36	53	catalytic modules	structure_element	The sequence identity of the GH5_34 catalytic modules with CtXyl5A ranged from 55 to 80% (supplemental Fig. S1).	RESULTS
+59	66	CtXyl5A	protein	The sequence identity of the GH5_34 catalytic modules with CtXyl5A ranged from 55 to 80% (supplemental Fig. S1).	RESULTS
+8	14	GH5_34	protein_type	All the GH5_34 enzymes were active on the arabinoxylans RAX, WAX, and CX but displayed no activity on BX (Table 1 and Fig. 6) and are thus defined as arabinoxylanases.	RESULTS
+42	55	arabinoxylans	chemical	All the GH5_34 enzymes were active on the arabinoxylans RAX, WAX, and CX but displayed no activity on BX (Table 1 and Fig. 6) and are thus defined as arabinoxylanases.	RESULTS
+56	59	RAX	chemical	All the GH5_34 enzymes were active on the arabinoxylans RAX, WAX, and CX but displayed no activity on BX (Table 1 and Fig. 6) and are thus defined as arabinoxylanases.	RESULTS
+61	64	WAX	chemical	All the GH5_34 enzymes were active on the arabinoxylans RAX, WAX, and CX but displayed no activity on BX (Table 1 and Fig. 6) and are thus defined as arabinoxylanases.	RESULTS
+70	72	CX	chemical	All the GH5_34 enzymes were active on the arabinoxylans RAX, WAX, and CX but displayed no activity on BX (Table 1 and Fig. 6) and are thus defined as arabinoxylanases.	RESULTS
+102	104	BX	chemical	All the GH5_34 enzymes were active on the arabinoxylans RAX, WAX, and CX but displayed no activity on BX (Table 1 and Fig. 6) and are thus defined as arabinoxylanases.	RESULTS
+150	166	arabinoxylanases	protein_type	All the GH5_34 enzymes were active on the arabinoxylans RAX, WAX, and CX but displayed no activity on BX (Table 1 and Fig. 6) and are thus defined as arabinoxylanases.	RESULTS
+32	39	CtXyl5A	protein	The limit products generated by CtXyl5A, AcGH5, and GpGH5 comprised a range of oligosaccharides with some high molecular weight material.	RESULTS
+41	46	AcGH5	protein	The limit products generated by CtXyl5A, AcGH5, and GpGH5 comprised a range of oligosaccharides with some high molecular weight material.	RESULTS
+52	57	GpGH5	protein	The limit products generated by CtXyl5A, AcGH5, and GpGH5 comprised a range of oligosaccharides with some high molecular weight material.	RESULTS
+79	95	oligosaccharides	chemical	The limit products generated by CtXyl5A, AcGH5, and GpGH5 comprised a range of oligosaccharides with some high molecular weight material.	RESULTS
+4	20	oligosaccharides	chemical	The oligosaccharides with low degrees of polymerization were absent in the VbGH5 reaction products.	RESULTS
+75	80	VbGH5	protein	The oligosaccharides with low degrees of polymerization were absent in the VbGH5 reaction products.	RESULTS
+48	57	arabinose	chemical	However, the enzyme generated a large amount of arabinose, which was not produced by the other arabinoxylanases.	RESULTS
+95	111	arabinoxylanases	protein_type	However, the enzyme generated a large amount of arabinose, which was not produced by the other arabinoxylanases.	RESULTS
+11	18	GH43_10	protein_type	Given that GH43_10 is predominantly an arabinofuranosidase subfamily of GH43, the arabinose generated by VbGH5 is likely mediated by the N-terminal catalytic module (see below).	RESULTS
+39	58	arabinofuranosidase	protein_type	Given that GH43_10 is predominantly an arabinofuranosidase subfamily of GH43, the arabinose generated by VbGH5 is likely mediated by the N-terminal catalytic module (see below).	RESULTS
+72	76	GH43	protein_type	Given that GH43_10 is predominantly an arabinofuranosidase subfamily of GH43, the arabinose generated by VbGH5 is likely mediated by the N-terminal catalytic module (see below).	RESULTS
+82	91	arabinose	chemical	Given that GH43_10 is predominantly an arabinofuranosidase subfamily of GH43, the arabinose generated by VbGH5 is likely mediated by the N-terminal catalytic module (see below).	RESULTS
+105	110	VbGH5	protein	Given that GH43_10 is predominantly an arabinofuranosidase subfamily of GH43, the arabinose generated by VbGH5 is likely mediated by the N-terminal catalytic module (see below).	RESULTS
+148	164	catalytic module	structure_element	Given that GH43_10 is predominantly an arabinofuranosidase subfamily of GH43, the arabinose generated by VbGH5 is likely mediated by the N-terminal catalytic module (see below).	RESULTS
+29	34	AcGH5	protein	Kinetic analysis showed that AcGH5 displayed similar activity to CtXyl5A against both WAX and RAX and was 2-fold less active against CX.	RESULTS
+65	72	CtXyl5A	protein	Kinetic analysis showed that AcGH5 displayed similar activity to CtXyl5A against both WAX and RAX and was 2-fold less active against CX.	RESULTS
+86	89	WAX	chemical	Kinetic analysis showed that AcGH5 displayed similar activity to CtXyl5A against both WAX and RAX and was 2-fold less active against CX.	RESULTS
+94	97	RAX	chemical	Kinetic analysis showed that AcGH5 displayed similar activity to CtXyl5A against both WAX and RAX and was 2-fold less active against CX.	RESULTS
+133	135	CX	chemical	Kinetic analysis showed that AcGH5 displayed similar activity to CtXyl5A against both WAX and RAX and was 2-fold less active against CX.	RESULTS
+41	50	wild type	protein_state	When initially measuring the activity of wild type VbGH5 against the different substrates, no clear data could be obtained, regardless of the concentration of enzyme used the reaction appeared to cease after a few minutes.	RESULTS
+51	56	VbGH5	protein	When initially measuring the activity of wild type VbGH5 against the different substrates, no clear data could be obtained, regardless of the concentration of enzyme used the reaction appeared to cease after a few minutes.	RESULTS
+36	43	GH43_10	protein_type	We hypothesized that the N-terminal GH43_10 rapidly removed single arabinose decorations from the arabinoxylans depleting the substrate available to the arabinoxylanase, explaining why this activity was short lived.	RESULTS
+67	76	arabinose	chemical	We hypothesized that the N-terminal GH43_10 rapidly removed single arabinose decorations from the arabinoxylans depleting the substrate available to the arabinoxylanase, explaining why this activity was short lived.	RESULTS
+98	111	arabinoxylans	chemical	We hypothesized that the N-terminal GH43_10 rapidly removed single arabinose decorations from the arabinoxylans depleting the substrate available to the arabinoxylanase, explaining why this activity was short lived.	RESULTS
+153	168	arabinoxylanase	protein_type	We hypothesized that the N-terminal GH43_10 rapidly removed single arabinose decorations from the arabinoxylans depleting the substrate available to the arabinoxylanase, explaining why this activity was short lived.	RESULTS
+29	38	conserved	protein_state	To test this hypothesis, the conserved catalytic base (Asp45) of the GH43_10 module of VbGH5 was substituted with alanine, which is predicted to inactivate this catalytic module.	RESULTS
+55	60	Asp45	residue_name_number	To test this hypothesis, the conserved catalytic base (Asp45) of the GH43_10 module of VbGH5 was substituted with alanine, which is predicted to inactivate this catalytic module.	RESULTS
+69	76	GH43_10	structure_element	To test this hypothesis, the conserved catalytic base (Asp45) of the GH43_10 module of VbGH5 was substituted with alanine, which is predicted to inactivate this catalytic module.	RESULTS
+87	92	VbGH5	protein	To test this hypothesis, the conserved catalytic base (Asp45) of the GH43_10 module of VbGH5 was substituted with alanine, which is predicted to inactivate this catalytic module.	RESULTS
+97	113	substituted with	experimental_method	To test this hypothesis, the conserved catalytic base (Asp45) of the GH43_10 module of VbGH5 was substituted with alanine, which is predicted to inactivate this catalytic module.	RESULTS
+114	121	alanine	residue_name	To test this hypothesis, the conserved catalytic base (Asp45) of the GH43_10 module of VbGH5 was substituted with alanine, which is predicted to inactivate this catalytic module.	RESULTS
+161	177	catalytic module	structure_element	To test this hypothesis, the conserved catalytic base (Asp45) of the GH43_10 module of VbGH5 was substituted with alanine, which is predicted to inactivate this catalytic module.	RESULTS
+4	8	D45A	mutant	The D45A mutant did not produce arabinose consistent with the arabinofuranosidase activity displayed by the GH43_10 module in the wild type enzyme (Fig. 6).	RESULTS
+9	15	mutant	protein_state	The D45A mutant did not produce arabinose consistent with the arabinofuranosidase activity displayed by the GH43_10 module in the wild type enzyme (Fig. 6).	RESULTS
+32	41	arabinose	chemical	The D45A mutant did not produce arabinose consistent with the arabinofuranosidase activity displayed by the GH43_10 module in the wild type enzyme (Fig. 6).	RESULTS
+62	81	arabinofuranosidase	protein_type	The D45A mutant did not produce arabinose consistent with the arabinofuranosidase activity displayed by the GH43_10 module in the wild type enzyme (Fig. 6).	RESULTS
+108	115	GH43_10	structure_element	The D45A mutant did not produce arabinose consistent with the arabinofuranosidase activity displayed by the GH43_10 module in the wild type enzyme (Fig. 6).	RESULTS
+130	139	wild type	protein_state	The D45A mutant did not produce arabinose consistent with the arabinofuranosidase activity displayed by the GH43_10 module in the wild type enzyme (Fig. 6).	RESULTS
+4	12	kinetics	evidence	The kinetics of the GH5_34 arabinoxylanase catalytic module was now measurable, and activities were determined to be between ∼6- and 10-fold lower than that of CtXyl5A.	RESULTS
+20	26	GH5_34	protein_type	The kinetics of the GH5_34 arabinoxylanase catalytic module was now measurable, and activities were determined to be between ∼6- and 10-fold lower than that of CtXyl5A.	RESULTS
+27	42	arabinoxylanase	protein_type	The kinetics of the GH5_34 arabinoxylanase catalytic module was now measurable, and activities were determined to be between ∼6- and 10-fold lower than that of CtXyl5A.	RESULTS
+43	59	catalytic module	structure_element	The kinetics of the GH5_34 arabinoxylanase catalytic module was now measurable, and activities were determined to be between ∼6- and 10-fold lower than that of CtXyl5A.	RESULTS
+160	167	CtXyl5A	protein	The kinetics of the GH5_34 arabinoxylanase catalytic module was now measurable, and activities were determined to be between ∼6- and 10-fold lower than that of CtXyl5A.	RESULTS
+19	25	fungal	taxonomy_domain	Interestingly, the fungal arabinoxylanase displays the highest activities against WAX and RAX, ∼4- and 6-fold higher, respectively, than CtXyl5A; however, there is very little difference in the activity between the eukaryotic and prokaryotic enzymes against CX.	RESULTS
+26	41	arabinoxylanase	protein_type	Interestingly, the fungal arabinoxylanase displays the highest activities against WAX and RAX, ∼4- and 6-fold higher, respectively, than CtXyl5A; however, there is very little difference in the activity between the eukaryotic and prokaryotic enzymes against CX.	RESULTS
+82	85	WAX	chemical	Interestingly, the fungal arabinoxylanase displays the highest activities against WAX and RAX, ∼4- and 6-fold higher, respectively, than CtXyl5A; however, there is very little difference in the activity between the eukaryotic and prokaryotic enzymes against CX.	RESULTS
+90	93	RAX	chemical	Interestingly, the fungal arabinoxylanase displays the highest activities against WAX and RAX, ∼4- and 6-fold higher, respectively, than CtXyl5A; however, there is very little difference in the activity between the eukaryotic and prokaryotic enzymes against CX.	RESULTS
+137	144	CtXyl5A	protein	Interestingly, the fungal arabinoxylanase displays the highest activities against WAX and RAX, ∼4- and 6-fold higher, respectively, than CtXyl5A; however, there is very little difference in the activity between the eukaryotic and prokaryotic enzymes against CX.	RESULTS
+215	225	eukaryotic	taxonomy_domain	Interestingly, the fungal arabinoxylanase displays the highest activities against WAX and RAX, ∼4- and 6-fold higher, respectively, than CtXyl5A; however, there is very little difference in the activity between the eukaryotic and prokaryotic enzymes against CX.	RESULTS
+230	241	prokaryotic	taxonomy_domain	Interestingly, the fungal arabinoxylanase displays the highest activities against WAX and RAX, ∼4- and 6-fold higher, respectively, than CtXyl5A; however, there is very little difference in the activity between the eukaryotic and prokaryotic enzymes against CX.	RESULTS
+258	260	CX	chemical	Interestingly, the fungal arabinoxylanase displays the highest activities against WAX and RAX, ∼4- and 6-fold higher, respectively, than CtXyl5A; however, there is very little difference in the activity between the eukaryotic and prokaryotic enzymes against CX.	RESULTS
+70	75	AcGH5	protein	Attempts to express individual modules of a variety of truncations of AcGH5 and VbGH5 were unsuccessful.	RESULTS
+80	85	VbGH5	protein	Attempts to express individual modules of a variety of truncations of AcGH5 and VbGH5 were unsuccessful.	RESULTS
+97	108	full-length	protein_state	This may indicate that the individual modules can only fold correctly when incorporated into the full-length enzyme, demonstrating the importance of intermodule interactions to maintain the structural integrity of these enzymes.	RESULTS
+30	36	GH5_34	protein_type	Products profile generated of GH5_34 enzymes.	FIG
+25	34	incubated	experimental_method	The enzymes at 1 μm were incubated with the four different xylans at 1% in 50 mm sodium phosphate buffer for 16 h at 37 °C (GpGH5, VbGH5, and AcGH5) or 60 °C.	FIG
+59	65	xylans	chemical	The enzymes at 1 μm were incubated with the four different xylans at 1% in 50 mm sodium phosphate buffer for 16 h at 37 °C (GpGH5, VbGH5, and AcGH5) or 60 °C.	FIG
+124	129	GpGH5	protein	The enzymes at 1 μm were incubated with the four different xylans at 1% in 50 mm sodium phosphate buffer for 16 h at 37 °C (GpGH5, VbGH5, and AcGH5) or 60 °C.	FIG
+131	136	VbGH5	protein	The enzymes at 1 μm were incubated with the four different xylans at 1% in 50 mm sodium phosphate buffer for 16 h at 37 °C (GpGH5, VbGH5, and AcGH5) or 60 °C.	FIG
+142	147	AcGH5	protein	The enzymes at 1 μm were incubated with the four different xylans at 1% in 50 mm sodium phosphate buffer for 16 h at 37 °C (GpGH5, VbGH5, and AcGH5) or 60 °C.	FIG
+37	40	TLC	experimental_method	The limit products were separated by TLC.	FIG
+4	23	xylooligosaccharide	chemical	The xylooligosaccharide standards (X) are indicated by their degrees of polymerization.	FIG
+52	57	plant	taxonomy_domain	A characteristic feature of enzymes that attack the plant cell wall is their complex molecular architecture.	DISCUSS
+4	8	CBMs	structure_element	The CBMs in these enzymes generally play a role in substrate targeting and are appended to the catalytic modules through flexible linker sequences.	DISCUSS
+95	112	catalytic modules	structure_element	The CBMs in these enzymes generally play a role in substrate targeting and are appended to the catalytic modules through flexible linker sequences.	DISCUSS
+121	146	flexible linker sequences	structure_element	The CBMs in these enzymes generally play a role in substrate targeting and are appended to the catalytic modules through flexible linker sequences.	DISCUSS
+0	7	CtXyl5A	protein	CtXyl5A provides a rare visualization of the structure of multiple modules within a single enzyme.	DISCUSS
+45	54	structure	evidence	CtXyl5A provides a rare visualization of the structure of multiple modules within a single enzyme.	DISCUSS
+78	82	CBMs	structure_element	The central feature of these data is the structural role played by two of the CBMs, CtCBM6 and CtCBM13, in maintaining the active conformation of the catalytic module, CtGH5.	DISCUSS
+84	90	CtCBM6	structure_element	The central feature of these data is the structural role played by two of the CBMs, CtCBM6 and CtCBM13, in maintaining the active conformation of the catalytic module, CtGH5.	DISCUSS
+95	102	CtCBM13	structure_element	The central feature of these data is the structural role played by two of the CBMs, CtCBM6 and CtCBM13, in maintaining the active conformation of the catalytic module, CtGH5.	DISCUSS
+123	129	active	protein_state	The central feature of these data is the structural role played by two of the CBMs, CtCBM6 and CtCBM13, in maintaining the active conformation of the catalytic module, CtGH5.	DISCUSS
+150	166	catalytic module	structure_element	The central feature of these data is the structural role played by two of the CBMs, CtCBM6 and CtCBM13, in maintaining the active conformation of the catalytic module, CtGH5.	DISCUSS
+168	173	CtGH5	structure_element	The central feature of these data is the structural role played by two of the CBMs, CtCBM6 and CtCBM13, in maintaining the active conformation of the catalytic module, CtGH5.	DISCUSS
+4	25	crystallographic data	evidence	The crystallographic data described here are supported by biochemical data showing either that these two modules do not bind to glycans (CtCBM13) or that the recognition of the non-reducing end of xylan or cellulose chains (CtCBM6) is unlikely to be biologically significant.	DISCUSS
+128	135	glycans	chemical	The crystallographic data described here are supported by biochemical data showing either that these two modules do not bind to glycans (CtCBM13) or that the recognition of the non-reducing end of xylan or cellulose chains (CtCBM6) is unlikely to be biologically significant.	DISCUSS
+137	144	CtCBM13	structure_element	The crystallographic data described here are supported by biochemical data showing either that these two modules do not bind to glycans (CtCBM13) or that the recognition of the non-reducing end of xylan or cellulose chains (CtCBM6) is unlikely to be biologically significant.	DISCUSS
+197	202	xylan	chemical	The crystallographic data described here are supported by biochemical data showing either that these two modules do not bind to glycans (CtCBM13) or that the recognition of the non-reducing end of xylan or cellulose chains (CtCBM6) is unlikely to be biologically significant.	DISCUSS
+206	215	cellulose	chemical	The crystallographic data described here are supported by biochemical data showing either that these two modules do not bind to glycans (CtCBM13) or that the recognition of the non-reducing end of xylan or cellulose chains (CtCBM6) is unlikely to be biologically significant.	DISCUSS
+224	230	CtCBM6	structure_element	The crystallographic data described here are supported by biochemical data showing either that these two modules do not bind to glycans (CtCBM13) or that the recognition of the non-reducing end of xylan or cellulose chains (CtCBM6) is unlikely to be biologically significant.	DISCUSS
+39	45	glycan	chemical	It should be emphasized, however, that glycan binding and substrate targeting may only be evident in the full-length enzyme acting on highly complex structures such as the plant cell wall, as observed recently by a CBM46 module in the Bacillus xyloglucanase/mixed linked glucanase BhCel5B.	DISCUSS
+105	116	full-length	protein_state	It should be emphasized, however, that glycan binding and substrate targeting may only be evident in the full-length enzyme acting on highly complex structures such as the plant cell wall, as observed recently by a CBM46 module in the Bacillus xyloglucanase/mixed linked glucanase BhCel5B.	DISCUSS
+172	177	plant	taxonomy_domain	It should be emphasized, however, that glycan binding and substrate targeting may only be evident in the full-length enzyme acting on highly complex structures such as the plant cell wall, as observed recently by a CBM46 module in the Bacillus xyloglucanase/mixed linked glucanase BhCel5B.	DISCUSS
+215	220	CBM46	structure_element	It should be emphasized, however, that glycan binding and substrate targeting may only be evident in the full-length enzyme acting on highly complex structures such as the plant cell wall, as observed recently by a CBM46 module in the Bacillus xyloglucanase/mixed linked glucanase BhCel5B.	DISCUSS
+235	243	Bacillus	taxonomy_domain	It should be emphasized, however, that glycan binding and substrate targeting may only be evident in the full-length enzyme acting on highly complex structures such as the plant cell wall, as observed recently by a CBM46 module in the Bacillus xyloglucanase/mixed linked glucanase BhCel5B.	DISCUSS
+244	257	xyloglucanase	protein_type	It should be emphasized, however, that glycan binding and substrate targeting may only be evident in the full-length enzyme acting on highly complex structures such as the plant cell wall, as observed recently by a CBM46 module in the Bacillus xyloglucanase/mixed linked glucanase BhCel5B.	DISCUSS
+258	280	mixed linked glucanase	protein_type	It should be emphasized, however, that glycan binding and substrate targeting may only be evident in the full-length enzyme acting on highly complex structures such as the plant cell wall, as observed recently by a CBM46 module in the Bacillus xyloglucanase/mixed linked glucanase BhCel5B.	DISCUSS
+281	288	BhCel5B	protein	It should be emphasized, however, that glycan binding and substrate targeting may only be evident in the full-length enzyme acting on highly complex structures such as the plant cell wall, as observed recently by a CBM46 module in the Bacillus xyloglucanase/mixed linked glucanase BhCel5B.	DISCUSS
+0	7	CtXyl5A	protein	CtXyl5A is a member of GH5 that contains 6644 members.	DISCUSS
+23	26	GH5	protein_type	CtXyl5A is a member of GH5 that contains 6644 members.	DISCUSS
+0	7	CtXyl5A	protein	CtXyl5A is a member of subfamily GH5_34.	DISCUSS
+33	39	GH5_34	protein_type	CtXyl5A is a member of subfamily GH5_34.	DISCUSS
+78	94	arabinoxylanases	protein_type	Despite differences in sequence identity all of the homologs were shown to be arabinoxylanases.	DISCUSS
+68	74	GH5_34	protein_type	Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the −2* subsite, which are 1,3-linked to the xylose positioned in the active site.	DISCUSS
+89	113	specificity determinants	site	Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the −2* subsite, which are 1,3-linked to the xylose positioned in the active site.	DISCUSS
+114	119	Glu68	residue_name_number	Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the −2* subsite, which are 1,3-linked to the xylose positioned in the active site.	DISCUSS
+121	126	Tyr92	residue_name_number	Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the −2* subsite, which are 1,3-linked to the xylose positioned in the active site.	DISCUSS
+132	138	Asn139	residue_name_number	Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the −2* subsite, which are 1,3-linked to the xylose positioned in the active site.	DISCUSS
+182	188	xylose	chemical	Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the −2* subsite, which are 1,3-linked to the xylose positioned in the active site.	DISCUSS
+192	201	arabinose	chemical	Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the −2* subsite, which are 1,3-linked to the xylose positioned in the active site.	DISCUSS
+209	220	−2* subsite	site	Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the −2* subsite, which are 1,3-linked to the xylose positioned in the active site.	DISCUSS
+250	256	xylose	chemical	Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the −2* subsite, which are 1,3-linked to the xylose positioned in the active site.	DISCUSS
+275	286	active site	site	Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the −2* subsite, which are 1,3-linked to the xylose positioned in the active site.	DISCUSS
+18	23	CBM62	structure_element	The presence of a CBM62 in CtXyl5A and AcGH5 suggests that these enzymes target highly complex xylans that contain d-galactose in their side chains.	DISCUSS
+27	34	CtXyl5A	protein	The presence of a CBM62 in CtXyl5A and AcGH5 suggests that these enzymes target highly complex xylans that contain d-galactose in their side chains.	DISCUSS
+39	44	AcGH5	protein	The presence of a CBM62 in CtXyl5A and AcGH5 suggests that these enzymes target highly complex xylans that contain d-galactose in their side chains.	DISCUSS
+95	101	xylans	chemical	The presence of a CBM62 in CtXyl5A and AcGH5 suggests that these enzymes target highly complex xylans that contain d-galactose in their side chains.	DISCUSS
+115	126	d-galactose	chemical	The presence of a CBM62 in CtXyl5A and AcGH5 suggests that these enzymes target highly complex xylans that contain d-galactose in their side chains.	DISCUSS
+4	14	absence of	protein_state	The absence of a “non-structural” CBM in GpGH5 may indicate that this arabinoxylanase is designed to target simpler arabinoxylans present in the endosperm of cereals.	DISCUSS
+34	37	CBM	structure_element	The absence of a “non-structural” CBM in GpGH5 may indicate that this arabinoxylanase is designed to target simpler arabinoxylans present in the endosperm of cereals.	DISCUSS
+41	46	GpGH5	protein	The absence of a “non-structural” CBM in GpGH5 may indicate that this arabinoxylanase is designed to target simpler arabinoxylans present in the endosperm of cereals.	DISCUSS
+70	85	arabinoxylanase	protein_type	The absence of a “non-structural” CBM in GpGH5 may indicate that this arabinoxylanase is designed to target simpler arabinoxylans present in the endosperm of cereals.	DISCUSS
+116	129	arabinoxylans	chemical	The absence of a “non-structural” CBM in GpGH5 may indicate that this arabinoxylanase is designed to target simpler arabinoxylans present in the endosperm of cereals.	DISCUSS
+158	165	cereals	taxonomy_domain	The absence of a “non-structural” CBM in GpGH5 may indicate that this arabinoxylanase is designed to target simpler arabinoxylans present in the endosperm of cereals.	DISCUSS
+48	54	GH5_34	protein_type	Although the characterization of all members of GH5_34 suggests that this subfamily is monospecific, differences in specificity are observed in other subfamilies of GHs including GH43 and GH5.	DISCUSS
+165	168	GHs	protein_type	Although the characterization of all members of GH5_34 suggests that this subfamily is monospecific, differences in specificity are observed in other subfamilies of GHs including GH43 and GH5.	DISCUSS
+179	183	GH43	protein_type	Although the characterization of all members of GH5_34 suggests that this subfamily is monospecific, differences in specificity are observed in other subfamilies of GHs including GH43 and GH5.	DISCUSS
+188	191	GH5	protein_type	Although the characterization of all members of GH5_34 suggests that this subfamily is monospecific, differences in specificity are observed in other subfamilies of GHs including GH43 and GH5.	DISCUSS
+24	30	GH5_34	protein_type	Thus, as new members of GH5_34 are identified from genomic sequence data and subsequently characterized, the specificity of this family may require reinterpretation.	DISCUSS
+25	30	VbGH5	protein	An intriguing feature of VbGH5 is that the limited products generated by this enzymes are much larger than those produced by the other arabinoxylanases.	DISCUSS
+135	151	arabinoxylanases	protein_type	An intriguing feature of VbGH5 is that the limited products generated by this enzymes are much larger than those produced by the other arabinoxylanases.	DISCUSS
+28	37	arabinose	chemical	This suggests that although arabinose decorations contribute to enzyme specificity (VbGH5 is not active on xylans lacking arabinose side chains), the enzyme requires other specificity determinants that occur less frequently in arabinoxylans.	DISCUSS
+84	89	VbGH5	protein	This suggests that although arabinose decorations contribute to enzyme specificity (VbGH5 is not active on xylans lacking arabinose side chains), the enzyme requires other specificity determinants that occur less frequently in arabinoxylans.	DISCUSS
+107	113	xylans	chemical	This suggests that although arabinose decorations contribute to enzyme specificity (VbGH5 is not active on xylans lacking arabinose side chains), the enzyme requires other specificity determinants that occur less frequently in arabinoxylans.	DISCUSS
+122	131	arabinose	chemical	This suggests that although arabinose decorations contribute to enzyme specificity (VbGH5 is not active on xylans lacking arabinose side chains), the enzyme requires other specificity determinants that occur less frequently in arabinoxylans.	DISCUSS
+227	240	arabinoxylans	chemical	This suggests that although arabinose decorations contribute to enzyme specificity (VbGH5 is not active on xylans lacking arabinose side chains), the enzyme requires other specificity determinants that occur less frequently in arabinoxylans.	DISCUSS
+50	54	GH98	protein_type	This has some resonance with a recently described GH98 xylanase that also exploits specificity determinants that occur infrequently and are only evident in highly complex xylans (e.g. CX).	DISCUSS
+55	63	xylanase	protein_type	This has some resonance with a recently described GH98 xylanase that also exploits specificity determinants that occur infrequently and are only evident in highly complex xylans (e.g. CX).	DISCUSS
+171	177	xylans	chemical	This has some resonance with a recently described GH98 xylanase that also exploits specificity determinants that occur infrequently and are only evident in highly complex xylans (e.g. CX).	DISCUSS
+184	186	CX	chemical	This has some resonance with a recently described GH98 xylanase that also exploits specificity determinants that occur infrequently and are only evident in highly complex xylans (e.g. CX).	DISCUSS
+86	102	arabinoxylanases	protein_type	To conclude, this study provides the molecular basis for the specificity displayed by arabinoxylanases.	DISCUSS
+42	48	pocket	site	Substrate specificity is dominated by the pocket that binds single arabinose or xylose side chains.	DISCUSS
+67	76	arabinose	chemical	Substrate specificity is dominated by the pocket that binds single arabinose or xylose side chains.	DISCUSS
+80	86	xylose	chemical	Substrate specificity is dominated by the pocket that binds single arabinose or xylose side chains.	DISCUSS
+4	8	open	protein_state	The open xylan binding cleft explains why the enzyme is able to attack highly decorated forms of the hemicellulose.	DISCUSS
+9	28	xylan binding cleft	site	The open xylan binding cleft explains why the enzyme is able to attack highly decorated forms of the hemicellulose.	DISCUSS
+101	114	hemicellulose	chemical	The open xylan binding cleft explains why the enzyme is able to attack highly decorated forms of the hemicellulose.	DISCUSS
+45	62	catalytic modules	structure_element	It is also evident that appending additional catalytic modules and CBMs onto the core components of these enzymes generates bespoke arabinoxylanases with activities optimized for specific functions.	DISCUSS
+67	71	CBMs	structure_element	It is also evident that appending additional catalytic modules and CBMs onto the core components of these enzymes generates bespoke arabinoxylanases with activities optimized for specific functions.	DISCUSS
+132	148	arabinoxylanases	protein_type	It is also evident that appending additional catalytic modules and CBMs onto the core components of these enzymes generates bespoke arabinoxylanases with activities optimized for specific functions.	DISCUSS
+25	41	arabinoxylanases	protein_type	The specificities of the arabinoxylanases described here are distinct from the classical endo-xylanases and thus have the potential to contribute to the toolbox of biocatalysts required by industries that exploit the plant cell wall as a sustainable substrate.	DISCUSS
+89	103	endo-xylanases	protein_type	The specificities of the arabinoxylanases described here are distinct from the classical endo-xylanases and thus have the potential to contribute to the toolbox of biocatalysts required by industries that exploit the plant cell wall as a sustainable substrate.	DISCUSS
+217	222	plant	taxonomy_domain	The specificities of the arabinoxylanases described here are distinct from the classical endo-xylanases and thus have the potential to contribute to the toolbox of biocatalysts required by industries that exploit the plant cell wall as a sustainable substrate.	DISCUSS
+0	41	Data collection and refinement statistics	evidence	Data collection and refinement statistics	TABLE
+1	10	CtXyl5A-D	mutant	"	CtXyl5A-D	GH5-CBM6-Arap	GH5-CBM6-Xylp	GH5-CBM6- (Araf-Xylp4)	 	Data collection					 	    Source	ESRF-ID14-1	Diamond I04–1	Diamond I24	Diamond I02	 	    Wavelength (Å)	0.9334	0.9173	0.9772	0.9791	 	    Space group	P21212	P212121	P212121	P212121	 	    Cell dimensions					 	        a, b, c (Å)	147.4, 191.7, 50.7	67.1, 72.4, 109.1	67.9, 72.5, 109.5	76.3, 123.2, 125.4	 	        α, β, γ (°)	90, 90, 90	90, 90, 90	90, 90, 90	90, 90, 90	 	    No. of measured reflections	244,475 (29,324)	224,842 (11,281)	152,004 (4,996)	463,237 (23,068)	 	    No. of independent reflections	42246 (5,920)	63,523 (3,175)	42,716 (2,334)	140,288 (6,879)	 	    Resolution (Å)	50.70–2.64 (2.78–2.64)	44.85–1.65 (1.68–1.65)	45.16–1.90 (1.94–1.90)	48.43–1.65 (1.68–1.65)	 	    Rmerge (%)	16.5 (69.5)	6.7 (65.1)	2.8 (8.4)	5.7 (74.9)	 	    CC1/2	0.985 (0.478)	0.998 (0.594)	0.999 (0.982)	0.998 (0.484)	 	    I/σI	8.0 (2.0)	13 (1.6)	26.6 (8.0)	11.2 (1.6)	 	    Completeness (%)	98.5 (96.4)	98.5 (99.4)	98.6 (85.0)	98.8 (99.4)	 	    Redundancy	5.8 (5.0)	3.5 (3.6)	3.6 (2.1)	3.3 (3.4)	 		 	Refinement					 	    Rwork/Rfree	23.7/27.8	12.2/17.0	12.9/16.1	14.5/19.9	 	    No. atoms					 	        Protein	5446	3790	3729	7333	 	        Ligand	19	20	20	92	 	        Water	227	579	601	923	 	    B-factors					 	        Protein	41.6	17.8	15.8	21.0	 	        Ligand	65.0	19.4	24.2	39.5	 	        Water	35.4	38.5	32.2	37.6	 	    R.m.s deviations					 	        Bond lengths (Å)	0.008	0.015	0.012	0.012	 	        Bond angles (°)	1.233	1.502	1.624	1.554	 	    Protein Data Bank code	5G56	5LA0	5LA1	2LA2	 	"	TABLE
+11	24	GH5-CBM6-Arap	complex_assembly	"	CtXyl5A-D	GH5-CBM6-Arap	GH5-CBM6-Xylp	GH5-CBM6- (Araf-Xylp4)	 	Data collection					 	    Source	ESRF-ID14-1	Diamond I04–1	Diamond I24	Diamond I02	 	    Wavelength (Å)	0.9334	0.9173	0.9772	0.9791	 	    Space group	P21212	P212121	P212121	P212121	 	    Cell dimensions					 	        a, b, c (Å)	147.4, 191.7, 50.7	67.1, 72.4, 109.1	67.9, 72.5, 109.5	76.3, 123.2, 125.4	 	        α, β, γ (°)	90, 90, 90	90, 90, 90	90, 90, 90	90, 90, 90	 	    No. of measured reflections	244,475 (29,324)	224,842 (11,281)	152,004 (4,996)	463,237 (23,068)	 	    No. of independent reflections	42246 (5,920)	63,523 (3,175)	42,716 (2,334)	140,288 (6,879)	 	    Resolution (Å)	50.70–2.64 (2.78–2.64)	44.85–1.65 (1.68–1.65)	45.16–1.90 (1.94–1.90)	48.43–1.65 (1.68–1.65)	 	    Rmerge (%)	16.5 (69.5)	6.7 (65.1)	2.8 (8.4)	5.7 (74.9)	 	    CC1/2	0.985 (0.478)	0.998 (0.594)	0.999 (0.982)	0.998 (0.484)	 	    I/σI	8.0 (2.0)	13 (1.6)	26.6 (8.0)	11.2 (1.6)	 	    Completeness (%)	98.5 (96.4)	98.5 (99.4)	98.6 (85.0)	98.8 (99.4)	 	    Redundancy	5.8 (5.0)	3.5 (3.6)	3.6 (2.1)	3.3 (3.4)	 		 	Refinement					 	    Rwork/Rfree	23.7/27.8	12.2/17.0	12.9/16.1	14.5/19.9	 	    No. atoms					 	        Protein	5446	3790	3729	7333	 	        Ligand	19	20	20	92	 	        Water	227	579	601	923	 	    B-factors					 	        Protein	41.6	17.8	15.8	21.0	 	        Ligand	65.0	19.4	24.2	39.5	 	        Water	35.4	38.5	32.2	37.6	 	    R.m.s deviations					 	        Bond lengths (Å)	0.008	0.015	0.012	0.012	 	        Bond angles (°)	1.233	1.502	1.624	1.554	 	    Protein Data Bank code	5G56	5LA0	5LA1	2LA2	 	"	TABLE
+25	38	GH5-CBM6-Xylp	complex_assembly	"	CtXyl5A-D	GH5-CBM6-Arap	GH5-CBM6-Xylp	GH5-CBM6- (Araf-Xylp4)	 	Data collection					 	    Source	ESRF-ID14-1	Diamond I04–1	Diamond I24	Diamond I02	 	    Wavelength (Å)	0.9334	0.9173	0.9772	0.9791	 	    Space group	P21212	P212121	P212121	P212121	 	    Cell dimensions					 	        a, b, c (Å)	147.4, 191.7, 50.7	67.1, 72.4, 109.1	67.9, 72.5, 109.5	76.3, 123.2, 125.4	 	        α, β, γ (°)	90, 90, 90	90, 90, 90	90, 90, 90	90, 90, 90	 	    No. of measured reflections	244,475 (29,324)	224,842 (11,281)	152,004 (4,996)	463,237 (23,068)	 	    No. of independent reflections	42246 (5,920)	63,523 (3,175)	42,716 (2,334)	140,288 (6,879)	 	    Resolution (Å)	50.70–2.64 (2.78–2.64)	44.85–1.65 (1.68–1.65)	45.16–1.90 (1.94–1.90)	48.43–1.65 (1.68–1.65)	 	    Rmerge (%)	16.5 (69.5)	6.7 (65.1)	2.8 (8.4)	5.7 (74.9)	 	    CC1/2	0.985 (0.478)	0.998 (0.594)	0.999 (0.982)	0.998 (0.484)	 	    I/σI	8.0 (2.0)	13 (1.6)	26.6 (8.0)	11.2 (1.6)	 	    Completeness (%)	98.5 (96.4)	98.5 (99.4)	98.6 (85.0)	98.8 (99.4)	 	    Redundancy	5.8 (5.0)	3.5 (3.6)	3.6 (2.1)	3.3 (3.4)	 		 	Refinement					 	    Rwork/Rfree	23.7/27.8	12.2/17.0	12.9/16.1	14.5/19.9	 	    No. atoms					 	        Protein	5446	3790	3729	7333	 	        Ligand	19	20	20	92	 	        Water	227	579	601	923	 	    B-factors					 	        Protein	41.6	17.8	15.8	21.0	 	        Ligand	65.0	19.4	24.2	39.5	 	        Water	35.4	38.5	32.2	37.6	 	    R.m.s deviations					 	        Bond lengths (Å)	0.008	0.015	0.012	0.012	 	        Bond angles (°)	1.233	1.502	1.624	1.554	 	    Protein Data Bank code	5G56	5LA0	5LA1	2LA2	 	"	TABLE
+39	61	GH5-CBM6- (Araf-Xylp4)	complex_assembly	"	CtXyl5A-D	GH5-CBM6-Arap	GH5-CBM6-Xylp	GH5-CBM6- (Araf-Xylp4)	 	Data collection					 	    Source	ESRF-ID14-1	Diamond I04–1	Diamond I24	Diamond I02	 	    Wavelength (Å)	0.9334	0.9173	0.9772	0.9791	 	    Space group	P21212	P212121	P212121	P212121	 	    Cell dimensions					 	        a, b, c (Å)	147.4, 191.7, 50.7	67.1, 72.4, 109.1	67.9, 72.5, 109.5	76.3, 123.2, 125.4	 	        α, β, γ (°)	90, 90, 90	90, 90, 90	90, 90, 90	90, 90, 90	 	    No. of measured reflections	244,475 (29,324)	224,842 (11,281)	152,004 (4,996)	463,237 (23,068)	 	    No. of independent reflections	42246 (5,920)	63,523 (3,175)	42,716 (2,334)	140,288 (6,879)	 	    Resolution (Å)	50.70–2.64 (2.78–2.64)	44.85–1.65 (1.68–1.65)	45.16–1.90 (1.94–1.90)	48.43–1.65 (1.68–1.65)	 	    Rmerge (%)	16.5 (69.5)	6.7 (65.1)	2.8 (8.4)	5.7 (74.9)	 	    CC1/2	0.985 (0.478)	0.998 (0.594)	0.999 (0.982)	0.998 (0.484)	 	    I/σI	8.0 (2.0)	13 (1.6)	26.6 (8.0)	11.2 (1.6)	 	    Completeness (%)	98.5 (96.4)	98.5 (99.4)	98.6 (85.0)	98.8 (99.4)	 	    Redundancy	5.8 (5.0)	3.5 (3.6)	3.6 (2.1)	3.3 (3.4)	 		 	Refinement					 	    Rwork/Rfree	23.7/27.8	12.2/17.0	12.9/16.1	14.5/19.9	 	    No. atoms					 	        Protein	5446	3790	3729	7333	 	        Ligand	19	20	20	92	 	        Water	227	579	601	923	 	    B-factors					 	        Protein	41.6	17.8	15.8	21.0	 	        Ligand	65.0	19.4	24.2	39.5	 	        Water	35.4	38.5	32.2	37.6	 	    R.m.s deviations					 	        Bond lengths (Å)	0.008	0.015	0.012	0.012	 	        Bond angles (°)	1.233	1.502	1.624	1.554	 	    Protein Data Bank code	5G56	5LA0	5LA1	2LA2	 	"	TABLE
+1079	1084	Rwork	evidence	"	CtXyl5A-D	GH5-CBM6-Arap	GH5-CBM6-Xylp	GH5-CBM6- (Araf-Xylp4)	 	Data collection					 	    Source	ESRF-ID14-1	Diamond I04–1	Diamond I24	Diamond I02	 	    Wavelength (Å)	0.9334	0.9173	0.9772	0.9791	 	    Space group	P21212	P212121	P212121	P212121	 	    Cell dimensions					 	        a, b, c (Å)	147.4, 191.7, 50.7	67.1, 72.4, 109.1	67.9, 72.5, 109.5	76.3, 123.2, 125.4	 	        α, β, γ (°)	90, 90, 90	90, 90, 90	90, 90, 90	90, 90, 90	 	    No. of measured reflections	244,475 (29,324)	224,842 (11,281)	152,004 (4,996)	463,237 (23,068)	 	    No. of independent reflections	42246 (5,920)	63,523 (3,175)	42,716 (2,334)	140,288 (6,879)	 	    Resolution (Å)	50.70–2.64 (2.78–2.64)	44.85–1.65 (1.68–1.65)	45.16–1.90 (1.94–1.90)	48.43–1.65 (1.68–1.65)	 	    Rmerge (%)	16.5 (69.5)	6.7 (65.1)	2.8 (8.4)	5.7 (74.9)	 	    CC1/2	0.985 (0.478)	0.998 (0.594)	0.999 (0.982)	0.998 (0.484)	 	    I/σI	8.0 (2.0)	13 (1.6)	26.6 (8.0)	11.2 (1.6)	 	    Completeness (%)	98.5 (96.4)	98.5 (99.4)	98.6 (85.0)	98.8 (99.4)	 	    Redundancy	5.8 (5.0)	3.5 (3.6)	3.6 (2.1)	3.3 (3.4)	 		 	Refinement					 	    Rwork/Rfree	23.7/27.8	12.2/17.0	12.9/16.1	14.5/19.9	 	    No. atoms					 	        Protein	5446	3790	3729	7333	 	        Ligand	19	20	20	92	 	        Water	227	579	601	923	 	    B-factors					 	        Protein	41.6	17.8	15.8	21.0	 	        Ligand	65.0	19.4	24.2	39.5	 	        Water	35.4	38.5	32.2	37.6	 	    R.m.s deviations					 	        Bond lengths (Å)	0.008	0.015	0.012	0.012	 	        Bond angles (°)	1.233	1.502	1.624	1.554	 	    Protein Data Bank code	5G56	5LA0	5LA1	2LA2	 	"	TABLE
+1085	1090	Rfree	evidence	"	CtXyl5A-D	GH5-CBM6-Arap	GH5-CBM6-Xylp	GH5-CBM6- (Araf-Xylp4)	 	Data collection					 	    Source	ESRF-ID14-1	Diamond I04–1	Diamond I24	Diamond I02	 	    Wavelength (Å)	0.9334	0.9173	0.9772	0.9791	 	    Space group	P21212	P212121	P212121	P212121	 	    Cell dimensions					 	        a, b, c (Å)	147.4, 191.7, 50.7	67.1, 72.4, 109.1	67.9, 72.5, 109.5	76.3, 123.2, 125.4	 	        α, β, γ (°)	90, 90, 90	90, 90, 90	90, 90, 90	90, 90, 90	 	    No. of measured reflections	244,475 (29,324)	224,842 (11,281)	152,004 (4,996)	463,237 (23,068)	 	    No. of independent reflections	42246 (5,920)	63,523 (3,175)	42,716 (2,334)	140,288 (6,879)	 	    Resolution (Å)	50.70–2.64 (2.78–2.64)	44.85–1.65 (1.68–1.65)	45.16–1.90 (1.94–1.90)	48.43–1.65 (1.68–1.65)	 	    Rmerge (%)	16.5 (69.5)	6.7 (65.1)	2.8 (8.4)	5.7 (74.9)	 	    CC1/2	0.985 (0.478)	0.998 (0.594)	0.999 (0.982)	0.998 (0.484)	 	    I/σI	8.0 (2.0)	13 (1.6)	26.6 (8.0)	11.2 (1.6)	 	    Completeness (%)	98.5 (96.4)	98.5 (99.4)	98.6 (85.0)	98.8 (99.4)	 	    Redundancy	5.8 (5.0)	3.5 (3.6)	3.6 (2.1)	3.3 (3.4)	 		 	Refinement					 	    Rwork/Rfree	23.7/27.8	12.2/17.0	12.9/16.1	14.5/19.9	 	    No. atoms					 	        Protein	5446	3790	3729	7333	 	        Ligand	19	20	20	92	 	        Water	227	579	601	923	 	    B-factors					 	        Protein	41.6	17.8	15.8	21.0	 	        Ligand	65.0	19.4	24.2	39.5	 	        Water	35.4	38.5	32.2	37.6	 	    R.m.s deviations					 	        Bond lengths (Å)	0.008	0.015	0.012	0.012	 	        Bond angles (°)	1.233	1.502	1.624	1.554	 	    Protein Data Bank code	5G56	5LA0	5LA1	2LA2	 	"	TABLE
+0	2	GH	protein_type	GH	SUPPL
+0	19	glycoside hydrolase	protein_type	glycoside hydrolase	SUPPL
+0	7	CtXyl5A	protein	CtXyl5A	SUPPL
+0	15	C. thermocellum	species	C. thermocellum arabinoxylanase	SUPPL
+16	31	arabinoxylanase	protein_type	C. thermocellum arabinoxylanase	SUPPL
+0	3	CBM	structure_element	CBM	SUPPL
+0	41	non-catalytic carbohydrate binding module	structure_element	non-catalytic carbohydrate binding module	SUPPL
+0	2	Fn	protein_type	Fn	SUPPL
+0	11	fibronectin	protein_type	fibronectin	SUPPL
+0	3	WAX	chemical	WAX	SUPPL
+0	5	wheat	taxonomy_domain	wheat arabinoxylan	SUPPL
+6	18	arabinoxylan	chemical	wheat arabinoxylan	SUPPL
+0	3	RAX	chemical	RAX	SUPPL
+0	3	rye	taxonomy_domain	rye arabinoxylan	SUPPL
+4	16	arabinoxylan	chemical	rye arabinoxylan	SUPPL
+0	2	CX	chemical	CX	SUPPL
+0	4	corn	taxonomy_domain	corn bran xylan	SUPPL
+10	15	xylan	chemical	corn bran xylan	SUPPL
+0	5	HPAEC	experimental_method	HPAEC	SUPPL
+0	46	high performance anion exchange chromatography	experimental_method	high performance anion exchange chromatography	SUPPL
+0	9	birchwood	taxonomy_domain	birchwood xylan	SUPPL
+10	15	xylan	chemical	birchwood xylan	SUPPL
+0	23	electrospray ionization	experimental_method	electrospray ionization.	SUPPL
diff --git a/annotation_CSV/PMC5173035.csv b/annotation_CSV/PMC5173035.csv
new file mode 100644
index 0000000000000000000000000000000000000000..9f77f252bd7c713baf225ab121edec9653a9d88e
--- /dev/null
+++ b/annotation_CSV/PMC5173035.csv
@@ -0,0 +1,886 @@
+anno_start	anno_end	anno_text	entity_type	sentence	section
+0	43	Biochemical and structural characterization	experimental_method	Biochemical and structural characterization of a DNA N6-adenine methyltransferase from Helicobacter pylori	TITLE
+49	81	DNA N6-adenine methyltransferase	protein_type	Biochemical and structural characterization of a DNA N6-adenine methyltransferase from Helicobacter pylori	TITLE
+87	106	Helicobacter pylori	species	Biochemical and structural characterization of a DNA N6-adenine methyltransferase from Helicobacter pylori	TITLE
+0	20	DNA N6-methyladenine	ptm	DNA N6-methyladenine modification plays an important role in regulating a variety of biological functions in bacteria.	ABSTRACT
+109	117	bacteria	taxonomy_domain	DNA N6-methyladenine modification plays an important role in regulating a variety of biological functions in bacteria.	ABSTRACT
+59	75	N6-methyladenine	ptm	However, the mechanism of sequence-specific recognition in N6-methyladenine modification remains elusive.	ABSTRACT
+0	9	M1.HpyAVI	protein	M1.HpyAVI, a DNA N6-adenine methyltransferase from Helicobacter pylori, shows more promiscuous substrate specificity than other enzymes.	ABSTRACT
+13	45	DNA N6-adenine methyltransferase	protein_type	M1.HpyAVI, a DNA N6-adenine methyltransferase from Helicobacter pylori, shows more promiscuous substrate specificity than other enzymes.	ABSTRACT
+51	70	Helicobacter pylori	species	M1.HpyAVI, a DNA N6-adenine methyltransferase from Helicobacter pylori, shows more promiscuous substrate specificity than other enzymes.	ABSTRACT
+21	39	crystal structures	evidence	Here, we present the crystal structures of cofactor-free and AdoMet-bound structures of this enzyme, which were determined at resolutions of 3.0 Å and 3.1 Å, respectively.	ABSTRACT
+43	56	cofactor-free	protein_state	Here, we present the crystal structures of cofactor-free and AdoMet-bound structures of this enzyme, which were determined at resolutions of 3.0 Å and 3.1 Å, respectively.	ABSTRACT
+61	73	AdoMet-bound	protein_state	Here, we present the crystal structures of cofactor-free and AdoMet-bound structures of this enzyme, which were determined at resolutions of 3.0 Å and 3.1 Å, respectively.	ABSTRACT
+74	84	structures	evidence	Here, we present the crystal structures of cofactor-free and AdoMet-bound structures of this enzyme, which were determined at resolutions of 3.0 Å and 3.1 Å, respectively.	ABSTRACT
+22	31	M1.HpyAVI	protein	The core structure of M1.HpyAVI resembles the canonical AdoMet-dependent MTase fold, while the putative DNA binding regions considerably differ from those of the other MTases, which may account for the substrate promiscuity of this enzyme.	ABSTRACT
+56	78	AdoMet-dependent MTase	protein_type	The core structure of M1.HpyAVI resembles the canonical AdoMet-dependent MTase fold, while the putative DNA binding regions considerably differ from those of the other MTases, which may account for the substrate promiscuity of this enzyme.	ABSTRACT
+104	123	DNA binding regions	site	The core structure of M1.HpyAVI resembles the canonical AdoMet-dependent MTase fold, while the putative DNA binding regions considerably differ from those of the other MTases, which may account for the substrate promiscuity of this enzyme.	ABSTRACT
+168	174	MTases	protein_type	The core structure of M1.HpyAVI resembles the canonical AdoMet-dependent MTase fold, while the putative DNA binding regions considerably differ from those of the other MTases, which may account for the substrate promiscuity of this enzyme.	ABSTRACT
+0	25	Site-directed mutagenesis	experimental_method	Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity.	ABSTRACT
+58	61	D29	residue_name_number	Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity.	ABSTRACT
+66	70	E216	residue_name_number	Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity.	ABSTRACT
+123	129	methyl	chemical	Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity.	ABSTRACT
+169	172	P41	residue_name_number	Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity.	ABSTRACT
+187	202	highly flexible	protein_state	Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity.	ABSTRACT
+203	207	loop	structure_element	Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity.	ABSTRACT
+66	98	DNA N6-adenine methyltransferase	protein_type	Taken together, our data revealed the structural basis underlying DNA N6-adenine methyltransferase substrate promiscuity.	ABSTRACT
+0	15	DNA methylation	ptm	DNA methylation is a common form of modification on nucleic acids occurring in both prokaryotes and eukaryotes.	INTRO
+84	95	prokaryotes	taxonomy_domain	DNA methylation is a common form of modification on nucleic acids occurring in both prokaryotes and eukaryotes.	INTRO
+100	110	eukaryotes	taxonomy_domain	DNA methylation is a common form of modification on nucleic acids occurring in both prokaryotes and eukaryotes.	INTRO
+60	63	DNA	chemical	Such a modification creates a signature motif recognized by DNA-interacting proteins and functions as a mechanism to regulate gene expression.	INTRO
+0	15	DNA methylation	ptm	DNA methylation is mediated by DNA methyltransferases (MTases), which catalyze the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to a given position of a particular DNA base within a specific DNA sequence.	INTRO
+31	53	DNA methyltransferases	protein_type	DNA methylation is mediated by DNA methyltransferases (MTases), which catalyze the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to a given position of a particular DNA base within a specific DNA sequence.	INTRO
+55	61	MTases	protein_type	DNA methylation is mediated by DNA methyltransferases (MTases), which catalyze the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to a given position of a particular DNA base within a specific DNA sequence.	INTRO
+97	103	methyl	chemical	DNA methylation is mediated by DNA methyltransferases (MTases), which catalyze the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to a given position of a particular DNA base within a specific DNA sequence.	INTRO
+115	139	S-adenosyl-L- methionine	chemical	DNA methylation is mediated by DNA methyltransferases (MTases), which catalyze the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to a given position of a particular DNA base within a specific DNA sequence.	INTRO
+141	147	AdoMet	chemical	DNA methylation is mediated by DNA methyltransferases (MTases), which catalyze the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to a given position of a particular DNA base within a specific DNA sequence.	INTRO
+185	188	DNA	chemical	DNA methylation is mediated by DNA methyltransferases (MTases), which catalyze the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to a given position of a particular DNA base within a specific DNA sequence.	INTRO
+212	215	DNA	chemical	DNA methylation is mediated by DNA methyltransferases (MTases), which catalyze the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to a given position of a particular DNA base within a specific DNA sequence.	INTRO
+17	27	DNA MTases	protein_type	Three classes of DNA MTases have been identified to transfer a methyl group to different positions of DNA bases.	INTRO
+63	69	methyl	chemical	Three classes of DNA MTases have been identified to transfer a methyl group to different positions of DNA bases.	INTRO
+102	105	DNA	chemical	Three classes of DNA MTases have been identified to transfer a methyl group to different positions of DNA bases.	INTRO
+0	18	C5-cytosine MTases	protein_type	C5-cytosine MTases, for example, methylate C5 of cytosine (m5C).	INTRO
+49	57	cytosine	residue_name	C5-cytosine MTases, for example, methylate C5 of cytosine (m5C).	INTRO
+59	62	m5C	ptm	C5-cytosine MTases, for example, methylate C5 of cytosine (m5C).	INTRO
+3	13	eukaryotes	taxonomy_domain	In eukaryotes, m5C plays an important role in gene expression, chromatin organization, genome maintenance and parental imprinting, and is involved in a variety of human diseases including cancer.	INTRO
+15	18	m5C	ptm	In eukaryotes, m5C plays an important role in gene expression, chromatin organization, genome maintenance and parental imprinting, and is involved in a variety of human diseases including cancer.	INTRO
+163	168	human	species	In eukaryotes, m5C plays an important role in gene expression, chromatin organization, genome maintenance and parental imprinting, and is involved in a variety of human diseases including cancer.	INTRO
+34	45	prokaryotic	taxonomy_domain	By contrast, the functions of the prokaryotic DNA cytosine MTase remain unknown.	INTRO
+46	64	DNA cytosine MTase	protein_type	By contrast, the functions of the prokaryotic DNA cytosine MTase remain unknown.	INTRO
+0	18	N4-cytosine MTases	protein_type	N4-cytosine MTases, which are frequently present in thermophilic or mesophilic bacteria, transfer a methyl group to the exocyclic amino group of cytosine (4mC).	INTRO
+52	64	thermophilic	taxonomy_domain	N4-cytosine MTases, which are frequently present in thermophilic or mesophilic bacteria, transfer a methyl group to the exocyclic amino group of cytosine (4mC).	INTRO
+68	78	mesophilic	taxonomy_domain	N4-cytosine MTases, which are frequently present in thermophilic or mesophilic bacteria, transfer a methyl group to the exocyclic amino group of cytosine (4mC).	INTRO
+79	87	bacteria	taxonomy_domain	N4-cytosine MTases, which are frequently present in thermophilic or mesophilic bacteria, transfer a methyl group to the exocyclic amino group of cytosine (4mC).	INTRO
+100	106	methyl	chemical	N4-cytosine MTases, which are frequently present in thermophilic or mesophilic bacteria, transfer a methyl group to the exocyclic amino group of cytosine (4mC).	INTRO
+145	153	cytosine	residue_name	N4-cytosine MTases, which are frequently present in thermophilic or mesophilic bacteria, transfer a methyl group to the exocyclic amino group of cytosine (4mC).	INTRO
+155	158	4mC	ptm	N4-cytosine MTases, which are frequently present in thermophilic or mesophilic bacteria, transfer a methyl group to the exocyclic amino group of cytosine (4mC).	INTRO
+0	14	N4 methylation	ptm	N4 methylation seems to be primarily a component of bacterial immune system against invasion by foreign DNA, such as conjugative plasmids and bacteriophages.	INTRO
+52	61	bacterial	taxonomy_domain	N4 methylation seems to be primarily a component of bacterial immune system against invasion by foreign DNA, such as conjugative plasmids and bacteriophages.	INTRO
+104	107	DNA	chemical	N4 methylation seems to be primarily a component of bacterial immune system against invasion by foreign DNA, such as conjugative plasmids and bacteriophages.	INTRO
+142	156	bacteriophages	taxonomy_domain	N4 methylation seems to be primarily a component of bacterial immune system against invasion by foreign DNA, such as conjugative plasmids and bacteriophages.	INTRO
+17	34	N6-adenine MTases	protein_type	The third group, N6-adenine MTases methylate the exocyclic amino groups of adenine (6mA), which exists in prokaryotes as a signal for genome defense, DNA replication and repair, regulation of gene expression, control of transposition and host-pathogen interactions.	INTRO
+75	82	adenine	residue_name	The third group, N6-adenine MTases methylate the exocyclic amino groups of adenine (6mA), which exists in prokaryotes as a signal for genome defense, DNA replication and repair, regulation of gene expression, control of transposition and host-pathogen interactions.	INTRO
+84	87	6mA	ptm	The third group, N6-adenine MTases methylate the exocyclic amino groups of adenine (6mA), which exists in prokaryotes as a signal for genome defense, DNA replication and repair, regulation of gene expression, control of transposition and host-pathogen interactions.	INTRO
+106	117	prokaryotes	taxonomy_domain	The third group, N6-adenine MTases methylate the exocyclic amino groups of adenine (6mA), which exists in prokaryotes as a signal for genome defense, DNA replication and repair, regulation of gene expression, control of transposition and host-pathogen interactions.	INTRO
+150	153	DNA	chemical	The third group, N6-adenine MTases methylate the exocyclic amino groups of adenine (6mA), which exists in prokaryotes as a signal for genome defense, DNA replication and repair, regulation of gene expression, control of transposition and host-pathogen interactions.	INTRO
+80	83	6mA	ptm	Recent studies utilizing new sequencing approaches have showed the existence of 6mA in several eukaryotic species.	INTRO
+95	105	eukaryotic	taxonomy_domain	Recent studies utilizing new sequencing approaches have showed the existence of 6mA in several eukaryotic species.	INTRO
+0	3	DNA	chemical	DNA 6mA modification is associated with important biological processes including nucleosome distribution close to the transcription start sites in Chlamydomonas, carrying heritable epigenetic information in C.elegans or controlling development of Drosophila.	INTRO
+4	7	6mA	ptm	DNA 6mA modification is associated with important biological processes including nucleosome distribution close to the transcription start sites in Chlamydomonas, carrying heritable epigenetic information in C.elegans or controlling development of Drosophila.	INTRO
+147	160	Chlamydomonas	taxonomy_domain	DNA 6mA modification is associated with important biological processes including nucleosome distribution close to the transcription start sites in Chlamydomonas, carrying heritable epigenetic information in C.elegans or controlling development of Drosophila.	INTRO
+207	216	C.elegans	species	DNA 6mA modification is associated with important biological processes including nucleosome distribution close to the transcription start sites in Chlamydomonas, carrying heritable epigenetic information in C.elegans or controlling development of Drosophila.	INTRO
+247	257	Drosophila	taxonomy_domain	DNA 6mA modification is associated with important biological processes including nucleosome distribution close to the transcription start sites in Chlamydomonas, carrying heritable epigenetic information in C.elegans or controlling development of Drosophila.	INTRO
+23	34	methylation	ptm	All the three types of methylation exist in prokaryotes, and most DNA MTases are components of the restriction-modification (R-M) systems.	INTRO
+44	55	prokaryotes	taxonomy_domain	All the three types of methylation exist in prokaryotes, and most DNA MTases are components of the restriction-modification (R-M) systems.	INTRO
+66	76	DNA MTases	protein_type	All the three types of methylation exist in prokaryotes, and most DNA MTases are components of the restriction-modification (R-M) systems.	INTRO
+17	41	restriction endonuclease	protein_type	“R” stands for a restriction endonuclease cleaving specific DNA sequences, while “M” symbolizes a modification methyltransferase rendering these sequences resistant to cleavage.	INTRO
+60	63	DNA	chemical	“R” stands for a restriction endonuclease cleaving specific DNA sequences, while “M” symbolizes a modification methyltransferase rendering these sequences resistant to cleavage.	INTRO
+98	128	modification methyltransferase	protein_type	“R” stands for a restriction endonuclease cleaving specific DNA sequences, while “M” symbolizes a modification methyltransferase rendering these sequences resistant to cleavage.	INTRO
+79	87	bacteria	taxonomy_domain	The cooperation of these two enzymes provides a defensive mechanism to protect bacteria from infection by bacteriophages.	INTRO
+106	120	bacteriophages	taxonomy_domain	The cooperation of these two enzymes provides a defensive mechanism to protect bacteria from infection by bacteriophages.	INTRO
+99	102	DNA	chemical	The R-M systems are classified into three types based on specific structural features, position of DNA cleavage and cofactor requirements.	INTRO
+24	65	DNA adenine or cytosine methyltransferase	protein_type	In types I and III, the DNA adenine or cytosine methyltransferase is part of a multi-subunit enzyme that catalyzes both restriction and modification.	INTRO
+68	92	restriction endonuclease	protein_type	By contrast, two separate enzymes exist in type II systems, where a restriction endonuclease and a DNA adenine or cytosine methyltransferase recognize the same targets.	INTRO
+99	140	DNA adenine or cytosine methyltransferase	protein_type	By contrast, two separate enzymes exist in type II systems, where a restriction endonuclease and a DNA adenine or cytosine methyltransferase recognize the same targets.	INTRO
+21	30	bacterial	taxonomy_domain	To date, a number of bacterial DNA MTases have been structurally characterized, covering enzymes from all the three classes.	INTRO
+31	41	DNA MTases	protein_type	To date, a number of bacterial DNA MTases have been structurally characterized, covering enzymes from all the three classes.	INTRO
+52	78	structurally characterized	experimental_method	To date, a number of bacterial DNA MTases have been structurally characterized, covering enzymes from all the three classes.	INTRO
+10	16	MTases	protein_type	All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base.	INTRO
+120	129	conserved	protein_state	All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base.	INTRO
+137	153	catalytic domain	structure_element	All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base.	INTRO
+168	179	active site	site	All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base.	INTRO
+184	190	methyl	chemical	All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base.	INTRO
+215	221	AdoMet	chemical	All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base.	INTRO
+245	276	target (DNA)-recognition domain	structure_element	All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base.	INTRO
+278	281	TRD	structure_element	All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base.	INTRO
+343	346	DNA	chemical	All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base.	INTRO
+387	390	DNA	chemical	All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base.	INTRO
+0	9	Conserved	protein_state	Conserved amino acid motifs have been identified from reported structures, including ten motifs (I-X) in cytosine MTases and nine motifs (I-VIII and X) in adenine MTases, all of which are arranged in an almost constant order.	INTRO
+63	73	structures	evidence	Conserved amino acid motifs have been identified from reported structures, including ten motifs (I-X) in cytosine MTases and nine motifs (I-VIII and X) in adenine MTases, all of which are arranged in an almost constant order.	INTRO
+97	100	I-X	structure_element	Conserved amino acid motifs have been identified from reported structures, including ten motifs (I-X) in cytosine MTases and nine motifs (I-VIII and X) in adenine MTases, all of which are arranged in an almost constant order.	INTRO
+105	120	cytosine MTases	protein_type	Conserved amino acid motifs have been identified from reported structures, including ten motifs (I-X) in cytosine MTases and nine motifs (I-VIII and X) in adenine MTases, all of which are arranged in an almost constant order.	INTRO
+138	144	I-VIII	structure_element	Conserved amino acid motifs have been identified from reported structures, including ten motifs (I-X) in cytosine MTases and nine motifs (I-VIII and X) in adenine MTases, all of which are arranged in an almost constant order.	INTRO
+149	150	X	structure_element	Conserved amino acid motifs have been identified from reported structures, including ten motifs (I-X) in cytosine MTases and nine motifs (I-VIII and X) in adenine MTases, all of which are arranged in an almost constant order.	INTRO
+155	169	adenine MTases	protein_type	Conserved amino acid motifs have been identified from reported structures, including ten motifs (I-X) in cytosine MTases and nine motifs (I-VIII and X) in adenine MTases, all of which are arranged in an almost constant order.	INTRO
+45	54	conserved	protein_state	According to the linear arrangement of three conserved domains, exocyclic amino MTases are subdivided into six groups (namely α, β, γ, ζ, δ and ε).	INTRO
+64	86	exocyclic amino MTases	protein_type	According to the linear arrangement of three conserved domains, exocyclic amino MTases are subdivided into six groups (namely α, β, γ, ζ, δ and ε).	INTRO
+126	127	α	protein_type	According to the linear arrangement of three conserved domains, exocyclic amino MTases are subdivided into six groups (namely α, β, γ, ζ, δ and ε).	INTRO
+129	130	β	protein_type	According to the linear arrangement of three conserved domains, exocyclic amino MTases are subdivided into six groups (namely α, β, γ, ζ, δ and ε).	INTRO
+132	133	γ	protein_type	According to the linear arrangement of three conserved domains, exocyclic amino MTases are subdivided into six groups (namely α, β, γ, ζ, δ and ε).	INTRO
+135	136	ζ	protein_type	According to the linear arrangement of three conserved domains, exocyclic amino MTases are subdivided into six groups (namely α, β, γ, ζ, δ and ε).	INTRO
+138	139	δ	protein_type	According to the linear arrangement of three conserved domains, exocyclic amino MTases are subdivided into six groups (namely α, β, γ, ζ, δ and ε).	INTRO
+144	145	ε	protein_type	According to the linear arrangement of three conserved domains, exocyclic amino MTases are subdivided into six groups (namely α, β, γ, ζ, δ and ε).	INTRO
+0	33	N6-adenine and N4-cytosine MTases	protein_type	N6-adenine and N4-cytosine MTases, in particular, are closely related by sharing common structural features.	RESULTS
+0	33	N6-adenine and N4-cytosine MTases	protein_type	N6-adenine and N4-cytosine MTases, in particular, are closely related by sharing common structural features.	RESULTS
+42	51	bacterial	taxonomy_domain	Despite the considerable similarity among bacterial MTases, some differences were observed among the enzymes from various species.	INTRO
+52	58	MTases	protein_type	Despite the considerable similarity among bacterial MTases, some differences were observed among the enzymes from various species.	INTRO
+39	45	MTases	protein_type	For example, the structural regions of MTases beyond the catalytic domain are rather variable, such as the C-terminal domain of M.TaqI, the extended arm of M.MboIIA and M.RsrI, the helix bundle of EcoDam, and so on.	INTRO
+57	73	catalytic domain	structure_element	For example, the structural regions of MTases beyond the catalytic domain are rather variable, such as the C-terminal domain of M.TaqI, the extended arm of M.MboIIA and M.RsrI, the helix bundle of EcoDam, and so on.	INTRO
+107	124	C-terminal domain	structure_element	For example, the structural regions of MTases beyond the catalytic domain are rather variable, such as the C-terminal domain of M.TaqI, the extended arm of M.MboIIA and M.RsrI, the helix bundle of EcoDam, and so on.	INTRO
+128	134	M.TaqI	protein	For example, the structural regions of MTases beyond the catalytic domain are rather variable, such as the C-terminal domain of M.TaqI, the extended arm of M.MboIIA and M.RsrI, the helix bundle of EcoDam, and so on.	INTRO
+156	164	M.MboIIA	protein	For example, the structural regions of MTases beyond the catalytic domain are rather variable, such as the C-terminal domain of M.TaqI, the extended arm of M.MboIIA and M.RsrI, the helix bundle of EcoDam, and so on.	INTRO
+169	175	M.RsrI	protein	For example, the structural regions of MTases beyond the catalytic domain are rather variable, such as the C-terminal domain of M.TaqI, the extended arm of M.MboIIA and M.RsrI, the helix bundle of EcoDam, and so on.	INTRO
+181	193	helix bundle	structure_element	For example, the structural regions of MTases beyond the catalytic domain are rather variable, such as the C-terminal domain of M.TaqI, the extended arm of M.MboIIA and M.RsrI, the helix bundle of EcoDam, and so on.	INTRO
+197	203	EcoDam	protein	For example, the structural regions of MTases beyond the catalytic domain are rather variable, such as the C-terminal domain of M.TaqI, the extended arm of M.MboIIA and M.RsrI, the helix bundle of EcoDam, and so on.	INTRO
+0	15	DNA methylation	ptm	DNA methylation is thought to influence bacterial virulence.	INTRO
+40	49	bacterial	taxonomy_domain	DNA methylation is thought to influence bacterial virulence.	INTRO
+0	29	DNA adenine methyltransferase	protein_type	DNA adenine methyltransferase has been shown to play a crucial role in colonization of deep tissue sites in Salmonella typhimurium and Aeromonas hydrophila.	INTRO
+108	130	Salmonella typhimurium	species	DNA adenine methyltransferase has been shown to play a crucial role in colonization of deep tissue sites in Salmonella typhimurium and Aeromonas hydrophila.	INTRO
+135	155	Aeromonas hydrophila	species	DNA adenine methyltransferase has been shown to play a crucial role in colonization of deep tissue sites in Salmonella typhimurium and Aeromonas hydrophila.	INTRO
+13	36	DNA adenine methylation	ptm	Importantly, DNA adenine methylation is a global regulator of genes expressed during infection and inhibitors of DNA adenine methylation are likely to have a broad antimicrobial action.	INTRO
+113	136	DNA adenine methylation	ptm	Importantly, DNA adenine methylation is a global regulator of genes expressed during infection and inhibitors of DNA adenine methylation are likely to have a broad antimicrobial action.	INTRO
+0	3	Dam	protein_type	Dam was considered a promising target for antimicrobial drug development.	INTRO
+0	19	Helicobacter pylori	species	Helicobacter pylori is a Gram-negative bacterium that persistently colonizes in human stomach worldwide.	INTRO
+25	48	Gram-negative bacterium	taxonomy_domain	Helicobacter pylori is a Gram-negative bacterium that persistently colonizes in human stomach worldwide.	INTRO
+80	85	human	species	Helicobacter pylori is a Gram-negative bacterium that persistently colonizes in human stomach worldwide.	INTRO
+0	9	H. pylori	species	H. pylori is involved in 90% of all gastric malignancies, infecting nearly 50% of the world's population and is the most crucial etiologic agent for gastric adenocarcinoma.	INTRO
+0	9	H. pylori	species	H. pylori strains possess a few R-M systems like other bacteria to function as defensive systems.	INTRO
+55	63	bacteria	taxonomy_domain	H. pylori strains possess a few R-M systems like other bacteria to function as defensive systems.	INTRO
+0	15	H. pylori 26695	species	H. pylori 26695, for example, has 23 R-M systems.	INTRO
+0	18	Methyltransferases	protein_type	Methyltransferases were suggested to be involved in H. pylori pathogenicity.	INTRO
+52	61	H. pylori	species	Methyltransferases were suggested to be involved in H. pylori pathogenicity.	INTRO
+0	9	M1.HpyAVI	protein	M1.HpyAVI is a DNA adenine MTase that belongs to the type II R-M system.	INTRO
+15	32	DNA adenine MTase	protein_type	M1.HpyAVI is a DNA adenine MTase that belongs to the type II R-M system.	INTRO
+25	35	DNA MTases	protein_type	This system contains two DNA MTases named M1.HpyAVI and M2.HpyAVI, and a putative restriction enzyme.	INTRO
+42	51	M1.HpyAVI	protein	This system contains two DNA MTases named M1.HpyAVI and M2.HpyAVI, and a putative restriction enzyme.	INTRO
+56	65	M2.HpyAVI	protein	This system contains two DNA MTases named M1.HpyAVI and M2.HpyAVI, and a putative restriction enzyme.	INTRO
+82	100	restriction enzyme	protein_type	This system contains two DNA MTases named M1.HpyAVI and M2.HpyAVI, and a putative restriction enzyme.	INTRO
+0	9	M1.HpyAVI	protein	M1.HpyAVI encoded by ORF hp0050 is an N6-adenine methyltransferase belonging to the β-class MTase.	INTRO
+25	31	hp0050	gene	M1.HpyAVI encoded by ORF hp0050 is an N6-adenine methyltransferase belonging to the β-class MTase.	INTRO
+38	66	N6-adenine methyltransferase	protein_type	M1.HpyAVI encoded by ORF hp0050 is an N6-adenine methyltransferase belonging to the β-class MTase.	INTRO
+84	97	β-class MTase	protein_type	M1.HpyAVI encoded by ORF hp0050 is an N6-adenine methyltransferase belonging to the β-class MTase.	INTRO
+74	85	5′-GAGG-3′,	chemical	It has been reported recently that this enzyme recognizes the sequence of 5′-GAGG-3′, 5′-GGAG-3′ or 5′-GAAG-3′ and methylates adenines in these sequences.	INTRO
+86	96	5′-GGAG-3′	chemical	It has been reported recently that this enzyme recognizes the sequence of 5′-GAGG-3′, 5′-GGAG-3′ or 5′-GAAG-3′ and methylates adenines in these sequences.	INTRO
+100	110	5′-GAAG-3′	chemical	It has been reported recently that this enzyme recognizes the sequence of 5′-GAGG-3′, 5′-GGAG-3′ or 5′-GAAG-3′ and methylates adenines in these sequences.	INTRO
+126	134	adenines	residue_name	It has been reported recently that this enzyme recognizes the sequence of 5′-GAGG-3′, 5′-GGAG-3′ or 5′-GAAG-3′ and methylates adenines in these sequences.	INTRO
+11	22	methylation	ptm	Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5′-GAAG-3′ seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5′-GAGG-3′. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.	INTRO
+39	47	adenines	residue_name	Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5′-GAAG-3′ seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5′-GAGG-3′. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.	INTRO
+102	119	N6-adenine MTases	protein_type	Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5′-GAAG-3′ seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5′-GAGG-3′. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.	INTRO
+125	136	methylation	ptm	Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5′-GAAG-3′ seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5′-GAGG-3′. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.	INTRO
+149	159	5′-GAAG-3′	chemical	Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5′-GAAG-3′ seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5′-GAGG-3′. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.	INTRO
+192	201	M1.HpyAVI	protein	Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5′-GAAG-3′ seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5′-GAGG-3′. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.	INTRO
+252	260	H.pylori	species	Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5′-GAAG-3′ seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5′-GAGG-3′. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.	INTRO
+293	303	5′-GAGG-3′	chemical	Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5′-GAAG-3′ seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5′-GAGG-3′. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.	INTRO
+449	458	structure	evidence	Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5′-GAAG-3′ seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5′-GAGG-3′. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.	INTRO
+20	37	crystal structure	evidence	Here, we report the crystal structure of M1.HpyAVI from H. pylori 26695, which is the first determined N6-adenine MTase structure in H. pylori.	INTRO
+41	50	M1.HpyAVI	protein	Here, we report the crystal structure of M1.HpyAVI from H. pylori 26695, which is the first determined N6-adenine MTase structure in H. pylori.	INTRO
+56	71	H. pylori 26695	species	Here, we report the crystal structure of M1.HpyAVI from H. pylori 26695, which is the first determined N6-adenine MTase structure in H. pylori.	INTRO
+103	119	N6-adenine MTase	protein_type	Here, we report the crystal structure of M1.HpyAVI from H. pylori 26695, which is the first determined N6-adenine MTase structure in H. pylori.	INTRO
+120	129	structure	evidence	Here, we report the crystal structure of M1.HpyAVI from H. pylori 26695, which is the first determined N6-adenine MTase structure in H. pylori.	INTRO
+133	142	H. pylori	species	Here, we report the crystal structure of M1.HpyAVI from H. pylori 26695, which is the first determined N6-adenine MTase structure in H. pylori.	INTRO
+4	13	structure	evidence	The structure reveals a similar architecture as the canonical fold of homologous proteins, but displays several differences in the loop regions and TRD.	INTRO
+131	135	loop	structure_element	The structure reveals a similar architecture as the canonical fold of homologous proteins, but displays several differences in the loop regions and TRD.	INTRO
+148	151	TRD	structure_element	The structure reveals a similar architecture as the canonical fold of homologous proteins, but displays several differences in the loop regions and TRD.	INTRO
+9	44	structural and biochemical analyses	experimental_method	Based on structural and biochemical analyses, we then identified two conserved amino acids, D29 at the catalytic site and E216 close to the C-terminus, as crucial residues for cofactor binding and methyltransferase activity of M1.HpyAVI.	INTRO
+69	78	conserved	protein_state	Based on structural and biochemical analyses, we then identified two conserved amino acids, D29 at the catalytic site and E216 close to the C-terminus, as crucial residues for cofactor binding and methyltransferase activity of M1.HpyAVI.	INTRO
+92	95	D29	residue_name_number	Based on structural and biochemical analyses, we then identified two conserved amino acids, D29 at the catalytic site and E216 close to the C-terminus, as crucial residues for cofactor binding and methyltransferase activity of M1.HpyAVI.	INTRO
+103	117	catalytic site	site	Based on structural and biochemical analyses, we then identified two conserved amino acids, D29 at the catalytic site and E216 close to the C-terminus, as crucial residues for cofactor binding and methyltransferase activity of M1.HpyAVI.	INTRO
+122	126	E216	residue_name_number	Based on structural and biochemical analyses, we then identified two conserved amino acids, D29 at the catalytic site and E216 close to the C-terminus, as crucial residues for cofactor binding and methyltransferase activity of M1.HpyAVI.	INTRO
+197	214	methyltransferase	protein_type	Based on structural and biochemical analyses, we then identified two conserved amino acids, D29 at the catalytic site and E216 close to the C-terminus, as crucial residues for cofactor binding and methyltransferase activity of M1.HpyAVI.	INTRO
+227	236	M1.HpyAVI	protein	Based on structural and biochemical analyses, we then identified two conserved amino acids, D29 at the catalytic site and E216 close to the C-terminus, as crucial residues for cofactor binding and methyltransferase activity of M1.HpyAVI.	INTRO
+15	28	non-conserved	protein_state	In addition, a non-conserved amino acid, P41, seems to play a key role in substrate recognition.	INTRO
+41	44	P41	residue_name_number	In addition, a non-conserved amino acid, P41, seems to play a key role in substrate recognition.	INTRO
+8	17	structure	evidence	Overall structure	RESULTS
+12	23	full-length	protein_state	Recombinant full-length M1.HpyAVI was produced as a soluble protein in Escherichia coli, but was quite unstable and tended to aggregate in low salt environment.	RESULTS
+24	33	M1.HpyAVI	protein	Recombinant full-length M1.HpyAVI was produced as a soluble protein in Escherichia coli, but was quite unstable and tended to aggregate in low salt environment.	RESULTS
+71	87	Escherichia coli	species	Recombinant full-length M1.HpyAVI was produced as a soluble protein in Escherichia coli, but was quite unstable and tended to aggregate in low salt environment.	RESULTS
+92	107	sodium chloride	chemical	The protein, however, remained fully soluble in a buffer containing higher concentration of sodium chloride (>300 mM), which prompted that M1.HpyAVI is likely a halophilic protein.	RESULTS
+139	148	M1.HpyAVI	protein	The protein, however, remained fully soluble in a buffer containing higher concentration of sodium chloride (>300 mM), which prompted that M1.HpyAVI is likely a halophilic protein.	RESULTS
+161	171	halophilic	protein_state	The protein, however, remained fully soluble in a buffer containing higher concentration of sodium chloride (>300 mM), which prompted that M1.HpyAVI is likely a halophilic protein.	RESULTS
+4	17	cofactor-free	protein_state	The cofactor-free and AdoMet-bound proteins were crystallized at different conditions.	RESULTS
+22	34	AdoMet-bound	protein_state	The cofactor-free and AdoMet-bound proteins were crystallized at different conditions.	RESULTS
+49	61	crystallized	experimental_method	The cofactor-free and AdoMet-bound proteins were crystallized at different conditions.	RESULTS
+5	15	structures	evidence	Both structures were determined by means of molecular replacement, and refined to 3.0 Å and 3.1 Å, respectively.	RESULTS
+44	65	molecular replacement	experimental_method	Both structures were determined by means of molecular replacement, and refined to 3.0 Å and 3.1 Å, respectively.	RESULTS
+14	35	X-ray data collection	experimental_method	Statistics of X-ray data collection and structure refinement were summarized in Table 1.	RESULTS
+40	60	structure refinement	experimental_method	Statistics of X-ray data collection and structure refinement were summarized in Table 1.	RESULTS
+20	51	structure refinement statistics	evidence	Data collection and structure refinement statistics of M1.HpyAVI	TABLE
+55	64	M1.HpyAVI	protein	Data collection and structure refinement statistics of M1.HpyAVI	TABLE
+1	10	M1.HpyAVI	protein	"	M1.HpyAVI	M1.HpyAVI-AdoMet complex	 	Data collection			 	Wavelength (Å)	1.0000	0.97772	 	Space group	P43212	P65	 	Unit-cell parameters (Å, ˚)	a = b = 69.73, c = 532.75α = β = γ = 90	a = b = 135.60, c = 265.15α = β = 90, γ = 120	 	Resolution range (Å) a	49.09-3.00 (3.09-3.00)	48.91-3.10 (3.18-3.10)	 	Unique reflections a	27243	49833	 	Multiplicity a	3.7 (3.8)	5.6 (4.0)	 	Completeness (%)a	98.7 (98.9)	99.7 (97.8)	 	Mean I/δ (I) a	12.1 (3.4)	14.0 (1.9)	 	Solvent content (%)	58.67	61.96	 	Rmergea	0.073 (0.378)	0.106 (0.769)	 	Structure refinement			 	Rwork	0.251	0.221	 	Rfree	0.308	0.276	 	R.m.s.d., bond lengths (Å)	0.007	0.007	 	R.m.s.d., bond angles (˚)	1.408	1.651	 	Ramachandran plot			 	Favoured region (%)	89.44	91.44	 	Allowed region (%)	9.58	7.11	 	Outliers (%)	0.99	1.45	 	"	TABLE
+11	27	M1.HpyAVI-AdoMet	complex_assembly	"	M1.HpyAVI	M1.HpyAVI-AdoMet complex	 	Data collection			 	Wavelength (Å)	1.0000	0.97772	 	Space group	P43212	P65	 	Unit-cell parameters (Å, ˚)	a = b = 69.73, c = 532.75α = β = γ = 90	a = b = 135.60, c = 265.15α = β = 90, γ = 120	 	Resolution range (Å) a	49.09-3.00 (3.09-3.00)	48.91-3.10 (3.18-3.10)	 	Unique reflections a	27243	49833	 	Multiplicity a	3.7 (3.8)	5.6 (4.0)	 	Completeness (%)a	98.7 (98.9)	99.7 (97.8)	 	Mean I/δ (I) a	12.1 (3.4)	14.0 (1.9)	 	Solvent content (%)	58.67	61.96	 	Rmergea	0.073 (0.378)	0.106 (0.769)	 	Structure refinement			 	Rwork	0.251	0.221	 	Rfree	0.308	0.276	 	R.m.s.d., bond lengths (Å)	0.007	0.007	 	R.m.s.d., bond angles (˚)	1.408	1.651	 	Ramachandran plot			 	Favoured region (%)	89.44	91.44	 	Allowed region (%)	9.58	7.11	 	Outliers (%)	0.99	1.45	 	"	TABLE
+594	601	R.m.s.d	evidence	"	M1.HpyAVI	M1.HpyAVI-AdoMet complex	 	Data collection			 	Wavelength (Å)	1.0000	0.97772	 	Space group	P43212	P65	 	Unit-cell parameters (Å, ˚)	a = b = 69.73, c = 532.75α = β = γ = 90	a = b = 135.60, c = 265.15α = β = 90, γ = 120	 	Resolution range (Å) a	49.09-3.00 (3.09-3.00)	48.91-3.10 (3.18-3.10)	 	Unique reflections a	27243	49833	 	Multiplicity a	3.7 (3.8)	5.6 (4.0)	 	Completeness (%)a	98.7 (98.9)	99.7 (97.8)	 	Mean I/δ (I) a	12.1 (3.4)	14.0 (1.9)	 	Solvent content (%)	58.67	61.96	 	Rmergea	0.073 (0.378)	0.106 (0.769)	 	Structure refinement			 	Rwork	0.251	0.221	 	Rfree	0.308	0.276	 	R.m.s.d., bond lengths (Å)	0.007	0.007	 	R.m.s.d., bond angles (˚)	1.408	1.651	 	Ramachandran plot			 	Favoured region (%)	89.44	91.44	 	Allowed region (%)	9.58	7.11	 	Outliers (%)	0.99	1.45	 	"	TABLE
+635	642	R.m.s.d	evidence	"	M1.HpyAVI	M1.HpyAVI-AdoMet complex	 	Data collection			 	Wavelength (Å)	1.0000	0.97772	 	Space group	P43212	P65	 	Unit-cell parameters (Å, ˚)	a = b = 69.73, c = 532.75α = β = γ = 90	a = b = 135.60, c = 265.15α = β = 90, γ = 120	 	Resolution range (Å) a	49.09-3.00 (3.09-3.00)	48.91-3.10 (3.18-3.10)	 	Unique reflections a	27243	49833	 	Multiplicity a	3.7 (3.8)	5.6 (4.0)	 	Completeness (%)a	98.7 (98.9)	99.7 (97.8)	 	Mean I/δ (I) a	12.1 (3.4)	14.0 (1.9)	 	Solvent content (%)	58.67	61.96	 	Rmergea	0.073 (0.378)	0.106 (0.769)	 	Structure refinement			 	Rwork	0.251	0.221	 	Rfree	0.308	0.276	 	R.m.s.d., bond lengths (Å)	0.007	0.007	 	R.m.s.d., bond angles (˚)	1.408	1.651	 	Ramachandran plot			 	Favoured region (%)	89.44	91.44	 	Allowed region (%)	9.58	7.11	 	Outliers (%)	0.99	1.45	 	"	TABLE
+23	31	monomers	oligomeric_state	Four and eight protein monomers resided in the asymmetric units of the two crystal structures.	RESULTS
+75	93	crystal structures	evidence	Four and eight protein monomers resided in the asymmetric units of the two crystal structures.	RESULTS
+48	53	loops	structure_element	Some amino acids, particularly those within two loops (residues 32-61 and 152-172) in both structures, were poorly defined in electron density and had to be omitted from the refined models.	RESULTS
+64	69	32-61	residue_range	Some amino acids, particularly those within two loops (residues 32-61 and 152-172) in both structures, were poorly defined in electron density and had to be omitted from the refined models.	RESULTS
+74	81	152-172	residue_range	Some amino acids, particularly those within two loops (residues 32-61 and 152-172) in both structures, were poorly defined in electron density and had to be omitted from the refined models.	RESULTS
+91	101	structures	evidence	Some amino acids, particularly those within two loops (residues 32-61 and 152-172) in both structures, were poorly defined in electron density and had to be omitted from the refined models.	RESULTS
+126	142	electron density	evidence	Some amino acids, particularly those within two loops (residues 32-61 and 152-172) in both structures, were poorly defined in electron density and had to be omitted from the refined models.	RESULTS
+8	18	structures	evidence	The two structures are very similar to each other (Figure 1) and could be well overlaid with an RMSD of 0.76 Å on 191 Cα atoms.	RESULTS
+96	100	RMSD	evidence	The two structures are very similar to each other (Figure 1) and could be well overlaid with an RMSD of 0.76 Å on 191 Cα atoms.	RESULTS
+28	37	M1.HpyAVI	protein	The overall architecture of M1.HpyAVI revealed in these structures resembles the AdoMet-dependent MTase fold in which a twisted seven-stranded β-sheet flanked by six α-helices forms the structural core.	RESULTS
+56	66	structures	evidence	The overall architecture of M1.HpyAVI revealed in these structures resembles the AdoMet-dependent MTase fold in which a twisted seven-stranded β-sheet flanked by six α-helices forms the structural core.	RESULTS
+81	103	AdoMet-dependent MTase	protein_type	The overall architecture of M1.HpyAVI revealed in these structures resembles the AdoMet-dependent MTase fold in which a twisted seven-stranded β-sheet flanked by six α-helices forms the structural core.	RESULTS
+143	150	β-sheet	structure_element	The overall architecture of M1.HpyAVI revealed in these structures resembles the AdoMet-dependent MTase fold in which a twisted seven-stranded β-sheet flanked by six α-helices forms the structural core.	RESULTS
+166	175	α-helices	structure_element	The overall architecture of M1.HpyAVI revealed in these structures resembles the AdoMet-dependent MTase fold in which a twisted seven-stranded β-sheet flanked by six α-helices forms the structural core.	RESULTS
+18	28	structures	evidence	Like the reported structures of the larger domain of MTases, three helices (αA, αB and αZ) are located at one face of the central β-sheet, while the other three αD, αE and αC sit at the other side.	RESULTS
+53	59	MTases	protein_type	Like the reported structures of the larger domain of MTases, three helices (αA, αB and αZ) are located at one face of the central β-sheet, while the other three αD, αE and αC sit at the other side.	RESULTS
+67	74	helices	structure_element	Like the reported structures of the larger domain of MTases, three helices (αA, αB and αZ) are located at one face of the central β-sheet, while the other three αD, αE and αC sit at the other side.	RESULTS
+76	78	αA	structure_element	Like the reported structures of the larger domain of MTases, three helices (αA, αB and αZ) are located at one face of the central β-sheet, while the other three αD, αE and αC sit at the other side.	RESULTS
+80	82	αB	structure_element	Like the reported structures of the larger domain of MTases, three helices (αA, αB and αZ) are located at one face of the central β-sheet, while the other three αD, αE and αC sit at the other side.	RESULTS
+87	89	αZ	structure_element	Like the reported structures of the larger domain of MTases, three helices (αA, αB and αZ) are located at one face of the central β-sheet, while the other three αD, αE and αC sit at the other side.	RESULTS
+130	137	β-sheet	structure_element	Like the reported structures of the larger domain of MTases, three helices (αA, αB and αZ) are located at one face of the central β-sheet, while the other three αD, αE and αC sit at the other side.	RESULTS
+161	163	αD	structure_element	Like the reported structures of the larger domain of MTases, three helices (αA, αB and αZ) are located at one face of the central β-sheet, while the other three αD, αE and αC sit at the other side.	RESULTS
+165	167	αE	structure_element	Like the reported structures of the larger domain of MTases, three helices (αA, αB and αZ) are located at one face of the central β-sheet, while the other three αD, αE and αC sit at the other side.	RESULTS
+172	174	αC	structure_element	Like the reported structures of the larger domain of MTases, three helices (αA, αB and αZ) are located at one face of the central β-sheet, while the other three αD, αE and αC sit at the other side.	RESULTS
+10	19	conserved	protein_state	All these conserved structural motifs form a typical α/β Rossmann fold.	RESULTS
+53	70	α/β Rossmann fold	structure_element	All these conserved structural motifs form a typical α/β Rossmann fold.	RESULTS
+4	19	catalytic motif	structure_element	The catalytic motif DPPY lies in a loop connecting αD and β4, and the cofactor AdoMet binds in a neighboring cavity.	RESULTS
+20	24	DPPY	structure_element	The catalytic motif DPPY lies in a loop connecting αD and β4, and the cofactor AdoMet binds in a neighboring cavity.	RESULTS
+35	39	loop	structure_element	The catalytic motif DPPY lies in a loop connecting αD and β4, and the cofactor AdoMet binds in a neighboring cavity.	RESULTS
+51	53	αD	structure_element	The catalytic motif DPPY lies in a loop connecting αD and β4, and the cofactor AdoMet binds in a neighboring cavity.	RESULTS
+58	60	β4	structure_element	The catalytic motif DPPY lies in a loop connecting αD and β4, and the cofactor AdoMet binds in a neighboring cavity.	RESULTS
+79	85	AdoMet	chemical	The catalytic motif DPPY lies in a loop connecting αD and β4, and the cofactor AdoMet binds in a neighboring cavity.	RESULTS
+109	115	cavity	site	The catalytic motif DPPY lies in a loop connecting αD and β4, and the cofactor AdoMet binds in a neighboring cavity.	RESULTS
+4	8	loop	structure_element	The loop (residues 136-166) located between β7 and αZ corresponds to a highly diverse region in other MTases that is involved in target DNA recognition.	RESULTS
+19	26	136-166	residue_range	The loop (residues 136-166) located between β7 and αZ corresponds to a highly diverse region in other MTases that is involved in target DNA recognition.	RESULTS
+44	46	β7	structure_element	The loop (residues 136-166) located between β7 and αZ corresponds to a highly diverse region in other MTases that is involved in target DNA recognition.	RESULTS
+51	53	αZ	structure_element	The loop (residues 136-166) located between β7 and αZ corresponds to a highly diverse region in other MTases that is involved in target DNA recognition.	RESULTS
+71	85	highly diverse	protein_state	The loop (residues 136-166) located between β7 and αZ corresponds to a highly diverse region in other MTases that is involved in target DNA recognition.	RESULTS
+102	108	MTases	protein_type	The loop (residues 136-166) located between β7 and αZ corresponds to a highly diverse region in other MTases that is involved in target DNA recognition.	RESULTS
+136	139	DNA	chemical	The loop (residues 136-166) located between β7 and αZ corresponds to a highly diverse region in other MTases that is involved in target DNA recognition.	RESULTS
+4	16	hairpin loop	structure_element	The hairpin loop (residues 101-133) bridging β6 and β7, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII.	RESULTS
+27	34	101-133	residue_range	The hairpin loop (residues 101-133) bridging β6 and β7, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII.	RESULTS
+45	47	β6	structure_element	The hairpin loop (residues 101-133) bridging β6 and β7, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII.	RESULTS
+52	54	β7	structure_element	The hairpin loop (residues 101-133) bridging β6 and β7, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII.	RESULTS
+82	85	DNA	chemical	The hairpin loop (residues 101-133) bridging β6 and β7, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII.	RESULTS
+93	105	minor groove	structure_element	The hairpin loop (residues 101-133) bridging β6 and β7, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII.	RESULTS
+160	168	M.MboIIA	protein	The hairpin loop (residues 101-133) bridging β6 and β7, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII.	RESULTS
+170	176	M.RsrI	protein	The hairpin loop (residues 101-133) bridging β6 and β7, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII.	RESULTS
+181	188	M.pvuII	protein	The hairpin loop (residues 101-133) bridging β6 and β7, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII.	RESULTS
+4	11	missing	protein_state	The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA.	RESULTS
+12	16	loop	structure_element	The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA.	RESULTS
+27	32	33-58	residue_range	The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA.	RESULTS
+41	50	structure	evidence	The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA.	RESULTS
+54	63	M1.HpyAVI	protein	The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA.	RESULTS
+79	85	loop I	structure_element	The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA.	RESULTS
+89	95	M.TaqI	protein	The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA.	RESULTS
+127	136	structure	evidence	The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA.	RESULTS
+137	148	without DNA	protein_state	The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA.	RESULTS
+5	9	loop	structure_element	This loop, however, was well ordered in an M.TaqI-DNA complex structure and was shown to play a crucial role in DNA methylation by contacting the flipping adenine and recognizing specific DNA sequence.	RESULTS
+24	36	well ordered	protein_state	This loop, however, was well ordered in an M.TaqI-DNA complex structure and was shown to play a crucial role in DNA methylation by contacting the flipping adenine and recognizing specific DNA sequence.	RESULTS
+43	71	M.TaqI-DNA complex structure	evidence	This loop, however, was well ordered in an M.TaqI-DNA complex structure and was shown to play a crucial role in DNA methylation by contacting the flipping adenine and recognizing specific DNA sequence.	RESULTS
+112	127	DNA methylation	ptm	This loop, however, was well ordered in an M.TaqI-DNA complex structure and was shown to play a crucial role in DNA methylation by contacting the flipping adenine and recognizing specific DNA sequence.	RESULTS
+155	162	adenine	residue_name	This loop, however, was well ordered in an M.TaqI-DNA complex structure and was shown to play a crucial role in DNA methylation by contacting the flipping adenine and recognizing specific DNA sequence.	RESULTS
+188	191	DNA	chemical	This loop, however, was well ordered in an M.TaqI-DNA complex structure and was shown to play a crucial role in DNA methylation by contacting the flipping adenine and recognizing specific DNA sequence.	RESULTS
+8	17	structure	evidence	Overall structure of M1.HpyAVI	FIG
+21	30	M1.HpyAVI	protein	Overall structure of M1.HpyAVI	FIG
+3	7	Free	protein_state	A. Free form B. AdoMet-bound form.	FIG
+16	28	AdoMet-bound	protein_state	A. Free form B. AdoMet-bound form.	FIG
+18	27	M1.HpyAVI	protein	Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.	FIG
+42	64	AdoMet-dependent MTase	protein_type	Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.	FIG
+95	102	β-sheet	structure_element	Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.	FIG
+118	127	α-helices	structure_element	Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.	FIG
+129	131	αA	structure_element	Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.	FIG
+133	135	αB	structure_element	Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.	FIG
+137	139	αZ	structure_element	Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.	FIG
+156	158	αD	structure_element	Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.	FIG
+160	162	αE	structure_element	Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.	FIG
+164	166	αC	structure_element	Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.	FIG
+199	205	AdoMet	chemical	Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.	FIG
+209	217	bound in	protein_state	Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.	FIG
+220	226	cavity	site	Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.	FIG
+236	245	conserved	protein_state	Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.	FIG
+268	272	DPPY	structure_element	Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.	FIG
+4	13	α-helices	structure_element	The α-helices and β-strands are labelled and numbered according to the commonly numbering rule for the known MTases.	FIG
+18	27	β-strands	structure_element	The α-helices and β-strands are labelled and numbered according to the commonly numbering rule for the known MTases.	FIG
+109	115	MTases	protein_type	The α-helices and β-strands are labelled and numbered according to the commonly numbering rule for the known MTases.	FIG
+4	10	AdoMet	chemical	The AdoMet molecule is shown in green.	FIG
+0	7	Dimeric	oligomeric_state	Dimeric state of M1.HpyAVI in crystal and solution	RESULTS
+17	26	M1.HpyAVI	protein	Dimeric state of M1.HpyAVI in crystal and solution	RESULTS
+30	37	crystal	evidence	Dimeric state of M1.HpyAVI in crystal and solution	RESULTS
+42	50	solution	experimental_method	Dimeric state of M1.HpyAVI in crystal and solution	RESULTS
+34	44	DNA MTases	protein_type	Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state.	RESULTS
+51	58	M.BamHI	protein	Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state.	RESULTS
+63	70	M.EcoRI	protein	Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state.	RESULTS
+81	88	monomer	oligomeric_state	Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state.	RESULTS
+136	139	DNA	chemical	Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state.	RESULTS
+164	169	MTase	protein_type	Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state.	RESULTS
+173	187	hemimethylated	protein_state	Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state.	RESULTS
+222	233	methylation	ptm	Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state.	RESULTS
+261	277	fully methylated	protein_state	Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state.	RESULTS
+21	28	dimeric	oligomeric_state	Increasing number of dimeric DNA MTases, however, has been identified from later studies.	RESULTS
+29	39	DNA MTases	protein_type	Increasing number of dimeric DNA MTases, however, has been identified from later studies.	RESULTS
+14	21	M.DpnII	protein	For instance, M.DpnII, M.RsrI, M.KpnI, and M.MboIIA have been found as dimers in solution.	RESULTS
+23	29	M.RsrI	protein	For instance, M.DpnII, M.RsrI, M.KpnI, and M.MboIIA have been found as dimers in solution.	RESULTS
+31	37	M.KpnI	protein	For instance, M.DpnII, M.RsrI, M.KpnI, and M.MboIIA have been found as dimers in solution.	RESULTS
+43	51	M.MboIIA	protein	For instance, M.DpnII, M.RsrI, M.KpnI, and M.MboIIA have been found as dimers in solution.	RESULTS
+71	77	dimers	oligomeric_state	For instance, M.DpnII, M.RsrI, M.KpnI, and M.MboIIA have been found as dimers in solution.	RESULTS
+21	27	MTases	protein_type	In addition, several MTases including M.MboIIA, M.RsrI and TTH0409 form tightly associated dimers in crystal structures.	RESULTS
+38	46	M.MboIIA	protein	In addition, several MTases including M.MboIIA, M.RsrI and TTH0409 form tightly associated dimers in crystal structures.	RESULTS
+48	54	M.RsrI	protein	In addition, several MTases including M.MboIIA, M.RsrI and TTH0409 form tightly associated dimers in crystal structures.	RESULTS
+59	66	TTH0409	protein	In addition, several MTases including M.MboIIA, M.RsrI and TTH0409 form tightly associated dimers in crystal structures.	RESULTS
+91	97	dimers	oligomeric_state	In addition, several MTases including M.MboIIA, M.RsrI and TTH0409 form tightly associated dimers in crystal structures.	RESULTS
+101	119	crystal structures	evidence	In addition, several MTases including M.MboIIA, M.RsrI and TTH0409 form tightly associated dimers in crystal structures.	RESULTS
+18	28	DNA MTases	protein_type	Nonetheless, some DNA MTases such as M.CcrMI and the Bacillus amyloliquefaciens MTase dissociate from dimer into monomer upon DNA-binding.	RESULTS
+37	44	M.CcrMI	protein	Nonetheless, some DNA MTases such as M.CcrMI and the Bacillus amyloliquefaciens MTase dissociate from dimer into monomer upon DNA-binding.	RESULTS
+53	79	Bacillus amyloliquefaciens	species	Nonetheless, some DNA MTases such as M.CcrMI and the Bacillus amyloliquefaciens MTase dissociate from dimer into monomer upon DNA-binding.	RESULTS
+80	85	MTase	protein_type	Nonetheless, some DNA MTases such as M.CcrMI and the Bacillus amyloliquefaciens MTase dissociate from dimer into monomer upon DNA-binding.	RESULTS
+102	107	dimer	oligomeric_state	Nonetheless, some DNA MTases such as M.CcrMI and the Bacillus amyloliquefaciens MTase dissociate from dimer into monomer upon DNA-binding.	RESULTS
+113	120	monomer	oligomeric_state	Nonetheless, some DNA MTases such as M.CcrMI and the Bacillus amyloliquefaciens MTase dissociate from dimer into monomer upon DNA-binding.	RESULTS
+126	129	DNA	chemical	Nonetheless, some DNA MTases such as M.CcrMI and the Bacillus amyloliquefaciens MTase dissociate from dimer into monomer upon DNA-binding.	RESULTS
+42	51	conserved	protein_state	According to the arrangement of the three conserved domains, M1.HpyAVI belongs to the β-subgroup, in which a conserved motif NXXTX9−11AXRXFSXXHX4WX6−9 YXFXLX3RX9−26NPX1−6NVWX29−34A has been identified at the dimerization interface in crystal structures.	RESULTS
+61	70	M1.HpyAVI	protein	According to the arrangement of the three conserved domains, M1.HpyAVI belongs to the β-subgroup, in which a conserved motif NXXTX9−11AXRXFSXXHX4WX6−9 YXFXLX3RX9−26NPX1−6NVWX29−34A has been identified at the dimerization interface in crystal structures.	RESULTS
+86	96	β-subgroup	protein_type	According to the arrangement of the three conserved domains, M1.HpyAVI belongs to the β-subgroup, in which a conserved motif NXXTX9−11AXRXFSXXHX4WX6−9 YXFXLX3RX9−26NPX1−6NVWX29−34A has been identified at the dimerization interface in crystal structures.	RESULTS
+109	118	conserved	protein_state	According to the arrangement of the three conserved domains, M1.HpyAVI belongs to the β-subgroup, in which a conserved motif NXXTX9−11AXRXFSXXHX4WX6−9 YXFXLX3RX9−26NPX1−6NVWX29−34A has been identified at the dimerization interface in crystal structures.	RESULTS
+125	180	NXXTX9−11AXRXFSXXHX4WX6−9 YXFXLX3RX9−26NPX1−6NVWX29−34A	structure_element	According to the arrangement of the three conserved domains, M1.HpyAVI belongs to the β-subgroup, in which a conserved motif NXXTX9−11AXRXFSXXHX4WX6−9 YXFXLX3RX9−26NPX1−6NVWX29−34A has been identified at the dimerization interface in crystal structures.	RESULTS
+208	230	dimerization interface	site	According to the arrangement of the three conserved domains, M1.HpyAVI belongs to the β-subgroup, in which a conserved motif NXXTX9−11AXRXFSXXHX4WX6−9 YXFXLX3RX9−26NPX1−6NVWX29−34A has been identified at the dimerization interface in crystal structures.	RESULTS
+234	252	crystal structures	evidence	According to the arrangement of the three conserved domains, M1.HpyAVI belongs to the β-subgroup, in which a conserved motif NXXTX9−11AXRXFSXXHX4WX6−9 YXFXLX3RX9−26NPX1−6NVWX29−34A has been identified at the dimerization interface in crystal structures.	RESULTS
+8	17	conserved	protein_state	Most of conserved amino acids within that motif are present in the sequence of M1.HpyAVI (Figure 2A), implying dimerization of this protein.	RESULTS
+79	88	M1.HpyAVI	protein	Most of conserved amino acids within that motif are present in the sequence of M1.HpyAVI (Figure 2A), implying dimerization of this protein.	RESULTS
+111	123	dimerization	oligomeric_state	Most of conserved amino acids within that motif are present in the sequence of M1.HpyAVI (Figure 2A), implying dimerization of this protein.	RESULTS
+16	21	dimer	oligomeric_state	In agreement, a dimer of M1.HpyAVI was observed in our crystal structures with the two monomers related by a two-fold axis (Figure 2B and 2C).	RESULTS
+25	34	M1.HpyAVI	protein	In agreement, a dimer of M1.HpyAVI was observed in our crystal structures with the two monomers related by a two-fold axis (Figure 2B and 2C).	RESULTS
+55	73	crystal structures	evidence	In agreement, a dimer of M1.HpyAVI was observed in our crystal structures with the two monomers related by a two-fold axis (Figure 2B and 2C).	RESULTS
+87	95	monomers	oligomeric_state	In agreement, a dimer of M1.HpyAVI was observed in our crystal structures with the two monomers related by a two-fold axis (Figure 2B and 2C).	RESULTS
+38	55	dimeric interface	site	An area of ~1900 Å2 was buried at the dimeric interface, taking up ca 17% of the total area.	RESULTS
+4	11	dimeric	oligomeric_state	The dimeric architecture was greatly stabilized by hydrogen bonds and salt bridges formed among residues R86, D93 and E96.	RESULTS
+51	65	hydrogen bonds	bond_interaction	The dimeric architecture was greatly stabilized by hydrogen bonds and salt bridges formed among residues R86, D93 and E96.	RESULTS
+70	82	salt bridges	bond_interaction	The dimeric architecture was greatly stabilized by hydrogen bonds and salt bridges formed among residues R86, D93 and E96.	RESULTS
+105	108	R86	residue_name_number	The dimeric architecture was greatly stabilized by hydrogen bonds and salt bridges formed among residues R86, D93 and E96.	RESULTS
+110	113	D93	residue_name_number	The dimeric architecture was greatly stabilized by hydrogen bonds and salt bridges formed among residues R86, D93 and E96.	RESULTS
+118	121	E96	residue_name_number	The dimeric architecture was greatly stabilized by hydrogen bonds and salt bridges formed among residues R86, D93 and E96.	RESULTS
+31	36	dimer	oligomeric_state	In addition, comparison of the dimer structure of M1.HpyAVI with some other β-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3).	RESULTS
+37	46	structure	evidence	In addition, comparison of the dimer structure of M1.HpyAVI with some other β-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3).	RESULTS
+50	59	M1.HpyAVI	protein	In addition, comparison of the dimer structure of M1.HpyAVI with some other β-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3).	RESULTS
+76	90	β-class MTases	protein_type	In addition, comparison of the dimer structure of M1.HpyAVI with some other β-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3).	RESULTS
+92	101	M1.MboIIA	protein	In addition, comparison of the dimer structure of M1.HpyAVI with some other β-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3).	RESULTS
+103	109	M.RsrI	protein	In addition, comparison of the dimer structure of M1.HpyAVI with some other β-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3).	RESULTS
+114	122	TTHA0409	protein	In addition, comparison of the dimer structure of M1.HpyAVI with some other β-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3).	RESULTS
+143	152	M1.HpyAVI	protein	In addition, comparison of the dimer structure of M1.HpyAVI with some other β-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3).	RESULTS
+153	158	dimer	oligomeric_state	In addition, comparison of the dimer structure of M1.HpyAVI with some other β-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3).	RESULTS
+0	9	M1.HpyAVI	protein	M1.HpyAVI exists as dimer in crystal and solution	FIG
+20	25	dimer	oligomeric_state	M1.HpyAVI exists as dimer in crystal and solution	FIG
+29	36	crystal	evidence	M1.HpyAVI exists as dimer in crystal and solution	FIG
+5	14	conserved	protein_state	A. A conserved interface area of β-class MTases is defined in M1.HpyAVI.	FIG
+15	29	interface area	site	A. A conserved interface area of β-class MTases is defined in M1.HpyAVI.	FIG
+33	47	β-class MTases	protein_type	A. A conserved interface area of β-class MTases is defined in M1.HpyAVI.	FIG
+62	71	M1.HpyAVI	protein	A. A conserved interface area of β-class MTases is defined in M1.HpyAVI.	FIG
+48	60	Dimerization	oligomeric_state	Residues that involved are signed in red color; Dimerization of free-form M1.HpyAVI B. and cofactor-bound M1.HpyAVI C. The two monomers are marked in green and blue, AdoMet molecules are marked in magenta.	FIG
+64	68	free	protein_state	Residues that involved are signed in red color; Dimerization of free-form M1.HpyAVI B. and cofactor-bound M1.HpyAVI C. The two monomers are marked in green and blue, AdoMet molecules are marked in magenta.	FIG
+74	83	M1.HpyAVI	protein	Residues that involved are signed in red color; Dimerization of free-form M1.HpyAVI B. and cofactor-bound M1.HpyAVI C. The two monomers are marked in green and blue, AdoMet molecules are marked in magenta.	FIG
+91	105	cofactor-bound	protein_state	Residues that involved are signed in red color; Dimerization of free-form M1.HpyAVI B. and cofactor-bound M1.HpyAVI C. The two monomers are marked in green and blue, AdoMet molecules are marked in magenta.	FIG
+106	115	M1.HpyAVI	protein	Residues that involved are signed in red color; Dimerization of free-form M1.HpyAVI B. and cofactor-bound M1.HpyAVI C. The two monomers are marked in green and blue, AdoMet molecules are marked in magenta.	FIG
+127	135	monomers	oligomeric_state	Residues that involved are signed in red color; Dimerization of free-form M1.HpyAVI B. and cofactor-bound M1.HpyAVI C. The two monomers are marked in green and blue, AdoMet molecules are marked in magenta.	FIG
+166	172	AdoMet	chemical	Residues that involved are signed in red color; Dimerization of free-form M1.HpyAVI B. and cofactor-bound M1.HpyAVI C. The two monomers are marked in green and blue, AdoMet molecules are marked in magenta.	FIG
+3	26	Gel-filtration analysis	experimental_method	D. Gel-filtration analysis revealed that M1.HpyAVI exist as a dimer in solution.	FIG
+41	50	M1.HpyAVI	protein	D. Gel-filtration analysis revealed that M1.HpyAVI exist as a dimer in solution.	FIG
+62	67	dimer	oligomeric_state	D. Gel-filtration analysis revealed that M1.HpyAVI exist as a dimer in solution.	FIG
+0	4	FPLC	experimental_method	FPLC system coupled to a Superdex 75 10/300 column.	FIG
+0	16	Elution profiles	evidence	Elution profiles at 280 nm (blue) and 260 nm (red) are: different concentration (0.05, 0.1, 0.2, 0.5 mg/ml) of M1.HpyAVI protein.	FIG
+111	120	M1.HpyAVI	protein	Elution profiles at 280 nm (blue) and 260 nm (red) are: different concentration (0.05, 0.1, 0.2, 0.5 mg/ml) of M1.HpyAVI protein.	FIG
+32	41	M1.HpyAVI	protein	To probe the oligomeric form of M1.HpyAVI in solution, different concentrations of purified enzyme was loaded onto a Superdex 75 10/300 column.	RESULTS
+94	101	dimeric	oligomeric_state	The protein was eluted at ~10 ml regardless of the protein concentrations, corresponding to a dimeric molecular mass of 54 kDa (Figure 2D).	RESULTS
+102	116	molecular mass	evidence	The protein was eluted at ~10 ml regardless of the protein concentrations, corresponding to a dimeric molecular mass of 54 kDa (Figure 2D).	RESULTS
+32	41	M1.HpyAVI	protein	Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other β-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution.	RESULTS
+50	55	dimer	oligomeric_state	Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other β-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution.	RESULTS
+64	71	crystal	evidence	Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other β-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution.	RESULTS
+94	108	β-class MTases	protein_type	Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other β-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution.	RESULTS
+170	194	dynamic light scattering	experimental_method	Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other β-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution.	RESULTS
+196	199	DLS	experimental_method	Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other β-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution.	RESULTS
+217	246	gel-filtration chromatography	experimental_method	Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other β-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution.	RESULTS
+264	273	M1.HpyAVI	protein	Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other β-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution.	RESULTS
+286	295	monomeric	oligomeric_state	Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other β-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution.	RESULTS
+55	63	arginine	chemical	This variance might be caused by an addition of 100 mM arginine before cell lysis to keep protein solubility and also by later replacement of arginine with 30% glycerol by dialysis.	RESULTS
+142	150	arginine	chemical	This variance might be caused by an addition of 100 mM arginine before cell lysis to keep protein solubility and also by later replacement of arginine with 30% glycerol by dialysis.	RESULTS
+160	168	glycerol	chemical	This variance might be caused by an addition of 100 mM arginine before cell lysis to keep protein solubility and also by later replacement of arginine with 30% glycerol by dialysis.	RESULTS
+0	21	Structure comparisons	experimental_method	Structure comparisons	RESULTS
+5	29	β-class N6 adenine MTase	protein_type	As a β-class N6 adenine MTase, the M1.HpyAVI structure displayed a good similarity with M.MboIIA (PDB ID 1G60) and M.RsrI (PDB ID 1NW7), which are falling into the same subgroup.	RESULTS
+35	44	M1.HpyAVI	protein	As a β-class N6 adenine MTase, the M1.HpyAVI structure displayed a good similarity with M.MboIIA (PDB ID 1G60) and M.RsrI (PDB ID 1NW7), which are falling into the same subgroup.	RESULTS
+45	54	structure	evidence	As a β-class N6 adenine MTase, the M1.HpyAVI structure displayed a good similarity with M.MboIIA (PDB ID 1G60) and M.RsrI (PDB ID 1NW7), which are falling into the same subgroup.	RESULTS
+88	96	M.MboIIA	protein	As a β-class N6 adenine MTase, the M1.HpyAVI structure displayed a good similarity with M.MboIIA (PDB ID 1G60) and M.RsrI (PDB ID 1NW7), which are falling into the same subgroup.	RESULTS
+115	121	M.RsrI	protein	As a β-class N6 adenine MTase, the M1.HpyAVI structure displayed a good similarity with M.MboIIA (PDB ID 1G60) and M.RsrI (PDB ID 1NW7), which are falling into the same subgroup.	RESULTS
+0	15	Superimposition	experimental_method	Superimposition of M1.HpyAVI onto them gave RMSDs of 1.63 Å and 1.9 Å on 168 and 190 Cα atoms, respectively.	RESULTS
+19	28	M1.HpyAVI	protein	Superimposition of M1.HpyAVI onto them gave RMSDs of 1.63 Å and 1.9 Å on 168 and 190 Cα atoms, respectively.	RESULTS
+44	49	RMSDs	evidence	Superimposition of M1.HpyAVI onto them gave RMSDs of 1.63 Å and 1.9 Å on 168 and 190 Cα atoms, respectively.	RESULTS
+67	70	TRD	structure_element	The most striking structural difference was found to locate on the TRD region (residues 133-163 in M1.HpyAVI) (Figure 3A–3C), where the secondary structures vary among these structures.	RESULTS
+88	95	133-163	residue_range	The most striking structural difference was found to locate on the TRD region (residues 133-163 in M1.HpyAVI) (Figure 3A–3C), where the secondary structures vary among these structures.	RESULTS
+99	108	M1.HpyAVI	protein	The most striking structural difference was found to locate on the TRD region (residues 133-163 in M1.HpyAVI) (Figure 3A–3C), where the secondary structures vary among these structures.	RESULTS
+89	98	α-helices	structure_element	By comparison with the other two enzymes that possess protruding arms containing several α-helices and/or β-strands, the TRD of M1.HpyAVI is much shorter in length (Figure S1), wrapping more closely around the structural core and lacking apparent secondary structures.	RESULTS
+106	115	β-strands	structure_element	By comparison with the other two enzymes that possess protruding arms containing several α-helices and/or β-strands, the TRD of M1.HpyAVI is much shorter in length (Figure S1), wrapping more closely around the structural core and lacking apparent secondary structures.	RESULTS
+121	124	TRD	structure_element	By comparison with the other two enzymes that possess protruding arms containing several α-helices and/or β-strands, the TRD of M1.HpyAVI is much shorter in length (Figure S1), wrapping more closely around the structural core and lacking apparent secondary structures.	RESULTS
+128	137	M1.HpyAVI	protein	By comparison with the other two enzymes that possess protruding arms containing several α-helices and/or β-strands, the TRD of M1.HpyAVI is much shorter in length (Figure S1), wrapping more closely around the structural core and lacking apparent secondary structures.	RESULTS
+230	237	lacking	protein_state	By comparison with the other two enzymes that possess protruding arms containing several α-helices and/or β-strands, the TRD of M1.HpyAVI is much shorter in length (Figure S1), wrapping more closely around the structural core and lacking apparent secondary structures.	RESULTS
+31	34	TRD	structure_element	Given the proposed role of the TRD for DNA interaction at the major groove, some differences of DNA recognition mode can be expected.	RESULTS
+39	42	DNA	chemical	Given the proposed role of the TRD for DNA interaction at the major groove, some differences of DNA recognition mode can be expected.	RESULTS
+62	74	major groove	structure_element	Given the proposed role of the TRD for DNA interaction at the major groove, some differences of DNA recognition mode can be expected.	RESULTS
+96	99	DNA	chemical	Given the proposed role of the TRD for DNA interaction at the major groove, some differences of DNA recognition mode can be expected.	RESULTS
+34	49	highly flexible	protein_state	Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.	RESULTS
+50	54	loop	structure_element	Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.	RESULTS
+63	65	β4	structure_element	Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.	RESULTS
+70	72	αD	structure_element	Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.	RESULTS
+83	88	33-58	residue_range	Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.	RESULTS
+93	102	M1.HpyAVI	protein	Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.	RESULTS
+131	141	structures	evidence	Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.	RESULTS
+161	171	structures	evidence	Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.	RESULTS
+175	183	M.MboIIA	protein	Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.	RESULTS
+188	194	M.RsrI	protein	Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.	RESULTS
+196	214	Sequence alignment	experimental_method	Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.	RESULTS
+244	253	M1.HpyAVI	protein	Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.	RESULTS
+351	360	H. pylori	species	Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.	RESULTS
+373	381	flexible	protein_state	Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.	RESULTS
+0	22	Structural comparisons	experimental_method	Structural comparisons between M1.HpyAVI and other DNA MTases	FIG
+31	40	M1.HpyAVI	protein	Structural comparisons between M1.HpyAVI and other DNA MTases	FIG
+51	61	DNA MTases	protein_type	Structural comparisons between M1.HpyAVI and other DNA MTases	FIG
+3	12	M1.HpyAVI	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+17	25	M.MboIIA	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+30	36	M.RsrI	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+41	49	TTHA0409	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+54	58	DpnM	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+63	69	M.TaqI	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+71	80	M1.HpyAVI	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+98	111	long disorder	protein_state	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+112	115	TRD	structure_element	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+142	156	structure-rich	protein_state	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+157	160	TRD	structure_element	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+164	172	M.MboIIA	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+174	180	M.RsrI	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+185	193	TTHA0409	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+208	226	DNA-binding domain	structure_element	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+230	234	DpnM	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+239	245	M.TaqI	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+278	281	TRD	structure_element	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+285	294	M1.HpyAVI	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+296	304	M.MboIIA	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+306	312	M.RsrI	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+317	325	TTHA0409	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+356	358	β4	structure_element	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+363	365	αD	structure_element	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+369	377	M.MboIIA	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+382	388	M.RsrI	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+402	420	DNA-binding domain	structure_element	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+424	428	DpnM	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+448	465	C-terminal domain	structure_element	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+469	475	M.TaqI	protein	A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.	FIG
+0	21	Structural comparison	experimental_method	Structural comparison between M1.HpyAVI and a putative β-class N4 cytosine MTase named TTHA0409 (PDB ID 2ZIF) showed a good similarity as well, giving an RMSD of 1.73 Å on 164 Cα atoms (Figure 3D).	RESULTS
+30	39	M1.HpyAVI	protein	Structural comparison between M1.HpyAVI and a putative β-class N4 cytosine MTase named TTHA0409 (PDB ID 2ZIF) showed a good similarity as well, giving an RMSD of 1.73 Å on 164 Cα atoms (Figure 3D).	RESULTS
+55	80	β-class N4 cytosine MTase	protein_type	Structural comparison between M1.HpyAVI and a putative β-class N4 cytosine MTase named TTHA0409 (PDB ID 2ZIF) showed a good similarity as well, giving an RMSD of 1.73 Å on 164 Cα atoms (Figure 3D).	RESULTS
+87	95	TTHA0409	protein	Structural comparison between M1.HpyAVI and a putative β-class N4 cytosine MTase named TTHA0409 (PDB ID 2ZIF) showed a good similarity as well, giving an RMSD of 1.73 Å on 164 Cα atoms (Figure 3D).	RESULTS
+154	158	RMSD	evidence	Structural comparison between M1.HpyAVI and a putative β-class N4 cytosine MTase named TTHA0409 (PDB ID 2ZIF) showed a good similarity as well, giving an RMSD of 1.73 Å on 164 Cα atoms (Figure 3D).	RESULTS
+81	84	TRD	structure_element	Exactly like the above comparison, the most significant difference exists in the TRD, where the structures vary in terms of length and presence of α-helices (Figure S1).	RESULTS
+96	106	structures	evidence	Exactly like the above comparison, the most significant difference exists in the TRD, where the structures vary in terms of length and presence of α-helices (Figure S1).	RESULTS
+147	156	α-helices	structure_element	Exactly like the above comparison, the most significant difference exists in the TRD, where the structures vary in terms of length and presence of α-helices (Figure S1).	RESULTS
+0	9	M1.HpyAVI	protein	M1.HpyAVI displayed a considerable structural dissimilarity in comparison with N6-adenine MTases from other subgroups including the α-class DpnM (PDB ID 2DPM) and the γ-class M.TaqI (PDB ID 2ADM).	RESULTS
+79	96	N6-adenine MTases	protein_type	M1.HpyAVI displayed a considerable structural dissimilarity in comparison with N6-adenine MTases from other subgroups including the α-class DpnM (PDB ID 2DPM) and the γ-class M.TaqI (PDB ID 2ADM).	RESULTS
+132	139	α-class	protein_type	M1.HpyAVI displayed a considerable structural dissimilarity in comparison with N6-adenine MTases from other subgroups including the α-class DpnM (PDB ID 2DPM) and the γ-class M.TaqI (PDB ID 2ADM).	RESULTS
+140	144	DpnM	protein	M1.HpyAVI displayed a considerable structural dissimilarity in comparison with N6-adenine MTases from other subgroups including the α-class DpnM (PDB ID 2DPM) and the γ-class M.TaqI (PDB ID 2ADM).	RESULTS
+167	174	γ-class	protein_type	M1.HpyAVI displayed a considerable structural dissimilarity in comparison with N6-adenine MTases from other subgroups including the α-class DpnM (PDB ID 2DPM) and the γ-class M.TaqI (PDB ID 2ADM).	RESULTS
+175	181	M.TaqI	protein	M1.HpyAVI displayed a considerable structural dissimilarity in comparison with N6-adenine MTases from other subgroups including the α-class DpnM (PDB ID 2DPM) and the γ-class M.TaqI (PDB ID 2ADM).	RESULTS
+22	27	RMSDs	evidence	Both comparisons gave RMSDs above 3.0 Å (Figure 3E and 3F).	RESULTS
+18	22	lack	protein_state	These two enzymes lack a counterpart loop present in the TRD of M1.HpyAVI, but instead rely on an extra domain for DNA binding and sequence recognition.	RESULTS
+25	41	counterpart loop	structure_element	These two enzymes lack a counterpart loop present in the TRD of M1.HpyAVI, but instead rely on an extra domain for DNA binding and sequence recognition.	RESULTS
+57	60	TRD	structure_element	These two enzymes lack a counterpart loop present in the TRD of M1.HpyAVI, but instead rely on an extra domain for DNA binding and sequence recognition.	RESULTS
+64	73	M1.HpyAVI	protein	These two enzymes lack a counterpart loop present in the TRD of M1.HpyAVI, but instead rely on an extra domain for DNA binding and sequence recognition.	RESULTS
+115	118	DNA	chemical	These two enzymes lack a counterpart loop present in the TRD of M1.HpyAVI, but instead rely on an extra domain for DNA binding and sequence recognition.	RESULTS
+14	23	M1.HpyAVI	protein	Collectively, M1.HpyAVI possesses a long disordered TRD, which is in sharp contrast to the secondary structure-rich TRD in other β-class N6 adenine or N4 cytosine MTases or the extra DNA binding domain present in DNA MTases from other subgroups.	RESULTS
+36	51	long disordered	protein_state	Collectively, M1.HpyAVI possesses a long disordered TRD, which is in sharp contrast to the secondary structure-rich TRD in other β-class N6 adenine or N4 cytosine MTases or the extra DNA binding domain present in DNA MTases from other subgroups.	RESULTS
+52	55	TRD	structure_element	Collectively, M1.HpyAVI possesses a long disordered TRD, which is in sharp contrast to the secondary structure-rich TRD in other β-class N6 adenine or N4 cytosine MTases or the extra DNA binding domain present in DNA MTases from other subgroups.	RESULTS
+91	115	secondary structure-rich	protein_state	Collectively, M1.HpyAVI possesses a long disordered TRD, which is in sharp contrast to the secondary structure-rich TRD in other β-class N6 adenine or N4 cytosine MTases or the extra DNA binding domain present in DNA MTases from other subgroups.	RESULTS
+116	119	TRD	structure_element	Collectively, M1.HpyAVI possesses a long disordered TRD, which is in sharp contrast to the secondary structure-rich TRD in other β-class N6 adenine or N4 cytosine MTases or the extra DNA binding domain present in DNA MTases from other subgroups.	RESULTS
+129	169	β-class N6 adenine or N4 cytosine MTases	protein_type	Collectively, M1.HpyAVI possesses a long disordered TRD, which is in sharp contrast to the secondary structure-rich TRD in other β-class N6 adenine or N4 cytosine MTases or the extra DNA binding domain present in DNA MTases from other subgroups.	RESULTS
+213	223	DNA MTases	protein_type	Collectively, M1.HpyAVI possesses a long disordered TRD, which is in sharp contrast to the secondary structure-rich TRD in other β-class N6 adenine or N4 cytosine MTases or the extra DNA binding domain present in DNA MTases from other subgroups.	RESULTS
+98	107	H. pylori	species	This striking difference may be a significant determinant of the wider substrate spectrum of this H. pylori enzyme.	RESULTS
+0	21	AdoMet-binding pocket	site	AdoMet-binding pocket	RESULTS
+4	27	cofactor binding pocket	site	The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases.	RESULTS
+31	40	M1.HpyAVI	protein	The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases.	RESULTS
+67	70	7-9	residue_range	The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases.	RESULTS
+72	77	29-31	residue_range	The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases.	RESULTS
+79	86	165-167	residue_range	The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases.	RESULTS
+88	95	216-218	residue_range	The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases.	RESULTS
+100	103	221	residue_number	The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases.	RESULTS
+127	136	conserved	protein_state	The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases.	RESULTS
+151	161	DNA MTases	protein_type	The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases.	RESULTS
+2	15	hydrogen bond	bond_interaction	A hydrogen bond between D29 in the catalytic motif DPPY and the amino group of bound AdoMet is preserved as other MTase structures.	RESULTS
+24	27	D29	residue_name_number	A hydrogen bond between D29 in the catalytic motif DPPY and the amino group of bound AdoMet is preserved as other MTase structures.	RESULTS
+35	50	catalytic motif	structure_element	A hydrogen bond between D29 in the catalytic motif DPPY and the amino group of bound AdoMet is preserved as other MTase structures.	RESULTS
+51	55	DPPY	structure_element	A hydrogen bond between D29 in the catalytic motif DPPY and the amino group of bound AdoMet is preserved as other MTase structures.	RESULTS
+79	84	bound	protein_state	A hydrogen bond between D29 in the catalytic motif DPPY and the amino group of bound AdoMet is preserved as other MTase structures.	RESULTS
+85	91	AdoMet	chemical	A hydrogen bond between D29 in the catalytic motif DPPY and the amino group of bound AdoMet is preserved as other MTase structures.	RESULTS
+114	119	MTase	protein_type	A hydrogen bond between D29 in the catalytic motif DPPY and the amino group of bound AdoMet is preserved as other MTase structures.	RESULTS
+120	130	structures	evidence	A hydrogen bond between D29 in the catalytic motif DPPY and the amino group of bound AdoMet is preserved as other MTase structures.	RESULTS
+9	11	D8	residue_name_number	Residues D8 and A9 from hydrogen-bonds with N6 and N1 of the purine ring, respectively, and E216 also locates at hydrogen bonding distance with O2′ and O3′ of the ribose.	RESULTS
+16	18	A9	residue_name_number	Residues D8 and A9 from hydrogen-bonds with N6 and N1 of the purine ring, respectively, and E216 also locates at hydrogen bonding distance with O2′ and O3′ of the ribose.	RESULTS
+24	38	hydrogen-bonds	bond_interaction	Residues D8 and A9 from hydrogen-bonds with N6 and N1 of the purine ring, respectively, and E216 also locates at hydrogen bonding distance with O2′ and O3′ of the ribose.	RESULTS
+61	67	purine	chemical	Residues D8 and A9 from hydrogen-bonds with N6 and N1 of the purine ring, respectively, and E216 also locates at hydrogen bonding distance with O2′ and O3′ of the ribose.	RESULTS
+92	96	E216	residue_name_number	Residues D8 and A9 from hydrogen-bonds with N6 and N1 of the purine ring, respectively, and E216 also locates at hydrogen bonding distance with O2′ and O3′ of the ribose.	RESULTS
+113	129	hydrogen bonding	bond_interaction	Residues D8 and A9 from hydrogen-bonds with N6 and N1 of the purine ring, respectively, and E216 also locates at hydrogen bonding distance with O2′ and O3′ of the ribose.	RESULTS
+163	169	ribose	chemical	Residues D8 and A9 from hydrogen-bonds with N6 and N1 of the purine ring, respectively, and E216 also locates at hydrogen bonding distance with O2′ and O3′ of the ribose.	RESULTS
+13	17	H168	residue_name_number	In addition, H168, T200 and S198 contact the terminal carboxyl of AdoMet.	RESULTS
+19	23	T200	residue_name_number	In addition, H168, T200 and S198 contact the terminal carboxyl of AdoMet.	RESULTS
+28	32	S198	residue_name_number	In addition, H168, T200 and S198 contact the terminal carboxyl of AdoMet.	RESULTS
+66	72	AdoMet	chemical	In addition, H168, T200 and S198 contact the terminal carboxyl of AdoMet.	RESULTS
+0	13	Superposition	experimental_method	Superposition of M1.HpyAVI with the five structures shown in Figure 3 reveals that the orientation of cofactor is rather conserved except for M.TaqI (Figure 4B).	RESULTS
+17	26	M1.HpyAVI	protein	Superposition of M1.HpyAVI with the five structures shown in Figure 3 reveals that the orientation of cofactor is rather conserved except for M.TaqI (Figure 4B).	RESULTS
+41	51	structures	evidence	Superposition of M1.HpyAVI with the five structures shown in Figure 3 reveals that the orientation of cofactor is rather conserved except for M.TaqI (Figure 4B).	RESULTS
+114	130	rather conserved	protein_state	Superposition of M1.HpyAVI with the five structures shown in Figure 3 reveals that the orientation of cofactor is rather conserved except for M.TaqI (Figure 4B).	RESULTS
+142	148	M.TaqI	protein	Superposition of M1.HpyAVI with the five structures shown in Figure 3 reveals that the orientation of cofactor is rather conserved except for M.TaqI (Figure 4B).	RESULTS
+34	39	bound	protein_state	The different conformation of the bound cofactor observed in M.TaqI might be attributable to the absence of corresponding residues of the conserved AdoMet-binding motif FXGXG in that structure.	RESULTS
+61	67	M.TaqI	protein	The different conformation of the bound cofactor observed in M.TaqI might be attributable to the absence of corresponding residues of the conserved AdoMet-binding motif FXGXG in that structure.	RESULTS
+97	107	absence of	protein_state	The different conformation of the bound cofactor observed in M.TaqI might be attributable to the absence of corresponding residues of the conserved AdoMet-binding motif FXGXG in that structure.	RESULTS
+138	147	conserved	protein_state	The different conformation of the bound cofactor observed in M.TaqI might be attributable to the absence of corresponding residues of the conserved AdoMet-binding motif FXGXG in that structure.	RESULTS
+148	154	AdoMet	chemical	The different conformation of the bound cofactor observed in M.TaqI might be attributable to the absence of corresponding residues of the conserved AdoMet-binding motif FXGXG in that structure.	RESULTS
+169	174	FXGXG	structure_element	The different conformation of the bound cofactor observed in M.TaqI might be attributable to the absence of corresponding residues of the conserved AdoMet-binding motif FXGXG in that structure.	RESULTS
+183	192	structure	evidence	The different conformation of the bound cofactor observed in M.TaqI might be attributable to the absence of corresponding residues of the conserved AdoMet-binding motif FXGXG in that structure.	RESULTS
+0	35	Structural and biochemical analyses	experimental_method	Structural and biochemical analyses define two conserved residues D29 and E216 to be the key sites for AdoMet binding	FIG
+47	56	conserved	protein_state	Structural and biochemical analyses define two conserved residues D29 and E216 to be the key sites for AdoMet binding	FIG
+66	69	D29	residue_name_number	Structural and biochemical analyses define two conserved residues D29 and E216 to be the key sites for AdoMet binding	FIG
+74	78	E216	residue_name_number	Structural and biochemical analyses define two conserved residues D29 and E216 to be the key sites for AdoMet binding	FIG
+103	109	AdoMet	chemical	Structural and biochemical analyses define two conserved residues D29 and E216 to be the key sites for AdoMet binding	FIG
+7	30	cofactor-binding cavity	site	A. The cofactor-binding cavity of M1.HpyAVI.	FIG
+34	43	M1.HpyAVI	protein	A. The cofactor-binding cavity of M1.HpyAVI.	FIG
+35	49	hydrogen bonds	bond_interaction	Residues (yellow) that form direct hydrogen bonds with AdoMet (green) are indicated, distance of the hydrogen bond is marked.	FIG
+55	61	AdoMet	chemical	Residues (yellow) that form direct hydrogen bonds with AdoMet (green) are indicated, distance of the hydrogen bond is marked.	FIG
+101	114	hydrogen bond	bond_interaction	Residues (yellow) that form direct hydrogen bonds with AdoMet (green) are indicated, distance of the hydrogen bond is marked.	FIG
+3	16	Superposition	experimental_method	B. Superposition of AdoMet in the structures of M1.HpyAVI (green), DpnM (yellow) and M.TaqI (orange).	FIG
+20	26	AdoMet	chemical	B. Superposition of AdoMet in the structures of M1.HpyAVI (green), DpnM (yellow) and M.TaqI (orange).	FIG
+34	44	structures	evidence	B. Superposition of AdoMet in the structures of M1.HpyAVI (green), DpnM (yellow) and M.TaqI (orange).	FIG
+48	57	M1.HpyAVI	protein	B. Superposition of AdoMet in the structures of M1.HpyAVI (green), DpnM (yellow) and M.TaqI (orange).	FIG
+67	71	DpnM	protein	B. Superposition of AdoMet in the structures of M1.HpyAVI (green), DpnM (yellow) and M.TaqI (orange).	FIG
+85	91	M.TaqI	protein	B. Superposition of AdoMet in the structures of M1.HpyAVI (green), DpnM (yellow) and M.TaqI (orange).	FIG
+4	10	AdoMet	chemical	The AdoMet terminal carboxyl of M.TaqI reveals different orientations.	FIG
+32	38	M.TaqI	protein	The AdoMet terminal carboxyl of M.TaqI reveals different orientations.	FIG
+3	28	Cofactor binding affinity	evidence	C. Cofactor binding affinity of wt-/mutants M1.HpyAVI proteins analyzed by microscale thermophoresis (MST).	FIG
+32	34	wt	protein_state	C. Cofactor binding affinity of wt-/mutants M1.HpyAVI proteins analyzed by microscale thermophoresis (MST).	FIG
+36	43	mutants	protein_state	C. Cofactor binding affinity of wt-/mutants M1.HpyAVI proteins analyzed by microscale thermophoresis (MST).	FIG
+44	53	M1.HpyAVI	protein	C. Cofactor binding affinity of wt-/mutants M1.HpyAVI proteins analyzed by microscale thermophoresis (MST).	FIG
+75	100	microscale thermophoresis	experimental_method	C. Cofactor binding affinity of wt-/mutants M1.HpyAVI proteins analyzed by microscale thermophoresis (MST).	FIG
+102	105	MST	experimental_method	C. Cofactor binding affinity of wt-/mutants M1.HpyAVI proteins analyzed by microscale thermophoresis (MST).	FIG
+4	20	binding affinity	evidence	The binding affinity was determined between fluorescently labelled M1.HpyAVI protein and unlabeled AdoMet.	FIG
+67	76	M1.HpyAVI	protein	The binding affinity was determined between fluorescently labelled M1.HpyAVI protein and unlabeled AdoMet.	FIG
+89	98	unlabeled	protein_state	The binding affinity was determined between fluorescently labelled M1.HpyAVI protein and unlabeled AdoMet.	FIG
+99	105	AdoMet	chemical	The binding affinity was determined between fluorescently labelled M1.HpyAVI protein and unlabeled AdoMet.	FIG
+0	6	AdoMet	chemical	AdoMet (15 nM to 1 mM) was titrated into a fixed concentration of M1.HpyAVI wt/mutant proteins (800 nM).	FIG
+27	35	titrated	experimental_method	AdoMet (15 nM to 1 mM) was titrated into a fixed concentration of M1.HpyAVI wt/mutant proteins (800 nM).	FIG
+66	75	M1.HpyAVI	protein	AdoMet (15 nM to 1 mM) was titrated into a fixed concentration of M1.HpyAVI wt/mutant proteins (800 nM).	FIG
+76	78	wt	protein_state	AdoMet (15 nM to 1 mM) was titrated into a fixed concentration of M1.HpyAVI wt/mutant proteins (800 nM).	FIG
+79	85	mutant	protein_state	AdoMet (15 nM to 1 mM) was titrated into a fixed concentration of M1.HpyAVI wt/mutant proteins (800 nM).	FIG
+4	25	dissociation constant	evidence	The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated.	FIG
+27	29	KD	evidence	The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated.	FIG
+87	95	isotherm	evidence	The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated.	FIG
+121	130	M1.HpyAVI	protein	The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated.	FIG
+131	133	wt	protein_state	The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated.	FIG
+146	159	M1.HpyAVI-D8A	mutant	The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated.	FIG
+174	188	M1.HpyAVI-D29A	mutant	The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated.	FIG
+197	212	M1.HpyAVI-H168A	mutant	The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated.	FIG
+228	243	M1.HpyAVI-S198A	mutant	The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated.	FIG
+259	274	M1.HpyAVI-T200A	mutant	The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated.	FIG
+290	305	M1.HpyAVI-E216A	mutant	The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated.	FIG
+3	24	DNA methyltransferase	protein_type	D. DNA methyltransferase activity of wide type protein and the mutants is quantified using radioactive assay.	FIG
+37	46	wide type	protein_state	D. DNA methyltransferase activity of wide type protein and the mutants is quantified using radioactive assay.	FIG
+63	70	mutants	protein_state	D. DNA methyltransferase activity of wide type protein and the mutants is quantified using radioactive assay.	FIG
+91	108	radioactive assay	experimental_method	D. DNA methyltransferase activity of wide type protein and the mutants is quantified using radioactive assay.	FIG
+0	11	[3H]-methyl	chemical	[3H]-methyl transferred to duplex DNA containing 5′-GAGG-3′ was quantified by Beckman LS6500 for 10 min, experiments were repeated for three times and data were corrected by subtraction of the background.	FIG
+34	37	DNA	chemical	[3H]-methyl transferred to duplex DNA containing 5′-GAGG-3′ was quantified by Beckman LS6500 for 10 min, experiments were repeated for three times and data were corrected by subtraction of the background.	FIG
+49	59	5′-GAGG-3′	chemical	[3H]-methyl transferred to duplex DNA containing 5′-GAGG-3′ was quantified by Beckman LS6500 for 10 min, experiments were repeated for three times and data were corrected by subtraction of the background.	FIG
+3	16	Superposition	experimental_method	E. Superposition of M1.HpyAVI (green) with M.MboIIA (cyan) and M.RsrI (magenta).	FIG
+20	29	M1.HpyAVI	protein	E. Superposition of M1.HpyAVI (green) with M.MboIIA (cyan) and M.RsrI (magenta).	FIG
+43	51	M.MboIIA	protein	E. Superposition of M1.HpyAVI (green) with M.MboIIA (cyan) and M.RsrI (magenta).	FIG
+63	69	M.RsrI	protein	E. Superposition of M1.HpyAVI (green) with M.MboIIA (cyan) and M.RsrI (magenta).	FIG
+9	12	D29	residue_name_number	Residues D29 and E216 are conserved through all the DNA MTases mentioned in Figure 3 (not shown in Figure 4).	FIG
+17	21	E216	residue_name_number	Residues D29 and E216 are conserved through all the DNA MTases mentioned in Figure 3 (not shown in Figure 4).	FIG
+26	35	conserved	protein_state	Residues D29 and E216 are conserved through all the DNA MTases mentioned in Figure 3 (not shown in Figure 4).	FIG
+52	62	DNA MTases	protein_type	Residues D29 and E216 are conserved through all the DNA MTases mentioned in Figure 3 (not shown in Figure 4).	FIG
+72	86	single mutants	experimental_method	To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay.	RESULTS
+90	99	replacing	experimental_method	To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay.	RESULTS
+100	102	D8	residue_name_number	To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay.	RESULTS
+104	107	D29	residue_name_number	To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay.	RESULTS
+109	113	H168	residue_name_number	To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay.	RESULTS
+115	119	S198	residue_name_number	To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay.	RESULTS
+121	125	T200	residue_name_number	To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay.	RESULTS
+127	131	E216	residue_name_number	To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay.	RESULTS
+137	144	alanine	residue_name	To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay.	RESULTS
+168	191	ligand binding affinity	evidence	To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay.	RESULTS
+198	223	microscale thermophoresis	experimental_method	To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay.	RESULTS
+225	228	MST	experimental_method	To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay.	RESULTS
+42	51	wild type	protein_state	As shown in Figure 4C, by contrast to the wild type enzyme, most mutants displayed variable reduction of KD value, among them the D29A and E216A mutants displayed no protein-AdoMet affinity at all.	RESULTS
+65	72	mutants	protein_state	As shown in Figure 4C, by contrast to the wild type enzyme, most mutants displayed variable reduction of KD value, among them the D29A and E216A mutants displayed no protein-AdoMet affinity at all.	RESULTS
+105	107	KD	evidence	As shown in Figure 4C, by contrast to the wild type enzyme, most mutants displayed variable reduction of KD value, among them the D29A and E216A mutants displayed no protein-AdoMet affinity at all.	RESULTS
+130	134	D29A	mutant	As shown in Figure 4C, by contrast to the wild type enzyme, most mutants displayed variable reduction of KD value, among them the D29A and E216A mutants displayed no protein-AdoMet affinity at all.	RESULTS
+139	144	E216A	mutant	As shown in Figure 4C, by contrast to the wild type enzyme, most mutants displayed variable reduction of KD value, among them the D29A and E216A mutants displayed no protein-AdoMet affinity at all.	RESULTS
+145	152	mutants	protein_state	As shown in Figure 4C, by contrast to the wild type enzyme, most mutants displayed variable reduction of KD value, among them the D29A and E216A mutants displayed no protein-AdoMet affinity at all.	RESULTS
+166	189	protein-AdoMet affinity	evidence	As shown in Figure 4C, by contrast to the wild type enzyme, most mutants displayed variable reduction of KD value, among them the D29A and E216A mutants displayed no protein-AdoMet affinity at all.	RESULTS
+31	45	hydrogen bonds	bond_interaction	The results suggested that the hydrogen bonds formed by D29 and E216 with AdoMet were most crucial interactions for cofactor binding.	RESULTS
+56	59	D29	residue_name_number	The results suggested that the hydrogen bonds formed by D29 and E216 with AdoMet were most crucial interactions for cofactor binding.	RESULTS
+64	68	E216	residue_name_number	The results suggested that the hydrogen bonds formed by D29 and E216 with AdoMet were most crucial interactions for cofactor binding.	RESULTS
+74	80	AdoMet	chemical	The results suggested that the hydrogen bonds formed by D29 and E216 with AdoMet were most crucial interactions for cofactor binding.	RESULTS
+0	8	Mutation	experimental_method	Mutation of the two residues may directly prevent the methyl transfer reaction of M1.HpyAVI.	RESULTS
+54	60	methyl	chemical	Mutation of the two residues may directly prevent the methyl transfer reaction of M1.HpyAVI.	RESULTS
+82	91	M1.HpyAVI	protein	Mutation of the two residues may directly prevent the methyl transfer reaction of M1.HpyAVI.	RESULTS
+18	21	D29	residue_name_number	The importance of D29 is preserved because it belongs to the catalytic active site DPPY, but the residue E216 has not been fully investigated even being a conserved amino acid throughout MTases (Figure 4E).	RESULTS
+61	82	catalytic active site	site	The importance of D29 is preserved because it belongs to the catalytic active site DPPY, but the residue E216 has not been fully investigated even being a conserved amino acid throughout MTases (Figure 4E).	RESULTS
+83	87	DPPY	structure_element	The importance of D29 is preserved because it belongs to the catalytic active site DPPY, but the residue E216 has not been fully investigated even being a conserved amino acid throughout MTases (Figure 4E).	RESULTS
+105	109	E216	residue_name_number	The importance of D29 is preserved because it belongs to the catalytic active site DPPY, but the residue E216 has not been fully investigated even being a conserved amino acid throughout MTases (Figure 4E).	RESULTS
+155	164	conserved	protein_state	The importance of D29 is preserved because it belongs to the catalytic active site DPPY, but the residue E216 has not been fully investigated even being a conserved amino acid throughout MTases (Figure 4E).	RESULTS
+165	175	amino acid	chemical	The importance of D29 is preserved because it belongs to the catalytic active site DPPY, but the residue E216 has not been fully investigated even being a conserved amino acid throughout MTases (Figure 4E).	RESULTS
+187	193	MTases	protein_type	The importance of D29 is preserved because it belongs to the catalytic active site DPPY, but the residue E216 has not been fully investigated even being a conserved amino acid throughout MTases (Figure 4E).	RESULTS
+0	4	E216	residue_name_number	E216 is the last residue of β2, which contacts the two hydroxyls of the ribose of AdoMet.	RESULTS
+28	30	β2	structure_element	E216 is the last residue of β2, which contacts the two hydroxyls of the ribose of AdoMet.	RESULTS
+72	78	ribose	chemical	E216 is the last residue of β2, which contacts the two hydroxyls of the ribose of AdoMet.	RESULTS
+82	88	AdoMet	chemical	E216 is the last residue of β2, which contacts the two hydroxyls of the ribose of AdoMet.	RESULTS
+0	11	Replacement	experimental_method	Replacement of this residue by alanine completely abolishes the key hydrogen bonds for AdoMet-binding, and very likely blocks the methyl transfer reaction.	RESULTS
+31	38	alanine	residue_name	Replacement of this residue by alanine completely abolishes the key hydrogen bonds for AdoMet-binding, and very likely blocks the methyl transfer reaction.	RESULTS
+68	82	hydrogen bonds	bond_interaction	Replacement of this residue by alanine completely abolishes the key hydrogen bonds for AdoMet-binding, and very likely blocks the methyl transfer reaction.	RESULTS
+87	93	AdoMet	chemical	Replacement of this residue by alanine completely abolishes the key hydrogen bonds for AdoMet-binding, and very likely blocks the methyl transfer reaction.	RESULTS
+130	136	methyl	chemical	Replacement of this residue by alanine completely abolishes the key hydrogen bonds for AdoMet-binding, and very likely blocks the methyl transfer reaction.	RESULTS
+24	53	[3H]AdoMet radiological assay	experimental_method	To confirm this notion, [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the mutants.	RESULTS
+82	88	methyl	chemical	To confirm this notion, [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the mutants.	RESULTS
+114	121	mutants	protein_state	To confirm this notion, [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the mutants.	RESULTS
+37	55	radiological assay	experimental_method	As shown in Figure 4D, the result of radiological assay agreed well with the MST measurement.	RESULTS
+77	80	MST	experimental_method	As shown in Figure 4D, the result of radiological assay agreed well with the MST measurement.	RESULTS
+4	8	D29A	mutant	The D29A and E216A mutants showed little or no methyl transfer activity, while other mutants exhibited reduced methyltransferase activity.	RESULTS
+13	18	E216A	mutant	The D29A and E216A mutants showed little or no methyl transfer activity, while other mutants exhibited reduced methyltransferase activity.	RESULTS
+19	26	mutants	protein_state	The D29A and E216A mutants showed little or no methyl transfer activity, while other mutants exhibited reduced methyltransferase activity.	RESULTS
+47	53	methyl	chemical	The D29A and E216A mutants showed little or no methyl transfer activity, while other mutants exhibited reduced methyltransferase activity.	RESULTS
+85	92	mutants	protein_state	The D29A and E216A mutants showed little or no methyl transfer activity, while other mutants exhibited reduced methyltransferase activity.	RESULTS
+111	128	methyltransferase	protein_type	The D29A and E216A mutants showed little or no methyl transfer activity, while other mutants exhibited reduced methyltransferase activity.	RESULTS
+25	30	FXGXG	structure_element	As mentioned previously, FXGXG is a conserved AdoMet-binding motif of DNA MTases.	RESULTS
+36	45	conserved	protein_state	As mentioned previously, FXGXG is a conserved AdoMet-binding motif of DNA MTases.	RESULTS
+46	52	AdoMet	chemical	As mentioned previously, FXGXG is a conserved AdoMet-binding motif of DNA MTases.	RESULTS
+70	80	DNA MTases	protein_type	As mentioned previously, FXGXG is a conserved AdoMet-binding motif of DNA MTases.	RESULTS
+13	20	mutants	protein_state	We also made mutants of “FMGSG” to alanine for every amino acid, and found that the F195A mutant was insoluble probably due to decreasing the local hydrophobicity upon this mutation.	RESULTS
+25	30	FMGSG	structure_element	We also made mutants of “FMGSG” to alanine for every amino acid, and found that the F195A mutant was insoluble probably due to decreasing the local hydrophobicity upon this mutation.	RESULTS
+35	42	alanine	residue_name	We also made mutants of “FMGSG” to alanine for every amino acid, and found that the F195A mutant was insoluble probably due to decreasing the local hydrophobicity upon this mutation.	RESULTS
+53	63	amino acid	chemical	We also made mutants of “FMGSG” to alanine for every amino acid, and found that the F195A mutant was insoluble probably due to decreasing the local hydrophobicity upon this mutation.	RESULTS
+84	89	F195A	mutant	We also made mutants of “FMGSG” to alanine for every amino acid, and found that the F195A mutant was insoluble probably due to decreasing the local hydrophobicity upon this mutation.	RESULTS
+90	96	mutant	protein_state	We also made mutants of “FMGSG” to alanine for every amino acid, and found that the F195A mutant was insoluble probably due to decreasing the local hydrophobicity upon this mutation.	RESULTS
+33	56	ligand binding affinity	evidence	We subsequently investigated the ligand binding affinity and methyl transfer reaction of the other mutants using MST and a radiological assay.	RESULTS
+61	67	methyl	chemical	We subsequently investigated the ligand binding affinity and methyl transfer reaction of the other mutants using MST and a radiological assay.	RESULTS
+99	106	mutants	protein_state	We subsequently investigated the ligand binding affinity and methyl transfer reaction of the other mutants using MST and a radiological assay.	RESULTS
+113	116	MST	experimental_method	We subsequently investigated the ligand binding affinity and methyl transfer reaction of the other mutants using MST and a radiological assay.	RESULTS
+123	141	radiological assay	experimental_method	We subsequently investigated the ligand binding affinity and methyl transfer reaction of the other mutants using MST and a radiological assay.	RESULTS
+14	18	G197	residue_name_number	We found that G197 played a crucial role in AdoMet-binding, while mutagenesis of M196 and G199 did not influence cofactor binding and catalytic activity (Figure S2A and B).	RESULTS
+44	50	AdoMet	chemical	We found that G197 played a crucial role in AdoMet-binding, while mutagenesis of M196 and G199 did not influence cofactor binding and catalytic activity (Figure S2A and B).	RESULTS
+66	77	mutagenesis	experimental_method	We found that G197 played a crucial role in AdoMet-binding, while mutagenesis of M196 and G199 did not influence cofactor binding and catalytic activity (Figure S2A and B).	RESULTS
+81	85	M196	residue_name_number	We found that G197 played a crucial role in AdoMet-binding, while mutagenesis of M196 and G199 did not influence cofactor binding and catalytic activity (Figure S2A and B).	RESULTS
+90	94	G199	residue_name_number	We found that G197 played a crucial role in AdoMet-binding, while mutagenesis of M196 and G199 did not influence cofactor binding and catalytic activity (Figure S2A and B).	RESULTS
+0	4	G197	residue_name_number	G197 is a conserved residue throughout the DNA MTases, and replacing by alanine at this site likely change the local conformation of cofactor-binding pocket.	RESULTS
+10	19	conserved	protein_state	G197 is a conserved residue throughout the DNA MTases, and replacing by alanine at this site likely change the local conformation of cofactor-binding pocket.	RESULTS
+43	53	DNA MTases	protein_type	G197 is a conserved residue throughout the DNA MTases, and replacing by alanine at this site likely change the local conformation of cofactor-binding pocket.	RESULTS
+59	68	replacing	experimental_method	G197 is a conserved residue throughout the DNA MTases, and replacing by alanine at this site likely change the local conformation of cofactor-binding pocket.	RESULTS
+72	79	alanine	residue_name	G197 is a conserved residue throughout the DNA MTases, and replacing by alanine at this site likely change the local conformation of cofactor-binding pocket.	RESULTS
+133	156	cofactor-binding pocket	site	G197 is a conserved residue throughout the DNA MTases, and replacing by alanine at this site likely change the local conformation of cofactor-binding pocket.	RESULTS
+0	11	Mutagenesis	experimental_method	Mutagenesis on this glycine residue in M.EcoKI or M.EcoP15I also abolished the AdoMet-binding activity.	RESULTS
+20	27	glycine	residue_name	Mutagenesis on this glycine residue in M.EcoKI or M.EcoP15I also abolished the AdoMet-binding activity.	RESULTS
+39	46	M.EcoKI	protein	Mutagenesis on this glycine residue in M.EcoKI or M.EcoP15I also abolished the AdoMet-binding activity.	RESULTS
+50	59	M.EcoP15I	protein	Mutagenesis on this glycine residue in M.EcoKI or M.EcoP15I also abolished the AdoMet-binding activity.	RESULTS
+79	85	AdoMet	chemical	Mutagenesis on this glycine residue in M.EcoKI or M.EcoP15I also abolished the AdoMet-binding activity.	RESULTS
+9	25	mutational study	experimental_method	Although mutational study could not tell the role of F195 in ligand binding due to the insolubility of the F195A mutant, structural analysis suggested the importance of this residue in AdoMet-binding.	RESULTS
+53	57	F195	residue_name_number	Although mutational study could not tell the role of F195 in ligand binding due to the insolubility of the F195A mutant, structural analysis suggested the importance of this residue in AdoMet-binding.	RESULTS
+107	112	F195A	mutant	Although mutational study could not tell the role of F195 in ligand binding due to the insolubility of the F195A mutant, structural analysis suggested the importance of this residue in AdoMet-binding.	RESULTS
+113	119	mutant	protein_state	Although mutational study could not tell the role of F195 in ligand binding due to the insolubility of the F195A mutant, structural analysis suggested the importance of this residue in AdoMet-binding.	RESULTS
+121	140	structural analysis	experimental_method	Although mutational study could not tell the role of F195 in ligand binding due to the insolubility of the F195A mutant, structural analysis suggested the importance of this residue in AdoMet-binding.	RESULTS
+185	191	AdoMet	chemical	Although mutational study could not tell the role of F195 in ligand binding due to the insolubility of the F195A mutant, structural analysis suggested the importance of this residue in AdoMet-binding.	RESULTS
+19	23	F195	residue_name_number	The phenyl ring of F195 forms a perpendicular π-stacking interaction with the purine ring of AdoMet, which stabilizes the orientation of AdoMet bound in the pocket of M1.HpyAVI (Figure S2C).	RESULTS
+46	68	π-stacking interaction	bond_interaction	The phenyl ring of F195 forms a perpendicular π-stacking interaction with the purine ring of AdoMet, which stabilizes the orientation of AdoMet bound in the pocket of M1.HpyAVI (Figure S2C).	RESULTS
+93	99	AdoMet	chemical	The phenyl ring of F195 forms a perpendicular π-stacking interaction with the purine ring of AdoMet, which stabilizes the orientation of AdoMet bound in the pocket of M1.HpyAVI (Figure S2C).	RESULTS
+137	143	AdoMet	chemical	The phenyl ring of F195 forms a perpendicular π-stacking interaction with the purine ring of AdoMet, which stabilizes the orientation of AdoMet bound in the pocket of M1.HpyAVI (Figure S2C).	RESULTS
+144	152	bound in	protein_state	The phenyl ring of F195 forms a perpendicular π-stacking interaction with the purine ring of AdoMet, which stabilizes the orientation of AdoMet bound in the pocket of M1.HpyAVI (Figure S2C).	RESULTS
+157	163	pocket	site	The phenyl ring of F195 forms a perpendicular π-stacking interaction with the purine ring of AdoMet, which stabilizes the orientation of AdoMet bound in the pocket of M1.HpyAVI (Figure S2C).	RESULTS
+167	176	M1.HpyAVI	protein	The phenyl ring of F195 forms a perpendicular π-stacking interaction with the purine ring of AdoMet, which stabilizes the orientation of AdoMet bound in the pocket of M1.HpyAVI (Figure S2C).	RESULTS
+24	35	mutagenesis	experimental_method	In a separate scenario, mutagenesis of this residue in M.EcoRV has been proven to play an important role in AdoMet binding.	RESULTS
+55	62	M.EcoRV	protein	In a separate scenario, mutagenesis of this residue in M.EcoRV has been proven to play an important role in AdoMet binding.	RESULTS
+108	114	AdoMet	chemical	In a separate scenario, mutagenesis of this residue in M.EcoRV has been proven to play an important role in AdoMet binding.	RESULTS
+10	27	DNA-binding sites	site	Potential DNA-binding sites	RESULTS
+13	31	DNA binding region	site	The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58).	RESULTS
+35	44	M1.HpyAVI	protein	The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58).	RESULTS
+58	70	hairpin loop	structure_element	The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58).	RESULTS
+80	87	101-133	residue_range	The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58).	RESULTS
+94	97	TRD	structure_element	The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58).	RESULTS
+108	115	136-166	residue_range	The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58).	RESULTS
+124	139	highly flexible	protein_state	The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58).	RESULTS
+140	144	loop	structure_element	The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58).	RESULTS
+155	160	33-58	residue_range	The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58).	RESULTS
+4	16	hairpin loop	structure_element	The hairpin loop between β6 and β7 strands that carries a conserved HRRY sequence signature in the middle is proposed to insert into the minor groove of the bound DNA.	RESULTS
+25	27	β6	structure_element	The hairpin loop between β6 and β7 strands that carries a conserved HRRY sequence signature in the middle is proposed to insert into the minor groove of the bound DNA.	RESULTS
+32	34	β7	structure_element	The hairpin loop between β6 and β7 strands that carries a conserved HRRY sequence signature in the middle is proposed to insert into the minor groove of the bound DNA.	RESULTS
+58	67	conserved	protein_state	The hairpin loop between β6 and β7 strands that carries a conserved HRRY sequence signature in the middle is proposed to insert into the minor groove of the bound DNA.	RESULTS
+68	72	HRRY	structure_element	The hairpin loop between β6 and β7 strands that carries a conserved HRRY sequence signature in the middle is proposed to insert into the minor groove of the bound DNA.	RESULTS
+137	149	minor groove	structure_element	The hairpin loop between β6 and β7 strands that carries a conserved HRRY sequence signature in the middle is proposed to insert into the minor groove of the bound DNA.	RESULTS
+157	162	bound	protein_state	The hairpin loop between β6 and β7 strands that carries a conserved HRRY sequence signature in the middle is proposed to insert into the minor groove of the bound DNA.	RESULTS
+163	166	DNA	chemical	The hairpin loop between β6 and β7 strands that carries a conserved HRRY sequence signature in the middle is proposed to insert into the minor groove of the bound DNA.	RESULTS
+23	26	TRD	structure_element	As aforementioned, the TRD of M1.HpyAVI shows striking difference from the other DNA MTases, and the relaxed specificity of substrate recognition may be at least partially attributable to the disordered TRD.	RESULTS
+30	39	M1.HpyAVI	protein	As aforementioned, the TRD of M1.HpyAVI shows striking difference from the other DNA MTases, and the relaxed specificity of substrate recognition may be at least partially attributable to the disordered TRD.	RESULTS
+81	91	DNA MTases	protein_type	As aforementioned, the TRD of M1.HpyAVI shows striking difference from the other DNA MTases, and the relaxed specificity of substrate recognition may be at least partially attributable to the disordered TRD.	RESULTS
+192	202	disordered	protein_state	As aforementioned, the TRD of M1.HpyAVI shows striking difference from the other DNA MTases, and the relaxed specificity of substrate recognition may be at least partially attributable to the disordered TRD.	RESULTS
+203	206	TRD	structure_element	As aforementioned, the TRD of M1.HpyAVI shows striking difference from the other DNA MTases, and the relaxed specificity of substrate recognition may be at least partially attributable to the disordered TRD.	RESULTS
+17	32	highly flexible	protein_state	In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either.	RESULTS
+33	37	loop	structure_element	In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either.	RESULTS
+64	68	DPPY	structure_element	In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either.	RESULTS
+78	87	M1.HpyAVI	protein	In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either.	RESULTS
+110	126	electron density	evidence	In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either.	RESULTS
+159	164	loops	structure_element	In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either.	RESULTS
+172	184	AdoMet-bound	protein_state	In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either.	RESULTS
+185	195	structures	evidence	In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either.	RESULTS
+199	206	M.PvuII	protein	In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either.	RESULTS
+208	212	DpnM	protein	In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either.	RESULTS
+216	222	M.TaqI	protein	In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either.	RESULTS
+5	9	loop	structure_element	This loop, however, was largely stabilized upon DNA binding, as observed in the protein-DNA complex structures of M.TaqI (PDB ID 2IBS), M.HhaI (PDB ID 1MHT) and M.HaeIII (PDB ID 1DCT).	RESULTS
+48	51	DNA	chemical	This loop, however, was largely stabilized upon DNA binding, as observed in the protein-DNA complex structures of M.TaqI (PDB ID 2IBS), M.HhaI (PDB ID 1MHT) and M.HaeIII (PDB ID 1DCT).	RESULTS
+80	110	protein-DNA complex structures	evidence	This loop, however, was largely stabilized upon DNA binding, as observed in the protein-DNA complex structures of M.TaqI (PDB ID 2IBS), M.HhaI (PDB ID 1MHT) and M.HaeIII (PDB ID 1DCT).	RESULTS
+114	120	M.TaqI	protein	This loop, however, was largely stabilized upon DNA binding, as observed in the protein-DNA complex structures of M.TaqI (PDB ID 2IBS), M.HhaI (PDB ID 1MHT) and M.HaeIII (PDB ID 1DCT).	RESULTS
+136	142	M.HhaI	protein	This loop, however, was largely stabilized upon DNA binding, as observed in the protein-DNA complex structures of M.TaqI (PDB ID 2IBS), M.HhaI (PDB ID 1MHT) and M.HaeIII (PDB ID 1DCT).	RESULTS
+161	169	M.HaeIII	protein	This loop, however, was largely stabilized upon DNA binding, as observed in the protein-DNA complex structures of M.TaqI (PDB ID 2IBS), M.HhaI (PDB ID 1MHT) and M.HaeIII (PDB ID 1DCT).	RESULTS
+4	16	well-ordered	protein_state	The well-ordered loop in those structures directly contacts the flipping adenine and forms hydrogen bond with neighboring bases.	RESULTS
+17	21	loop	structure_element	The well-ordered loop in those structures directly contacts the flipping adenine and forms hydrogen bond with neighboring bases.	RESULTS
+31	41	structures	evidence	The well-ordered loop in those structures directly contacts the flipping adenine and forms hydrogen bond with neighboring bases.	RESULTS
+73	80	adenine	residue_name	The well-ordered loop in those structures directly contacts the flipping adenine and forms hydrogen bond with neighboring bases.	RESULTS
+91	104	hydrogen bond	bond_interaction	The well-ordered loop in those structures directly contacts the flipping adenine and forms hydrogen bond with neighboring bases.	RESULTS
+50	54	loop	structure_element	These observations implied that the corresponding loop in other MTases, e.g. M1.HpyAVI, is likely responsible for reducing sequence recognition specificity and thus plays crucial roles in catalysis.	RESULTS
+64	70	MTases	protein_type	These observations implied that the corresponding loop in other MTases, e.g. M1.HpyAVI, is likely responsible for reducing sequence recognition specificity and thus plays crucial roles in catalysis.	RESULTS
+77	86	M1.HpyAVI	protein	These observations implied that the corresponding loop in other MTases, e.g. M1.HpyAVI, is likely responsible for reducing sequence recognition specificity and thus plays crucial roles in catalysis.	RESULTS
+33	42	M1.HpyAVI	protein	Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment.	RESULTS
+75	91	N6 adenine MTase	protein_type	Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment.	RESULTS
+115	122	adenine	residue_name	Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment.	RESULTS
+126	136	5′-GAGG-3′	chemical	Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment.	RESULTS
+137	147	5′-GGAG-3′	chemical	Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment.	RESULTS
+160	168	adenines	residue_name	Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment.	RESULTS
+172	182	5′-GAAG-3′	chemical	Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment.	RESULTS
+258	265	adenine	residue_name	Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment.	RESULTS
+269	279	5′-GAGG-3′	chemical	Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment.	RESULTS
+295	304	M1.HpyAVI	protein	Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment.	RESULTS
+339	342	DNA	chemical	Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment.	RESULTS
+384	393	M1.HpyAVI	protein	Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment.	RESULTS
+421	430	H. pylori	species	Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment.	RESULTS
+443	470	multiple sequence alignment	experimental_method	Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment.	RESULTS
+9	28	sequence comparison	experimental_method	Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A).	RESULTS
+33	52	structural analysis	experimental_method	Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A).	RESULTS
+78	81	P41	residue_name_number	Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A).	RESULTS
+83	87	N111	residue_name_number	Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A).	RESULTS
+89	93	K165	residue_name_number	Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A).	RESULTS
+98	102	T166	residue_name_number	Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A).	RESULTS
+121	129	replaced	experimental_method	Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A).	RESULTS
+133	139	serine	residue_name	Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A).	RESULTS
+141	150	threonine	residue_name	Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A).	RESULTS
+152	161	threonine	residue_name	Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A).	RESULTS
+166	172	valine	residue_name	Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A).	RESULTS
+8	37	[3H]AdoMet radiological assay	experimental_method	Then, a [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the wide type protein and the mutants.	RESULTS
+66	72	methyl	chemical	Then, a [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the wide type protein and the mutants.	RESULTS
+98	107	wide type	protein_state	Then, a [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the wide type protein and the mutants.	RESULTS
+124	131	mutants	protein_state	Then, a [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the wide type protein and the mutants.	RESULTS
+41	44	DNA	chemical	As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.	RESULTS
+54	64	5′-GAGG-3′	chemical	As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.	RESULTS
+68	79	5′-GAAG-3′,	chemical	As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.	RESULTS
+88	95	mutants	protein_state	As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.	RESULTS
+129	135	methyl	chemical	As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.	RESULTS
+170	172	wt	protein_state	As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.	RESULTS
+173	182	M1.HpyAVI	protein	As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.	RESULTS
+222	233	5′-GGAG-3′,	chemical	As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.	RESULTS
+238	244	methyl	chemical	As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.	RESULTS
+270	274	P41S	mutant	As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.	RESULTS
+275	281	mutant	protein_state	As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.	RESULTS
+324	333	wild type	protein_state	As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.	RESULTS
+334	343	M1.HpyAVI	protein	As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.	RESULTS
+0	18	Sequence alignment	experimental_method	Sequence alignment, structural analysis and radioactive methyl transfer activity define the key residue for wider substrate specificity of M1.HpyAVI	FIG
+20	39	structural analysis	experimental_method	Sequence alignment, structural analysis and radioactive methyl transfer activity define the key residue for wider substrate specificity of M1.HpyAVI	FIG
+44	80	radioactive methyl transfer activity	experimental_method	Sequence alignment, structural analysis and radioactive methyl transfer activity define the key residue for wider substrate specificity of M1.HpyAVI	FIG
+139	148	M1.HpyAVI	protein	Sequence alignment, structural analysis and radioactive methyl transfer activity define the key residue for wider substrate specificity of M1.HpyAVI	FIG
+3	21	Sequence alignment	experimental_method	A. Sequence alignment of M1.HpyAVI from 50 H. pylori strains including 26695 revealed several variant residues.	FIG
+25	34	M1.HpyAVI	protein	A. Sequence alignment of M1.HpyAVI from 50 H. pylori strains including 26695 revealed several variant residues.	FIG
+43	52	H. pylori	species	A. Sequence alignment of M1.HpyAVI from 50 H. pylori strains including 26695 revealed several variant residues.	FIG
+9	12	P41	residue_name_number	Residues P41, N111, K165 and T166 of M1.HpyAVI from strain 26695 were chosen based on structural analysis and sequence alignment (shown in red arrow).	FIG
+14	18	N111	residue_name_number	Residues P41, N111, K165 and T166 of M1.HpyAVI from strain 26695 were chosen based on structural analysis and sequence alignment (shown in red arrow).	FIG
+20	24	K165	residue_name_number	Residues P41, N111, K165 and T166 of M1.HpyAVI from strain 26695 were chosen based on structural analysis and sequence alignment (shown in red arrow).	FIG
+29	33	T166	residue_name_number	Residues P41, N111, K165 and T166 of M1.HpyAVI from strain 26695 were chosen based on structural analysis and sequence alignment (shown in red arrow).	FIG
+37	46	M1.HpyAVI	protein	Residues P41, N111, K165 and T166 of M1.HpyAVI from strain 26695 were chosen based on structural analysis and sequence alignment (shown in red arrow).	FIG
+59	64	26695	species	Residues P41, N111, K165 and T166 of M1.HpyAVI from strain 26695 were chosen based on structural analysis and sequence alignment (shown in red arrow).	FIG
+86	105	structural analysis	experimental_method	Residues P41, N111, K165 and T166 of M1.HpyAVI from strain 26695 were chosen based on structural analysis and sequence alignment (shown in red arrow).	FIG
+110	128	sequence alignment	experimental_method	Residues P41, N111, K165 and T166 of M1.HpyAVI from strain 26695 were chosen based on structural analysis and sequence alignment (shown in red arrow).	FIG
+42	49	WebLogo	experimental_method	Amino-acid conservation is depicted using WebLogo (Crooks et al, 2004).	FIG
+11	17	Methyl	chemical	B., C., D. Methyl transfer reactions were performed using wt-M1.HpyAVI, M1.HpyAVI-P41S, M1.HpyAVI-N111T, and M1.HpyAVI-K165R T166V, respectively.	FIG
+58	60	wt	protein_state	B., C., D. Methyl transfer reactions were performed using wt-M1.HpyAVI, M1.HpyAVI-P41S, M1.HpyAVI-N111T, and M1.HpyAVI-K165R T166V, respectively.	FIG
+61	70	M1.HpyAVI	protein	B., C., D. Methyl transfer reactions were performed using wt-M1.HpyAVI, M1.HpyAVI-P41S, M1.HpyAVI-N111T, and M1.HpyAVI-K165R T166V, respectively.	FIG
+72	86	M1.HpyAVI-P41S	mutant	B., C., D. Methyl transfer reactions were performed using wt-M1.HpyAVI, M1.HpyAVI-P41S, M1.HpyAVI-N111T, and M1.HpyAVI-K165R T166V, respectively.	FIG
+88	103	M1.HpyAVI-N111T	mutant	B., C., D. Methyl transfer reactions were performed using wt-M1.HpyAVI, M1.HpyAVI-P41S, M1.HpyAVI-N111T, and M1.HpyAVI-K165R T166V, respectively.	FIG
+109	130	M1.HpyAVI-K165R T166V	mutant	B., C., D. Methyl transfer reactions were performed using wt-M1.HpyAVI, M1.HpyAVI-P41S, M1.HpyAVI-N111T, and M1.HpyAVI-K165R T166V, respectively.	FIG
+43	46	DNA	chemical	Radioactivity incorporated into the duplex DNA containing 5′-GAGG-3′, 5′-GAAG-3′ or 5′-GGAG-3′ was quantified by Beckman LS6500 for 10 min.	FIG
+58	68	5′-GAGG-3′	chemical	Radioactivity incorporated into the duplex DNA containing 5′-GAGG-3′, 5′-GAAG-3′ or 5′-GGAG-3′ was quantified by Beckman LS6500 for 10 min.	FIG
+70	80	5′-GAAG-3′	chemical	Radioactivity incorporated into the duplex DNA containing 5′-GAGG-3′, 5′-GAAG-3′ or 5′-GGAG-3′ was quantified by Beckman LS6500 for 10 min.	FIG
+84	94	5′-GGAG-3′	chemical	Radioactivity incorporated into the duplex DNA containing 5′-GAGG-3′, 5′-GAAG-3′ or 5′-GGAG-3′ was quantified by Beckman LS6500 for 10 min.	FIG
+33	36	P41	residue_name_number	Our experimental data identified P41 as a key residue determining the recognition of GGAG of M1.HpyAVI.	RESULTS
+85	89	GGAG	structure_element	Our experimental data identified P41 as a key residue determining the recognition of GGAG of M1.HpyAVI.	RESULTS
+93	102	M1.HpyAVI	protein	Our experimental data identified P41 as a key residue determining the recognition of GGAG of M1.HpyAVI.	RESULTS
+31	46	highly flexible	protein_state	This amino acid locates in the highly flexible loop between residues 33 and 58, which is involved in DNA binding and substrate recognition as shown above.	RESULTS
+47	51	loop	structure_element	This amino acid locates in the highly flexible loop between residues 33 and 58, which is involved in DNA binding and substrate recognition as shown above.	RESULTS
+69	78	33 and 58	residue_range	This amino acid locates in the highly flexible loop between residues 33 and 58, which is involved in DNA binding and substrate recognition as shown above.	RESULTS
+101	104	DNA	chemical	This amino acid locates in the highly flexible loop between residues 33 and 58, which is involved in DNA binding and substrate recognition as shown above.	RESULTS
+0	11	Replacement	experimental_method	Replacement by serine at this position definitely changes the local conformation and hydrophobicity, and probably some structural properties of the whole loop, which may in turn result in reduced specificity for sequence recognition of the enzyme from strain 26695.	RESULTS
+15	21	serine	residue_name	Replacement by serine at this position definitely changes the local conformation and hydrophobicity, and probably some structural properties of the whole loop, which may in turn result in reduced specificity for sequence recognition of the enzyme from strain 26695.	RESULTS
+154	158	loop	structure_element	Replacement by serine at this position definitely changes the local conformation and hydrophobicity, and probably some structural properties of the whole loop, which may in turn result in reduced specificity for sequence recognition of the enzyme from strain 26695.	RESULTS
+259	264	26695	species	Replacement by serine at this position definitely changes the local conformation and hydrophobicity, and probably some structural properties of the whole loop, which may in turn result in reduced specificity for sequence recognition of the enzyme from strain 26695.	RESULTS
+13	22	DNA-bound	protein_state	Although the DNA-bound structure of previous investigation on a γ-class N6-adenine MTase revealed that the target adenine was rotated out of DNA helix, details of the methyl transfer process were still unclear.	DISCUSS
+23	32	structure	evidence	Although the DNA-bound structure of previous investigation on a γ-class N6-adenine MTase revealed that the target adenine was rotated out of DNA helix, details of the methyl transfer process were still unclear.	DISCUSS
+64	88	γ-class N6-adenine MTase	protein_type	Although the DNA-bound structure of previous investigation on a γ-class N6-adenine MTase revealed that the target adenine was rotated out of DNA helix, details of the methyl transfer process were still unclear.	DISCUSS
+114	121	adenine	residue_name	Although the DNA-bound structure of previous investigation on a γ-class N6-adenine MTase revealed that the target adenine was rotated out of DNA helix, details of the methyl transfer process were still unclear.	DISCUSS
+141	144	DNA	chemical	Although the DNA-bound structure of previous investigation on a γ-class N6-adenine MTase revealed that the target adenine was rotated out of DNA helix, details of the methyl transfer process were still unclear.	DISCUSS
+167	173	methyl	chemical	Although the DNA-bound structure of previous investigation on a γ-class N6-adenine MTase revealed that the target adenine was rotated out of DNA helix, details of the methyl transfer process were still unclear.	DISCUSS
+56	72	N6-methyladenine	ptm	Additionally, recent studies reported the importance of N6-methyladenine in some eukaryotic species, but until now there has not been any N6-adenine MTases being identified in eukaryotes.	DISCUSS
+81	91	eukaryotic	taxonomy_domain	Additionally, recent studies reported the importance of N6-methyladenine in some eukaryotic species, but until now there has not been any N6-adenine MTases being identified in eukaryotes.	DISCUSS
+138	155	N6-adenine MTases	protein_type	Additionally, recent studies reported the importance of N6-methyladenine in some eukaryotic species, but until now there has not been any N6-adenine MTases being identified in eukaryotes.	DISCUSS
+176	186	eukaryotes	taxonomy_domain	Additionally, recent studies reported the importance of N6-methyladenine in some eukaryotic species, but until now there has not been any N6-adenine MTases being identified in eukaryotes.	DISCUSS
+0	43	Biochemical and structural characterization	experimental_method	Biochemical and structural characterization of M1.HpyAVI provides a new model for uncovering the methyl transfer mechanism and for investigating the N6-methyladenine in eukaryotes.	DISCUSS
+47	56	M1.HpyAVI	protein	Biochemical and structural characterization of M1.HpyAVI provides a new model for uncovering the methyl transfer mechanism and for investigating the N6-methyladenine in eukaryotes.	DISCUSS
+97	103	methyl	chemical	Biochemical and structural characterization of M1.HpyAVI provides a new model for uncovering the methyl transfer mechanism and for investigating the N6-methyladenine in eukaryotes.	DISCUSS
+149	165	N6-methyladenine	ptm	Biochemical and structural characterization of M1.HpyAVI provides a new model for uncovering the methyl transfer mechanism and for investigating the N6-methyladenine in eukaryotes.	DISCUSS
+169	179	eukaryotes	taxonomy_domain	Biochemical and structural characterization of M1.HpyAVI provides a new model for uncovering the methyl transfer mechanism and for investigating the N6-methyladenine in eukaryotes.	DISCUSS
+20	30	DNA MTases	protein_type	Oligomeric state of DNA MTases was long accepted as monomer, but our study indicated here that M1.HpyAVI exists as a dimer both in crystal and solution.	DISCUSS
+52	59	monomer	oligomeric_state	Oligomeric state of DNA MTases was long accepted as monomer, but our study indicated here that M1.HpyAVI exists as a dimer both in crystal and solution.	DISCUSS
+95	104	M1.HpyAVI	protein	Oligomeric state of DNA MTases was long accepted as monomer, but our study indicated here that M1.HpyAVI exists as a dimer both in crystal and solution.	DISCUSS
+117	122	dimer	oligomeric_state	Oligomeric state of DNA MTases was long accepted as monomer, but our study indicated here that M1.HpyAVI exists as a dimer both in crystal and solution.	DISCUSS
+131	138	crystal	evidence	Oligomeric state of DNA MTases was long accepted as monomer, but our study indicated here that M1.HpyAVI exists as a dimer both in crystal and solution.	DISCUSS
+26	54	β-class DNA exocyclic MTases	protein_type	Interestingly, some other β-class DNA exocyclic MTases showed similar oligomeric state in crystal and in solution, indicating that dimer may be the functional state shared by a subgroup of DNA MTases.	DISCUSS
+90	97	crystal	evidence	Interestingly, some other β-class DNA exocyclic MTases showed similar oligomeric state in crystal and in solution, indicating that dimer may be the functional state shared by a subgroup of DNA MTases.	DISCUSS
+131	136	dimer	oligomeric_state	Interestingly, some other β-class DNA exocyclic MTases showed similar oligomeric state in crystal and in solution, indicating that dimer may be the functional state shared by a subgroup of DNA MTases.	DISCUSS
+189	199	DNA MTases	protein_type	Interestingly, some other β-class DNA exocyclic MTases showed similar oligomeric state in crystal and in solution, indicating that dimer may be the functional state shared by a subgroup of DNA MTases.	DISCUSS
+4	19	highly flexible	protein_state	The highly flexible region (residues 33-58) and TRD (residues 133-163) of M1.HpyAVI are supposed to interact with DNA at minor and major grooves, respectively.	DISCUSS
+37	42	33-58	residue_range	The highly flexible region (residues 33-58) and TRD (residues 133-163) of M1.HpyAVI are supposed to interact with DNA at minor and major grooves, respectively.	DISCUSS
+48	51	TRD	structure_element	The highly flexible region (residues 33-58) and TRD (residues 133-163) of M1.HpyAVI are supposed to interact with DNA at minor and major grooves, respectively.	DISCUSS
+62	69	133-163	residue_range	The highly flexible region (residues 33-58) and TRD (residues 133-163) of M1.HpyAVI are supposed to interact with DNA at minor and major grooves, respectively.	DISCUSS
+74	83	M1.HpyAVI	protein	The highly flexible region (residues 33-58) and TRD (residues 133-163) of M1.HpyAVI are supposed to interact with DNA at minor and major grooves, respectively.	DISCUSS
+114	117	DNA	chemical	The highly flexible region (residues 33-58) and TRD (residues 133-163) of M1.HpyAVI are supposed to interact with DNA at minor and major grooves, respectively.	DISCUSS
+121	144	minor and major grooves	structure_element	The highly flexible region (residues 33-58) and TRD (residues 133-163) of M1.HpyAVI are supposed to interact with DNA at minor and major grooves, respectively.	DISCUSS
+12	15	P41	residue_name_number	And residue P41 might be a key residue partially determining the substrate spectrum of M1.HpyAVI.	DISCUSS
+87	96	M1.HpyAVI	protein	And residue P41 might be a key residue partially determining the substrate spectrum of M1.HpyAVI.	DISCUSS
+4	11	missing	protein_state	The missing loop between residues 33 and 58 may need DNA binding so as to form a stable conformation, which is similar to the condition of M.TaqI. Crystallization of M1.HpyAVI-DNA complex warrants future investigations, with the purpose of revealing the mechanism behind the wider substrate specificity of this enzyme.	DISCUSS
+12	16	loop	structure_element	The missing loop between residues 33 and 58 may need DNA binding so as to form a stable conformation, which is similar to the condition of M.TaqI. Crystallization of M1.HpyAVI-DNA complex warrants future investigations, with the purpose of revealing the mechanism behind the wider substrate specificity of this enzyme.	DISCUSS
+34	43	33 and 58	residue_range	The missing loop between residues 33 and 58 may need DNA binding so as to form a stable conformation, which is similar to the condition of M.TaqI. Crystallization of M1.HpyAVI-DNA complex warrants future investigations, with the purpose of revealing the mechanism behind the wider substrate specificity of this enzyme.	DISCUSS
+53	56	DNA	chemical	The missing loop between residues 33 and 58 may need DNA binding so as to form a stable conformation, which is similar to the condition of M.TaqI. Crystallization of M1.HpyAVI-DNA complex warrants future investigations, with the purpose of revealing the mechanism behind the wider substrate specificity of this enzyme.	DISCUSS
+81	87	stable	protein_state	The missing loop between residues 33 and 58 may need DNA binding so as to form a stable conformation, which is similar to the condition of M.TaqI. Crystallization of M1.HpyAVI-DNA complex warrants future investigations, with the purpose of revealing the mechanism behind the wider substrate specificity of this enzyme.	DISCUSS
+139	145	M.TaqI	protein	The missing loop between residues 33 and 58 may need DNA binding so as to form a stable conformation, which is similar to the condition of M.TaqI. Crystallization of M1.HpyAVI-DNA complex warrants future investigations, with the purpose of revealing the mechanism behind the wider substrate specificity of this enzyme.	DISCUSS
+147	162	Crystallization	experimental_method	The missing loop between residues 33 and 58 may need DNA binding so as to form a stable conformation, which is similar to the condition of M.TaqI. Crystallization of M1.HpyAVI-DNA complex warrants future investigations, with the purpose of revealing the mechanism behind the wider substrate specificity of this enzyme.	DISCUSS
+166	179	M1.HpyAVI-DNA	complex_assembly	The missing loop between residues 33 and 58 may need DNA binding so as to form a stable conformation, which is similar to the condition of M.TaqI. Crystallization of M1.HpyAVI-DNA complex warrants future investigations, with the purpose of revealing the mechanism behind the wider substrate specificity of this enzyme.	DISCUSS
+0	15	DNA methylation	ptm	DNA methylation plays an important role in bacterial pathogenicity.	DISCUSS
+43	52	bacterial	taxonomy_domain	DNA methylation plays an important role in bacterial pathogenicity.	DISCUSS
+0	23	DNA adenine methylation	ptm	DNA adenine methylation was known to regulate the expression of some virulence genes in bacteria including H.pylori.	DISCUSS
+88	96	bacteria	taxonomy_domain	DNA adenine methylation was known to regulate the expression of some virulence genes in bacteria including H.pylori.	DISCUSS
+107	115	H.pylori	species	DNA adenine methylation was known to regulate the expression of some virulence genes in bacteria including H.pylori.	DISCUSS
+14	37	DNA adenine methylation	ptm	Inhibitors of DNA adenine methylation may have a broad antimicrobial action by targeting DNA adenine methyltransferase.	DISCUSS
+89	118	DNA adenine methyltransferase	protein_type	Inhibitors of DNA adenine methylation may have a broad antimicrobial action by targeting DNA adenine methyltransferase.	DISCUSS
+41	56	DNA methylation	ptm	As an important biological modification, DNA methylation directly influences bacterial survival.	DISCUSS
+77	86	bacterial	taxonomy_domain	As an important biological modification, DNA methylation directly influences bacterial survival.	DISCUSS
+0	11	Knockout of	experimental_method	Knockout of M1.HpyAVI largely prevents the growth of H. pylori.	DISCUSS
+12	21	M1.HpyAVI	protein	Knockout of M1.HpyAVI largely prevents the growth of H. pylori.	DISCUSS
+53	62	H. pylori	species	Knockout of M1.HpyAVI largely prevents the growth of H. pylori.	DISCUSS
+13	22	H. pylori	species	Importantly, H. pylori is involved in 90% of all gastric malignancies.	DISCUSS
+83	91	H.pylori	species	Appropriate antibiotic regimens could successfully cure gastric diseases caused by H.pylori infection.	DISCUSS
+24	33	H. pylori	species	However, eradication of H. pylori infection remains a big challenge for the significantly increasing prevalence of its resistance to antibiotics.	DISCUSS
+39	53	adenine MTases	protein_type	The development of new drugs targeting adenine MTases such as M1.HpyAVI offers a new opportunity for inhibition of H. pylori infection.	DISCUSS
+62	71	M1.HpyAVI	protein	The development of new drugs targeting adenine MTases such as M1.HpyAVI offers a new opportunity for inhibition of H. pylori infection.	DISCUSS
+115	124	H. pylori	species	The development of new drugs targeting adenine MTases such as M1.HpyAVI offers a new opportunity for inhibition of H. pylori infection.	DISCUSS
+61	64	D29	residue_name_number	Residues that play crucial roles for catalytic activity like D29 or E216 may influence the H.pylori survival.	DISCUSS
+68	72	E216	residue_name_number	Residues that play crucial roles for catalytic activity like D29 or E216 may influence the H.pylori survival.	DISCUSS
+91	99	H.pylori	species	Residues that play crucial roles for catalytic activity like D29 or E216 may influence the H.pylori survival.	DISCUSS
+32	48	highly conserved	protein_state	Small molecules targeting these highly conserved residues are likely to emerge less drug resistance.	DISCUSS
+16	25	structure	evidence	In summary, the structure of M1.HpyAVI is featured with a disordered TRD and a key residue P41that located in the putative DNA binding region that may associate with the wider substrate specificity.	DISCUSS
+29	38	M1.HpyAVI	protein	In summary, the structure of M1.HpyAVI is featured with a disordered TRD and a key residue P41that located in the putative DNA binding region that may associate with the wider substrate specificity.	DISCUSS
+58	68	disordered	protein_state	In summary, the structure of M1.HpyAVI is featured with a disordered TRD and a key residue P41that located in the putative DNA binding region that may associate with the wider substrate specificity.	DISCUSS
+69	72	TRD	structure_element	In summary, the structure of M1.HpyAVI is featured with a disordered TRD and a key residue P41that located in the putative DNA binding region that may associate with the wider substrate specificity.	DISCUSS
+91	94	P41	residue_name_number	In summary, the structure of M1.HpyAVI is featured with a disordered TRD and a key residue P41that located in the putative DNA binding region that may associate with the wider substrate specificity.	DISCUSS
+123	141	DNA binding region	site	In summary, the structure of M1.HpyAVI is featured with a disordered TRD and a key residue P41that located in the putative DNA binding region that may associate with the wider substrate specificity.	DISCUSS
+9	12	D29	residue_name_number	Residues D29 and E216 were identified to play a crucial role in cofactor binding.	DISCUSS
+17	21	E216	residue_name_number	Residues D29 and E216 were identified to play a crucial role in cofactor binding.	DISCUSS
+13	30	crystal structure	evidence	As the first crystal structure of N6-adenine MTase in H.pylori, this model may shed light on design of new antibiotics to interfere the growth and pathogenesis of H.pylori in human.	DISCUSS
+34	50	N6-adenine MTase	protein_type	As the first crystal structure of N6-adenine MTase in H.pylori, this model may shed light on design of new antibiotics to interfere the growth and pathogenesis of H.pylori in human.	DISCUSS
+54	62	H.pylori	species	As the first crystal structure of N6-adenine MTase in H.pylori, this model may shed light on design of new antibiotics to interfere the growth and pathogenesis of H.pylori in human.	DISCUSS
+163	171	H.pylori	species	As the first crystal structure of N6-adenine MTase in H.pylori, this model may shed light on design of new antibiotics to interfere the growth and pathogenesis of H.pylori in human.	DISCUSS
+175	180	human	species	As the first crystal structure of N6-adenine MTase in H.pylori, this model may shed light on design of new antibiotics to interfere the growth and pathogenesis of H.pylori in human.	DISCUSS
diff --git a/annotation_CSV/PMC5603727.csv b/annotation_CSV/PMC5603727.csv
new file mode 100644
index 0000000000000000000000000000000000000000..45d50c7b4289c4004970ccb429d676077ba6625d
--- /dev/null
+++ b/annotation_CSV/PMC5603727.csv
@@ -0,0 +1,1354 @@
+anno_start	anno_end	anno_text	entity_type	sentence	section
+0	6	Roquin	protein	Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40	TITLE
+34	42	hexaloop	structure_element	Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40	TITLE
+60	66	3′-UTR	structure_element	Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40	TITLE
+70	74	Ox40	protein	Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40	TITLE
+4	23	RNA-binding protein	protein_type	The RNA-binding protein Roquin is required to prevent autoimmunity.	ABSTRACT
+24	30	Roquin	protein	The RNA-binding protein Roquin is required to prevent autoimmunity.	ABSTRACT
+0	6	Roquin	protein	Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40).	ABSTRACT
+99	122	costimulatory receptors	protein_type	Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40).	ABSTRACT
+131	175	tumor necrosis factor receptor superfamily 4	protein	Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40).	ABSTRACT
+177	183	Tnfrs4	protein	Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40).	ABSTRACT
+187	191	Ox40	protein	Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40).	ABSTRACT
+2	28	constitutive decay element	structure_element	A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin.	ABSTRACT
+30	33	CDE	structure_element	A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin.	ABSTRACT
+57	72	triloop hairpin	structure_element	A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin.	ABSTRACT
+114	120	Roquin	protein	A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin.	ABSTRACT
+12	24	SELEX assays	experimental_method	Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE).	ABSTRACT
+45	66	U-rich hexaloop motif	structure_element	Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE).	ABSTRACT
+84	109	alternative decay element	structure_element	Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE).	ABSTRACT
+111	114	ADE	structure_element	Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE).	ABSTRACT
+0	18	Crystal structures	evidence	Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes.	ABSTRACT
+23	26	NMR	experimental_method	Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes.	ABSTRACT
+46	54	Roquin-1	protein	Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes.	ABSTRACT
+55	58	ROQ	structure_element	Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes.	ABSTRACT
+77	86	hexaloops	structure_element	Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes.	ABSTRACT
+94	99	SELEX	experimental_method	Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes.	ABSTRACT
+108	111	ADE	structure_element	Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes.	ABSTRACT
+122	125	ADE	structure_element	Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes.	ABSTRACT
+154	158	Ox40	protein	Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes.	ABSTRACT
+159	165	3′-UTR	structure_element	Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes.	ABSTRACT
+10	13	ADE	structure_element	In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin.	ABSTRACT
+23	26	CDE	structure_element	In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin.	ABSTRACT
+70	74	Ox40	protein	In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin.	ABSTRACT
+78	84	Roquin	protein	In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin.	ABSTRACT
+45	66	hexaloop cis elements	structure_element	Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin.	ABSTRACT
+116	130	messenger RNAs	chemical	Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin.	ABSTRACT
+134	140	Roquin	protein	Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin.	ABSTRACT
+1	7	Roquin	protein	 Roquin is an RNA-binding protein that prevents autoimmunity by limiting expression of receptors such as Ox40.	ABSTRACT
+14	33	RNA-binding protein	protein_type	 Roquin is an RNA-binding protein that prevents autoimmunity by limiting expression of receptors such as Ox40.	ABSTRACT
+105	109	Ox40	protein	 Roquin is an RNA-binding protein that prevents autoimmunity by limiting expression of receptors such as Ox40.	ABSTRACT
+30	33	RNA	chemical	Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques.	ABSTRACT
+34	43	structure	evidence	Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques.	ABSTRACT
+69	94	alternative decay element	structure_element	Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques.	ABSTRACT
+139	145	Roquin	protein	Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques.	ABSTRACT
+152	189	structural and biochemical techniques	experimental_method	Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques.	ABSTRACT
+4	10	Roquin	protein	The Roquin protein is essential in T cells for the prevention of autoimmune disease.	INTRO
+56	64	Roquin-1	protein	This is evident from the so-called sanroque mutation in Roquin-1, a single amino acid exchange from Met199 to Arg that causes the development of systemic lupus erythematosus-like symptoms in homozygous mice.	INTRO
+100	106	Met199	residue_name_number	This is evident from the so-called sanroque mutation in Roquin-1, a single amino acid exchange from Met199 to Arg that causes the development of systemic lupus erythematosus-like symptoms in homozygous mice.	INTRO
+110	113	Arg	residue_name	This is evident from the so-called sanroque mutation in Roquin-1, a single amino acid exchange from Met199 to Arg that causes the development of systemic lupus erythematosus-like symptoms in homozygous mice.	INTRO
+202	206	mice	taxonomy_domain	This is evident from the so-called sanroque mutation in Roquin-1, a single amino acid exchange from Met199 to Arg that causes the development of systemic lupus erythematosus-like symptoms in homozygous mice.	INTRO
+4	9	Rc3h1	gene	The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells.	INTRO
+14	19	Rc3h2	gene	The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells.	INTRO
+40	48	Roquin-1	protein	The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells.	INTRO
+53	61	Roquin-2	protein	The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells.	INTRO
+74	85	vertebrates	taxonomy_domain	The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells.	INTRO
+158	162	mice	taxonomy_domain	The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells.	INTRO
+54	65	deletion of	experimental_method	Consistently, CD4+ and CD8+ T cells with the combined deletion of Roquin-encoding genes are spontaneously activated and CD4+ T-helper cells preferentially differentiate into the Th1, Tfh or Th17 subsets.	INTRO
+66	72	Roquin	protein	Consistently, CD4+ and CD8+ T cells with the combined deletion of Roquin-encoding genes are spontaneously activated and CD4+ T-helper cells preferentially differentiate into the Th1, Tfh or Th17 subsets.	INTRO
+0	8	Roquin-1	protein	Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs).	INTRO
+81	105	co-stimulatory receptors	protein_type	Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs).	INTRO
+114	118	Icos	protein	Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs).	INTRO
+120	124	Ox40	protein	Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs).	INTRO
+129	135	CTLA-4	protein	Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs).	INTRO
+141	150	cytokines	protein_type	Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs).	INTRO
+159	177	interleukin (IL)-6	protein	Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs).	INTRO
+182	204	tumour necrosis factor	protein	Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs).	INTRO
+212	233	transcription factors	protein_type	Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs).	INTRO
+242	246	IRF4	protein	Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs).	INTRO
+248	253	IκBNS	protein	Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs).	INTRO
+258	262	IκBζ	protein	Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs).	INTRO
+26	56	structural and functional data	evidence	We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref).	INTRO
+64	72	Roquin-1	protein	We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref).	INTRO
+73	76	ROQ	structure_element	We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref).	INTRO
+84	92	bound to	protein_state	We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref).	INTRO
+105	131	constitutive decay element	structure_element	We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref).	INTRO
+133	136	CDE	structure_element	We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref).	INTRO
+141	156	short stem loop	structure_element	We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref).	INTRO
+158	160	SL	structure_element	We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref).	INTRO
+192	195	RNA	chemical	We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref).	INTRO
+211	234	3′-untranslated regions	structure_element	We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref).	INTRO
+236	243	3′-UTRs	structure_element	We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref).	INTRO
+269	272	Tnf	protein	We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref).	INTRO
+4	7	ROQ	structure_element	The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA.	INTRO
+25	51	extended winged helix fold	structure_element	The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA.	INTRO
+109	112	RNA	chemical	The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA.	INTRO
+171	174	Tnf	protein	The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA.	INTRO
+175	178	CDE	structure_element	The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA.	INTRO
+179	182	RNA	chemical	The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA.	INTRO
+4	19	structural data	evidence	The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin.	INTRO
+24	43	mutational analysis	experimental_method	The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin.	INTRO
+120	124	loop	structure_element	The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin.	INTRO
+129	133	stem	structure_element	The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin.	INTRO
+141	144	CDE	structure_element	The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin.	INTRO
+184	190	Roquin	protein	The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin.	INTRO
+43	60	crystal structure	evidence	At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain.	INTRO
+124	127	CDE	structure_element	At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain.	INTRO
+133	135	SL	structure_element	At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain.	INTRO
+152	171	second binding site	site	At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain.	INTRO
+178	197	double-stranded RNA	chemical	At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain.	INTRO
+199	204	dsRNA	chemical	At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain.	INTRO
+216	224	extended	protein_state	At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain.	INTRO
+225	228	ROQ	structure_element	At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain.	INTRO
+25	28	CDE	structure_element	The structural basis for CDE recognition by the Roquin-2 ROQ domain has also been recently reported.	INTRO
+48	56	Roquin-2	protein	The structural basis for CDE recognition by the Roquin-2 ROQ domain has also been recently reported.	INTRO
+57	60	ROQ	structure_element	The structural basis for CDE recognition by the Roquin-2 ROQ domain has also been recently reported.	INTRO
+50	58	Roquin-1	protein	We found that the posttranscriptional activity of Roquin-1 and Roquin-2 is regulated through cleavage by the paracaspase MALT1 (refs).	INTRO
+63	71	Roquin-2	protein	We found that the posttranscriptional activity of Roquin-1 and Roquin-2 is regulated through cleavage by the paracaspase MALT1 (refs).	INTRO
+109	120	paracaspase	protein_type	We found that the posttranscriptional activity of Roquin-1 and Roquin-2 is regulated through cleavage by the paracaspase MALT1 (refs).	INTRO
+121	126	MALT1	protein	We found that the posttranscriptional activity of Roquin-1 and Roquin-2 is regulated through cleavage by the paracaspase MALT1 (refs).	INTRO
+9	14	MALT1	protein	Enhanced MALT1-dependent cleavage and inactivation of Roquin, and thus less effective repression of target genes, result from increased strength of antigen recognition in T cells.	INTRO
+54	60	Roquin	protein	Enhanced MALT1-dependent cleavage and inactivation of Roquin, and thus less effective repression of target genes, result from increased strength of antigen recognition in T cells.	INTRO
+215	221	Roquin	protein	These findings suggest that dependent on the strength of cognate antigen recognition differential gene expression and cell fate decisions can be established in naive T cells by a graded cleavage and inactivation of Roquin.	INTRO
+51	67	binding affinity	evidence	In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology.	INTRO
+86	88	SL	structure_element	In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology.	INTRO
+105	112	3′-UTRs	structure_element	In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology.	INTRO
+123	128	mRNAs	chemical	In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology.	INTRO
+200	206	Roquin	protein	In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology.	INTRO
+221	223	SL	structure_element	In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology.	INTRO
+224	227	RNA	chemical	In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology.	INTRO
+262	268	Roquin	protein	In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology.	INTRO
+356	362	Roquin	protein	In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology.	INTRO
+88	91	RNA	chemical	Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain.	INTRO
+123	129	Roquin	protein	Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain.	INTRO
+159	174	U-rich hexaloop	structure_element	Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain.	INTRO
+175	177	SL	structure_element	Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain.	INTRO
+185	191	3′-UTR	structure_element	Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain.	INTRO
+195	199	Ox40	protein	Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain.	INTRO
+200	208	bound to	protein_state	Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain.	INTRO
+213	221	Roquin-1	protein	Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain.	INTRO
+222	225	ROQ	structure_element	Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain.	INTRO
+34	38	Ox40	protein	We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments.	INTRO
+73	76	CDE	structure_element	We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments.	INTRO
+86	94	hexaloop	structure_element	We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments.	INTRO
+95	97	SL	structure_element	We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments.	INTRO
+98	102	RNAs	chemical	We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments.	INTRO
+128	185	Systematic Evolution of Ligands by Exponential Enrichment	experimental_method	We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments.	INTRO
+187	192	SELEX	experimental_method	We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments.	INTRO
+4	26	X-ray crystallographic	experimental_method	Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells.	INTRO
+28	31	NMR	experimental_method	Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells.	INTRO
+33	64	biochemical and functional data	evidence	Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells.	INTRO
+79	98	mutational analysis	experimental_method	Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells.	INTRO
+121	128	triloop	structure_element	Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells.	INTRO
+133	141	hexaloop	structure_element	Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells.	INTRO
+142	144	SL	structure_element	Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells.	INTRO
+145	149	RNAs	chemical	Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells.	INTRO
+191	197	Roquin	protein	Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells.	INTRO
+0	5	SELEX	experimental_method	SELEX identifies novel RNA ligands of Roquin-1	RESULTS
+23	26	RNA	chemical	SELEX identifies novel RNA ligands of Roquin-1	RESULTS
+38	46	Roquin-1	protein	SELEX identifies novel RNA ligands of Roquin-1	RESULTS
+23	35	Roquin-bound	protein_state	We set out to identify Roquin-bound RNA motifs in an unbiased manner by performing SELEX experiments.	RESULTS
+36	39	RNA	chemical	We set out to identify Roquin-bound RNA motifs in an unbiased manner by performing SELEX experiments.	RESULTS
+83	88	SELEX	experimental_method	We set out to identify Roquin-bound RNA motifs in an unbiased manner by performing SELEX experiments.	RESULTS
+2	14	biotinylated	protein_state	A biotinylated amino-terminal protein fragment of Roquin-1 (residues 2–440) was used to enrich RNAs from a library containing 47 random nucleotides over three sequential selection rounds.	RESULTS
+50	58	Roquin-1	protein	A biotinylated amino-terminal protein fragment of Roquin-1 (residues 2–440) was used to enrich RNAs from a library containing 47 random nucleotides over three sequential selection rounds.	RESULTS
+69	74	2–440	residue_range	A biotinylated amino-terminal protein fragment of Roquin-1 (residues 2–440) was used to enrich RNAs from a library containing 47 random nucleotides over three sequential selection rounds.	RESULTS
+95	99	RNAs	chemical	A biotinylated amino-terminal protein fragment of Roquin-1 (residues 2–440) was used to enrich RNAs from a library containing 47 random nucleotides over three sequential selection rounds.	RESULTS
+0	26	Next-generation sequencing	experimental_method	Next-generation sequencing (NGS) of the RNA before and after each selection round revealed that the starting pool represented about 99.6% unique reads in ∼4.2 × 106 sequences.	RESULTS
+28	31	NGS	experimental_method	Next-generation sequencing (NGS) of the RNA before and after each selection round revealed that the starting pool represented about 99.6% unique reads in ∼4.2 × 106 sequences.	RESULTS
+40	43	RNA	chemical	Next-generation sequencing (NGS) of the RNA before and after each selection round revealed that the starting pool represented about 99.6% unique reads in ∼4.2 × 106 sequences.	RESULTS
+0	22	Bioinformatic analysis	experimental_method	Bioinformatic analysis of NGS data sets derived from the starting pool and enriched selection rounds revealed that the complexity was reduced to 78.6% unique reads in 3.7 × 106 sequences that were analysed after 3 rounds of selection and enrichment.	RESULTS
+26	29	NGS	experimental_method	Bioinformatic analysis of NGS data sets derived from the starting pool and enriched selection rounds revealed that the complexity was reduced to 78.6% unique reads in 3.7 × 106 sequences that were analysed after 3 rounds of selection and enrichment.	RESULTS
+4	7	NGS	experimental_method	For NGS data analysis, the COMPAS software (AptaIT, Munich, Germany) was applied.	RESULTS
+9	33	sequences were clustered	experimental_method	Enriched sequences were clustered into so-called patterns with highly homologous sequences.	RESULTS
+24	46	co-occurrence approach	experimental_method	Based on this so-called co-occurrence approach, patterns on the basis of frequent motifs were generated and were searched for prominent hexamer sequences (Supplementary Fig. 1a).	RESULTS
+14	27	5′-CGTTTT-3′,	chemical	We identified 5′-CGTTTT-3′, 5′-GCGTTT-3′, 5′-TGCGTT-3′ and 5′-GTTTTA-3′ motifs that were also reconfirmed in an independent experiment (Supplementary Fig. 1a) and are located within highly similar sequences (Fig. 1a and Supplementary Fig. 1b).	RESULTS
+28	40	5′-GCGTTT-3′	chemical	We identified 5′-CGTTTT-3′, 5′-GCGTTT-3′, 5′-TGCGTT-3′ and 5′-GTTTTA-3′ motifs that were also reconfirmed in an independent experiment (Supplementary Fig. 1a) and are located within highly similar sequences (Fig. 1a and Supplementary Fig. 1b).	RESULTS
+42	54	5′-TGCGTT-3′	chemical	We identified 5′-CGTTTT-3′, 5′-GCGTTT-3′, 5′-TGCGTT-3′ and 5′-GTTTTA-3′ motifs that were also reconfirmed in an independent experiment (Supplementary Fig. 1a) and are located within highly similar sequences (Fig. 1a and Supplementary Fig. 1b).	RESULTS
+59	71	5′-GTTTTA-3′	chemical	We identified 5′-CGTTTT-3′, 5′-GCGTTT-3′, 5′-TGCGTT-3′ and 5′-GTTTTA-3′ motifs that were also reconfirmed in an independent experiment (Supplementary Fig. 1a) and are located within highly similar sequences (Fig. 1a and Supplementary Fig. 1b).	RESULTS
+51	68	sanroque mutation	mutant	Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b).	RESULTS
+85	88	RNA	chemical	Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b).	RESULTS
+100	106	Roquin	protein	Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b).	RESULTS
+149	154	SELEX	experimental_method	Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b).	RESULTS
+188	196	Roquin-1	protein	Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b).	RESULTS
+221	226	M199R	mutant	Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b).	RESULTS
+13	18	SELEX	experimental_method	Notably, our SELEX approach did not reveal the previously identified CDE sequence.	RESULTS
+69	72	CDE	structure_element	Notably, our SELEX approach did not reveal the previously identified CDE sequence.	RESULTS
+54	57	CDE	structure_element	We assume that the region of sequence identity in the CDE is too short for our sequence clustering algorithm.	RESULTS
+79	108	sequence clustering algorithm	experimental_method	We assume that the region of sequence identity in the CDE is too short for our sequence clustering algorithm.	RESULTS
+45	50	SELEX	experimental_method	Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5–8 nt stem, with one base in the stem not being paired (Supplementary Fig. 1c).	RESULTS
+86	88	SL	structure_element	Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5–8 nt stem, with one base in the stem not being paired (Supplementary Fig. 1c).	RESULTS
+134	138	loop	structure_element	Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5–8 nt stem, with one base in the stem not being paired (Supplementary Fig. 1c).	RESULTS
+160	164	stem	structure_element	Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5–8 nt stem, with one base in the stem not being paired (Supplementary Fig. 1c).	RESULTS
+187	191	stem	structure_element	Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5–8 nt stem, with one base in the stem not being paired (Supplementary Fig. 1c).	RESULTS
+14	21	3′-UTRs	structure_element	Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b).	RESULTS
+31	37	Roquin	protein	Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b).	RESULTS
+65	83	5′-TGCGTTTTAGGA-3′	chemical	Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b).	RESULTS
+97	126	Motif-based sequence analysis	experimental_method	Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b).	RESULTS
+128	132	MEME	experimental_method	Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b).	RESULTS
+195	203	hexaloop	structure_element	Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b).	RESULTS
+221	227	3′-UTR	structure_element	Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b).	RESULTS
+231	235	Ox40	protein	Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b).	RESULTS
+57	64	3′-UTRs	structure_element	Importantly, this motif is present across species in the 3′-UTRs of respective mRNAs and showed highest conservation in the loop and the upper stem sequences with a drop of conservation towards the boundaries of the motif (Fig. 1c,d).	RESULTS
+79	84	mRNAs	chemical	Importantly, this motif is present across species in the 3′-UTRs of respective mRNAs and showed highest conservation in the loop and the upper stem sequences with a drop of conservation towards the boundaries of the motif (Fig. 1c,d).	RESULTS
+124	128	loop	structure_element	Importantly, this motif is present across species in the 3′-UTRs of respective mRNAs and showed highest conservation in the loop and the upper stem sequences with a drop of conservation towards the boundaries of the motif (Fig. 1c,d).	RESULTS
+143	147	stem	structure_element	Importantly, this motif is present across species in the 3′-UTRs of respective mRNAs and showed highest conservation in the loop and the upper stem sequences with a drop of conservation towards the boundaries of the motif (Fig. 1c,d).	RESULTS
+14	16	SL	structure_element	The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA.	RESULTS
+35	40	SELEX	experimental_method	The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA.	RESULTS
+84	109	alternative decay element	structure_element	The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA.	RESULTS
+110	112	SL	structure_element	The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA.	RESULTS
+114	117	ADE	structure_element	The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA.	RESULTS
+118	120	SL	structure_element	The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA.	RESULTS
+127	130	ADE	structure_element	The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA.	RESULTS
+136	138	SL	structure_element	The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA.	RESULTS
+168	171	CDE	structure_element	The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA.	RESULTS
+177	179	SL	structure_element	The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA.	RESULTS
+187	193	3′-UTR	structure_element	The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA.	RESULTS
+197	201	Ox40	protein	The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA.	RESULTS
+202	206	mRNA	chemical	The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA.	RESULTS
+5	8	CDE	structure_element	This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d).	RESULTS
+14	16	SL	structure_element	This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d).	RESULTS
+78	81	CDE	structure_element	This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d).	RESULTS
+91	97	3′-UTR	structure_element	This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d).	RESULTS
+101	104	Tnf	protein	This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d).	RESULTS
+105	109	mRNA	chemical	This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d).	RESULTS
+111	114	CDE	structure_element	This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d).	RESULTS
+115	117	SL	structure_element	This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d).	RESULTS
+0	3	NMR	experimental_method	NMR analysis of Roquin-bound SL RNAs	RESULTS
+16	28	Roquin-bound	protein_state	NMR analysis of Roquin-bound SL RNAs	RESULTS
+29	31	SL	structure_element	NMR analysis of Roquin-bound SL RNAs	RESULTS
+32	36	RNAs	chemical	NMR analysis of Roquin-bound SL RNAs	RESULTS
+8	11	NMR	experimental_method	We used NMR to analyse the secondary structure of Roquin-1-binding motifs derived from SELEX.	RESULTS
+50	73	Roquin-1-binding motifs	structure_element	We used NMR to analyse the secondary structure of Roquin-1-binding motifs derived from SELEX.	RESULTS
+87	92	SELEX	experimental_method	We used NMR to analyse the secondary structure of Roquin-1-binding motifs derived from SELEX.	RESULTS
+0	74	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy	experimental_method	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+76	81	NOESY	experimental_method	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+83	86	NMR	experimental_method	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+87	94	spectra	evidence	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+102	106	free	protein_state	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+107	110	RNA	chemical	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+120	128	bound to	protein_state	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+133	141	Roquin-1	protein	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+142	145	ROQ	structure_element	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+175	178	ADE	structure_element	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+179	181	SL	structure_element	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+187	190	ADE	structure_element	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+196	198	SL	structure_element	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+206	212	3′-UTR	structure_element	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+216	220	Ox40	protein	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+251	255	Ox40	protein	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+256	259	CDE	structure_element	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+265	267	SL	structure_element	Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).	RESULTS
+4	7	NMR	experimental_method	The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs.	RESULTS
+20	24	free	protein_state	The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs.	RESULTS
+25	29	RNAs	chemical	The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs.	RESULTS
+79	91	stem regions	structure_element	The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs.	RESULTS
+99	116	hexa- and triloop	structure_element	The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs.	RESULTS
+117	119	SL	structure_element	The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs.	RESULTS
+177	181	RNAs	chemical	The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs.	RESULTS
+62	65	G15	residue_name_number	Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson–Crick G–G base pair at this position (Fig. 2a).	RESULTS
+75	77	G6	residue_name_number	Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson–Crick G–G base pair at this position (Fig. 2a).	RESULTS
+86	89	ADE	structure_element	Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson–Crick G–G base pair at this position (Fig. 2a).	RESULTS
+90	92	SL	structure_element	Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson–Crick G–G base pair at this position (Fig. 2a).	RESULTS
+107	137	non-Watson–Crick G–G base pair	bond_interaction	Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson–Crick G–G base pair at this position (Fig. 2a).	RESULTS
+12	40	chemical shift perturbations	evidence	Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red).	RESULTS
+42	46	CSPs	evidence	Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red).	RESULTS
+104	107	ROQ	structure_element	Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red).	RESULTS
+156	159	RNA	chemical	Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red).	RESULTS
+195	198	ROQ	structure_element	Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red).	RESULTS
+213	224	stem region	structure_element	Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red).	RESULTS
+232	235	RNA	chemical	Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red).	RESULTS
+53	76	Watson–Crick base pairs	bond_interaction	No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic.	RESULTS
+98	101	ADE	structure_element	No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic.	RESULTS
+102	104	SL	structure_element	No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic.	RESULTS
+113	117	Ox40	protein	No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic.	RESULTS
+118	121	ADE	structure_element	No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic.	RESULTS
+127	129	SL	structure_element	No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic.	RESULTS
+130	134	RNAs	chemical	No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic.	RESULTS
+155	156	A	residue_name	No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic.	RESULTS
+157	158	U	residue_name	No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic.	RESULTS
+182	187	bulge	structure_element	No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic.	RESULTS
+195	199	Ox40	protein	No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic.	RESULTS
+200	203	ADE	structure_element	No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic.	RESULTS
+209	211	SL	structure_element	No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic.	RESULTS
+212	215	RNA	chemical	No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic.	RESULTS
+58	62	Ox40	protein	In contrast, all expected base pairs are observed for the Ox40 CDE-like SL RNA (Fig. 2c; see also Supplementary Notes).	RESULTS
+63	66	CDE	structure_element	In contrast, all expected base pairs are observed for the Ox40 CDE-like SL RNA (Fig. 2c; see also Supplementary Notes).	RESULTS
+72	74	SL	structure_element	In contrast, all expected base pairs are observed for the Ox40 CDE-like SL RNA (Fig. 2c; see also Supplementary Notes).	RESULTS
+75	78	RNA	chemical	In contrast, all expected base pairs are observed for the Ox40 CDE-like SL RNA (Fig. 2c; see also Supplementary Notes).	RESULTS
+0	10	Structures	evidence	Structures of ROQ bound to ADE SL RNAs	RESULTS
+14	17	ROQ	structure_element	Structures of ROQ bound to ADE SL RNAs	RESULTS
+18	26	bound to	protein_state	Structures of ROQ bound to ADE SL RNAs	RESULTS
+27	30	ADE	structure_element	Structures of ROQ bound to ADE SL RNAs	RESULTS
+31	33	SL	structure_element	Structures of ROQ bound to ADE SL RNAs	RESULTS
+34	38	RNAs	chemical	Structures of ROQ bound to ADE SL RNAs	RESULTS
+17	23	Roquin	protein	To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements.	RESULTS
+48	50	SL	structure_element	To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements.	RESULTS
+78	83	SELEX	experimental_method	To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements.	RESULTS
+97	103	solved	experimental_method	To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements.	RESULTS
+104	122	crystal structures	evidence	To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements.	RESULTS
+130	138	Roquin-1	protein	To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements.	RESULTS
+139	142	ROQ	structure_element	To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements.	RESULTS
+150	158	bound to	protein_state	To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements.	RESULTS
+179	182	RNA	chemical	To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements.	RESULTS
+4	14	structures	evidence	The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively.	RESULTS
+18	21	ROQ	structure_element	The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively.	RESULTS
+22	30	bound to	protein_state	The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively.	RESULTS
+42	45	ADE	structure_element	The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively.	RESULTS
+46	48	SL	structure_element	The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively.	RESULTS
+91	95	Ox40	protein	The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively.	RESULTS
+96	99	ADE	structure_element	The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively.	RESULTS
+105	107	SL	structure_element	The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively.	RESULTS
+108	112	RNAs	chemical	The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively.	RESULTS
+8	18	structures	evidence	In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b).	RESULTS
+23	26	RNA	chemical	In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b).	RESULTS
+37	39	SL	structure_element	In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b).	RESULTS
+56	64	hexaloop	structure_element	In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b).	RESULTS
+123	126	ROQ	structure_element	In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b).	RESULTS
+127	132	helix	structure_element	In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b).	RESULTS
+133	135	α4	structure_element	In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b).	RESULTS
+163	165	β3	structure_element	In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b).	RESULTS
+4	9	dsRNA	chemical	The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e).	RESULTS
+10	14	stem	structure_element	The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e).	RESULTS
+76	79	Tnf	protein	The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e).	RESULTS
+80	83	CDE	structure_element	The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e).	RESULTS
+84	86	SL	structure_element	The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e).	RESULTS
+87	90	RNA	chemical	The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e).	RESULTS
+43	51	hexaloop	structure_element	As may be expected, the recognition of the hexaloop is significantly different from the triloop in the CDE RNA (Fig. 3b,c and Supplementary Fig. 2b).	RESULTS
+88	95	triloop	structure_element	As may be expected, the recognition of the hexaloop is significantly different from the triloop in the CDE RNA (Fig. 3b,c and Supplementary Fig. 2b).	RESULTS
+103	106	CDE	structure_element	As may be expected, the recognition of the hexaloop is significantly different from the triloop in the CDE RNA (Fig. 3b,c and Supplementary Fig. 2b).	RESULTS
+107	110	RNA	chemical	As may be expected, the recognition of the hexaloop is significantly different from the triloop in the CDE RNA (Fig. 3b,c and Supplementary Fig. 2b).	RESULTS
+45	48	ADE	structure_element	Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e).	RESULTS
+49	51	SL	structure_element	Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e).	RESULTS
+56	59	ADE	structure_element	Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e).	RESULTS
+65	67	SL	structure_element	Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e).	RESULTS
+68	72	RNAs	chemical	Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e).	RESULTS
+100	110	structures	evidence	Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e).	RESULTS
+123	126	RNA	chemical	Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e).	RESULTS
+27	30	C19	residue_name_number	The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g).	RESULTS
+31	36	bulge	structure_element	The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g).	RESULTS
+42	58	non-Watson–Crick	bond_interaction	The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g).	RESULTS
+59	61	G6	residue_name_number	The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g).	RESULTS
+62	65	G15	residue_name_number	The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g).	RESULTS
+66	75	base pair	bond_interaction	The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g).	RESULTS
+99	101	U1	residue_name_number	The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g).	RESULTS
+107	113	Trp184	residue_name_number	The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g).	RESULTS
+118	124	Phe194	residue_name_number	The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g).	RESULTS
+132	135	ADE	structure_element	The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g).	RESULTS
+141	143	SL	structure_element	The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g).	RESULTS
+144	147	RNA	chemical	The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g).	RESULTS
+82	91	structure	evidence	Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution.	RESULTS
+99	103	Ox40	protein	Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution.	RESULTS
+104	107	ADE	structure_element	Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution.	RESULTS
+113	115	SL	structure_element	Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution.	RESULTS
+116	119	RNA	chemical	Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution.	RESULTS
+151	155	Ox40	protein	Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution.	RESULTS
+156	162	3′-UTR	structure_element	Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution.	RESULTS
+47	67	double-stranded stem	structure_element	The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop.	RESULTS
+75	79	Ox40	protein	The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop.	RESULTS
+80	83	ADE	structure_element	The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop.	RESULTS
+89	91	SL	structure_element	The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop.	RESULTS
+110	113	CDE	structure_element	The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop.	RESULTS
+114	121	triloop	structure_element	The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop.	RESULTS
+13	28	U-rich hexaloop	structure_element	Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts.	RESULTS
+36	40	Ox40	protein	Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts.	RESULTS
+41	44	ADE	structure_element	Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts.	RESULTS
+50	52	SL	structure_element	Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts.	RESULTS
+53	56	RNA	chemical	Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts.	RESULTS
+78	85	surface	site	Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts.	RESULTS
+93	96	ROQ	structure_element	Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts.	RESULTS
+135	138	CDE	structure_element	Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts.	RESULTS
+139	146	triloop	structure_element	Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts.	RESULTS
+37	43	Phe255	residue_name_number	For example, the main chain atoms of Phe255 form two hydrogen bonds with the Watson–Crick face of the U11 base (Fig. 3d).	RESULTS
+53	67	hydrogen bonds	bond_interaction	For example, the main chain atoms of Phe255 form two hydrogen bonds with the Watson–Crick face of the U11 base (Fig. 3d).	RESULTS
+102	105	U11	residue_name_number	For example, the main chain atoms of Phe255 form two hydrogen bonds with the Watson–Crick face of the U11 base (Fig. 3d).	RESULTS
+16	25	structure	evidence	Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e).	RESULTS
+33	36	Tnf	protein	Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e).	RESULTS
+37	40	CDE	structure_element	Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e).	RESULTS
+41	48	triloop	structure_element	Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e).	RESULTS
+53	59	Tyr250	residue_name_number	Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e).	RESULTS
+88	101	hydrogen bond	bond_interaction	Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e).	RESULTS
+128	131	G12	residue_name_number	Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e).	RESULTS
+183	191	hexaloop	structure_element	Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e).	RESULTS
+232	238	Tyr250	residue_name_number	Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e).	RESULTS
+271	274	U11	residue_name_number	Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e).	RESULTS
+341	362	stacking interactions	bond_interaction	Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e).	RESULTS
+372	375	U10	residue_name_number	Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e).	RESULTS
+380	383	U11	residue_name_number	Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e).	RESULTS
+17	23	Tyr250	residue_name_number	In addition, the Tyr250 main-chain carbonyl interacts with U13 imino proton (Fig. 3d,e).	RESULTS
+59	62	U13	residue_name_number	In addition, the Tyr250 main-chain carbonyl interacts with U13 imino proton (Fig. 3d,e).	RESULTS
+0	6	Val257	residue_name_number	Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop.	RESULTS
+11	17	Lys259	residue_name_number	Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop.	RESULTS
+21	27	strand	structure_element	Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop.	RESULTS
+28	30	β3	structure_element	Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop.	RESULTS
+58	61	UGU	structure_element	Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop.	RESULTS
+62	69	triloop	structure_element	Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop.	RESULTS
+77	80	Tnf	protein	Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop.	RESULTS
+81	84	CDE	structure_element	Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop.	RESULTS
+85	88	RNA	chemical	Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop.	RESULTS
+139	147	hexaloop	structure_element	Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop.	RESULTS
+18	24	Lys259	residue_name_number	The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f).	RESULTS
+31	45	hydrogen bonds	bond_interaction	The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f).	RESULTS
+75	78	U10	residue_name_number	The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f).	RESULTS
+83	86	U11	residue_name_number	The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f).	RESULTS
+133	139	Val257	residue_name_number	The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f).	RESULTS
+140	146	stacks	bond_interaction	The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f).	RESULTS
+156	159	U11	residue_name_number	The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f).	RESULTS
+4	7	RNA	chemical	The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA).	RESULTS
+8	12	stem	structure_element	The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA).	RESULTS
+28	50	Watson–Crick base pair	bond_interaction	The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA).	RESULTS
+52	54	C8	residue_name_number	The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA).	RESULTS
+55	58	G15	residue_name_number	The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA).	RESULTS
+66	74	hexaloop	structure_element	The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA).	RESULTS
+75	77	SL	structure_element	The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA).	RESULTS
+78	81	RNA	chemical	The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA).	RESULTS
+19	21	G9	residue_name_number	Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b).	RESULTS
+27	33	stacks	bond_interaction	Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b).	RESULTS
+131	134	G12	residue_name_number	Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b).	RESULTS
+142	145	CDE	structure_element	Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b).	RESULTS
+146	153	triloop	structure_element	Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b).	RESULTS
+4	6	G9	residue_name_number	The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex.	RESULTS
+43	46	A14	residue_name_number	The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex.	RESULTS
+62	65	A14	residue_name_number	The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex.	RESULTS
+86	98	minor groove	site	The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex.	RESULTS
+106	109	RNA	chemical	The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex.	RESULTS
+38	58	stacking interaction	bond_interaction	This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e).	RESULTS
+62	64	G9	residue_name_number	This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e).	RESULTS
+66	69	U10	residue_name_number	This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e).	RESULTS
+74	80	Tyr250	residue_name_number	This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e).	RESULTS
+88	91	ROQ	structure_element	This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e).	RESULTS
+121	124	RNA	chemical	This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e).	RESULTS
+125	129	stem	structure_element	This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e).	RESULTS
+4	7	U11	residue_name_number	The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f).	RESULTS
+12	15	U13	residue_name_number	The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f).	RESULTS
+22	27	stack	bond_interaction	The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f).	RESULTS
+67	70	ROQ	structure_element	The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f).	RESULTS
+78	82	wing	structure_element	The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f).	RESULTS
+38	41	C12	residue_name_number	This is possible by exposing the base C12 of the Ox-40 ADE-like SL towards the solvent, which accordingly does not show any contacts to the protein.	RESULTS
+49	54	Ox-40	protein	This is possible by exposing the base C12 of the Ox-40 ADE-like SL towards the solvent, which accordingly does not show any contacts to the protein.	RESULTS
+55	58	ADE	structure_element	This is possible by exposing the base C12 of the Ox-40 ADE-like SL towards the solvent, which accordingly does not show any contacts to the protein.	RESULTS
+64	66	SL	structure_element	This is possible by exposing the base C12 of the Ox-40 ADE-like SL towards the solvent, which accordingly does not show any contacts to the protein.	RESULTS
+27	30	CDE	structure_element	In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts.	RESULTS
+31	33	SL	structure_element	In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts.	RESULTS
+44	47	ADE	structure_element	In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts.	RESULTS
+48	50	SL	structure_element	In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts.	RESULTS
+55	58	ADE	structure_element	In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts.	RESULTS
+64	66	SL	structure_element	In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts.	RESULTS
+67	71	RNAs	chemical	In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts.	RESULTS
+58	61	ROQ	structure_element	However, these involve an extended binding surface on the ROQ domain with a number of additional residues compared with the triloop RNA.	RESULTS
+132	135	RNA	chemical	However, these involve an extended binding surface on the ROQ domain with a number of additional residues compared with the triloop RNA.	RESULTS
+0	3	NMR	experimental_method	NMR analysis of ROQ interactions with ADE SLs	RESULTS
+16	19	ROQ	structure_element	NMR analysis of ROQ interactions with ADE SLs	RESULTS
+38	41	ADE	structure_element	NMR analysis of ROQ interactions with ADE SLs	RESULTS
+42	45	SLs	structure_element	NMR analysis of ROQ interactions with ADE SLs	RESULTS
+13	29	NMR spectroscopy	experimental_method	We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution.	RESULTS
+45	48	ROQ	structure_element	We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution.	RESULTS
+71	74	ADE	structure_element	We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution.	RESULTS
+84	87	CDE	structure_element	We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution.	RESULTS
+93	95	SL	structure_element	We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution.	RESULTS
+96	100	RNAs	chemical	We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution.	RESULTS
+0	4	CSPs	evidence	CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b).	RESULTS
+32	35	ROQ	structure_element	CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b).	RESULTS
+61	65	Ox40	protein	CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b).	RESULTS
+66	69	ADE	structure_element	CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b).	RESULTS
+75	77	SL	structure_element	CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b).	RESULTS
+78	81	RNA	chemical	CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b).	RESULTS
+150	153	CDE	structure_element	CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b).	RESULTS
+154	157	SLs	structure_element	CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b).	RESULTS
+167	173	Lys220	residue_name_number	CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b).	RESULTS
+175	181	Lys239	residue_name_number	CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b).	RESULTS
+182	188	Thr240	residue_name_number	CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b).	RESULTS
+193	199	Lys259	residue_name_number	CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b).	RESULTS
+200	206	Arg260	residue_name_number	CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b).	RESULTS
+63	80	crystal structure	evidence	This is fully consistent with the interactions observed in the crystal structure (Supplementary Fig. 2c–e) and indicates a similar binding surface.	RESULTS
+131	146	binding surface	site	This is fully consistent with the interactions observed in the crystal structure (Supplementary Fig. 2c–e) and indicates a similar binding surface.	RESULTS
+32	47	CSP differences	evidence	However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).	RESULTS
+78	81	ROQ	structure_element	However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).	RESULTS
+92	96	Ox40	protein	However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).	RESULTS
+97	100	ADE	structure_element	However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).	RESULTS
+106	108	SL	structure_element	However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).	RESULTS
+109	113	RNAs	chemical	However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).	RESULTS
+125	128	CDE	structure_element	However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).	RESULTS
+134	136	SL	structure_element	However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).	RESULTS
+137	140	RNA	chemical	However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).	RESULTS
+148	152	Ox40	protein	However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).	RESULTS
+153	159	3′-UTR	structure_element	However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).	RESULTS
+181	184	Tnf	protein	However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).	RESULTS
+185	188	CDE	structure_element	However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).	RESULTS
+189	191	SL	structure_element	However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).	RESULTS
+192	195	RNA	chemical	However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).	RESULTS
+13	19	Ser253	residue_name_number	For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d).	RESULTS
+64	68	Ox40	protein	For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d).	RESULTS
+69	72	ADE	structure_element	For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d).	RESULTS
+78	80	SL	structure_element	For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d).	RESULTS
+134	142	hexaloop	structure_element	For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d).	RESULTS
+33	36	ROQ	structure_element	On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs.	RESULTS
+61	64	ADE	structure_element	On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs.	RESULTS
+78	81	ADE	structure_element	On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs.	RESULTS
+87	89	SL	structure_element	On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs.	RESULTS
+90	94	RNAs	chemical	On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs.	RESULTS
+122	125	NMR	experimental_method	On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs.	RESULTS
+126	133	spectra	evidence	On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs.	RESULTS
+138	142	CSPs	evidence	On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs.	RESULTS
+73	76	RNA	chemical	This is consistent with the very similar structural features and mode of RNA recognition of the ROQ domain with these RNAs (Supplementary Fig. 2a,d,e).	RESULTS
+96	99	ROQ	structure_element	This is consistent with the very similar structural features and mode of RNA recognition of the ROQ domain with these RNAs (Supplementary Fig. 2a,d,e).	RESULTS
+118	122	RNAs	chemical	This is consistent with the very similar structural features and mode of RNA recognition of the ROQ domain with these RNAs (Supplementary Fig. 2a,d,e).	RESULTS
+0	19	Mutational analysis	experimental_method	Mutational analysis of the ROQ-ADE interaction	RESULTS
+27	30	ROQ	structure_element	Mutational analysis of the ROQ-ADE interaction	RESULTS
+31	34	ADE	structure_element	Mutational analysis of the ROQ-ADE interaction	RESULTS
+43	46	ROQ	structure_element	To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4).	RESULTS
+105	142	electrophoretic mobility shift assays	experimental_method	To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4).	RESULTS
+144	149	EMSAs	experimental_method	To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4).	RESULTS
+172	175	ROQ	structure_element	To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4).	RESULTS
+191	195	Ox40	protein	To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4).	RESULTS
+196	199	ADE	structure_element	To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4).	RESULTS
+205	208	RNA	chemical	To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4).	RESULTS
+33	42	wild-type	protein_state	Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2).	RESULTS
+43	46	ROQ	structure_element	Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2).	RESULTS
+68	76	affinity	evidence	Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2).	RESULTS
+107	110	Tnf	protein	Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2).	RESULTS
+111	114	CDE	structure_element	Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2).	RESULTS
+110	127	crystal structure	evidence	We next tested a set of mutants (Supplementary Fig. 4), which were designed based on contacts observed in the crystal structure (Fig. 3) and the NMR CSPs (Fig. 4a,b).	RESULTS
+145	148	NMR	experimental_method	We next tested a set of mutants (Supplementary Fig. 4), which were designed based on contacts observed in the crystal structure (Fig. 3) and the NMR CSPs (Fig. 4a,b).	RESULTS
+149	153	CSPs	evidence	We next tested a set of mutants (Supplementary Fig. 4), which were designed based on contacts observed in the crystal structure (Fig. 3) and the NMR CSPs (Fig. 4a,b).	RESULTS
+31	42	ROQ-Tnf CDE	complex_assembly	In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine.	RESULTS
+109	128	structural analysis	experimental_method	In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine.	RESULTS
+147	153	Lys220	residue_name_number	In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine.	RESULTS
+155	161	Lys239	residue_name_number	In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine.	RESULTS
+163	169	Lys259	residue_name_number	In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine.	RESULTS
+174	180	Arg260	residue_name_number	In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine.	RESULTS
+222	233	replacement	experimental_method	In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine.	RESULTS
+237	244	alanine	residue_name	In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine.	RESULTS
+58	63	Y250A	mutant	We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a).	RESULTS
+64	70	mutant	protein_state	We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a).	RESULTS
+78	86	hexaloop	structure_element	We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a).	RESULTS
+87	89	SL	structure_element	We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a).	RESULTS
+90	93	RNA	chemical	We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a).	RESULTS
+127	130	Tnf	protein	We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a).	RESULTS
+131	134	CDE	structure_element	We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a).	RESULTS
+36	42	Tyr250	residue_name_number	This underlines the central role of Tyr250 for stabilization of the hexaloop structure and recognition by stacking interactions (Fig. 3b,e).	RESULTS
+68	76	hexaloop	structure_element	This underlines the central role of Tyr250 for stabilization of the hexaloop structure and recognition by stacking interactions (Fig. 3b,e).	RESULTS
+106	127	stacking interactions	bond_interaction	This underlines the central role of Tyr250 for stabilization of the hexaloop structure and recognition by stacking interactions (Fig. 3b,e).	RESULTS
+0	8	Mutation	experimental_method	Mutation of Ser253, which shows large CSPs in the NMR titrations (Fig. 4a,b), does not significantly impair complex formation (Supplementary Fig. 4).	RESULTS
+12	18	Ser253	residue_name_number	Mutation of Ser253, which shows large CSPs in the NMR titrations (Fig. 4a,b), does not significantly impair complex formation (Supplementary Fig. 4).	RESULTS
+38	42	CSPs	evidence	Mutation of Ser253, which shows large CSPs in the NMR titrations (Fig. 4a,b), does not significantly impair complex formation (Supplementary Fig. 4).	RESULTS
+50	64	NMR titrations	experimental_method	Mutation of Ser253, which shows large CSPs in the NMR titrations (Fig. 4a,b), does not significantly impair complex formation (Supplementary Fig. 4).	RESULTS
+10	31	chemical shift change	evidence	The large chemical shift change is probably caused by ring current effects induced by the close proximity of the U11 and U13 bases.	RESULTS
+113	116	U11	residue_name_number	The large chemical shift change is probably caused by ring current effects induced by the close proximity of the U11 and U13 bases.	RESULTS
+121	124	U13	residue_name_number	The large chemical shift change is probably caused by ring current effects induced by the close proximity of the U11 and U13 bases.	RESULTS
+11	17	mutant	protein_state	Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4).	RESULTS
+25	29	wing	structure_element	Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4).	RESULTS
+37	40	ROQ	structure_element	Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4).	RESULTS
+49	54	S265Y	mutant	Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4).	RESULTS
+152	155	Tnf	protein	Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4).	RESULTS
+156	159	CDE	structure_element	Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4).	RESULTS
+20	31	replacement	experimental_method	This indicates that replacement by Tyr does not strongly affect the RNA interaction, and that some conformational variations are tolerated.	RESULTS
+35	38	Tyr	residue_name	This indicates that replacement by Tyr does not strongly affect the RNA interaction, and that some conformational variations are tolerated.	RESULTS
+68	71	RNA	chemical	This indicates that replacement by Tyr does not strongly affect the RNA interaction, and that some conformational variations are tolerated.	RESULTS
+10	29	mutational analysis	experimental_method	Thus, the mutational analysis is fully consistent with the recognition of the hexaloop observed in our crystal structures.	RESULTS
+78	86	hexaloop	structure_element	Thus, the mutational analysis is fully consistent with the recognition of the hexaloop observed in our crystal structures.	RESULTS
+103	121	crystal structures	evidence	Thus, the mutational analysis is fully consistent with the recognition of the hexaloop observed in our crystal structures.	RESULTS
+45	51	Tyr250	residue_name_number	To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells.	RESULTS
+55	63	Roquin-1	protein	To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells.	RESULTS
+67	71	Ox40	protein	To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells.	RESULTS
+72	76	mRNA	chemical	To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells.	RESULTS
+117	149	retroviral reconstitution system	experimental_method	To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells.	RESULTS
+153	159	Roquin	protein	To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells.	RESULTS
+27	32	Rc3h1	gene	Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion.	RESULTS
+33	36	2fl	gene	Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion.	RESULTS
+37	39	fl	gene	Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion.	RESULTS
+60	64	mice	taxonomy_domain	Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion.	RESULTS
+83	91	Roquin-1	protein	Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion.	RESULTS
+92	93	2	protein	Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion.	RESULTS
+114	123	tamoxifen	chemical	Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion.	RESULTS
+158	204	reverse tetracycline-controlled transactivator	protein_type	Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion.	RESULTS
+205	209	rtTA	protein	Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion.	RESULTS
+237	256	4-hydroxy tamoxifen	chemical	Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion.	RESULTS
+36	47	doxycycline	chemical	The cells were then transduced with doxycycline-inducible retroviral vectors to reconstitute Roquin-1 expression (Fig. 5b).	RESULTS
+93	101	Roquin-1	protein	The cells were then transduced with doxycycline-inducible retroviral vectors to reconstitute Roquin-1 expression (Fig. 5b).	RESULTS
+13	19	Roquin	protein	Depletion of Roquin proteins on tamoxifen treatment (Supplementary Fig. 5a) strongly increased surface expression of Ox40 and Icos (Fig. 5c).	RESULTS
+32	41	tamoxifen	chemical	Depletion of Roquin proteins on tamoxifen treatment (Supplementary Fig. 5a) strongly increased surface expression of Ox40 and Icos (Fig. 5c).	RESULTS
+117	121	Ox40	protein	Depletion of Roquin proteins on tamoxifen treatment (Supplementary Fig. 5a) strongly increased surface expression of Ox40 and Icos (Fig. 5c).	RESULTS
+126	130	Icos	protein	Depletion of Roquin proteins on tamoxifen treatment (Supplementary Fig. 5a) strongly increased surface expression of Ox40 and Icos (Fig. 5c).	RESULTS
+44	67	costimulatory receptors	protein_type	This increase in surface expression of both costimulatory receptors was partially corrected by the doxycycline-induced reconstitution with Roquin-1 WT protein (Fig. 5c left panels).	RESULTS
+99	110	doxycycline	chemical	This increase in surface expression of both costimulatory receptors was partially corrected by the doxycycline-induced reconstitution with Roquin-1 WT protein (Fig. 5c left panels).	RESULTS
+139	147	Roquin-1	protein	This increase in surface expression of both costimulatory receptors was partially corrected by the doxycycline-induced reconstitution with Roquin-1 WT protein (Fig. 5c left panels).	RESULTS
+148	150	WT	protein_state	This increase in surface expression of both costimulatory receptors was partially corrected by the doxycycline-induced reconstitution with Roquin-1 WT protein (Fig. 5c left panels).	RESULTS
+57	62	Y250A	mutant	Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels).	RESULTS
+63	69	mutant	protein_state	Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels).	RESULTS
+73	81	Roquin-1	protein	Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels).	RESULTS
+89	94	K220A	mutant	Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels).	RESULTS
+96	101	K239A	mutant	Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels).	RESULTS
+106	110	R260	mutant	Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels).	RESULTS
+111	117	mutant	protein_state	Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels).	RESULTS
+149	152	CDE	structure_element	Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels).	RESULTS
+153	155	SL	structure_element	Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels).	RESULTS
+45	59	overexpression	experimental_method	However, it is also possible that continuous overexpression of targets following Roquin deletion induces a hyperactivated state in the T cells.	RESULTS
+81	87	Roquin	protein	However, it is also possible that continuous overexpression of targets following Roquin deletion induces a hyperactivated state in the T cells.	RESULTS
+127	131	Icos	protein	This hyperactivation, compared with the actual posttranscriptional derepression, may contribute even stronger to the increased Icos and Ox40 expression levels.	RESULTS
+136	140	Ox40	protein	This hyperactivation, compared with the actual posttranscriptional derepression, may contribute even stronger to the increased Icos and Ox40 expression levels.	RESULTS
+11	38	structure–function analyses	experimental_method	Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos.	RESULTS
+66	70	Y250	residue_name_number	Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos.	RESULTS
+96	102	Roquin	protein	Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos.	RESULTS
+133	137	Ox40	protein	Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos.	RESULTS
+170	176	Roquin	protein	Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos.	RESULTS
+193	197	Icos	protein	Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos.	RESULTS
+63	67	Ox40	protein	We also investigated the role of individual nucleotides in the Ox40 ADE-like SL for complex formation with the ROQ domain.	RESULTS
+68	71	ADE	structure_element	We also investigated the role of individual nucleotides in the Ox40 ADE-like SL for complex formation with the ROQ domain.	RESULTS
+77	79	SL	structure_element	We also investigated the role of individual nucleotides in the Ox40 ADE-like SL for complex formation with the ROQ domain.	RESULTS
+111	114	ROQ	structure_element	We also investigated the role of individual nucleotides in the Ox40 ADE-like SL for complex formation with the ROQ domain.	RESULTS
+141	161	co-crystal structure	evidence	We designed four mutants (Mut1–4, see Supplementary Fig. 6) that were expected to disrupt key interactions with the protein according to our co-crystal structure (Fig. 3d–f and Supplementary Fig. 2).	RESULTS
+0	3	NMR	experimental_method	NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6).	RESULTS
+32	38	mutant	protein_state	NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6).	RESULTS
+39	43	RNAs	chemical	NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6).	RESULTS
+78	89	stem region	structure_element	NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6).	RESULTS
+108	117	wild-type	protein_state	NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6).	RESULTS
+118	121	ADE	structure_element	NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6).	RESULTS
+127	129	SL	structure_element	NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6).	RESULTS
+13	38	surface plasmon resonance	experimental_method	We next used surface plasmon resonance experiments to determine dissociation constants for the ROQ-RNA interaction (Table 2 and Supplementary Fig. 7).	RESULTS
+64	86	dissociation constants	evidence	We next used surface plasmon resonance experiments to determine dissociation constants for the ROQ-RNA interaction (Table 2 and Supplementary Fig. 7).	RESULTS
+95	98	ROQ	structure_element	We next used surface plasmon resonance experiments to determine dissociation constants for the ROQ-RNA interaction (Table 2 and Supplementary Fig. 7).	RESULTS
+99	102	RNA	chemical	We next used surface plasmon resonance experiments to determine dissociation constants for the ROQ-RNA interaction (Table 2 and Supplementary Fig. 7).	RESULTS
+13	24	replacement	experimental_method	Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance.	RESULTS
+30	32	C8	residue_name_number	Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance.	RESULTS
+33	36	G15	residue_name_number	Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance.	RESULTS
+58	59	A	residue_name	Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance.	RESULTS
+60	61	U	residue_name	Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance.	RESULTS
+63	68	Mut 4	mutant	Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance.	RESULTS
+87	95	affinity	evidence	Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance.	RESULTS
+120	124	loop	structure_element	Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance.	RESULTS
+137	141	A14C	mutant	Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance.	RESULTS
+142	148	mutant	protein_state	Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance.	RESULTS
+150	155	Mut 1	mutant	Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance.	RESULTS
+182	190	affinity	evidence	Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance.	RESULTS
+222	247	surface plasmon resonance	experimental_method	Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance.	RESULTS
+26	31	Mut 1	mutant	As intended, the mutation Mut 1 allows the formation of an additional base pair and thus leads to the formation of a tetraloop with a new G-C closing base pair (Supplementary Fig. 6a).	RESULTS
+117	126	tetraloop	structure_element	As intended, the mutation Mut 1 allows the formation of an additional base pair and thus leads to the formation of a tetraloop with a new G-C closing base pair (Supplementary Fig. 6a).	RESULTS
+138	139	G	residue_name	As intended, the mutation Mut 1 allows the formation of an additional base pair and thus leads to the formation of a tetraloop with a new G-C closing base pair (Supplementary Fig. 6a).	RESULTS
+140	141	C	residue_name	As intended, the mutation Mut 1 allows the formation of an additional base pair and thus leads to the formation of a tetraloop with a new G-C closing base pair (Supplementary Fig. 6a).	RESULTS
+20	39	structural analysis	experimental_method	Consistent with the structural analysis, we assume that this variant alters the hexaloop conformation and thus reduces the interaction with ROQ.	RESULTS
+80	88	hexaloop	structure_element	Consistent with the structural analysis, we assume that this variant alters the hexaloop conformation and thus reduces the interaction with ROQ.	RESULTS
+140	143	ROQ	structure_element	Consistent with the structural analysis, we assume that this variant alters the hexaloop conformation and thus reduces the interaction with ROQ.	RESULTS
+14	35	stacking interactions	bond_interaction	Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7).	RESULTS
+44	47	G15	residue_name_number	Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7).	RESULTS
+49	51	G9	residue_name_number	Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7).	RESULTS
+56	60	Y250	residue_name_number	Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7).	RESULTS
+68	71	G9C	mutant	Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7).	RESULTS
+72	78	mutant	protein_state	Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7).	RESULTS
+80	85	Mut 2	mutant	Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7).	RESULTS
+119	122	ROQ	structure_element	Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7).	RESULTS
+130	132	SL	structure_element	Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7).	RESULTS
+133	136	RNA	chemical	Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7).	RESULTS
+36	44	U11AU13G	mutant	No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d).	RESULTS
+45	58	double mutant	protein_state	No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d).	RESULTS
+60	65	Mut 3	mutant	No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d).	RESULTS
+153	156	U11	residue_name_number	No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d).	RESULTS
+161	164	U13	residue_name_number	No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d).	RESULTS
+172	180	hexaloop	structure_element	No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d).	RESULTS
+186	189	ROQ	structure_element	No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d).	RESULTS
+20	25	SELEX	experimental_method	Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ.	RESULTS
+65	74	mutations	experimental_method	Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ.	RESULTS
+78	87	conserved	protein_state	Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ.	RESULTS
+88	99	nucleotides	chemical	Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ.	RESULTS
+107	111	loop	structure_element	Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ.	RESULTS
+151	154	ROQ	structure_element	Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ.	RESULTS
+19	27	affinity	evidence	Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7).	RESULTS
+35	44	wild-type	protein_state	Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7).	RESULTS
+45	48	Tnf	protein	Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7).	RESULTS
+49	52	CDE	structure_element	Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7).	RESULTS
+61	65	Ox40	protein	Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7).	RESULTS
+66	69	ADE	structure_element	Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7).	RESULTS
+75	78	SLs	structure_element	Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7).	RESULTS
+82	85	ROQ	structure_element	Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7).	RESULTS
+0	6	Roquin	protein	Roquin binding to different SLs in the Ox40 3′-UTR	RESULTS
+28	31	SLs	structure_element	Roquin binding to different SLs in the Ox40 3′-UTR	RESULTS
+39	43	Ox40	protein	Roquin binding to different SLs in the Ox40 3′-UTR	RESULTS
+44	50	3′-UTR	structure_element	Roquin binding to different SLs in the Ox40 3′-UTR	RESULTS
+28	36	Roquin-1	protein	We have recently shown that Roquin-1 binds to a CDE-like motif in the 3′-UTR of Ox40 mRNA (Figs 1d and 4c).	RESULTS
+48	51	CDE	structure_element	We have recently shown that Roquin-1 binds to a CDE-like motif in the 3′-UTR of Ox40 mRNA (Figs 1d and 4c).	RESULTS
+70	76	3′-UTR	structure_element	We have recently shown that Roquin-1 binds to a CDE-like motif in the 3′-UTR of Ox40 mRNA (Figs 1d and 4c).	RESULTS
+80	84	Ox40	protein	We have recently shown that Roquin-1 binds to a CDE-like motif in the 3′-UTR of Ox40 mRNA (Figs 1d and 4c).	RESULTS
+85	89	mRNA	chemical	We have recently shown that Roquin-1 binds to a CDE-like motif in the 3′-UTR of Ox40 mRNA (Figs 1d and 4c).	RESULTS
+60	63	CDE	structure_element	We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR.	RESULTS
+77	80	ADE	structure_element	We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR.	RESULTS
+86	88	SL	structure_element	We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR.	RESULTS
+89	93	RNAs	chemical	We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR.	RESULTS
+113	121	Roquin-1	protein	We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR.	RESULTS
+152	163	full-length	protein_state	We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR.	RESULTS
+164	168	Ox40	protein	We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR.	RESULTS
+169	175	3′-UTR	structure_element	We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR.	RESULTS
+4	22	binding affinities	evidence	The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2).	RESULTS
+47	64	N-terminal domain	structure_element	The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2).	RESULTS
+68	76	Roquin-1	protein	The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2).	RESULTS
+87	92	2–440	residue_range	The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2).	RESULTS
+127	130	ROQ	structure_element	The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2).	RESULTS
+138	143	alone	protein_state	The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2).	RESULTS
+4	26	dissociation constants	evidence	The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2).	RESULTS
+35	38	ROQ	structure_element	The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2).	RESULTS
+60	64	Ox40	protein	The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2).	RESULTS
+65	68	CDE	structure_element	The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2).	RESULTS
+74	76	SL	structure_element	The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2).	RESULTS
+85	88	ADE	structure_element	The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2).	RESULTS
+94	96	SL	structure_element	The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2).	RESULTS
+97	101	RNAs	chemical	The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2).	RESULTS
+37	54	binding interface	site	This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR.	RESULTS
+101	109	hexaloop	structure_element	This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR.	RESULTS
+154	162	hexaloop	structure_element	This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR.	RESULTS
+163	165	SL	structure_element	This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR.	RESULTS
+166	169	RNA	chemical	This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR.	RESULTS
+177	181	Ox40	protein	This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR.	RESULTS
+182	188	3′-UTR	structure_element	This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR.	RESULTS
+38	44	3′-UTR	structure_element	We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a).	RESULTS
+48	65	point mutagenesis	experimental_method	We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a).	RESULTS
+97	108	stem region	structure_element	We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a).	RESULTS
+142	144	SL	structure_element	We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a).	RESULTS
+145	148	RNA	chemical	We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a).	RESULTS
+161	168	mutated	experimental_method	We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a).	RESULTS
+179	187	Roquin-1	protein	We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a).	RESULTS
+6	10	RNAs	chemical	These RNAs were then tested in EMSAs with the Roquin-1 N terminus (residues 2–440) (Fig. 6b).	RESULTS
+31	36	EMSAs	experimental_method	These RNAs were then tested in EMSAs with the Roquin-1 N terminus (residues 2–440) (Fig. 6b).	RESULTS
+46	54	Roquin-1	protein	These RNAs were then tested in EMSAs with the Roquin-1 N terminus (residues 2–440) (Fig. 6b).	RESULTS
+76	81	2–440	residue_range	These RNAs were then tested in EMSAs with the Roquin-1 N terminus (residues 2–440) (Fig. 6b).	RESULTS
+0	16	Gel shift assays	experimental_method	Gel shift assays show that binding to the wild-type 3′-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively.	RESULTS
+42	51	wild-type	protein_state	Gel shift assays show that binding to the wild-type 3′-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively.	RESULTS
+52	58	3′-UTR	structure_element	Gel shift assays show that binding to the wild-type 3′-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively.	RESULTS
+108	118	titrations	experimental_method	Gel shift assays show that binding to the wild-type 3′-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively.	RESULTS
+165	168	RNA	chemical	Gel shift assays show that binding to the wild-type 3′-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively.	RESULTS
+126	129	SLs	structure_element	Consistent with this, both bands are strongly reduced when mutations are introduced that interfere with the formation of both SLs.	RESULTS
+82	84	SL	structure_element	Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL.	RESULTS
+85	88	RNA	chemical	Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL.	RESULTS
+117	123	Roquin	protein	Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL.	RESULTS
+178	187	wild-type	protein_state	Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL.	RESULTS
+188	190	SL	structure_element	Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL.	RESULTS
+22	28	Roquin	protein	We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR.	RESULTS
+53	55	SL	structure_element	We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR.	RESULTS
+56	59	RNA	chemical	We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR.	RESULTS
+89	100	full-length	protein_state	We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR.	RESULTS
+101	105	Ox40	protein	We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR.	RESULTS
+106	112	3′-UTR	structure_element	We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR.	RESULTS
+14	18	Ox40	protein	Regulation of Ox40 expression via two motifs in its 3′-UTR	RESULTS
+52	58	3′-UTR	structure_element	Regulation of Ox40 expression via two motifs in its 3′-UTR	RESULTS
+35	38	ADE	structure_element	To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells.	RESULTS
+60	64	mRNA	chemical	To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells.	RESULTS
+80	90	introduced	experimental_method	To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells.	RESULTS
+91	95	Ox40	protein	To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells.	RESULTS
+96	100	mRNA	chemical	To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells.	RESULTS
+121	128	altered	protein_state	To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells.	RESULTS
+129	136	3′-UTRs	structure_element	To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells.	RESULTS
+39	42	ADE	structure_element	Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c).	RESULTS
+52	55	CDE	structure_element	Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c).	RESULTS
+61	63	SL	structure_element	Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c).	RESULTS
+64	68	RNAs	chemical	Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c).	RESULTS
+76	82	3′-UTR	structure_element	Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c).	RESULTS
+117	123	Roquin	protein	Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c).	RESULTS
+167	171	Ox40	protein	Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c).	RESULTS
+256	259	RNA	chemical	Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c).	RESULTS
+272	302	deletion and point mutagenesis	experimental_method	Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c).	RESULTS
+303	313	abrogating	protein_state	Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c).	RESULTS
+314	326	base pairing	bond_interaction	Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c).	RESULTS
+334	345	stem region	structure_element	Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c).	RESULTS
+20	32	retroviruses	taxonomy_domain	Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts.	RESULTS
+47	51	Ox40	protein	Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts.	RESULTS
+112	119	3′-UTRs	structure_element	Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts.	RESULTS
+125	133	Roquin-1	protein	Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts.	RESULTS
+134	135	2	protein	Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts.	RESULTS
+146	151	mouse	taxonomy_domain	Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts.	RESULTS
+0	11	Doxycycline	chemical	Doxycycline treatment of cells from this cell line enabled ectopic Roquin-1 and co-translational mCherry expression due to the stable integration of an inducible lentiviral vector (Supplementary Fig. 8c).	RESULTS
+67	75	Roquin-1	protein	Doxycycline treatment of cells from this cell line enabled ectopic Roquin-1 and co-translational mCherry expression due to the stable integration of an inducible lentiviral vector (Supplementary Fig. 8c).	RESULTS
+162	172	lentiviral	taxonomy_domain	Doxycycline treatment of cells from this cell line enabled ectopic Roquin-1 and co-translational mCherry expression due to the stable integration of an inducible lentiviral vector (Supplementary Fig. 8c).	RESULTS
+18	22	Ox40	protein	The expression of Ox40 in cells with and without doxycycline treatment was then quantified by flow cytometry (Supplementary Fig. 8c).	RESULTS
+49	60	doxycycline	chemical	The expression of Ox40 in cells with and without doxycycline treatment was then quantified by flow cytometry (Supplementary Fig. 8c).	RESULTS
+94	108	flow cytometry	experimental_method	The expression of Ox40 in cells with and without doxycycline treatment was then quantified by flow cytometry (Supplementary Fig. 8c).	RESULTS
+23	27	Ox40	protein	Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c).	RESULTS
+28	57	mean fluorescence intensities	evidence	Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c).	RESULTS
+84	95	doxycycline	chemical	Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c).	RESULTS
+157	161	Ox40	protein	Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c).	RESULTS
+173	180	without	protein_state	Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c).	RESULTS
+181	187	3′-UTR	structure_element	Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c).	RESULTS
+14	43	deletion or point mutagenesis	experimental_method	In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40.	RESULTS
+104	110	3′-UTR	structure_element	In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40.	RESULTS
+111	115	1–80	residue_range	In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40.	RESULTS
+120	130	double mut	protein_state	In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40.	RESULTS
+144	150	Roquin	protein	In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40.	RESULTS
+175	179	Ox40	protein	In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40.	RESULTS
+24	33	mutations	experimental_method	In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c).	RESULTS
+48	56	hexaloop	structure_element	In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c).	RESULTS
+58	64	3′-UTR	structure_element	In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c).	RESULTS
+65	70	1–120	residue_range	In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c).	RESULTS
+74	81	CDE mut	mutant	In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c).	RESULTS
+90	93	CDE	structure_element	In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c).	RESULTS
+99	106	triloop	structure_element	In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c).	RESULTS
+107	113	intact	protein_state	In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c).	RESULTS
+128	134	Roquin	protein	In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c).	RESULTS
+214	225	full-length	protein_state	In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c).	RESULTS
+226	232	3′-UTR	structure_element	In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c).	RESULTS
+50	56	Roquin	protein	To further analyse the functional consequences of Roquin binding to the 3′-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d).	RESULTS
+72	78	3′-UTR	structure_element	To further analyse the functional consequences of Roquin binding to the 3′-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d).	RESULTS
+97	113	mRNA decay rates	evidence	To further analyse the functional consequences of Roquin binding to the 3′-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d).	RESULTS
+146	150	Ox40	protein	To further analyse the functional consequences of Roquin binding to the 3′-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d).	RESULTS
+278	289	doxycycline	chemical	To further analyse the functional consequences of Roquin binding to the 3′-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d).	RESULTS
+0	38	Quantitative reverse transcriptase–PCR	experimental_method	Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min).	RESULTS
+78	82	Ox40	protein	Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min).	RESULTS
+83	87	mRNA	chemical	Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min).	RESULTS
+91	102	deletion of	experimental_method	Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min).	RESULTS
+107	113	3′-UTR	structure_element	Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min).	RESULTS
+115	118	CDS	structure_element	Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min).	RESULTS
+119	123	t1/2	evidence	Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min).	RESULTS
+135	146	full-length	protein_state	Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min).	RESULTS
+147	151	t1/2	evidence	Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min).	RESULTS
+43	60	combined mutation	experimental_method	A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min).	RESULTS
+68	71	CDE	structure_element	A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min).	RESULTS
+85	88	ADE	structure_element	A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min).	RESULTS
+94	97	SLs	structure_element	A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min).	RESULTS
+99	102	ADE	structure_element	A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min).	RESULTS
+103	106	CDE	structure_element	A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min).	RESULTS
+112	115	mut	protein_state	A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min).	RESULTS
+116	120	t1/2	evidence	A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min).	RESULTS
+11	20	mutations	experimental_method	Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively.	RESULTS
+35	38	ADE	structure_element	Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively.	RESULTS
+51	54	CDE	structure_element	Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively.	RESULTS
+60	63	SLs	structure_element	Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively.	RESULTS
+93	96	ADE	structure_element	Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively.	RESULTS
+102	105	mut	protein_state	Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively.	RESULTS
+106	110	t1/2	evidence	Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively.	RESULTS
+120	123	CDE	structure_element	Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively.	RESULTS
+129	132	mut	protein_state	Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively.	RESULTS
+133	137	t1/2	evidence	Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively.	RESULTS
+133	137	Ox40	protein	These findings underscore the importance of both structural motifs and reveal that they have an additive effect on the regulation of Ox40 mRNA expression in cells.	RESULTS
+138	142	mRNA	chemical	These findings underscore the importance of both structural motifs and reveal that they have an additive effect on the regulation of Ox40 mRNA expression in cells.	RESULTS
+7	40	structural and functional studies	experimental_method	Recent structural and functional studies have provided first insight into the RNA binding of Roquin.	DISCUSS
+78	81	RNA	chemical	Recent structural and functional studies have provided first insight into the RNA binding of Roquin.	DISCUSS
+93	99	Roquin	protein	Recent structural and functional studies have provided first insight into the RNA binding of Roquin.	DISCUSS
+0	10	Structures	evidence	Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop.	DISCUSS
+14	20	Roquin	protein	Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop.	DISCUSS
+21	29	bound to	protein_state	Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop.	DISCUSS
+30	33	CDE	structure_element	Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop.	DISCUSS
+34	36	SL	structure_element	Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop.	DISCUSS
+37	41	RNAs	chemical	Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop.	DISCUSS
+84	86	SL	structure_element	Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop.	DISCUSS
+87	90	RNA	chemical	Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop.	DISCUSS
+108	114	A-site	site	Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop.	DISCUSS
+122	139	N-terminal region	structure_element	Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop.	DISCUSS
+147	153	Roquin	protein	Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop.	DISCUSS
+221	257	pyrimidine–purine–pyrimidine triloop	structure_element	Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop.	DISCUSS
+21	24	CDE	structure_element	Considering that the CDE RNA recognition is mostly structure specific and not sequence dependent, a wide spectrum of target mRNA might be recognized by Roquin.	DISCUSS
+25	28	RNA	chemical	Considering that the CDE RNA recognition is mostly structure specific and not sequence dependent, a wide spectrum of target mRNA might be recognized by Roquin.	DISCUSS
+124	128	mRNA	chemical	Considering that the CDE RNA recognition is mostly structure specific and not sequence dependent, a wide spectrum of target mRNA might be recognized by Roquin.	DISCUSS
+152	158	Roquin	protein	Considering that the CDE RNA recognition is mostly structure specific and not sequence dependent, a wide spectrum of target mRNA might be recognized by Roquin.	DISCUSS
+18	30	SELEX assays	experimental_method	Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes.	DISCUSS
+51	72	RNA recognition motif	structure_element	Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes.	DISCUSS
+76	84	Roquin-1	protein	Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes.	DISCUSS
+110	114	Ox40	protein	Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes.	DISCUSS
+115	121	3′-UTR	structure_element	Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes.	DISCUSS
+166	173	3′-UTRs	structure_element	Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes.	DISCUSS
+31	36	SELEX	experimental_method	Our experiments show that this SELEX-derived ADE shows functional activity comparable to the previously established CDE motif.	DISCUSS
+45	48	ADE	structure_element	Our experiments show that this SELEX-derived ADE shows functional activity comparable to the previously established CDE motif.	DISCUSS
+116	119	CDE	structure_element	Our experiments show that this SELEX-derived ADE shows functional activity comparable to the previously established CDE motif.	DISCUSS
+4	7	ADE	structure_element	The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop.	DISCUSS
+12	16	Ox40	protein	The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop.	DISCUSS
+17	20	ADE	structure_element	The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop.	DISCUSS
+26	28	SL	structure_element	The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop.	DISCUSS
+29	33	RNAs	chemical	The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop.	DISCUSS
+40	42	SL	structure_element	The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop.	DISCUSS
+56	64	hexaloop	structure_element	The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop.	DISCUSS
+78	85	triloop	structure_element	The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop.	DISCUSS
+43	67	RNA-helical stem regions	structure_element	Notably, the recognition of the respective RNA-helical stem regions by the ROQ domain is identical for the triloop and hexaloop motifs.	DISCUSS
+75	78	ROQ	structure_element	Notably, the recognition of the respective RNA-helical stem regions by the ROQ domain is identical for the triloop and hexaloop motifs.	DISCUSS
+107	114	triloop	structure_element	Notably, the recognition of the respective RNA-helical stem regions by the ROQ domain is identical for the triloop and hexaloop motifs.	DISCUSS
+119	127	hexaloop	structure_element	Notably, the recognition of the respective RNA-helical stem regions by the ROQ domain is identical for the triloop and hexaloop motifs.	DISCUSS
+13	29	U-rich hexaloops	structure_element	However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f).	DISCUSS
+37	40	ADE	structure_element	However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f).	DISCUSS
+45	48	ADE	structure_element	However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f).	DISCUSS
+54	56	SL	structure_element	However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f).	DISCUSS
+57	61	RNAs	chemical	However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f).	DISCUSS
+111	116	helix	structure_element	However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f).	DISCUSS
+117	119	α4	structure_element	However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f).	DISCUSS
+124	130	strand	structure_element	However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f).	DISCUSS
+131	133	β3	structure_element	However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f).	DISCUSS
+141	144	ROQ	structure_element	However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f).	DISCUSS
+175	182	triloop	structure_element	However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f).	DISCUSS
+183	186	CDE	structure_element	However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f).	DISCUSS
+33	41	hexaloop	structure_element	Of particular importance for the hexaloop recognition is Tyr250, which acts as a stabilizing element for the integrity of a defined loop conformation.	DISCUSS
+57	63	Tyr250	residue_name_number	Of particular importance for the hexaloop recognition is Tyr250, which acts as a stabilizing element for the integrity of a defined loop conformation.	DISCUSS
+132	136	loop	structure_element	Of particular importance for the hexaloop recognition is Tyr250, which acts as a stabilizing element for the integrity of a defined loop conformation.	DISCUSS
+3	9	stacks	bond_interaction	It stacks with nucleotides in the hexaloop but not the CDE triloop (Fig. 3b,c).	DISCUSS
+34	42	hexaloop	structure_element	It stacks with nucleotides in the hexaloop but not the CDE triloop (Fig. 3b,c).	DISCUSS
+55	58	CDE	structure_element	It stacks with nucleotides in the hexaloop but not the CDE triloop (Fig. 3b,c).	DISCUSS
+59	66	triloop	structure_element	It stacks with nucleotides in the hexaloop but not the CDE triloop (Fig. 3b,c).	DISCUSS
+23	29	Tyr250	residue_name_number	The functional role of Tyr250 for ADE-mediated mRNA regulation by Roquin-1 is thus explained by our experiments (Fig. 5b,c).	DISCUSS
+34	37	ADE	structure_element	The functional role of Tyr250 for ADE-mediated mRNA regulation by Roquin-1 is thus explained by our experiments (Fig. 5b,c).	DISCUSS
+47	51	mRNA	chemical	The functional role of Tyr250 for ADE-mediated mRNA regulation by Roquin-1 is thus explained by our experiments (Fig. 5b,c).	DISCUSS
+66	74	Roquin-1	protein	The functional role of Tyr250 for ADE-mediated mRNA regulation by Roquin-1 is thus explained by our experiments (Fig. 5b,c).	DISCUSS
+19	35	U-rich hexaloops	structure_element	The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL.	DISCUSS
+83	86	ROQ	structure_element	The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL.	DISCUSS
+92	95	U10	residue_name_number	The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL.	DISCUSS
+97	100	U11	residue_name_number	The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL.	DISCUSS
+105	108	U13	residue_name_number	The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL.	DISCUSS
+116	120	Ox40	protein	The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL.	DISCUSS
+121	124	ADE	structure_element	The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL.	DISCUSS
+130	132	SL	structure_element	The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL.	DISCUSS
+30	33	ROQ	structure_element	Consistent with this, loss of ROQ binding is observed on replacement of U11 and U13 by other bases (Table 2).	DISCUSS
+57	68	replacement	experimental_method	Consistent with this, loss of ROQ binding is observed on replacement of U11 and U13 by other bases (Table 2).	DISCUSS
+72	75	U11	residue_name_number	Consistent with this, loss of ROQ binding is observed on replacement of U11 and U13 by other bases (Table 2).	DISCUSS
+80	83	U13	residue_name_number	Consistent with this, loss of ROQ binding is observed on replacement of U11 and U13 by other bases (Table 2).	DISCUSS
+53	56	RNA	chemical	In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively.	DISCUSS
+90	96	Roquin	protein	In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively.	DISCUSS
+122	125	ADE	structure_element	In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively.	DISCUSS
+130	133	CDE	structure_element	In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively.	DISCUSS
+139	143	RNAs	chemical	In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively.	DISCUSS
+209	212	RNA	chemical	In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively.	DISCUSS
+213	217	stem	structure_element	In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively.	DISCUSS
+254	272	hexa- and triloops	structure_element	In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively.	DISCUSS
+24	29	bound	protein_state	A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c).	DISCUSS
+30	33	RNA	chemical	A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c).	DISCUSS
+34	43	structure	evidence	A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c).	DISCUSS
+60	78	tri- and hexaloops	structure_element	A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c).	DISCUSS
+87	95	stacking	bond_interaction	A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c).	DISCUSS
+9	24	structural data	evidence	Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops.	DISCUSS
+79	85	Roquin	protein	Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops.	DISCUSS
+111	113	SL	structure_element	Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops.	DISCUSS
+114	117	RNA	chemical	Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops.	DISCUSS
+150	155	loops	structure_element	Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops.	DISCUSS
+19	24	SELEX	experimental_method	Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work.	DISCUSS
+53	66	U-rich motifs	structure_element	Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work.	DISCUSS
+141	153	U-rich loops	structure_element	Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work.	DISCUSS
+190	226	crosslinking and immunoprecipitation	experimental_method	Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work.	DISCUSS
+230	238	Roquin-1	protein	Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work.	DISCUSS
+245	253	PAR-CLIP	experimental_method	Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work.	DISCUSS
+306	321	U-rich hexaloop	structure_element	Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work.	DISCUSS
+112	118	Roquin	protein	Most probably, the experimental setup of Murakawa et al. revealed both high- and low-affinity target motifs for Roquin, whereas our structural study reports on a high-affinity binding motif.	DISCUSS
+132	148	structural study	experimental_method	Most probably, the experimental setup of Murakawa et al. revealed both high- and low-affinity target motifs for Roquin, whereas our structural study reports on a high-affinity binding motif.	DISCUSS
+43	49	Roquin	protein	Notably, Murakawa et al. neither found the Roquin-regulated Ox40 nor the Tnf 3′-UTRs, as both genes are not expressed in HEK 293 cells.	DISCUSS
+60	64	Ox40	protein	Notably, Murakawa et al. neither found the Roquin-regulated Ox40 nor the Tnf 3′-UTRs, as both genes are not expressed in HEK 293 cells.	DISCUSS
+73	76	Tnf	protein	Notably, Murakawa et al. neither found the Roquin-regulated Ox40 nor the Tnf 3′-UTRs, as both genes are not expressed in HEK 293 cells.	DISCUSS
+77	84	3′-UTRs	structure_element	Notably, Murakawa et al. neither found the Roquin-regulated Ox40 nor the Tnf 3′-UTRs, as both genes are not expressed in HEK 293 cells.	DISCUSS
+46	48	SL	structure_element	However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement.	DISCUSS
+60	66	3′-UTR	structure_element	However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement.	DISCUSS
+70	73	A20	protein	However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement.	DISCUSS
+74	78	mRNA	chemical	However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement.	DISCUSS
+108	114	Roquin	protein	However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement.	DISCUSS
+177	180	CDE	structure_element	However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement.	DISCUSS
+181	188	triloop	structure_element	However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement.	DISCUSS
+73	76	A20	protein	It remains to be seen which exact features govern the recognition of the A20 SL by Roquin.	DISCUSS
+77	79	SL	structure_element	It remains to be seen which exact features govern the recognition of the A20 SL by Roquin.	DISCUSS
+83	89	Roquin	protein	It remains to be seen which exact features govern the recognition of the A20 SL by Roquin.	DISCUSS
+15	31	cis RNA elements	structure_element	The regulatory cis RNA elements in 3′-UTRs may also be targeted by additional trans-acting factors.	DISCUSS
+35	42	3′-UTRs	structure_element	The regulatory cis RNA elements in 3′-UTRs may also be targeted by additional trans-acting factors.	DISCUSS
+32	44	endonuclease	protein_type	We have recently identified the endonuclease Regnase-1 as a cofactor of Roquin function that shares an overlapping set of target mRNAs.	DISCUSS
+45	54	Regnase-1	protein	We have recently identified the endonuclease Regnase-1 as a cofactor of Roquin function that shares an overlapping set of target mRNAs.	DISCUSS
+72	78	Roquin	protein	We have recently identified the endonuclease Regnase-1 as a cofactor of Roquin function that shares an overlapping set of target mRNAs.	DISCUSS
+129	134	mRNAs	chemical	We have recently identified the endonuclease Regnase-1 as a cofactor of Roquin function that shares an overlapping set of target mRNAs.	DISCUSS
+125	143	lipopolysaccharide	chemical	In another study, the overlap in targets was confirmed, but a mutually exclusive regulation was proposed based on studies in lipopolysaccharide (LPS)-stimulated myeloid cells.	DISCUSS
+145	148	LPS	chemical	In another study, the overlap in targets was confirmed, but a mutually exclusive regulation was proposed based on studies in lipopolysaccharide (LPS)-stimulated myeloid cells.	DISCUSS
+16	22	Roquin	protein	In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target.	DISCUSS
+31	35	mRNA	chemical	In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target.	DISCUSS
+67	75	inactive	protein_state	In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target.	DISCUSS
+76	81	mRNAs	chemical	In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target.	DISCUSS
+89	98	Regnase-1	protein	In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target.	DISCUSS
+107	111	mRNA	chemical	In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target.	DISCUSS
+17	21	Ox40	protein	In CD4+ T cells, Ox40 does not show derepression in individual knockouts of Roquin-1 or Roquin-2 encoding genes, but is strongly induced upon combined deficiency of both genes.	DISCUSS
+76	84	Roquin-1	protein	In CD4+ T cells, Ox40 does not show derepression in individual knockouts of Roquin-1 or Roquin-2 encoding genes, but is strongly induced upon combined deficiency of both genes.	DISCUSS
+88	96	Roquin-2	protein	In CD4+ T cells, Ox40 does not show derepression in individual knockouts of Roquin-1 or Roquin-2 encoding genes, but is strongly induced upon combined deficiency of both genes.	DISCUSS
+151	161	deficiency	experimental_method	In CD4+ T cells, Ox40 does not show derepression in individual knockouts of Roquin-1 or Roquin-2 encoding genes, but is strongly induced upon combined deficiency of both genes.	DISCUSS
+25	36	deletion of	experimental_method	In addition, conditional deletion of the Regnase-1-encoding gene induced Ox40 expression in these cells.	DISCUSS
+41	50	Regnase-1	protein	In addition, conditional deletion of the Regnase-1-encoding gene induced Ox40 expression in these cells.	DISCUSS
+73	77	Ox40	protein	In addition, conditional deletion of the Regnase-1-encoding gene induced Ox40 expression in these cells.	DISCUSS
+25	29	Ox40	protein	Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes.	DISCUSS
+30	34	mRNA	chemical	Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes.	DISCUSS
+38	44	Roquin	protein	Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes.	DISCUSS
+48	55	Regnase	protein_type	Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes.	DISCUSS
+243	270	double and triple knockouts	experimental_method	Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes.	DISCUSS
+38	42	Ox40	protein	However, in cultures of CD4+ T cells, Ox40 is translated on day 4–5 and is expressed much higher in T cells with combined deficiency of Roquin-1 and Roquin-2.	DISCUSS
+136	144	Roquin-1	protein	However, in cultures of CD4+ T cells, Ox40 is translated on day 4–5 and is expressed much higher in T cells with combined deficiency of Roquin-1 and Roquin-2.	DISCUSS
+149	157	Roquin-2	protein	However, in cultures of CD4+ T cells, Ox40 is translated on day 4–5 and is expressed much higher in T cells with combined deficiency of Roquin-1 and Roquin-2.	DISCUSS
+45	59	reconstitution	experimental_method	At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c).	DISCUSS
+65	67	WT	protein_state	At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c).	DISCUSS
+68	76	Roquin-1	protein	At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c).	DISCUSS
+102	106	Ox40	protein	At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c).	DISCUSS
+169	175	active	protein_state	At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c).	DISCUSS
+176	180	mRNA	chemical	At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c).	DISCUSS
+184	192	Roquin-1	protein	At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c).	DISCUSS
+44	52	Roquin-1	protein	Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays.	DISCUSS
+56	64	Roquin-2	protein	Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays.	DISCUSS
+98	102	Ox40	protein	Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays.	DISCUSS
+103	107	mRNA	chemical	Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays.	DISCUSS
+111	116	EMSAs	experimental_method	Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays.	DISCUSS
+125	131	3′-UTR	structure_element	Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays.	DISCUSS
+135	139	Ox40	protein	Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays.	DISCUSS
+197	218	filter binding assays	experimental_method	Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays.	DISCUSS
+27	30	RNA	chemical	Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3′-UTR by both proteins.	DISCUSS
+95	98	ADE	structure_element	Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3′-UTR by both proteins.	DISCUSS
+103	106	CDE	structure_element	Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3′-UTR by both proteins.	DISCUSS
+121	125	Ox40	protein	Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3′-UTR by both proteins.	DISCUSS
+126	132	3′-UTR	structure_element	Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3′-UTR by both proteins.	DISCUSS
+39	48	Regnase-1	protein	In contrast, structural details on how Regnase-1 can interact with these SL RNAs are currently missing.	DISCUSS
+73	75	SL	structure_element	In contrast, structural details on how Regnase-1 can interact with these SL RNAs are currently missing.	DISCUSS
+76	80	RNAs	chemical	In contrast, structural details on how Regnase-1 can interact with these SL RNAs are currently missing.	DISCUSS
+44	67	Regnase-1-binding sites	site	Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene.	DISCUSS
+71	119	crosslinking and immunoprecipitation experiments	experimental_method	Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene.	DISCUSS
+140	147	triloop	structure_element	Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene.	DISCUSS
+164	198	pyrimidine–purine–pyrimidine loops	structure_element	Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene.	DISCUSS
+218	223	stems	structure_element	Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene.	DISCUSS
+244	252	hexaloop	structure_element	Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene.	DISCUSS
+270	275	Ptgs2	gene	Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene.	DISCUSS
+23	32	Regnase-1	protein	Both were required for Regnase-1-mediated repression.	DISCUSS
+52	61	Regnase-1	protein	These findings therefore raise the possibility that Regnase-1 interacts with ADE-like hexaloop structures either in a direct or indirect manner.	DISCUSS
+77	80	ADE	structure_element	These findings therefore raise the possibility that Regnase-1 interacts with ADE-like hexaloop structures either in a direct or indirect manner.	DISCUSS
+86	94	hexaloop	structure_element	These findings therefore raise the possibility that Regnase-1 interacts with ADE-like hexaloop structures either in a direct or indirect manner.	DISCUSS
+46	58	cis-elements	structure_element	Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation.	DISCUSS
+93	96	SLs	structure_element	Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation.	DISCUSS
+103	107	Ox40	protein	Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation.	DISCUSS
+111	115	Icos	protein	Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation.	DISCUSS
+231	235	mRNA	chemical	Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation.	DISCUSS
+10	16	3′-UTR	structure_element	The novel 3′-UTR loop motif that we have identified as a bona fide target of Roquin now expands this multilayer mode of co-regulation.	DISCUSS
+17	27	loop motif	structure_element	The novel 3′-UTR loop motif that we have identified as a bona fide target of Roquin now expands this multilayer mode of co-regulation.	DISCUSS
+77	83	Roquin	protein	The novel 3′-UTR loop motif that we have identified as a bona fide target of Roquin now expands this multilayer mode of co-regulation.	DISCUSS
+43	47	mRNA	chemical	We suggest that differential regulation of mRNA expression is not only achieved through multiple regulators with individual preferences for a given motif or variants thereof, but that regulators may also identify and use distinct motifs, as long as they exhibit some basic features regarding shape, size and sequence.	DISCUSS
+35	42	3′-UTRs	structure_element	The presence of distinct motifs in 3′-UTRs offers a broader variability for gene regulation by RNA cis elements.	DISCUSS
+95	98	RNA	chemical	The presence of distinct motifs in 3′-UTRs offers a broader variability for gene regulation by RNA cis elements.	DISCUSS
+99	111	cis elements	structure_element	The presence of distinct motifs in 3′-UTRs offers a broader variability for gene regulation by RNA cis elements.	DISCUSS
+132	135	RNA	chemical	Their accessibility can be modulated by trans-acting factors that may bind regulatory motifs, unfold higher-order structures in the RNA or maintain a preference for duplex structures as was shown recently for mRNAs that are recognized by Staufen-1 (ref.).	DISCUSS
+209	214	mRNAs	chemical	Their accessibility can be modulated by trans-acting factors that may bind regulatory motifs, unfold higher-order structures in the RNA or maintain a preference for duplex structures as was shown recently for mRNAs that are recognized by Staufen-1 (ref.).	DISCUSS
+238	247	Staufen-1	protein	Their accessibility can be modulated by trans-acting factors that may bind regulatory motifs, unfold higher-order structures in the RNA or maintain a preference for duplex structures as was shown recently for mRNAs that are recognized by Staufen-1 (ref.).	DISCUSS
+7	13	3′-UTR	structure_element	In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain.	DISCUSS
+21	25	Ox40	protein	In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain.	DISCUSS
+26	30	mRNA	chemical	In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain.	DISCUSS
+44	47	ADE	structure_element	In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain.	DISCUSS
+61	64	CDE	structure_element	In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain.	DISCUSS
+70	72	SL	structure_element	In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain.	DISCUSS
+102	105	ROQ	structure_element	In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain.	DISCUSS
+27	33	Roquin	protein	The exact stoichiometry of Roquin bound to the Ox40 3′-UTR is unknown.	DISCUSS
+34	42	bound to	protein_state	The exact stoichiometry of Roquin bound to the Ox40 3′-UTR is unknown.	DISCUSS
+47	51	Ox40	protein	The exact stoichiometry of Roquin bound to the Ox40 3′-UTR is unknown.	DISCUSS
+52	58	3′-UTR	structure_element	The exact stoichiometry of Roquin bound to the Ox40 3′-UTR is unknown.	DISCUSS
+24	46	secondary binding site	site	The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs.	DISCUSS
+51	56	dsRNA	chemical	The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs.	DISCUSS
+60	66	Roquin	protein	The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs.	DISCUSS
+68	74	B-site	site	The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs.	DISCUSS
+128	133	dsRNA	chemical	The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs.	DISCUSS
+168	174	Roquin	protein	The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs.	DISCUSS
+186	190	RNAs	chemical	The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs.	DISCUSS
+218	226	affinity	evidence	The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs.	DISCUSS
+227	230	SLs	structure_element	The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs.	DISCUSS
+65	68	RNA	chemical	In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site.	DISCUSS
+87	90	Tnf	protein	In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site.	DISCUSS
+91	94	CDE	structure_element	In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site.	DISCUSS
+99	102	ADE	structure_element	In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site.	DISCUSS
+103	105	SL	structure_element	In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site.	DISCUSS
+106	110	RNAs	chemical	In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site.	DISCUSS
+139	154	crystal lattice	evidence	In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site.	DISCUSS
+208	213	dsRNA	chemical	In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site.	DISCUSS
+221	227	B site	site	In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site.	DISCUSS
+36	42	Roquin	protein	This opens the possibility that one Roquin molecule may cluster two motifs in a given 3′-UTR and/or cluster motifs from distinct 3′-UTRs to enhance downstream processing.	DISCUSS
+86	92	3′-UTR	structure_element	This opens the possibility that one Roquin molecule may cluster two motifs in a given 3′-UTR and/or cluster motifs from distinct 3′-UTRs to enhance downstream processing.	DISCUSS
+129	136	3′-UTRs	structure_element	This opens the possibility that one Roquin molecule may cluster two motifs in a given 3′-UTR and/or cluster motifs from distinct 3′-UTRs to enhance downstream processing.	DISCUSS
+19	21	SL	structure_element	Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA.	DISCUSS
+22	25	RNA	chemical	Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA.	DISCUSS
+70	76	Roquin	protein	Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA.	DISCUSS
+110	116	3′-UTR	structure_element	Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA.	DISCUSS
+124	130	Nfkbid	protein	Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA.	DISCUSS
+131	135	mRNA	chemical	Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA.	DISCUSS
+58	71	binding sites	site	We therefore hypothesize that the combination of multiple binding sites may be more commonly used to enhance the functional activity of Roquin.	DISCUSS
+136	142	Roquin	protein	We therefore hypothesize that the combination of multiple binding sites may be more commonly used to enhance the functional activity of Roquin.	DISCUSS
+37	49	cis elements	structure_element	At the same time, the combination of cis elements may be important for differential gene regulation, as composite cis elements with lower affinity may be less sensitive to Roquin.	DISCUSS
+114	126	cis elements	structure_element	At the same time, the combination of cis elements may be important for differential gene regulation, as composite cis elements with lower affinity may be less sensitive to Roquin.	DISCUSS
+138	146	affinity	evidence	At the same time, the combination of cis elements may be important for differential gene regulation, as composite cis elements with lower affinity may be less sensitive to Roquin.	DISCUSS
+172	178	Roquin	protein	At the same time, the combination of cis elements may be important for differential gene regulation, as composite cis elements with lower affinity may be less sensitive to Roquin.	DISCUSS
+143	149	Roquin	protein	This will lead to less effective repression in T cells when antigen recognition is of moderate signal strength and only incomplete cleavage of Roquin by MALT1 occurs.	DISCUSS
+153	158	MALT1	protein	This will lead to less effective repression in T cells when antigen recognition is of moderate signal strength and only incomplete cleavage of Roquin by MALT1 occurs.	DISCUSS
+46	52	3′-UTR	structure_element	For understanding the intricate complexity of 3′-UTR regulation, future work will be necessary by combining large-scale approaches, such as cross-linking and immunoprecipitation experiments to identify RNA-binding sites, and structural biology to dissect the underlying molecular mechanisms.	DISCUSS
+140	189	cross-linking and immunoprecipitation experiments	experimental_method	For understanding the intricate complexity of 3′-UTR regulation, future work will be necessary by combining large-scale approaches, such as cross-linking and immunoprecipitation experiments to identify RNA-binding sites, and structural biology to dissect the underlying molecular mechanisms.	DISCUSS
+202	219	RNA-binding sites	site	For understanding the intricate complexity of 3′-UTR regulation, future work will be necessary by combining large-scale approaches, such as cross-linking and immunoprecipitation experiments to identify RNA-binding sites, and structural biology to dissect the underlying molecular mechanisms.	DISCUSS
+225	243	structural biology	experimental_method	For understanding the intricate complexity of 3′-UTR regulation, future work will be necessary by combining large-scale approaches, such as cross-linking and immunoprecipitation experiments to identify RNA-binding sites, and structural biology to dissect the underlying molecular mechanisms.	DISCUSS
+0	5	SELEX	experimental_method	SELEX identifies a novel SL RNA ligand of Roquin-1.	FIG
+25	27	SL	structure_element	SELEX identifies a novel SL RNA ligand of Roquin-1.	FIG
+28	31	RNA	chemical	SELEX identifies a novel SL RNA ligand of Roquin-1.	FIG
+42	50	Roquin-1	protein	SELEX identifies a novel SL RNA ligand of Roquin-1.	FIG
+41	49	Roquin-1	protein	(a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences.	FIG
+71	76	2–440	residue_range	(a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences.	FIG
+81	95	Roquin-1 M199R	mutant	(a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences.	FIG
+117	122	2–440	residue_range	(a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences.	FIG
+164	167	ADE	structure_element	(a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences.	FIG
+190	194	Ox40	protein	(a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences.	FIG
+195	201	3′-UTR	structure_element	(a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences.	FIG
+224	228	MEME	experimental_method	(a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences.	FIG
+244	249	SELEX	experimental_method	(a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences.	FIG
+259	262	RNA	chemical	(a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences.	FIG
+39	43	Ox40	protein	(c) Conservation of the motif found in Ox40 3′-UTRs for various species as indicated.	FIG
+44	51	3′-UTRs	structure_element	(c) Conservation of the motif found in Ox40 3′-UTRs for various species as indicated.	FIG
+0	3	rn5	gene	rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL.	FIG
+41	44	rat	taxonomy_domain	rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL.	FIG
+46	62	Rattus novegicus	species	rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL.	FIG
+111	116	SELEX	experimental_method	rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL.	FIG
+135	137	SL	structure_element	rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL.	FIG
+139	142	ADE	structure_element	rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL.	FIG
+151	155	Ox40	protein	rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL.	FIG
+156	159	ADE	structure_element	rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL.	FIG
+165	173	hexaloop	structure_element	rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL.	FIG
+174	176	SL	structure_element	rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL.	FIG
+49	52	ADE	structure_element	The broken line between the G–G base pair in the ADE SL indicates a putative non-Watson–Crick pairing.	FIG
+53	55	SL	structure_element	The broken line between the G–G base pair in the ADE SL indicates a putative non-Watson–Crick pairing.	FIG
+77	101	non-Watson–Crick pairing	bond_interaction	The broken line between the G–G base pair in the ADE SL indicates a putative non-Watson–Crick pairing.	FIG
+4	8	Ox40	protein	The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison.	FIG
+9	12	CDE	structure_element	The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison.	FIG
+18	20	SL	structure_element	The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison.	FIG
+29	32	Tnf	protein	The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison.	FIG
+33	36	CDE	structure_element	The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison.	FIG
+37	39	SL	structure_element	The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison.	FIG
+0	3	NMR	experimental_method	NMR analysis of the SL RNAs used in this study.	FIG
+20	22	SL	structure_element	NMR analysis of the SL RNAs used in this study.	FIG
+23	27	RNAs	chemical	NMR analysis of the SL RNAs used in this study.	FIG
+40	46	1H NMR	experimental_method	Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).	FIG
+47	54	spectra	evidence	Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).	FIG
+66	69	ADE	structure_element	Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).	FIG
+70	72	SL	structure_element	Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).	FIG
+82	86	Ox40	protein	Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).	FIG
+87	90	ADE	structure_element	Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).	FIG
+96	98	SL	structure_element	Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).	FIG
+111	115	Ox40	protein	Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).	FIG
+116	119	CDE	structure_element	Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).	FIG
+125	127	SL	structure_element	Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).	FIG
+142	146	free	protein_state	Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).	FIG
+147	151	RNAs	chemical	Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).	FIG
+164	179	in complex with	protein_state	Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).	FIG
+184	192	Roquin-1	protein	Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).	FIG
+193	196	ROQ	structure_element	Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).	FIG
+15	17	SL	structure_element	The respective SL RNAs and their base pairs are indicated.	FIG
+18	22	RNAs	chemical	The respective SL RNAs and their base pairs are indicated.	FIG
+23	26	NMR	experimental_method	Red asterisks indicate NMR signals of the protein.	FIG
+82	85	NMR	experimental_method	Green lines in the secondary structure schemes on the left refer to visible imino NMR signals and thus experimental confirmation of the base pairs indicated.	FIG
+86	93	signals	evidence	Green lines in the secondary structure schemes on the left refer to visible imino NMR signals and thus experimental confirmation of the base pairs indicated.	FIG
+30	32	G6	residue_name_number	The dotted green line between G6 and G15 in a highlights a G–G base pair.	FIG
+37	40	G15	residue_name_number	The dotted green line between G6 and G15 in a highlights a G–G base pair.	FIG
+59	60	G	residue_name	The dotted green line between G6 and G15 in a highlights a G–G base pair.	FIG
+61	62	G	residue_name	The dotted green line between G6 and G15 in a highlights a G–G base pair.	FIG
+0	9	Structure	evidence	Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA.	FIG
+17	25	Roquin-1	protein	Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA.	FIG
+26	29	ROQ	structure_element	Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA.	FIG
+37	45	bound to	protein_state	Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA.	FIG
+46	50	Ox40	protein	Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA.	FIG
+51	54	ADE	structure_element	Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA.	FIG
+60	63	RNA	chemical	Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA.	FIG
+32	49	crystal structure	evidence	(a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta).	FIG
+57	60	ROQ	structure_element	(a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta).	FIG
+78	85	174–325	residue_range	(a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta).	FIG
+101	105	Ox40	protein	(a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta).	FIG
+106	109	ADE	structure_element	(a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta).	FIG
+115	117	SL	structure_element	(a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta).	FIG
+118	121	RNA	chemical	(a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta).	FIG
+9	12	RNA	chemical	Selected RNA bases and protein secondary structure elements are labelled.	FIG
+25	29	Ox40	protein	(b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green).	FIG
+30	33	ADE	structure_element	(b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green).	FIG
+39	41	SL	structure_element	(b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green).	FIG
+56	59	RNA	chemical	(b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green).	FIG
+60	68	hexaloop	structure_element	(b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green).	FIG
+123	132	structure	evidence	(b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green).	FIG
+140	151	ROQ-Tnf CDE	complex_assembly	(b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green).	FIG
+182	185	RNA	chemical	(b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green).	FIG
+5	29	RNA-interacting residues	site	Only RNA-interacting residues that are different in both structures are shown.	FIG
+57	67	structures	evidence	Only RNA-interacting residues that are different in both structures are shown.	FIG
+48	52	RNAs	chemical	Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA.	FIG
+173	176	ROQ	structure_element	Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA.	FIG
+200	203	U11	residue_name_number	Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA.	FIG
+208	211	U13	residue_name_number	Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA.	FIG
+219	223	Ox40	protein	Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA.	FIG
+224	227	ADE	structure_element	Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA.	FIG
+233	235	SL	structure_element	Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA.	FIG
+236	239	RNA	chemical	Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA.	FIG
+52	57	helix	structure_element	The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257).	FIG
+58	60	α4	structure_element	The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257).	FIG
+62	68	Tyr250	residue_name_number	The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257).	FIG
+73	79	Ser253	residue_name_number	The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257).	FIG
+108	114	strand	structure_element	The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257).	FIG
+115	117	β3	structure_element	The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257).	FIG
+119	125	Phe255	residue_name_number	The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257).	FIG
+130	136	Val257	residue_name_number	The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257).	FIG
+48	51	RNA	chemical	The protein chain is shown in turquoise and the RNA is shown in grey.	FIG
+46	49	ROQ	structure_element	(e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop.	FIG
+73	76	U10	residue_name_number	(e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop.	FIG
+78	81	U11	residue_name_number	(e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop.	FIG
+86	89	U13	residue_name_number	(e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop.	FIG
+97	100	RNA	chemical	(e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop.	FIG
+101	109	hexaloop	structure_element	(e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop.	FIG
+0	3	U11	residue_name_number	U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247.	FIG
+8	11	U13	residue_name_number	U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247.	FIG
+42	47	helix	structure_element	U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247.	FIG
+48	50	α4	structure_element	U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247.	FIG
+61	67	Tyr250	residue_name_number	U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247.	FIG
+72	78	Gln247	residue_name_number	U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247.	FIG
+18	24	Tyr250	residue_name_number	The side chain of Tyr250 makes hydrophobic interactions with the pyrimidine side chain of U10 on one side and U11 on the other side.	FIG
+31	55	hydrophobic interactions	bond_interaction	The side chain of Tyr250 makes hydrophobic interactions with the pyrimidine side chain of U10 on one side and U11 on the other side.	FIG
+90	93	U10	residue_name_number	The side chain of Tyr250 makes hydrophobic interactions with the pyrimidine side chain of U10 on one side and U11 on the other side.	FIG
+110	113	U11	residue_name_number	The side chain of Tyr250 makes hydrophobic interactions with the pyrimidine side chain of U10 on one side and U11 on the other side.	FIG
+0	6	Lys259	residue_name_number	Lys259 interacts with the phosphate groups of U10 and U11.	FIG
+46	49	U10	residue_name_number	Lys259 interacts with the phosphate groups of U10 and U11.	FIG
+54	57	U11	residue_name_number	Lys259 interacts with the phosphate groups of U10 and U11.	FIG
+25	48	hydrophobic interaction	bond_interaction	(f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11.	FIG
+57	63	Val257	residue_name_number	(f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11.	FIG
+68	71	U11	residue_name_number	(f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11.	FIG
+95	108	hydrogen bond	bond_interaction	(f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11.	FIG
+112	118	Lys259	residue_name_number	(f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11.	FIG
+144	147	U10	residue_name_number	(f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11.	FIG
+152	155	U11	residue_name_number	(f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11.	FIG
+0	3	NMR	experimental_method	NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA.	FIG
+16	19	ROQ	structure_element	NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA.	FIG
+49	53	Ox40	protein	NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA.	FIG
+54	57	ADE	structure_element	NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA.	FIG
+63	71	hexaloop	structure_element	NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA.	FIG
+72	75	RNA	chemical	NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA.	FIG
+4	11	Overlay	experimental_method	(a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red).	FIG
+15	26	1H,15N HSQC	experimental_method	(a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red).	FIG
+27	34	spectra	evidence	(a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red).	FIG
+49	53	free	protein_state	(a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red).	FIG
+54	57	ROQ	structure_element	(a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red).	FIG
+66	73	171–326	residue_range	(a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red).	FIG
+85	100	in complex with	protein_state	(a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red).	FIG
+131	135	Ox40	protein	(a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red).	FIG
+136	139	ADE	structure_element	(a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red).	FIG
+145	147	SL	structure_element	(a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red).	FIG
+12	33	chemical shift change	evidence	(b) Plot of chemical shift change versus residue number in the ROQ domain (residues 171–326) from a. Grey negative bars indicate missing assignments in one of the spectra.	FIG
+63	66	ROQ	structure_element	(b) Plot of chemical shift change versus residue number in the ROQ domain (residues 171–326) from a. Grey negative bars indicate missing assignments in one of the spectra.	FIG
+84	91	171–326	residue_range	(b) Plot of chemical shift change versus residue number in the ROQ domain (residues 171–326) from a. Grey negative bars indicate missing assignments in one of the spectra.	FIG
+163	170	spectra	evidence	(b) Plot of chemical shift change versus residue number in the ROQ domain (residues 171–326) from a. Grey negative bars indicate missing assignments in one of the spectra.	FIG
+14	22	prolines	residue_name	Gaps indicate prolines.	FIG
+4	11	Overlay	experimental_method	(c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green).	FIG
+19	22	ROQ	structure_element	(c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green).	FIG
+30	35	alone	protein_state	(c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green).	FIG
+47	62	in complex with	protein_state	(c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green).	FIG
+67	71	Ox40	protein	(c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green).	FIG
+72	75	ADE	structure_element	(c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green).	FIG
+81	83	SL	structure_element	(c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green).	FIG
+97	101	Ox40	protein	(c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green).	FIG
+102	105	CDE	structure_element	(c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green).	FIG
+111	113	SL	structure_element	(c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green).	FIG
+0	19	Mutational analysis	experimental_method	Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR.	FIG
+23	31	Roquin-1	protein	Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR.	FIG
+50	54	Ox40	protein	Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR.	FIG
+55	58	ADE	structure_element	Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR.	FIG
+64	66	SL	structure_element	Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR.	FIG
+71	75	Ox40	protein	Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR.	FIG
+76	82	3′-UTR	structure_element	Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR.	FIG
+4	14	EMSA assay	experimental_method	(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right).	FIG
+40	49	wild-type	protein_state	(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right).	FIG
+61	66	Y250A	mutant	(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right).	FIG
+67	73	mutant	protein_state	(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right).	FIG
+74	77	ROQ	structure_element	(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right).	FIG
+104	108	Ox40	protein	(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right).	FIG
+109	112	ADE	structure_element	(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right).	FIG
+118	120	SL	structure_element	(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right).	FIG
+156	159	Tnf	protein	(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right).	FIG
+160	163	CDE	structure_element	(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right).	FIG
+164	166	SL	structure_element	(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right).	FIG
+160	174	Flow cytometry	experimental_method	A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam).	FIG
+178	182	Ox40	protein	A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam).	FIG
+187	191	Icos	protein	A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam).	FIG
+234	239	Rc3h1	gene	A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam).	FIG
+240	243	2fl	gene	A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam).	FIG
+244	246	fl	gene	A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam).	FIG
+267	271	mice	taxonomy_domain	A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam).	FIG
+285	294	tamoxifen	chemical	A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam).	FIG
+312	317	Rc3h1	gene	A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam).	FIG
+318	321	2fl	gene	A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam).	FIG
+322	324	fl	gene	A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam).	FIG
+325	333	deletion	experimental_method	A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam).	FIG
+74	84	retrovirus	taxonomy_domain	The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).	FIG
+98	109	doxycycline	chemical	The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).	FIG
+141	149	Roquin-1	protein	The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).	FIG
+150	152	WT	protein_state	The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).	FIG
+154	162	Roquin-1	protein	The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).	FIG
+163	168	Y250A	mutant	The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).	FIG
+172	180	Roquin-1	protein	The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).	FIG
+181	186	K220A	mutant	The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).	FIG
+188	193	K239A	mutant	The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).	FIG
+198	203	R260A	mutant	The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).	FIG
+204	211	mutants	protein_state	The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).	FIG
+25	33	Roquin-1	protein	Functional importance of Roquin-1 target motifs in cells.	FIG
+20	24	Ox40	protein	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+25	31	3′-UTR	structure_element	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+36	45	truncated	protein_state	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+46	53	mutated	protein_state	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+83	87	EMSA	experimental_method	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+134	138	Ox40	protein	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+155	159	EMSA	experimental_method	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+208	216	Roquin-1	protein	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+245	250	2–440	residue_range	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+276	285	wild-type	protein_state	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+286	290	Ox40	protein	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+291	297	3′-UTR	structure_element	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+315	324	mutations	experimental_method	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+332	335	CDE	structure_element	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+341	343	SL	structure_element	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+349	352	ADE	structure_element	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+358	360	SL	structure_element	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+369	372	SLs	structure_element	(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).	FIG
+148	156	Roquin-1	protein	It is noteworthy that the higher bands observed at large protein concentrations are probably additional nonspecific, lower-affinity interactions of Roquin-1 with the 3′-UTR or protein aggregates.	FIG
+166	172	3′-UTR	structure_element	It is noteworthy that the higher bands observed at large protein concentrations are probably additional nonspecific, lower-affinity interactions of Roquin-1 with the 3′-UTR or protein aggregates.	FIG
+13	17	Ox40	protein	(c) Relative Ox40 MFI normalized to expression levels from the Ox40 CDS construct.	FIG
+18	53	MFI normalized to expression levels	evidence	(c) Relative Ox40 MFI normalized to expression levels from the Ox40 CDS construct.	FIG
+63	67	Ox40	protein	(c) Relative Ox40 MFI normalized to expression levels from the Ox40 CDS construct.	FIG
+68	71	CDS	structure_element	(c) Relative Ox40 MFI normalized to expression levels from the Ox40 CDS construct.	FIG
+31	34	CDS	structure_element	Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments.	FIG
+36	40	1–40	residue_range	Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments.	FIG
+42	46	1–80	residue_range	Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments.	FIG
+48	53	1–120	residue_range	Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments.	FIG
+58	69	full-length	protein_state	Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments.	FIG
+77	80	ADE	structure_element	Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments.	FIG
+86	89	mut	protein_state	Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments.	FIG
+94	97	CDE	structure_element	Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments.	FIG
+98	101	mut	protein_state	Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments.	FIG
+113	123	double mut	protein_state	Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments.	FIG
+43	71	one-way analysis of variance	experimental_method	Statistical significance was calculated by one-way analysis of variance (ANOVA) Kruskal–Wallis test followed by Dunn’s multiple comparison test (**P<0.01).	FIG
+73	78	ANOVA	experimental_method	Statistical significance was calculated by one-way analysis of variance (ANOVA) Kruskal–Wallis test followed by Dunn’s multiple comparison test (**P<0.01).	FIG
+80	99	Kruskal–Wallis test	experimental_method	Statistical significance was calculated by one-way analysis of variance (ANOVA) Kruskal–Wallis test followed by Dunn’s multiple comparison test (**P<0.01).	FIG
+112	143	Dunn’s multiple comparison test	experimental_method	Statistical significance was calculated by one-way analysis of variance (ANOVA) Kruskal–Wallis test followed by Dunn’s multiple comparison test (**P<0.01).	FIG
+4	21	mRNA decay curves	evidence	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+67	79	retroviruses	taxonomy_domain	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+91	95	Ox40	protein	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+96	99	CDS	structure_element	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+108	114	3′-UTR	structure_element	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+116	119	CDS	structure_element	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+132	136	Ox40	protein	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+137	140	CDS	structure_element	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+150	159	wild-type	protein_state	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+160	166	3′-UTR	structure_element	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+168	179	full length	protein_state	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+194	198	Ox40	protein	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+199	210	full length	protein_state	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+216	223	mutated	protein_state	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+224	227	ADE	structure_element	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+240	243	ADE	structure_element	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+249	252	mut	protein_state	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+266	270	Ox40	protein	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+271	282	full length	protein_state	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+288	295	mutated	protein_state	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+296	299	CDE	structure_element	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+312	315	CDE	structure_element	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+321	324	mut	protein_state	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+341	345	Ox40	protein	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+346	357	full length	protein_state	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+363	370	mutated	protein_state	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+371	374	ADE	structure_element	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+379	382	CDE	structure_element	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+391	401	Double mut	protein_state	(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).	FIG
+0	20	mRNA half-life times	evidence	mRNA half-life times were calculated with Graph Pad Prism.	FIG
+0	41	Data collection and refinement statistics	evidence	Data collection and refinement statistics.	TABLE
+2	5	ROQ	structure_element	" 	ROQ-Ox40ADE-like SL	ROQ-ADE SL	 	Data collection	 	 space group	P21212	P212121	 	 	 	 	 	 Cell dimensions	 	 a, b, c (Å)	89.66, 115.79, 42.61	72.90, 89.30, 144.70	 	 α, β, γ (°)	90, 90, 90	90, 90, 90	 	 Resolution (Å)	50–2.23 (2.29–2.23)	50–3.0 (3.08–3.00)	 	 Rmerge	5.9 (68.3)	14.8 (93.8)	 	 I/σI	14.9 (2.1)	16.7 (3.1)	 	 Completeness (%)	98.7 (97.7)	99.9 (99.9)	 	 Redundancy	3.9 (3.7)	13.2 (12.7)	 	 	 	 	 	Refinement	 	 Resolution (Å)	2.23	3.00	 	 No. reflections	21,018	18,598	 	 Rwork/Rfree	21.8/25.7	18.6/23.4	 	 	 	 	 	 No. atoms	 	 Protein	2,404	4,820	 	 Ligand/ion	894	1,708	 	 Water	99	49	 	 B-factor overall	47.2	60.4	 	 	 	 	 	Root mean squared deviations	 	 Bond lengths (Å)	0.006	0.014	 	 Bond angles (°)	1.07	1.77	 	 	 	 	 	Ramachandran plot	 	 Most favoured (%)	98.6	99.8	 	 Additional allowed (%)	1.4	0.2	 	"	TABLE
+6	10	Ox40	protein	" 	ROQ-Ox40ADE-like SL	ROQ-ADE SL	 	Data collection	 	 space group	P21212	P212121	 	 	 	 	 	 Cell dimensions	 	 a, b, c (Å)	89.66, 115.79, 42.61	72.90, 89.30, 144.70	 	 α, β, γ (°)	90, 90, 90	90, 90, 90	 	 Resolution (Å)	50–2.23 (2.29–2.23)	50–3.0 (3.08–3.00)	 	 Rmerge	5.9 (68.3)	14.8 (93.8)	 	 I/σI	14.9 (2.1)	16.7 (3.1)	 	 Completeness (%)	98.7 (97.7)	99.9 (99.9)	 	 Redundancy	3.9 (3.7)	13.2 (12.7)	 	 	 	 	 	Refinement	 	 Resolution (Å)	2.23	3.00	 	 No. reflections	21,018	18,598	 	 Rwork/Rfree	21.8/25.7	18.6/23.4	 	 	 	 	 	 No. atoms	 	 Protein	2,404	4,820	 	 Ligand/ion	894	1,708	 	 Water	99	49	 	 B-factor overall	47.2	60.4	 	 	 	 	 	Root mean squared deviations	 	 Bond lengths (Å)	0.006	0.014	 	 Bond angles (°)	1.07	1.77	 	 	 	 	 	Ramachandran plot	 	 Most favoured (%)	98.6	99.8	 	 Additional allowed (%)	1.4	0.2	 	"	TABLE
+10	13	ADE	structure_element	" 	ROQ-Ox40ADE-like SL	ROQ-ADE SL	 	Data collection	 	 space group	P21212	P212121	 	 	 	 	 	 Cell dimensions	 	 a, b, c (Å)	89.66, 115.79, 42.61	72.90, 89.30, 144.70	 	 α, β, γ (°)	90, 90, 90	90, 90, 90	 	 Resolution (Å)	50–2.23 (2.29–2.23)	50–3.0 (3.08–3.00)	 	 Rmerge	5.9 (68.3)	14.8 (93.8)	 	 I/σI	14.9 (2.1)	16.7 (3.1)	 	 Completeness (%)	98.7 (97.7)	99.9 (99.9)	 	 Redundancy	3.9 (3.7)	13.2 (12.7)	 	 	 	 	 	Refinement	 	 Resolution (Å)	2.23	3.00	 	 No. reflections	21,018	18,598	 	 Rwork/Rfree	21.8/25.7	18.6/23.4	 	 	 	 	 	 No. atoms	 	 Protein	2,404	4,820	 	 Ligand/ion	894	1,708	 	 Water	99	49	 	 B-factor overall	47.2	60.4	 	 	 	 	 	Root mean squared deviations	 	 Bond lengths (Å)	0.006	0.014	 	 Bond angles (°)	1.07	1.77	 	 	 	 	 	Ramachandran plot	 	 Most favoured (%)	98.6	99.8	 	 Additional allowed (%)	1.4	0.2	 	"	TABLE
+19	21	SL	structure_element	" 	ROQ-Ox40ADE-like SL	ROQ-ADE SL	 	Data collection	 	 space group	P21212	P212121	 	 	 	 	 	 Cell dimensions	 	 a, b, c (Å)	89.66, 115.79, 42.61	72.90, 89.30, 144.70	 	 α, β, γ (°)	90, 90, 90	90, 90, 90	 	 Resolution (Å)	50–2.23 (2.29–2.23)	50–3.0 (3.08–3.00)	 	 Rmerge	5.9 (68.3)	14.8 (93.8)	 	 I/σI	14.9 (2.1)	16.7 (3.1)	 	 Completeness (%)	98.7 (97.7)	99.9 (99.9)	 	 Redundancy	3.9 (3.7)	13.2 (12.7)	 	 	 	 	 	Refinement	 	 Resolution (Å)	2.23	3.00	 	 No. reflections	21,018	18,598	 	 Rwork/Rfree	21.8/25.7	18.6/23.4	 	 	 	 	 	 No. atoms	 	 Protein	2,404	4,820	 	 Ligand/ion	894	1,708	 	 Water	99	49	 	 B-factor overall	47.2	60.4	 	 	 	 	 	Root mean squared deviations	 	 Bond lengths (Å)	0.006	0.014	 	 Bond angles (°)	1.07	1.77	 	 	 	 	 	Ramachandran plot	 	 Most favoured (%)	98.6	99.8	 	 Additional allowed (%)	1.4	0.2	 	"	TABLE
+22	25	ROQ	structure_element	" 	ROQ-Ox40ADE-like SL	ROQ-ADE SL	 	Data collection	 	 space group	P21212	P212121	 	 	 	 	 	 Cell dimensions	 	 a, b, c (Å)	89.66, 115.79, 42.61	72.90, 89.30, 144.70	 	 α, β, γ (°)	90, 90, 90	90, 90, 90	 	 Resolution (Å)	50–2.23 (2.29–2.23)	50–3.0 (3.08–3.00)	 	 Rmerge	5.9 (68.3)	14.8 (93.8)	 	 I/σI	14.9 (2.1)	16.7 (3.1)	 	 Completeness (%)	98.7 (97.7)	99.9 (99.9)	 	 Redundancy	3.9 (3.7)	13.2 (12.7)	 	 	 	 	 	Refinement	 	 Resolution (Å)	2.23	3.00	 	 No. reflections	21,018	18,598	 	 Rwork/Rfree	21.8/25.7	18.6/23.4	 	 	 	 	 	 No. atoms	 	 Protein	2,404	4,820	 	 Ligand/ion	894	1,708	 	 Water	99	49	 	 B-factor overall	47.2	60.4	 	 	 	 	 	Root mean squared deviations	 	 Bond lengths (Å)	0.006	0.014	 	 Bond angles (°)	1.07	1.77	 	 	 	 	 	Ramachandran plot	 	 Most favoured (%)	98.6	99.8	 	 Additional allowed (%)	1.4	0.2	 	"	TABLE
+26	29	ADE	structure_element	" 	ROQ-Ox40ADE-like SL	ROQ-ADE SL	 	Data collection	 	 space group	P21212	P212121	 	 	 	 	 	 Cell dimensions	 	 a, b, c (Å)	89.66, 115.79, 42.61	72.90, 89.30, 144.70	 	 α, β, γ (°)	90, 90, 90	90, 90, 90	 	 Resolution (Å)	50–2.23 (2.29–2.23)	50–3.0 (3.08–3.00)	 	 Rmerge	5.9 (68.3)	14.8 (93.8)	 	 I/σI	14.9 (2.1)	16.7 (3.1)	 	 Completeness (%)	98.7 (97.7)	99.9 (99.9)	 	 Redundancy	3.9 (3.7)	13.2 (12.7)	 	 	 	 	 	Refinement	 	 Resolution (Å)	2.23	3.00	 	 No. reflections	21,018	18,598	 	 Rwork/Rfree	21.8/25.7	18.6/23.4	 	 	 	 	 	 No. atoms	 	 Protein	2,404	4,820	 	 Ligand/ion	894	1,708	 	 Water	99	49	 	 B-factor overall	47.2	60.4	 	 	 	 	 	Root mean squared deviations	 	 Bond lengths (Å)	0.006	0.014	 	 Bond angles (°)	1.07	1.77	 	 	 	 	 	Ramachandran plot	 	 Most favoured (%)	98.6	99.8	 	 Additional allowed (%)	1.4	0.2	 	"	TABLE
+30	32	SL	structure_element	" 	ROQ-Ox40ADE-like SL	ROQ-ADE SL	 	Data collection	 	 space group	P21212	P212121	 	 	 	 	 	 Cell dimensions	 	 a, b, c (Å)	89.66, 115.79, 42.61	72.90, 89.30, 144.70	 	 α, β, γ (°)	90, 90, 90	90, 90, 90	 	 Resolution (Å)	50–2.23 (2.29–2.23)	50–3.0 (3.08–3.00)	 	 Rmerge	5.9 (68.3)	14.8 (93.8)	 	 I/σI	14.9 (2.1)	16.7 (3.1)	 	 Completeness (%)	98.7 (97.7)	99.9 (99.9)	 	 Redundancy	3.9 (3.7)	13.2 (12.7)	 	 	 	 	 	Refinement	 	 Resolution (Å)	2.23	3.00	 	 No. reflections	21,018	18,598	 	 Rwork/Rfree	21.8/25.7	18.6/23.4	 	 	 	 	 	 No. atoms	 	 Protein	2,404	4,820	 	 Ligand/ion	894	1,708	 	 Water	99	49	 	 B-factor overall	47.2	60.4	 	 	 	 	 	Root mean squared deviations	 	 Bond lengths (Å)	0.006	0.014	 	 Bond angles (°)	1.07	1.77	 	 	 	 	 	Ramachandran plot	 	 Most favoured (%)	98.6	99.8	 	 Additional allowed (%)	1.4	0.2	 	"	TABLE
+642	670	Root mean squared deviations	evidence	" 	ROQ-Ox40ADE-like SL	ROQ-ADE SL	 	Data collection	 	 space group	P21212	P212121	 	 	 	 	 	 Cell dimensions	 	 a, b, c (Å)	89.66, 115.79, 42.61	72.90, 89.30, 144.70	 	 α, β, γ (°)	90, 90, 90	90, 90, 90	 	 Resolution (Å)	50–2.23 (2.29–2.23)	50–3.0 (3.08–3.00)	 	 Rmerge	5.9 (68.3)	14.8 (93.8)	 	 I/σI	14.9 (2.1)	16.7 (3.1)	 	 Completeness (%)	98.7 (97.7)	99.9 (99.9)	 	 Redundancy	3.9 (3.7)	13.2 (12.7)	 	 	 	 	 	Refinement	 	 Resolution (Å)	2.23	3.00	 	 No. reflections	21,018	18,598	 	 Rwork/Rfree	21.8/25.7	18.6/23.4	 	 	 	 	 	 No. atoms	 	 Protein	2,404	4,820	 	 Ligand/ion	894	1,708	 	 Water	99	49	 	 B-factor overall	47.2	60.4	 	 	 	 	 	Root mean squared deviations	 	 Bond lengths (Å)	0.006	0.014	 	 Bond angles (°)	1.07	1.77	 	 	 	 	 	Ramachandran plot	 	 Most favoured (%)	98.6	99.8	 	 Additional allowed (%)	1.4	0.2	 	"	TABLE
+0	3	ADE	structure_element	ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop.	TABLE
+5	30	alternative decay element	structure_element	ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop.	TABLE
+32	35	CDE	structure_element	ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop.	TABLE
+37	63	constitutive decay element	structure_element	ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop.	TABLE
+65	67	SL	structure_element	ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop.	TABLE
+69	78	stem loop	structure_element	ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop.	TABLE
+28	35	crystal	evidence	For each data set, only one crystal has been used.	TABLE
+0	2	KD	evidence	KD for selected RNAs obtained from SPR measurements with immobilized ROQ domain of Roquin-1.	TABLE
+16	20	RNAs	chemical	KD for selected RNAs obtained from SPR measurements with immobilized ROQ domain of Roquin-1.	TABLE
+35	51	SPR measurements	experimental_method	KD for selected RNAs obtained from SPR measurements with immobilized ROQ domain of Roquin-1.	TABLE
+69	72	ROQ	structure_element	KD for selected RNAs obtained from SPR measurements with immobilized ROQ domain of Roquin-1.	TABLE
+83	91	Roquin-1	protein	KD for selected RNAs obtained from SPR measurements with immobilized ROQ domain of Roquin-1.	TABLE
diff --git a/annotation_IOB/PMC4806292.tsv b/annotation_IOB/PMC4806292.tsv
new file mode 100644
index 0000000000000000000000000000000000000000..24c1dc622cd21b6ea4bbe55242f8b5fd61ec3b57
--- /dev/null
+++ b/annotation_IOB/PMC4806292.tsv
@@ -0,0 +1,4800 @@
+Structural	O
+insights	O
+and	O
+in	B-experimental_method
+vitro	I-experimental_method
+reconstitution	I-experimental_method
+of	O
+membrane	O
+targeting	O
+and	O
+activation	O
+of	O
+human	B-species
+PI4KB	B-protein
+by	O
+the	O
+ACBD3	B-protein
+protein	O
+
+Phosphatidylinositol	B-protein
+4	I-protein
+-	I-protein
+kinase	I-protein
+beta	I-protein
+(	O
+PI4KB	B-protein
+)	O
+is	O
+one	O
+of	O
+four	O
+human	B-species
+PI4K	B-protein_type
+enzymes	O
+that	O
+generate	O
+phosphatidylinositol	B-chemical
+4	I-chemical
+-	I-chemical
+phosphate	I-chemical
+(	O
+PI4P	B-chemical
+),	O
+a	O
+minor	O
+but	O
+essential	O
+regulatory	O
+lipid	O
+found	O
+in	O
+all	O
+eukaryotic	B-taxonomy_domain
+cells	O
+.	O
+
+To	O
+convert	O
+their	O
+lipid	O
+substrates	O
+,	O
+PI4Ks	B-protein_type
+must	O
+be	O
+recruited	O
+to	O
+the	O
+correct	O
+membrane	O
+compartment	O
+.	O
+
+PI4KB	B-protein
+is	O
+critical	O
+for	O
+the	O
+maintenance	O
+of	O
+the	O
+Golgi	O
+and	O
+trans	O
+Golgi	O
+network	O
+(	O
+TGN	O
+)	O
+PI4P	B-chemical
+pools	O
+,	O
+however	O
+,	O
+the	O
+actual	O
+targeting	O
+mechanism	O
+of	O
+PI4KB	B-protein
+to	O
+the	O
+Golgi	O
+and	O
+TGN	O
+membranes	O
+is	O
+unknown	O
+.	O
+
+Here	O
+,	O
+we	O
+present	O
+an	O
+NMR	B-experimental_method
+structure	B-evidence
+of	O
+the	O
+complex	O
+of	O
+PI4KB	B-protein
+and	O
+its	O
+interacting	O
+partner	O
+,	O
+Golgi	B-protein_type
+adaptor	I-protein_type
+protein	I-protein_type
+acyl	B-protein
+-	I-protein
+coenzyme	I-protein
+A	I-protein
+binding	I-protein
+domain	I-protein
+containing	I-protein
+protein	I-protein
+3	I-protein
+(	O
+ACBD3	B-protein
+).	O
+
+We	O
+show	O
+that	O
+ACBD3	B-protein
+is	O
+capable	O
+of	O
+recruiting	O
+PI4KB	B-protein
+to	O
+membranes	O
+both	O
+in	O
+vitro	O
+and	O
+in	O
+vivo	O
+,	O
+and	O
+that	O
+membrane	O
+recruitment	O
+of	O
+PI4KB	B-protein
+by	O
+ACBD3	B-protein
+increases	O
+its	O
+enzymatic	B-evidence
+activity	I-evidence
+and	O
+that	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+complex	O
+formation	O
+is	O
+essential	O
+for	O
+proper	O
+function	O
+of	O
+the	O
+Golgi	O
+.	O
+
+Phosphatidylinositol	B-protein
+4	I-protein
+-	I-protein
+kinase	I-protein
+beta	I-protein
+(	O
+PI4KB	B-protein
+,	O
+also	O
+known	O
+as	O
+PI4K	B-protein
+IIIβ	I-protein
+)	O
+is	O
+a	O
+soluble	O
+cytosolic	O
+protein	O
+yet	O
+its	O
+function	O
+is	O
+to	O
+phosphorylate	O
+membrane	O
+lipids	O
+.	O
+
+It	O
+is	O
+one	O
+of	O
+four	O
+human	B-species
+PI4K	B-protein_type
+enzymes	O
+that	O
+phosphorylate	O
+phosphatidylinositol	B-chemical
+(	O
+PI	B-chemical
+)	O
+to	O
+generate	O
+phosphatidylinositol	B-chemical
+4	I-chemical
+-	I-chemical
+phosphate	I-chemical
+(	O
+PI4P	B-chemical
+).	O
+
+PI4P	B-chemical
+is	O
+an	O
+essential	O
+lipid	O
+found	O
+in	O
+various	O
+membrane	O
+compartments	O
+including	O
+the	O
+Golgi	O
+and	O
+trans	O
+-	O
+Golgi	O
+network	O
+(	O
+TGN	O
+),	O
+the	O
+plasma	O
+membrane	O
+and	O
+the	O
+endocytic	O
+compartments	O
+.	O
+
+In	O
+these	O
+locations	O
+,	O
+PI4P	B-chemical
+plays	O
+an	O
+important	O
+role	O
+in	O
+cell	O
+signaling	O
+and	O
+lipid	O
+transport	O
+,	O
+and	O
+serves	O
+as	O
+a	O
+precursor	O
+for	O
+higher	O
+phosphoinositides	B-chemical
+or	O
+as	O
+a	O
+docking	O
+site	O
+for	O
+clathrin	B-protein_type
+adaptor	O
+or	O
+lipid	O
+transfer	O
+proteins	O
+.	O
+
+A	O
+wide	O
+range	O
+of	O
+positive	B-taxonomy_domain
+-	I-taxonomy_domain
+sense	I-taxonomy_domain
+single	I-taxonomy_domain
+-	I-taxonomy_domain
+stranded	I-taxonomy_domain
+RNA	I-taxonomy_domain
+viruses	I-taxonomy_domain
+(+	O
+RNA	B-taxonomy_domain
+viruses	I-taxonomy_domain
+),	O
+including	O
+many	O
+that	O
+are	O
+important	O
+human	B-species
+pathogens	O
+,	O
+hijack	O
+human	B-species
+PI4KA	B-protein
+or	O
+PI4KB	B-protein
+enzymes	O
+to	O
+generate	O
+specific	O
+PI4P	B-chemical
+-	O
+enriched	O
+organelles	O
+called	O
+membranous	O
+webs	O
+or	O
+replication	O
+factories	O
+.	O
+
+These	O
+structures	B-evidence
+are	O
+essential	O
+for	O
+effective	O
+viral	B-taxonomy_domain
+replication	O
+.	O
+
+Recently	O
+,	O
+highly	O
+specific	O
+PI4KB	B-protein
+inhibitors	O
+were	O
+developed	O
+as	O
+potential	O
+antivirals	O
+.	O
+
+PI4K	B-protein_type
+kinases	B-protein_type
+must	O
+be	O
+recruited	O
+to	O
+the	O
+correct	O
+membrane	O
+type	O
+to	O
+fulfill	O
+their	O
+enzymatic	O
+functions	O
+.	O
+
+Type	B-protein_type
+II	I-protein_type
+PI4Ks	I-protein_type
+(	O
+PI4K2A	B-protein
+and	O
+PI4K2B	B-protein
+)	O
+are	O
+heavily	B-protein_state
+palmitoylated	I-protein_state
+and	O
+thus	O
+behave	O
+as	O
+membrane	B-protein
+proteins	I-protein
+.	O
+
+In	O
+contrast	O
+,	O
+type	B-protein_type
+III	I-protein_type
+PI4Ks	I-protein_type
+(	O
+PI4KA	B-protein
+and	O
+PI4KB	B-protein
+)	O
+are	O
+soluble	O
+cytosolic	O
+proteins	O
+that	O
+are	O
+recruited	O
+to	O
+appropriate	O
+membranes	O
+indirectly	O
+via	O
+protein	O
+-	O
+protein	O
+interactions	O
+.	O
+
+The	O
+recruitment	O
+of	O
+PI4KA	B-protein
+to	O
+the	O
+plasma	O
+membrane	O
+by	O
+EFR3	B-protein
+and	O
+TTC7	B-protein
+is	O
+relatively	O
+well	O
+understood	O
+even	O
+at	O
+the	O
+structural	O
+level	O
+,	O
+but	O
+,	O
+the	O
+actual	O
+molecular	O
+mechanism	O
+of	O
+PI4KB	B-protein
+recruitment	O
+to	O
+the	O
+Golgi	O
+is	O
+still	O
+poorly	O
+understood	O
+.	O
+
+Acyl	B-protein
+-	I-protein
+coenzyme	I-protein
+A	I-protein
+binding	I-protein
+domain	I-protein
+containing	I-protein
+protein	I-protein
+3	I-protein
+(	O
+ACBD3	B-protein
+,	O
+also	O
+known	O
+as	O
+GCP60	B-protein
+and	O
+PAP7	B-protein
+)	O
+is	O
+a	O
+Golgi	O
+resident	O
+protein	O
+.	O
+
+Its	O
+membrane	O
+localization	O
+is	O
+mediated	O
+by	O
+the	O
+interaction	O
+with	O
+the	O
+Golgi	O
+integral	O
+protein	O
+golgin	B-protein
+B1	I-protein
+/	O
+giantin	B-protein
+.	O
+
+ACBD3	B-protein
+functions	O
+as	O
+an	O
+adaptor	O
+protein	O
+and	O
+signaling	O
+hub	O
+across	O
+cellular	O
+signaling	O
+pathways	O
+.	O
+
+ACBD3	B-protein
+can	O
+interact	O
+with	O
+a	O
+number	O
+of	O
+proteins	O
+including	O
+golgin	B-protein
+A3	I-protein
+/	O
+golgin	B-protein
+-	I-protein
+160	I-protein
+to	O
+regulate	O
+apoptosis	O
+,	O
+Numb	B-protein_type
+proteins	I-protein_type
+to	O
+control	O
+asymmetric	O
+cell	O
+division	O
+and	O
+neuronal	O
+differentiation	O
+,	O
+metal	B-protein_type
+transporter	I-protein_type
+DMT1	B-protein
+and	O
+monomeric	B-oligomeric_state
+G	B-protein_type
+protein	I-protein_type
+Dexras1	B-protein
+to	O
+maintain	O
+iron	B-chemical
+homeostasis	O
+,	O
+and	O
+the	O
+lipid	B-protein_type
+kinase	I-protein_type
+PI4KB	B-protein
+to	O
+regulate	O
+lipid	O
+homeostasis	O
+.	O
+
+ACBD3	B-protein
+has	O
+been	O
+also	O
+implicated	O
+in	O
+the	O
+pathology	O
+of	O
+neurodegenerative	O
+diseases	O
+such	O
+as	O
+Huntington	O
+’	O
+s	O
+disease	O
+due	O
+to	O
+its	O
+interactions	O
+with	O
+a	O
+polyglutamine	B-structure_element
+repeat	I-structure_element
+-	O
+containing	O
+mutant	B-protein_state
+huntingtin	B-protein
+and	O
+the	O
+striatal	O
+-	O
+selective	O
+monomeric	B-oligomeric_state
+G	B-protein_type
+protein	I-protein_type
+Rhes	B-protein
+/	O
+Dexras2	B-protein
+.	O
+
+ACBD3	B-protein
+is	O
+a	O
+binding	O
+partner	O
+of	O
+viral	B-taxonomy_domain
+non	B-protein_type
+-	I-protein_type
+structural	I-protein_type
+3A	I-protein_type
+proteins	I-protein_type
+and	O
+a	O
+host	O
+factor	O
+of	O
+several	O
+picornaviruses	B-taxonomy_domain
+including	O
+poliovirus	B-taxonomy_domain
+,	O
+coxsackievirus	B-taxonomy_domain
+B3	I-taxonomy_domain
+,	O
+and	O
+Aichi	B-taxonomy_domain
+virus	I-taxonomy_domain
+.	O
+
+We	O
+present	O
+a	O
+biochemical	B-experimental_method
+and	I-experimental_method
+structural	I-experimental_method
+characterization	I-experimental_method
+of	O
+the	O
+molecular	O
+complex	O
+composed	O
+of	O
+the	O
+ACBD3	B-protein
+protein	O
+and	O
+the	O
+PI4KB	B-protein
+enzyme	O
+.	O
+
+We	O
+show	O
+that	O
+ACBD3	B-protein
+can	O
+recruit	O
+PI4KB	B-protein
+to	O
+model	O
+membranes	O
+as	O
+well	O
+as	O
+redirect	O
+PI4KB	B-protein
+to	O
+cellular	O
+membranes	O
+where	O
+it	O
+is	O
+not	O
+naturally	O
+found	O
+.	O
+
+Our	O
+data	O
+also	O
+show	O
+that	O
+ACBD3	B-protein
+regulates	O
+the	O
+enzymatic	B-evidence
+activity	I-evidence
+of	O
+PI4KB	B-protein
+kinase	B-protein_type
+through	O
+membrane	O
+recruitment	O
+rather	O
+than	O
+allostery	O
+.	O
+
+ACBD3	B-protein
+and	O
+PI4KB	B-protein
+interact	O
+with	O
+1	O
+:	O
+1	O
+stoichiometry	O
+with	O
+submicromolar	O
+affinity	O
+
+In	O
+order	O
+to	O
+verify	O
+the	O
+interactions	O
+between	O
+ACBD3	B-protein
+and	O
+PI4KB	B-protein
+we	O
+expressed	B-experimental_method
+and	I-experimental_method
+purified	I-experimental_method
+both	O
+proteins	O
+.	O
+
+To	O
+increase	O
+yields	O
+of	O
+bacterial	B-experimental_method
+expression	I-experimental_method
+the	O
+intrinsically	B-structure_element
+disordered	I-structure_element
+region	I-structure_element
+of	O
+PI4KB	B-protein
+(	O
+residues	O
+423	B-residue_range
+–	I-residue_range
+522	I-residue_range
+)	O
+was	O
+removed	B-experimental_method
+(	O
+Fig	O
+.	O
+1A	O
+).	O
+
+This	O
+internal	O
+deletion	B-experimental_method
+does	O
+not	O
+significantly	O
+affect	O
+the	O
+kinase	B-protein_type
+activity	O
+(	O
+SI	O
+Fig	O
+.	O
+1A	O
+)	O
+or	O
+interaction	O
+with	O
+ACBD3	B-protein
+(	O
+SI	O
+Fig	O
+.	O
+1B	O
+,	O
+C	O
+).	O
+
+In	O
+an	O
+in	B-experimental_method
+vitro	I-experimental_method
+binding	I-experimental_method
+assay	I-experimental_method
+,	O
+ACBD3	B-protein
+co	B-experimental_method
+-	I-experimental_method
+purified	I-experimental_method
+with	I-experimental_method
+the	I-experimental_method
+NiNTA	I-experimental_method
+-	I-experimental_method
+immobilized	I-experimental_method
+N	O
+-	O
+terminal	O
+His6GB1	B-protein_state
+-	I-protein_state
+tagged	I-protein_state
+PI4KB	B-protein
+(	O
+Fig	O
+.	O
+1B	O
+,	O
+left	O
+panel	O
+),	O
+suggesting	O
+a	O
+direct	O
+interaction	O
+.	O
+
+Using	O
+a	O
+mammalian	B-experimental_method
+two	I-experimental_method
+-	I-experimental_method
+hybrid	I-experimental_method
+assay	I-experimental_method
+Greninger	O
+and	O
+colleagues	O
+localized	B-evidence
+this	O
+interaction	O
+to	O
+the	O
+Q	B-structure_element
+domain	I-structure_element
+of	O
+ACBD3	B-protein
+(	O
+named	O
+according	O
+to	O
+its	O
+high	O
+content	O
+of	O
+glutamine	B-residue_name
+residues	O
+)	O
+and	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+PI4KB	B-protein
+preceding	O
+its	O
+helical	B-structure_element
+domain	I-structure_element
+.	O
+
+We	O
+expressed	B-experimental_method
+the	O
+Q	B-structure_element
+domain	I-structure_element
+of	O
+ACBD3	B-protein
+(	O
+residues	O
+241	B-residue_range
+–	I-residue_range
+308	I-residue_range
+)	O
+and	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+PI4KB	B-protein
+(	O
+residues	O
+1	B-residue_range
+–	I-residue_range
+68	I-residue_range
+)	O
+in	O
+E	B-species
+.	I-species
+coli	I-species
+and	O
+using	O
+purified	O
+recombinant	O
+proteins	O
+,	O
+we	O
+confirmed	O
+that	O
+these	O
+two	O
+domains	O
+are	O
+sufficient	O
+to	O
+maintain	O
+the	O
+interaction	O
+(	O
+Fig	O
+.	O
+1B	O
+,	O
+middle	O
+and	O
+right	O
+panel	O
+).	O
+
+Because	O
+it	O
+has	O
+been	O
+reported	O
+that	O
+ACBD3	B-protein
+can	O
+dimerize	B-oligomeric_state
+in	O
+a	O
+mammalian	B-experimental_method
+two	I-experimental_method
+-	I-experimental_method
+hybrid	I-experimental_method
+assay	I-experimental_method
+,	O
+we	O
+were	O
+interested	O
+in	O
+determining	O
+the	O
+stoichiometry	O
+of	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+protein	O
+complex	O
+.	O
+
+The	O
+sedimentation	B-evidence
+coefficients	I-evidence
+of	O
+ACBD3	B-protein
+and	O
+PI4KB	B-protein
+alone	B-protein_state
+,	O
+or	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+complex	O
+were	O
+determined	O
+by	O
+analytical	B-experimental_method
+ultracentrifugation	I-experimental_method
+and	O
+found	O
+to	O
+be	O
+3	O
+.	O
+1	O
+S	O
+,	O
+4	O
+.	O
+1	O
+S	O
+,	O
+and	O
+5	O
+.	O
+1	O
+S	O
+.	O
+These	O
+values	O
+correspond	O
+to	O
+molecular	B-evidence
+weights	I-evidence
+of	O
+approximately	O
+55	O
+kDa	O
+,	O
+80	O
+kDa	O
+,	O
+and	O
+130	O
+kDa	O
+,	O
+respectively	O
+.	O
+
+This	O
+result	O
+suggests	O
+that	O
+both	O
+proteins	O
+are	O
+monomeric	B-oligomeric_state
+and	O
+the	O
+stoichiometry	O
+of	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+protein	O
+complex	O
+is	O
+1	O
+:	O
+1	O
+(	O
+Fig	O
+.	O
+1C	O
+,	O
+left	O
+panel	O
+).	O
+
+Similar	O
+results	O
+were	O
+obtained	O
+for	O
+the	O
+complex	O
+of	O
+the	O
+Q	B-structure_element
+domain	I-structure_element
+of	O
+ACBD3	B-protein
+and	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+PI4KB	B-protein
+(	O
+Fig	O
+.	O
+1C	O
+,	O
+right	O
+panel	O
+).	O
+
+We	O
+also	O
+determined	O
+the	O
+strength	O
+of	O
+the	O
+interaction	O
+between	O
+recombinant	O
+full	B-protein_state
+length	I-protein_state
+ACBD3	B-protein
+and	O
+PI4KB	B-protein
+using	O
+surface	B-experimental_method
+plasmon	I-experimental_method
+resonance	I-experimental_method
+(	O
+SPR	B-experimental_method
+).	O
+
+SPR	B-experimental_method
+measurements	O
+revealed	O
+a	O
+strong	O
+interaction	O
+with	O
+a	O
+Kd	B-evidence
+value	O
+of	O
+320	O
++/−	O
+130	O
+nM	O
+(	O
+Fig	O
+.	O
+1D	O
+,	O
+SI	O
+Fig	O
+.	O
+1D	O
+).	O
+
+We	O
+concluded	O
+that	O
+ACBD3	B-protein
+and	O
+PI4KB	B-protein
+interact	O
+directly	O
+through	O
+the	O
+Q	B-structure_element
+domain	I-structure_element
+of	O
+ACBD3	B-protein
+and	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+PI4KB	B-protein
+forming	O
+a	O
+1	O
+:	O
+1	O
+complex	O
+with	O
+a	O
+dissociation	B-evidence
+constant	I-evidence
+in	O
+the	O
+submicromolar	O
+range	O
+.	O
+
+Structural	B-experimental_method
+analysis	I-experimental_method
+of	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+complex	O
+
+Full	B-protein_state
+length	I-protein_state
+ACBD3	B-protein
+and	O
+PI4KB	B-protein
+both	O
+contain	O
+large	O
+intrinsically	B-structure_element
+disordered	I-structure_element
+regions	I-structure_element
+that	O
+impede	O
+crystallization	O
+.	O
+
+We	O
+used	O
+hydrogen	B-experimental_method
+-	I-experimental_method
+deuterium	I-experimental_method
+exchange	I-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+(	O
+HDX	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+)	O
+analysis	O
+of	O
+the	O
+complex	O
+to	O
+determine	O
+which	O
+parts	O
+of	O
+the	O
+complex	O
+are	O
+well	B-protein_state
+folded	I-protein_state
+(	O
+SI	O
+Fig	O
+.	O
+2	O
+).	O
+
+However	O
+,	O
+we	O
+were	O
+unable	O
+to	O
+obtain	O
+crystals	B-evidence
+even	O
+when	O
+using	O
+significantly	O
+truncated	B-protein_state
+constructs	O
+that	O
+included	O
+only	O
+the	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+and	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+PI4KB	B-protein
+.	O
+
+For	O
+this	O
+reason	O
+,	O
+we	O
+produced	O
+an	O
+isotopically	B-protein_state
+labeled	I-protein_state
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+and	O
+isotopically	B-protein_state
+labeled	I-protein_state
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+:	O
+PI4KB	B-protein
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+protein	O
+complex	O
+and	O
+used	O
+NMR	B-experimental_method
+spectroscopy	I-experimental_method
+for	O
+structural	O
+characterization	O
+.	O
+
+As	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+protein	O
+complex	O
+was	O
+prepared	O
+by	O
+co	B-experimental_method
+-	I-experimental_method
+expression	I-experimental_method
+of	O
+both	O
+proteins	O
+,	O
+the	O
+samples	O
+consisted	O
+of	O
+an	O
+equimolar	O
+mixture	O
+of	O
+two	O
+uniformly	O
+15N	B-chemical
+/	O
+13C	B-chemical
+labelled	B-protein_state
+molecules	O
+.	O
+
+Comprehensive	O
+backbone	O
+and	O
+side	O
+-	O
+chain	O
+resonance	O
+assignments	O
+for	O
+the	O
+free	B-protein_state
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+and	O
+the	O
+complex	O
+,	O
+as	O
+illustrated	O
+by	O
+the	O
+2D	B-experimental_method
+15N	I-experimental_method
+/	I-experimental_method
+1H	I-experimental_method
+HSQC	I-experimental_method
+spectra	B-evidence
+(	O
+SI	O
+Figs	O
+3	O
+and	O
+4	O
+),	O
+were	O
+obtained	O
+using	O
+a	O
+standard	O
+combination	O
+of	O
+triple	B-experimental_method
+-	I-experimental_method
+resonance	I-experimental_method
+experiments	I-experimental_method
+,	O
+as	O
+described	O
+previously	O
+.	O
+
+Backbone	O
+amide	O
+signals	O
+(	O
+15N	B-chemical
+and	O
+1H	B-chemical
+)	O
+for	O
+the	O
+free	B-protein_state
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+were	O
+nearly	O
+completely	O
+assigned	O
+apart	O
+from	O
+the	O
+first	O
+four	O
+N	O
+-	O
+terminal	O
+residues	O
+(	O
+Met1	B-residue_range
+-	I-residue_range
+Lys4	I-residue_range
+)	O
+and	O
+Gln44	B-residue_name_number
+.	O
+
+Over	O
+93	O
+%	O
+of	O
+non	O
+-	O
+exchangeable	O
+side	O
+-	O
+chain	O
+signals	O
+were	O
+assigned	O
+for	O
+the	O
+free	B-protein_state
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+.	O
+
+Apart	O
+from	O
+the	O
+four	O
+N	O
+-	O
+terminal	O
+residues	O
+,	O
+the	O
+side	O
+-	O
+chain	O
+assignments	O
+were	O
+missing	O
+for	O
+Gln	B-residue_name
+(	O
+Hg3	O
+),	O
+Gln	B-residue_name
+(	O
+Ha	O
+/	O
+Hb	O
+/	O
+Hg	O
+),	O
+Gln44	B-residue_name_number
+(	O
+Ha	O
+/	O
+Hb	O
+/	O
+Hg	O
+)	O
+and	O
+Gln48	B-residue_name_number
+(	O
+Hg	O
+)	O
+mainly	O
+due	O
+to	O
+extensive	O
+overlaps	O
+within	O
+the	O
+spectral	O
+regions	O
+populated	O
+by	O
+highly	O
+abundant	O
+glutamine	B-residue_name
+side	O
+-	O
+chain	O
+resonances	O
+.	O
+
+The	O
+protein	O
+complex	O
+yielded	O
+relatively	O
+well	O
+resolved	O
+spectra	B-evidence
+(	O
+SI	O
+Fig	O
+.	O
+4	O
+)	O
+that	O
+resulted	O
+in	O
+assignment	O
+of	O
+backbone	O
+amide	O
+signals	O
+for	O
+all	O
+residues	O
+apart	O
+from	O
+Gln	B-residue_name
+(	O
+ACBD3	B-protein
+)	O
+and	O
+Ala2	B-residue_name_number
+(	O
+PI4KB	B-protein
+).	O
+
+The	O
+essentially	O
+complete	O
+15N	B-chemical
+,	O
+13C	B-chemical
+and	O
+1H	B-chemical
+resonance	O
+assignments	O
+allowed	O
+automated	O
+assignment	O
+of	O
+the	O
+NOEs	B-evidence
+identified	O
+in	O
+the	O
+3D	B-experimental_method
+15N	I-experimental_method
+/	I-experimental_method
+1H	I-experimental_method
+NOESY	I-experimental_method
+-	I-experimental_method
+HSQC	I-experimental_method
+and	O
+13C	B-experimental_method
+/	I-experimental_method
+1H	I-experimental_method
+HMQC	I-experimental_method
+-	I-experimental_method
+NOESY	I-experimental_method
+spectra	B-evidence
+that	O
+were	O
+subsequently	O
+used	O
+in	O
+structural	B-experimental_method
+calculation	I-experimental_method
+.	O
+
+Structural	B-evidence
+statistics	I-evidence
+for	O
+the	O
+final	O
+water	O
+-	O
+refined	O
+sets	O
+of	O
+structures	B-evidence
+are	O
+shown	O
+in	O
+SI	O
+Table	O
+1	O
+.	O
+
+This	O
+structure	B-evidence
+revealed	O
+that	O
+the	O
+Q	B-structure_element
+domain	I-structure_element
+forms	O
+a	O
+two	B-structure_element
+helix	I-structure_element
+hairpin	I-structure_element
+.	O
+
+The	O
+first	O
+helix	B-structure_element
+bends	O
+sharply	O
+over	O
+the	O
+second	O
+helix	B-structure_element
+and	O
+creates	O
+a	O
+fold	O
+resembling	O
+a	O
+three	B-structure_element
+helix	I-structure_element
+bundle	I-structure_element
+that	O
+serves	O
+as	O
+a	O
+nest	O
+for	O
+one	O
+helix	B-structure_element
+of	O
+the	O
+PI4KB	B-protein
+N	O
+-	O
+terminus	O
+(	O
+residues	O
+44	B-residue_range
+–	I-residue_range
+64	I-residue_range
+,	O
+from	O
+this	O
+point	O
+on	O
+referred	O
+to	O
+as	O
+the	O
+kinase	B-structure_element
+helix	I-structure_element
+)	O
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+
+Preceding	O
+the	O
+kinase	B-structure_element
+helix	I-structure_element
+are	O
+three	O
+ordered	O
+residues	O
+(	O
+Val42	B-residue_name_number
+,	O
+Ile43	B-residue_name_number
+,	O
+and	O
+Asp44	B-residue_name_number
+)	O
+that	O
+also	O
+contribute	O
+to	O
+the	O
+interaction	O
+(	O
+Fig	O
+.	O
+2B	O
+).	O
+
+The	O
+remaining	O
+part	O
+of	O
+the	O
+PI4KB	B-protein
+N	O
+-	O
+termini	O
+,	O
+however	O
+,	O
+is	O
+disordered	O
+(	O
+SI	O
+Fig	O
+.	O
+5	O
+).	O
+
+Almost	O
+all	O
+of	O
+the	O
+PI4KB	B-complex_assembly
+:	I-complex_assembly
+ACBD3	I-complex_assembly
+interactions	B-bond_interaction
+are	I-bond_interaction
+hydrophobic	I-bond_interaction
+with	O
+the	O
+exception	O
+of	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+between	O
+the	O
+side	O
+chains	O
+of	O
+ACBD3	B-protein
+Tyr261	B-residue_name_number
+and	O
+PI4KB	B-protein
+His63	B-residue_name_number
+,	O
+and	O
+between	O
+the	O
+sidechain	O
+of	O
+ACBD3	B-protein
+Tyr288	B-residue_name_number
+and	O
+the	O
+PI4KB	B-protein
+backbone	O
+(	O
+Asp44	B-residue_name_number
+)	O
+(	O
+Fig	O
+.	O
+2B	O
+).	O
+
+Interestingly	O
+,	O
+we	O
+noted	O
+that	O
+the	O
+PI4KB	B-protein
+helix	B-structure_element
+is	O
+amphipathic	B-protein_state
+and	O
+its	O
+hydrophobic	B-site
+surface	I-site
+leans	O
+on	O
+the	O
+Q	B-structure_element
+domain	I-structure_element
+(	O
+Fig	O
+.	O
+2C	O
+).	O
+
+To	O
+corroborate	O
+the	O
+structural	B-evidence
+data	I-evidence
+,	O
+we	O
+introduced	B-experimental_method
+a	O
+number	O
+of	O
+point	B-experimental_method
+mutations	I-experimental_method
+and	O
+validated	O
+their	O
+effect	O
+on	O
+complex	O
+formation	O
+using	O
+an	O
+in	B-experimental_method
+vitro	I-experimental_method
+pull	I-experimental_method
+-	I-experimental_method
+down	I-experimental_method
+assay	I-experimental_method
+(	O
+Fig	O
+.	O
+2D	O
+).	O
+
+Wild	B-protein_state
+type	I-protein_state
+ACBD3	B-protein
+protein	O
+co	B-experimental_method
+-	I-experimental_method
+purified	I-experimental_method
+together	O
+with	O
+the	O
+NiNTA	O
+-	O
+immobilized	O
+His6	B-protein_state
+-	I-protein_state
+tagged	I-protein_state
+wild	B-protein_state
+type	I-protein_state
+PI4KB	B-protein
+as	O
+well	O
+as	O
+with	O
+the	O
+PI4KB	B-protein
+V42A	B-mutant
+and	O
+V47A	B-mutant
+mutants	B-protein_state
+,	O
+but	O
+not	O
+with	O
+mutants	B-protein_state
+within	O
+the	O
+imminent	O
+binding	B-site
+interface	I-site
+(	O
+I43A	B-mutant
+,	O
+V55A	B-mutant
+,	O
+L56A	B-mutant
+).	O
+
+As	O
+predicted	O
+,	O
+wild	B-protein_state
+type	I-protein_state
+PI4KB	B-protein
+interacted	O
+with	O
+the	O
+ACBD3	B-protein
+Y266A	B-mutant
+mutant	B-protein_state
+and	O
+slightly	O
+with	O
+the	O
+Y285A	B-mutant
+mutant	B-protein_state
+,	O
+but	O
+not	O
+with	O
+the	O
+F258A	B-mutant
+,	O
+H284A	B-mutant
+,	O
+and	O
+Y288A	B-mutant
+mutants	B-protein_state
+(	O
+Fig	O
+.	O
+2D	O
+).	O
+
+ACBD3	B-protein
+efficiently	O
+recruits	O
+the	O
+PI4KB	B-protein
+enzyme	O
+to	O
+membranes	O
+
+We	O
+next	O
+sought	O
+to	O
+determine	O
+if	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+interaction	O
+drives	O
+membrane	O
+localization	O
+of	O
+the	O
+PI4KB	B-protein
+enzyme	O
+.	O
+
+To	O
+do	O
+this	O
+,	O
+we	O
+first	O
+established	O
+an	O
+in	B-experimental_method
+vitro	I-experimental_method
+membrane	I-experimental_method
+recruitment	I-experimental_method
+system	I-experimental_method
+using	O
+Giant	B-experimental_method
+Unilamellar	I-experimental_method
+Vesicles	I-experimental_method
+(	O
+GUVs	B-experimental_method
+)	O
+containing	O
+the	O
+PI4KB	B-protein
+substrate	O
+–	O
+the	O
+PI	B-chemical
+lipid	O
+.	O
+
+We	O
+observed	O
+that	O
+PI4KB	B-protein
+kinase	B-protein_type
+was	O
+not	O
+membrane	O
+localized	B-evidence
+when	O
+added	O
+to	O
+the	O
+GUVs	B-experimental_method
+at	O
+600	O
+nM	O
+concentration	O
+,	O
+whereas	O
+non	O
+-	O
+covalent	O
+tethering	O
+of	O
+ACBD3	B-protein
+to	O
+the	O
+surface	O
+of	O
+the	O
+GUVs	B-experimental_method
+,	O
+using	O
+the	O
+His6	O
+tag	O
+on	O
+ACBD3	B-protein
+and	O
+the	O
+DGS	B-chemical
+-	I-chemical
+NTA	I-chemical
+(	I-chemical
+Ni	I-chemical
+)	I-chemical
+lipid	I-chemical
+,	O
+led	O
+to	O
+efficient	O
+PI4KB	B-protein
+membrane	O
+localization	O
+(	O
+Fig	O
+.	O
+3A	O
+).	O
+
+We	O
+hypothesized	O
+that	O
+if	O
+ACBD3	B-protein
+is	O
+one	O
+of	O
+the	O
+main	O
+Golgi	O
+localization	B-evidence
+signals	I-evidence
+for	O
+PI4KB	B-protein
+,	O
+overexpression	B-experimental_method
+of	O
+the	O
+Q	B-structure_element
+domain	I-structure_element
+should	O
+decrease	O
+the	O
+amount	O
+of	O
+the	O
+endogenous	O
+kinase	B-protein_type
+on	O
+the	O
+Golgi	O
+.	O
+Indeed	O
+,	O
+we	O
+observed	O
+loss	O
+for	O
+endogenous	O
+PI4KB	B-protein
+signal	O
+on	O
+the	O
+Golgi	O
+in	O
+cells	O
+overexpressing	B-experimental_method
+the	O
+GFP	B-experimental_method
+–	O
+Q	B-structure_element
+domain	I-structure_element
+construct	O
+(	O
+Fig	O
+.	O
+3B	O
+upper	O
+panel	O
+).	O
+
+We	O
+attribute	O
+the	O
+loss	O
+of	O
+signal	B-evidence
+to	O
+the	O
+immunostaining	O
+protocol	O
+-	O
+the	O
+kinase	B-protein_type
+that	O
+is	O
+not	O
+bound	O
+to	O
+Golgi	O
+is	O
+lost	O
+during	O
+the	O
+permeabilization	O
+step	O
+and	O
+hence	O
+the	O
+“	O
+disappearance	O
+”	O
+of	O
+the	O
+signal	B-evidence
+because	O
+overexpression	B-experimental_method
+of	O
+GFP	B-experimental_method
+alone	O
+or	O
+a	O
+non	B-protein_state
+-	I-protein_state
+binding	I-protein_state
+Q	B-structure_element
+domain	I-structure_element
+mutant	B-protein_state
+has	O
+no	O
+effect	O
+on	O
+the	O
+localization	B-evidence
+of	O
+the	O
+endogenous	O
+PI4KB	B-protein
+(	O
+Fig	O
+.	O
+3B	O
+).	O
+
+Given	O
+this	O
+result	O
+,	O
+overexpression	B-experimental_method
+of	O
+the	O
+Q	B-structure_element
+domain	I-structure_element
+should	O
+also	O
+interfere	O
+with	O
+the	O
+PI4KB	B-protein
+dependent	O
+Golgi	O
+functions	O
+.	O
+
+Ceramide	B-chemical
+transport	O
+and	O
+accumulation	O
+in	O
+Golgi	O
+is	O
+a	O
+well	O
+-	O
+known	O
+PI4KB	B-protein
+dependent	O
+process	O
+.	O
+
+We	O
+have	O
+used	O
+fluorescently	B-protein_state
+labeled	I-protein_state
+ceramide	B-chemical
+and	O
+analyzed	O
+its	O
+trafficking	O
+in	O
+non	O
+-	O
+transfected	O
+cells	O
+and	O
+cell	O
+overexpressing	B-experimental_method
+the	O
+Q	B-structure_element
+domain	I-structure_element
+.	O
+
+As	O
+expected	O
+,	O
+the	O
+Golgi	O
+accumulation	O
+of	O
+ceramide	B-chemical
+was	O
+not	O
+observed	O
+in	O
+cells	O
+expressing	B-experimental_method
+the	O
+wt	B-protein_state
+Q	B-structure_element
+domain	I-structure_element
+while	O
+cells	O
+expressing	O
+RFP	B-experimental_method
+or	O
+the	O
+mutant	B-protein_state
+Q	B-structure_element
+domain	I-structure_element
+accumulated	O
+ceramide	B-chemical
+normally	O
+(	O
+Fig	O
+.	O
+3C	O
+)	O
+suggesting	O
+that	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+complex	O
+formation	O
+is	O
+crucial	O
+for	O
+the	O
+normal	O
+function	O
+of	O
+Golgi	O
+.	O
+
+We	O
+further	O
+analyzed	O
+the	O
+function	O
+of	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+interaction	O
+in	O
+membrane	O
+recruitment	O
+of	O
+PI4KB	B-protein
+in	O
+living	O
+cells	O
+using	O
+fluorescently	B-protein_state
+tagged	I-protein_state
+proteins	O
+.	O
+
+We	O
+used	O
+the	O
+rapamycin	B-chemical
+-	O
+inducible	O
+heteromerization	O
+of	O
+FKBP12	B-protein
+(	O
+FK506	B-protein
+binding	I-protein
+protein	I-protein
+12	I-protein
+)	O
+and	O
+FRB	B-structure_element
+(	O
+fragment	B-structure_element
+of	O
+mTOR	B-protein
+that	O
+binds	O
+rapamycin	B-chemical
+)	O
+system	O
+.	O
+
+We	O
+fused	B-experimental_method
+the	O
+FRB	B-structure_element
+to	O
+residues	O
+34	B-residue_range
+–	I-residue_range
+63	I-residue_range
+of	O
+the	O
+mitochondrial	B-structure_element
+localization	I-structure_element
+signal	I-structure_element
+from	O
+mitochondrial	B-protein
+A	I-protein
+-	I-protein
+kinase	I-protein
+anchor	I-protein
+protein	I-protein
+1	I-protein
+(	O
+AKAP1	B-protein
+)	O
+and	O
+CFP	B-experimental_method
+.	O
+
+The	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+was	O
+then	O
+fused	B-experimental_method
+to	I-experimental_method
+FKBP12	B-protein
+and	O
+mRFP	B-experimental_method
+(	O
+Fig	O
+.	O
+3D	O
+).	O
+
+We	O
+analyzed	O
+localization	B-evidence
+of	O
+the	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+and	O
+GFP	B-experimental_method
+–	O
+PI4KB	B-protein
+before	O
+and	O
+after	O
+the	O
+addition	O
+of	O
+rapamycin	B-chemical
+.	O
+
+As	O
+a	O
+control	O
+we	O
+used	O
+H284A	B-mutant
+mutant	B-protein_state
+of	O
+the	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+that	O
+does	O
+not	O
+significantly	O
+bind	O
+PI4KB	B-protein
+kinase	B-protein_type
+.	O
+
+In	O
+every	O
+case	O
+the	O
+ACDB3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+was	O
+rapidly	O
+(	O
+within	O
+5	O
+minutes	O
+)	O
+recruited	O
+to	O
+the	O
+mitochondrial	O
+membrane	O
+upon	O
+addition	O
+of	O
+rapamycin	B-chemical
+,	O
+but	O
+only	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+protein	O
+effectively	O
+directed	O
+the	O
+kinase	B-protein_type
+to	O
+the	O
+mitochondria	O
+(	O
+Fig	O
+.	O
+3E	O
+,	O
+Movie	O
+1	O
+and	O
+2	O
+).	O
+
+Notably	O
+,	O
+we	O
+observed	O
+that	O
+when	O
+the	O
+GFP	B-experimental_method
+-	O
+PI4KB	B-protein
+kinase	B-protein_type
+is	O
+co	B-experimental_method
+-	I-experimental_method
+expressed	I-experimental_method
+with	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+ACDB3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+it	O
+loses	O
+its	O
+typical	O
+Golgi	O
+localization	B-evidence
+(	O
+Fig	O
+.	O
+3E	O
+upper	O
+panel	O
+).	O
+
+However	O
+,	O
+PI4KB	B-protein
+retains	O
+it	O
+Golgi	O
+localization	B-evidence
+when	O
+co	B-experimental_method
+-	I-experimental_method
+expressed	I-experimental_method
+with	O
+the	O
+non	B-protein_state
+-	I-protein_state
+interacting	I-protein_state
+Q	B-structure_element
+domain	I-structure_element
+mutant	B-protein_state
+(	O
+Fig	O
+.	O
+3E	O
+lower	O
+panel	O
+).	O
+
+ACBD3	B-protein
+increases	O
+PI4KB	B-protein
+enzymatic	B-evidence
+activity	I-evidence
+by	O
+recruiting	O
+PI4KB	B-protein
+to	O
+close	O
+vicinity	O
+of	O
+its	O
+substrate	O
+
+To	O
+test	O
+whether	O
+ACBD3	B-protein
+can	O
+stimulate	O
+PI4KB	B-protein
+kinase	B-protein_type
+enzymatic	B-evidence
+activity	I-evidence
+we	O
+performed	O
+a	O
+standard	O
+luminescent	B-experimental_method
+kinase	I-experimental_method
+assay	I-experimental_method
+using	O
+PI	B-chemical
+-	O
+containing	O
+micelles	O
+as	O
+the	O
+substrate	O
+.	O
+
+We	O
+observed	O
+no	O
+effect	O
+on	O
+the	O
+kinase	B-protein_type
+activity	O
+of	O
+PI4KB	B-protein
+(	O
+Fig	O
+.	O
+4A	O
+)	O
+suggesting	O
+that	O
+ACBD3	B-protein
+does	O
+not	O
+directly	O
+affect	O
+the	O
+enzyme	O
+(	O
+e	O
+.	O
+g	O
+.	O
+induction	O
+of	O
+a	O
+conformation	O
+change	O
+).	O
+
+However	O
+,	O
+in	O
+vivo	O
+ACBD3	B-protein
+is	O
+located	O
+at	O
+the	O
+Golgi	O
+membranes	O
+,	O
+whereas	O
+in	O
+this	O
+experiment	O
+,	O
+ACBD3	B-protein
+was	O
+located	O
+in	O
+the	O
+solution	O
+and	O
+PI	B-chemical
+is	O
+provided	O
+as	O
+micelles	O
+.	O
+
+For	O
+this	O
+,	O
+we	O
+again	O
+turned	O
+to	O
+the	O
+GUV	B-experimental_method
+system	O
+with	O
+ACBD3	B-protein
+localized	B-evidence
+to	O
+the	O
+GUV	B-experimental_method
+membrane	O
+.	O
+
+The	O
+GUVs	B-experimental_method
+contained	O
+10	O
+%	O
+PI	B-chemical
+to	O
+serve	O
+as	O
+a	O
+substrate	O
+for	O
+PI4KB	B-protein
+kinase	B-protein_type
+.	O
+
+The	O
+buffer	O
+also	O
+contained	O
+CFP	B-experimental_method
+-	O
+SidC	B-protein
+,	O
+which	O
+binds	O
+to	O
+PI4P	B-chemical
+with	O
+nanomolar	O
+affinity	O
+.	O
+
+This	O
+enabled	O
+visualization	O
+of	O
+the	O
+kinase	B-protein_type
+reaction	O
+using	O
+a	O
+confocal	B-experimental_method
+microscope	I-experimental_method
+.	O
+
+We	O
+compared	O
+the	O
+efficiency	O
+of	O
+the	O
+phosphorylation	B-ptm
+reaction	O
+of	O
+the	O
+kinase	B-protein_type
+alone	B-protein_state
+with	O
+that	O
+of	O
+kinase	B-protein_type
+recruited	O
+to	O
+the	O
+surface	O
+of	O
+the	O
+GUVs	B-experimental_method
+by	O
+ACBD3	B-protein
+.	O
+
+Reaction	O
+was	O
+also	O
+performed	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+ATP	B-chemical
+as	O
+a	O
+negative	O
+control	O
+(	O
+Fig	O
+.	O
+4B	O
+).	O
+
+These	O
+experiments	O
+showed	O
+that	O
+PI4KB	B-protein
+enzymatic	B-evidence
+activity	I-evidence
+increases	O
+when	O
+ACBD3	B-protein
+is	O
+membrane	O
+localized	O
+(	O
+Fig	O
+.	O
+4C	O
+,	O
+SI	O
+Fig	O
+.	O
+6	O
+).	O
+
+Membrane	O
+recruitment	O
+of	O
+PI4KB	B-protein
+enzyme	O
+is	O
+crucial	O
+to	O
+ensure	O
+its	O
+proper	O
+function	O
+at	O
+the	O
+Golgi	O
+and	O
+TGN	O
+.	O
+
+However	O
+,	O
+the	O
+molecular	O
+mechanism	O
+and	O
+structural	O
+basis	O
+for	O
+PI4KB	B-protein
+interaction	O
+with	O
+the	O
+membrane	O
+is	O
+poorly	O
+understood	O
+.	O
+
+In	O
+principle	O
+,	O
+any	O
+of	O
+the	O
+binding	O
+partners	O
+of	O
+PI4KB	B-protein
+could	O
+play	O
+a	O
+role	O
+in	O
+membrane	O
+recruitment	O
+.	O
+
+To	O
+date	O
+,	O
+several	O
+PI4KB	B-protein
+interacting	O
+proteins	O
+have	O
+been	O
+reported	O
+,	O
+including	O
+the	O
+small	B-protein_type
+GTPases	I-protein_type
+Rab11	B-protein
+and	O
+Arf1	B-protein
+,	O
+the	O
+Golgi	O
+resident	O
+acyl	B-protein
+-	I-protein
+CoA	I-protein
+binding	I-protein
+domain	I-protein
+containing	I-protein
+3	I-protein
+(	O
+ACBD3	B-protein
+)	O
+protein	O
+,	O
+neuronal	B-protein
+calcium	I-protein
+sensor	I-protein
+-	I-protein
+1	I-protein
+(	O
+NCS	B-protein
+-	I-protein
+1	I-protein
+also	O
+known	O
+as	O
+frequenin	B-protein
+in	O
+yeast	B-taxonomy_domain
+)	O
+and	O
+the	O
+14	B-protein_type
+-	I-protein_type
+3	I-protein_type
+-	I-protein_type
+3	I-protein_type
+proteins	I-protein_type
+.	O
+
+The	O
+monomeric	B-oligomeric_state
+G	B-protein_type
+protein	I-protein_type
+Rab11	B-protein
+binds	O
+mammalian	B-taxonomy_domain
+PI4KB	B-protein
+through	O
+the	O
+helical	B-structure_element
+domain	I-structure_element
+of	O
+the	O
+kinase	B-protein_type
+.	O
+
+Although	O
+Rab11	B-protein
+does	O
+not	O
+appear	O
+to	O
+be	O
+required	O
+for	O
+recruitment	O
+of	O
+PI4KB	B-protein
+to	O
+the	O
+Golgi	O
+,	O
+PI4KB	B-protein
+is	O
+required	O
+for	O
+Golgi	O
+recruitment	O
+of	O
+Rab11	B-protein
+.	O
+
+Arf1	B-protein
+,	O
+the	O
+other	O
+small	B-protein_type
+GTP	I-protein_type
+binding	I-protein_type
+protein	I-protein_type
+,	O
+is	O
+known	O
+to	O
+influence	O
+the	O
+activity	O
+and	O
+localization	O
+of	O
+PI4KB	B-protein
+,	O
+but	O
+it	O
+does	O
+not	O
+appear	O
+to	O
+interact	O
+directly	O
+with	O
+PI4KB	B-protein
+(	O
+our	O
+unpublished	O
+data	O
+).	O
+
+The	O
+yeast	B-taxonomy_domain
+homologue	O
+of	O
+NCS1	B-protein
+called	O
+frequenin	B-protein
+has	O
+been	O
+shown	O
+to	O
+interact	O
+with	O
+Pik1p	B-protein
+,	O
+the	O
+yeast	B-taxonomy_domain
+orthologue	O
+of	O
+PI4KB	B-protein
+and	O
+regulate	O
+its	O
+activity	O
+and	O
+perhaps	O
+its	O
+membrane	O
+association	O
+,	O
+but	O
+the	O
+role	O
+of	O
+NCS	B-protein
+-	I-protein
+1	I-protein
+in	O
+PI4KB	B-protein
+recruitment	O
+in	O
+mammalian	B-taxonomy_domain
+cells	O
+is	O
+unclear	O
+.	O
+
+NCS	B-protein
+-	I-protein
+1	I-protein
+is	O
+an	O
+N	O
+-	O
+terminally	O
+myristoylated	B-protein_state
+protein	O
+that	O
+participates	O
+in	O
+exocytosis	O
+.	O
+
+It	O
+is	O
+expressed	O
+only	O
+in	O
+certain	O
+cell	O
+types	O
+,	O
+suggesting	O
+that	O
+if	O
+it	O
+contributes	O
+to	O
+PI4KB	B-protein
+membrane	O
+recruitment	O
+,	O
+it	O
+does	O
+so	O
+in	O
+a	O
+tissues	O
+specific	O
+manner	O
+.	O
+
+The	O
+interaction	O
+of	O
+PI4KB	B-protein
+with	O
+14	B-protein_type
+-	I-protein_type
+3	I-protein_type
+-	I-protein_type
+3	I-protein_type
+proteins	I-protein_type
+,	O
+promoted	O
+by	O
+phosphorylation	B-ptm
+of	O
+PI4KB	B-protein
+by	O
+protein	B-protein
+kinase	I-protein
+D	I-protein
+,	O
+influences	O
+the	O
+activity	O
+of	O
+PI4KB	B-protein
+by	O
+stabilizing	O
+its	O
+active	B-protein_state
+conformation	O
+.	O
+
+However	O
+,	O
+14	B-protein_type
+-	I-protein_type
+3	I-protein_type
+-	I-protein_type
+3	I-protein_type
+proteins	I-protein_type
+do	O
+not	O
+appear	O
+to	O
+interfere	O
+with	O
+membrane	O
+recruitment	O
+of	O
+this	O
+kinase	B-protein_type
+.	O
+
+ACBD3	B-protein
+is	O
+a	O
+Golgi	O
+resident	O
+protein	O
+,	O
+conserved	B-protein_state
+among	O
+vertebrates	B-taxonomy_domain
+(	O
+SI	O
+Fig	O
+.	O
+7	O
+),	O
+that	O
+interacts	O
+directly	O
+with	O
+PI4KB	B-protein
+(	O
+see	O
+also	O
+SI	O
+Fig	O
+.	O
+8	O
+and	O
+SI	O
+Discussion	O
+),	O
+and	O
+whose	O
+genetic	O
+inactivation	O
+interferes	O
+with	O
+the	O
+Golgi	O
+localization	O
+of	O
+the	O
+kinase	B-protein_type
+.	O
+
+For	O
+these	O
+reasons	O
+we	O
+focused	O
+on	O
+the	O
+interaction	O
+of	O
+the	O
+PI4KB	B-protein
+enzyme	O
+with	O
+the	O
+Golgi	O
+resident	O
+ACBD3	B-protein
+protein	O
+in	O
+this	O
+study	O
+.	O
+
+Here	O
+we	O
+present	O
+the	O
+mechanism	O
+for	O
+membrane	O
+recruitment	O
+of	O
+PI4KB	B-protein
+by	O
+the	O
+Golgi	O
+resident	O
+ACBD3	B-protein
+protein	O
+.	O
+
+We	O
+show	O
+that	O
+these	O
+proteins	O
+interact	O
+directly	O
+with	O
+a	O
+Kd	B-evidence
+value	O
+in	O
+the	O
+submicromolar	O
+range	O
+.	O
+
+The	O
+interaction	O
+is	O
+sufficient	O
+to	O
+recruit	O
+PI4KB	B-protein
+to	O
+model	O
+membranes	O
+in	O
+vitro	O
+as	O
+well	O
+as	O
+to	O
+the	O
+mitochondria	O
+where	O
+PI4KB	B-protein
+is	O
+never	O
+naturally	O
+found	O
+.	O
+
+To	O
+understand	O
+this	O
+process	O
+at	O
+the	O
+atomic	O
+level	O
+we	O
+solved	B-experimental_method
+the	O
+solution	B-evidence
+structure	I-evidence
+of	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+sub	O
+complex	O
+(	O
+Fig	O
+.	O
+1A	O
+)	O
+and	O
+found	O
+that	O
+the	O
+PI4KB	B-protein
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+contains	O
+a	O
+short	B-structure_element
+amphipatic	I-structure_element
+helix	I-structure_element
+(	O
+residues	O
+44	B-residue_range
+–	I-residue_range
+64	I-residue_range
+)	O
+that	O
+binds	O
+the	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+.	O
+
+The	O
+Q	B-structure_element
+domain	I-structure_element
+adopts	O
+a	O
+helical	B-structure_element
+hairpin	I-structure_element
+fold	I-structure_element
+that	O
+is	O
+further	O
+stabilized	O
+upon	O
+binding	O
+the	O
+kinase	B-structure_element
+helix	I-structure_element
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+
+Our	O
+data	O
+strongly	O
+suggest	O
+that	O
+formation	O
+of	O
+the	O
+complex	O
+does	O
+not	O
+directly	O
+influence	O
+the	O
+catalytic	O
+abilities	O
+of	O
+the	O
+kinase	B-protein_type
+but	O
+experiments	O
+with	O
+model	O
+membranes	O
+revealed	O
+that	O
+ACBD3	B-protein
+enhances	O
+catalytic	O
+activity	O
+of	O
+the	O
+kinase	B-protein_type
+by	O
+a	O
+recruitment	O
+based	O
+mechanism	O
+;	O
+it	O
+recruits	O
+the	O
+kinase	B-protein_type
+to	O
+the	O
+membrane	O
+and	O
+thus	O
+increases	O
+the	O
+local	O
+concentration	O
+of	O
+the	O
+substrate	O
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+kinase	B-protein_type
+.	O
+
+Based	O
+on	O
+our	O
+and	O
+previously	O
+published	O
+structures	B-evidence
+we	O
+built	O
+a	O
+pseudoatomic	B-evidence
+model	I-evidence
+of	O
+PI4KB	B-protein
+multi	O
+-	O
+protein	O
+assembly	O
+on	O
+the	O
+membrane	O
+(	O
+Fig	O
+.	O
+5	O
+)	O
+that	O
+illustrates	O
+how	O
+the	O
+enzyme	O
+is	O
+recruited	O
+and	O
+positioned	O
+towards	O
+its	O
+lipidic	O
+substrate	O
+and	O
+how	O
+it	O
+in	O
+turn	O
+recruits	O
+Rab11	B-protein
+.	O
+
++	B-taxonomy_domain
+RNA	I-taxonomy_domain
+viruses	I-taxonomy_domain
+replicate	O
+at	O
+specific	O
+PI4P	B-chemical
+-	O
+enriched	O
+membranous	O
+compartments	O
+.	O
+
+These	O
+are	O
+called	O
+replication	O
+factories	O
+(	O
+because	O
+they	O
+enhance	O
+viral	B-taxonomy_domain
+replication	O
+)	O
+or	O
+membranous	O
+webs	O
+(	O
+because	O
+of	O
+their	O
+appearance	O
+under	O
+the	O
+electron	O
+microscope	O
+).	O
+
+To	O
+generate	O
+replication	O
+factories	O
+,	O
+viruses	B-taxonomy_domain
+hijack	O
+several	O
+host	O
+factors	O
+including	O
+the	O
+PI4K	B-protein_type
+kinases	B-protein_type
+to	O
+secure	O
+high	O
+content	O
+of	O
+the	O
+PI4P	B-chemical
+lipid	B-chemical
+.	O
+
+Non	B-protein_type
+-	I-protein_type
+structural	I-protein_type
+3A	I-protein_type
+proteins	I-protein_type
+from	O
+many	O
+picornaviruses	B-taxonomy_domain
+from	O
+the	O
+Enterovirus	B-taxonomy_domain
+(	O
+e	O
+.	O
+g	O
+.	O
+poliovirus	B-species
+,	O
+coxsackievirus	B-species
+-	I-species
+B3	I-species
+,	O
+rhinovirus	B-species
+-	I-species
+14	I-species
+)	O
+and	O
+Kobuvirus	B-taxonomy_domain
+(	O
+e	O
+.	O
+g	O
+.	O
+Aichi	B-species
+virus	I-species
+-	I-species
+1	I-species
+)	O
+genera	O
+directly	O
+interact	O
+with	O
+ACBD3	B-protein
+.	O
+
+Our	O
+data	O
+suggest	O
+that	O
+they	O
+could	O
+do	O
+this	O
+via	O
+3A	B-complex_assembly
+:	I-complex_assembly
+ACBD3	I-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+complex	O
+formation	O
+.	O
+
+The	O
+structure	B-evidence
+of	O
+the	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+and	O
+the	O
+kinase	B-structure_element
+helix	I-structure_element
+described	O
+here	O
+provides	O
+a	O
+novel	O
+opportunity	O
+for	O
+further	O
+research	O
+on	O
+the	O
+role	O
+of	O
+ACBD3	B-protein
+,	O
+PI4KB	B-protein
+,	O
+and	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+interaction	O
+in	O
+picornaviral	B-taxonomy_domain
+replication	O
+.	O
+
+This	O
+could	O
+eventually	O
+have	O
+implications	O
+for	O
+therapeutic	O
+intervention	O
+to	O
+combat	O
+picornaviruses	B-taxonomy_domain
+-	O
+mediated	O
+diseases	O
+ranging	O
+from	O
+polio	O
+to	O
+the	O
+common	O
+cold	O
+.	O
+
+Biochemical	B-experimental_method
+characterization	I-experimental_method
+of	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+complex	O
+.	O
+
+(	O
+A	O
+)	O
+Schematic	O
+representation	O
+of	O
+the	O
+ACBD3	B-protein
+and	O
+PI4KB	B-protein
+constructs	O
+used	O
+for	O
+the	O
+experiments	O
+.	O
+
+ACBD3	B-protein
+contains	O
+the	O
+acyl	B-structure_element
+-	I-structure_element
+CoA	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+(	O
+ACBD	B-structure_element
+),	O
+charged	B-structure_element
+amino	I-structure_element
+acids	I-structure_element
+region	I-structure_element
+(	O
+CAR	B-structure_element
+),	O
+glutamine	B-structure_element
+rich	I-structure_element
+region	I-structure_element
+(	O
+Q	B-structure_element
+),	O
+and	O
+Golgi	B-structure_element
+dynamics	I-structure_element
+domain	I-structure_element
+(	O
+GOLD	B-structure_element
+).	O
+
+PI4KB	B-protein
+is	O
+composed	O
+of	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+,	O
+helical	B-structure_element
+domain	I-structure_element
+,	O
+and	O
+kinase	B-structure_element
+domain	I-structure_element
+which	O
+can	O
+be	O
+divided	O
+into	O
+N	B-structure_element
+-	I-structure_element
+and	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+lobes	I-structure_element
+.	O
+
+(	O
+B	O
+)	O
+In	B-experimental_method
+vitro	I-experimental_method
+pull	I-experimental_method
+-	I-experimental_method
+down	I-experimental_method
+assay	I-experimental_method
+.	O
+
+Pull	B-experimental_method
+-	I-experimental_method
+down	I-experimental_method
+assays	I-experimental_method
+were	O
+performed	O
+using	O
+NiNTA	O
+-	O
+immobilized	O
+N	O
+-	O
+terminal	O
+His6GB1	B-protein_state
+-	I-protein_state
+tagged	I-protein_state
+proteins	O
+as	O
+indicated	O
+and	O
+untagged	B-protein_state
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+PI4KB	B-protein
+or	O
+ACBD3	B-protein
+.	O
+
+The	O
+inputs	O
+and	O
+bound	O
+proteins	O
+were	O
+analyzed	O
+on	O
+SDS	B-experimental_method
+gels	I-experimental_method
+stained	O
+with	O
+Coomassie	O
+Blue	O
+.	O
+
+Please	O
+,	O
+see	O
+SI	O
+Fig	O
+.	O
+9	O
+for	O
+original	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+gels	O
+.	O
+(	O
+C	O
+)	O
+Analytical	B-experimental_method
+Ultracentrifugation	I-experimental_method
+.	O
+
+AUC	B-experimental_method
+analysis	O
+of	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+complex	O
+at	O
+the	O
+concentration	O
+of	O
+5	O
+μM	O
+(	O
+both	O
+proteins	O
+,	O
+left	O
+panel	O
+)	O
+and	O
+ACBD3	B-complex_assembly
+Q	I-complex_assembly
+domain	I-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+N	I-complex_assembly
+terminal	I-complex_assembly
+region	I-complex_assembly
+complex	O
+at	O
+the	O
+concentration	O
+of	O
+35	O
+μM	O
+(	O
+both	O
+proteins	O
+,	O
+right	O
+panel	O
+).	O
+(	O
+D	O
+)	O
+Surface	B-experimental_method
+plasmon	I-experimental_method
+resonance	I-experimental_method
+.	O
+
+SPR	B-experimental_method
+analysis	O
+of	O
+the	O
+PI4KB	B-protein
+binding	O
+to	O
+immobilized	O
+ACBD3	B-protein
+.	O
+
+Sensorgrams	B-evidence
+for	O
+four	O
+concentrations	O
+of	O
+PI4KB	B-protein
+are	O
+shown	O
+.	O
+
+Structural	B-experimental_method
+analysis	I-experimental_method
+of	O
+the	O
+ACBD3	B-complex_assembly
+:	I-complex_assembly
+PI4KB	I-complex_assembly
+complex	O
+.	O
+
+(	O
+A	O
+)	O
+Overall	O
+structure	B-evidence
+of	O
+the	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+by	O
+itself	O
+and	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+PI4KB	B-protein
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+.	O
+
+Superposition	B-experimental_method
+of	O
+the	O
+30	O
+converged	O
+structures	B-evidence
+obtained	O
+for	O
+the	O
+Q	B-structure_element
+domain	I-structure_element
+(	O
+top	O
+)	O
+and	O
+the	O
+45	O
+converged	O
+structures	B-evidence
+obtained	O
+for	O
+the	O
+complex	O
+(	O
+bottom	O
+),	O
+with	O
+only	O
+the	O
+folded	B-protein_state
+part	O
+of	O
+PI4KB	B-protein
+shown	O
+(	O
+see	O
+SI	O
+Fig	O
+.	O
+2	O
+for	O
+the	O
+complete	O
+view	O
+).	O
+(	O
+B	O
+)	O
+Detailed	O
+view	O
+of	O
+the	O
+complex	O
+.	O
+
+The	O
+interaction	O
+is	O
+facilitated	O
+by	O
+only	O
+two	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+(	O
+ACBD3	B-protein
+Tyr261	B-residue_name_number
+:	O
+PI4KB	B-protein
+His63	B-residue_name_number
+and	O
+ACBD3	B-protein
+Tyr288	B-residue_name_number
+:	O
+PI4KB	B-protein
+Asp44	B-residue_name_number
+),	O
+while	O
+the	O
+hydrophobic	B-site
+surface	I-site
+of	O
+the	O
+kinase	B-structure_element
+helix	I-structure_element
+nests	O
+in	O
+the	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+.	O
+
+ACBD3	B-protein
+is	O
+shown	O
+in	O
+magenta	O
+and	O
+PI4KB	B-protein
+in	O
+orange	O
+.	O
+
+(	O
+C	O
+)	O
+Top	O
+view	O
+of	O
+the	O
+kinase	B-structure_element
+helix	I-structure_element
+.	O
+
+The	O
+kinase	B-structure_element
+helix	I-structure_element
+is	O
+amphipathic	B-protein_state
+and	O
+its	O
+hydrophobic	B-site
+surface	I-site
+overlaps	O
+with	O
+the	O
+ACBD3	B-protein
+binding	B-site
+surface	I-site
+(	O
+shown	O
+in	O
+magenta	O
+).	O
+
+Strong	O
+and	O
+weak	O
+hydrophobes	O
+are	O
+in	O
+green	O
+and	O
+cyan	O
+respectively	O
+,	O
+basic	O
+residues	O
+in	O
+blue	O
+,	O
+acidic	O
+residues	O
+in	O
+red	O
+and	O
+nonpolar	O
+hydrophilic	O
+residues	O
+in	O
+orange	O
+.	O
+(	O
+D	O
+)	O
+Pull	B-experimental_method
+-	I-experimental_method
+down	I-experimental_method
+assay	I-experimental_method
+with	O
+a	O
+NiNTA	O
+-	O
+immobilized	O
+N	O
+-	O
+terminally	O
+His6GB1	B-protein_state
+-	I-protein_state
+tagged	I-protein_state
+PI4KB	B-protein
+kinase	B-protein_type
+and	O
+untagged	B-protein_state
+ACBD3	B-protein
+protein	O
+.	O
+
+Wild	B-protein_state
+type	I-protein_state
+proteins	O
+and	O
+selected	O
+point	O
+mutants	B-protein_state
+of	O
+both	O
+PI4KB	B-protein
+and	O
+ACBD3	B-protein
+were	O
+used	O
+.	O
+
+Please	O
+,	O
+see	O
+SI	O
+Fig	O
+.	O
+9	O
+for	O
+original	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+gels	O
+.	O
+
+ACBD3	B-protein
+is	O
+sufficient	O
+to	O
+recruit	O
+the	O
+PI4KB	B-protein
+kinase	B-protein_type
+to	O
+membranes	O
+.	O
+
+(	O
+A	O
+)	O
+GUVs	B-experimental_method
+recruitment	I-experimental_method
+assay	I-experimental_method
+.	O
+
+Top	O
+–	O
+Virtually	O
+no	O
+membrane	O
+bound	O
+kinase	B-protein_type
+was	O
+observed	O
+when	O
+600	O
+nM	O
+PI4KB	B-protein
+was	O
+added	O
+to	O
+the	O
+GUVs	B-experimental_method
+.	O
+
+Bottom	O
+–	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+600	O
+nM	O
+GUV	B-protein_state
+tethered	I-protein_state
+ACBD3	B-protein
+a	O
+significant	O
+signal	O
+of	O
+the	O
+kinase	B-protein_type
+is	O
+detected	O
+on	O
+the	O
+surface	O
+of	O
+GUVs	B-experimental_method
+.	O
+
+(	O
+B	O
+)	O
+Golgi	B-experimental_method
+displacement	I-experimental_method
+experiment	I-experimental_method
+.	O
+
+Upper	O
+panel	O
+:	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+fused	O
+to	O
+GFP	B-experimental_method
+was	O
+overexpressed	B-experimental_method
+and	O
+the	O
+endogenous	O
+PI4KB	B-protein
+was	O
+immunostained	B-experimental_method
+.	O
+
+Middle	O
+panel	O
+:	O
+The	O
+same	O
+experiment	O
+performed	O
+with	O
+GFP	B-experimental_method
+alone	O
+.	O
+
+Lower	O
+panel	O
+:	O
+The	O
+same	O
+experiment	O
+performed	O
+with	O
+mutant	B-protein_state
+Q	B-structure_element
+domain	I-structure_element
+(	O
+F258A	B-mutant
+,	O
+H284A	B-mutant
+,	O
+Y288A	B-mutant
+)	O
+that	O
+does	O
+not	O
+bind	O
+the	O
+PI4KB	B-protein
+.	O
+(	O
+C	O
+)	O
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+overexpression	B-experimental_method
+inhibits	O
+ceramide	B-chemical
+transport	O
+to	O
+Golgi	O
+–	O
+COS	O
+-	O
+7	O
+cells	O
+transfected	O
+with	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+ACBD3	B-protein
+Q	B-structure_element
+domain	I-structure_element
+-	O
+FKBP	B-protein
+-	O
+mRFP	B-experimental_method
+were	O
+loaded	O
+with	O
+0	O
+.	O
+05	O
+μM	O
+Bodipy	B-chemical
+FL	I-chemical
+-	I-chemical
+Ceramide	I-chemical
+for	O
+20	O
+min	O
+,	O
+then	O
+washed	O
+and	O
+depicted	O
+after	O
+20	O
+min	O
+.	O
+
+Middle	O
+panel	O
+–	O
+The	O
+same	O
+experiment	O
+performed	O
+with	O
+mRFP	B-experimental_method
+-	O
+FKBP	B-protein
+alone	O
+.	O
+
+Lower	O
+panel	O
+–	O
+The	O
+same	O
+experiment	O
+performed	O
+with	O
+mutant	B-protein_state
+Q	B-structure_element
+domain	I-structure_element
+(	O
+F258A	B-mutant
+,	O
+H284A	B-mutant
+,	O
+Y288A	B-mutant
+)	O
+that	O
+does	O
+not	O
+bind	O
+the	O
+PI4KB	B-protein
+.	O
+(	O
+D	O
+)	O
+Scheme	O
+of	O
+the	O
+mitochondria	B-experimental_method
+recruitment	I-experimental_method
+experiment	I-experimental_method
+.	O
+
+–	O
+The	O
+AKAP1	B-protein
+-	O
+FRB	B-structure_element
+-	O
+CFP	B-experimental_method
+construct	O
+is	O
+localized	B-evidence
+at	O
+the	O
+outer	O
+mitochondrial	O
+membrane	O
+,	O
+while	O
+the	O
+GFP	B-experimental_method
+-	O
+PI4KB	B-protein
+and	O
+Q	B-structure_element
+domain	I-structure_element
+-	O
+FKBP	B-protein
+-	O
+mRFP	B-experimental_method
+constructs	O
+are	O
+localized	B-evidence
+in	O
+the	O
+cytoplasm	O
+where	O
+they	O
+can	O
+form	O
+a	O
+complex	O
+.	O
+
+Upon	O
+addition	O
+of	O
+rapamycin	B-chemical
+the	O
+Q	B-structure_element
+domain	I-structure_element
+-	O
+FKBP	B-protein
+-	O
+mRFP	B-experimental_method
+construct	O
+translocates	O
+to	O
+the	O
+mitochondria	O
+and	O
+takes	O
+GFP	B-experimental_method
+-	O
+PI4KB	B-protein
+with	O
+it	O
+.	O
+(	O
+E	O
+)	O
+Mitochondria	B-experimental_method
+recruitment	I-experimental_method
+experiment	I-experimental_method
+.	O
+
+Left	O
+–	O
+cells	O
+transfected	O
+with	O
+AKAP1	B-protein
+-	O
+FRB	B-structure_element
+-	O
+CFP	B-experimental_method
+,	O
+GFP	B-experimental_method
+-	O
+PI4KB	B-protein
+and	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+Q	B-structure_element
+domain	I-structure_element
+-	O
+FKBP	B-protein
+-	O
+mRFP	B-experimental_method
+constructs	O
+before	O
+and	O
+five	O
+minutes	O
+after	O
+addition	O
+of	O
+rapamycin	B-chemical
+.	O
+
+Right	O
+–	O
+The	O
+same	O
+experiment	O
+performed	O
+using	O
+the	O
+H264A	B-mutant
+Q	B-structure_element
+domain	I-structure_element
+mutant	B-protein_state
+.	O
+
+ACBD3	B-protein
+indirectly	O
+increases	O
+the	O
+activity	O
+of	O
+PI4KB	B-protein
+.	O
+
+(	O
+A	O
+)	O
+Micelles	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+kinase	I-experimental_method
+assay	I-experimental_method
+–	O
+PI	B-chemical
+in	O
+TX100	O
+micelles	O
+was	O
+used	O
+in	O
+a	O
+luminescent	B-experimental_method
+kinase	I-experimental_method
+assay	I-experimental_method
+and	O
+the	O
+production	O
+of	O
+PI4P	B-chemical
+was	O
+measured	O
+.	O
+
+Bar	O
+graph	O
+presents	O
+the	O
+mean	O
+values	O
+of	O
+PI4P	B-chemical
+generated	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+proteins	O
+as	O
+indicated	O
+,	O
+normalized	O
+to	O
+the	O
+amount	O
+of	O
+PI4P	B-chemical
+generated	O
+by	O
+PI4KB	B-protein
+alone	O
+.	O
+
+Error	O
+bars	O
+are	O
+standard	B-evidence
+errors	I-evidence
+of	I-evidence
+the	I-evidence
+mean	I-evidence
+(	O
+SEM	B-evidence
+)	O
+based	O
+on	O
+three	O
+independent	O
+experiments	O
+.	O
+(	O
+B	O
+)	O
+GUV	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+phosphorylation	I-experimental_method
+assay	I-experimental_method
+–	O
+GUVs	B-experimental_method
+containing	O
+10	O
+%	O
+PI	B-chemical
+were	O
+used	O
+as	O
+a	O
+substrate	O
+and	O
+the	O
+production	O
+of	O
+PI4P	B-chemical
+was	O
+measured	O
+using	O
+the	O
+CFP	B-experimental_method
+-	I-experimental_method
+SidC	I-experimental_method
+biosensor	I-experimental_method
+.	O
+
+(	O
+C	O
+)–	O
+Quantification	O
+of	O
+the	O
+GUV	B-experimental_method
+phosphorylation	I-experimental_method
+assay	I-experimental_method
+–	O
+Mean	B-evidence
+membrane	I-evidence
+fluorescence	I-evidence
+intensity	I-evidence
+of	O
+the	O
+PI4P	B-chemical
+reporter	O
+(	O
+SidC	B-protein
+-	O
+label	O
+)	O
+under	O
+different	O
+protein	O
+/	O
+ATP	B-chemical
+conditions	O
+.	O
+
+The	O
+mean	B-evidence
+membrane	I-evidence
+intensity	I-evidence
+value	O
+is	O
+relative	O
+to	O
+the	O
+background	O
+signal	O
+and	O
+the	O
+difference	O
+between	O
+the	O
+membrane	O
+and	O
+background	O
+signal	O
+in	O
+the	O
+reference	O
+system	O
+lacking	O
+ATP	B-chemical
+.	O
+
+The	O
+error	O
+bars	O
+stand	O
+for	O
+SEM	B-evidence
+based	O
+on	O
+three	O
+independent	O
+experiments	O
+(	O
+also	O
+SI	O
+Fig	O
+.	O
+6	O
+).	O
+
+Pseudoatomic	B-evidence
+model	I-evidence
+of	O
+the	O
+PI4KB	B-protein
+multiprotein	O
+complex	O
+assembly	O
+.	O
+
+PI4KB	B-protein
+in	O
+orange	O
+,	O
+Rab11	B-protein
+in	O
+purple	O
+,	O
+ACBD3	B-protein
+in	O
+blue	O
+.	O
+
+The	O
+model	O
+is	O
+based	O
+on	O
+our	O
+NMR	B-experimental_method
+structure	B-evidence
+and	O
+a	O
+previously	O
+published	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+PI4KB	B-complex_assembly
+:	I-complex_assembly
+Rab11	I-complex_assembly
+complex	O
+(	O
+PDB	O
+code	O
+4D0L	O
+),	O
+ACBD	B-structure_element
+and	O
+GOLD	B-structure_element
+domain	O
+were	O
+homology	B-experimental_method
+modeled	I-experimental_method
+based	O
+on	O
+high	O
+sequence	O
+identity	O
+structures	B-evidence
+produced	O
+by	O
+the	O
+Phyre2	B-experimental_method
+web	O
+server	O
+.	O
+
+The	O
+GOLD	B-structure_element
+domain	O
+is	O
+tethered	O
+to	O
+the	O
+membrane	O
+by	O
+GolginB1	B-protein
+(	O
+also	O
+known	O
+as	O
+Giantin	B-protein
+)	O
+which	O
+is	O
+not	O
+shown	O
+for	O
+clarity	O
+.	O
+
+Intrinsically	B-structure_element
+disordered	I-structure_element
+linkers	I-structure_element
+are	O
+modeled	O
+in	O
+an	O
+arbitrary	O
+but	O
+physically	O
+plausible	O
+conformation	O
+.	O
+
diff --git a/annotation_IOB/PMC4817029.tsv b/annotation_IOB/PMC4817029.tsv
new file mode 100644
index 0000000000000000000000000000000000000000..b54f4236e1cec7731479d922b436dc99461cf415
--- /dev/null
+++ b/annotation_IOB/PMC4817029.tsv
@@ -0,0 +1,6158 @@
+Molecular	O
+characterization	O
+of	O
+a	O
+family	B-protein_type
+5	I-protein_type
+glycoside	I-protein_type
+hydrolase	I-protein_type
+suggests	O
+an	O
+induced	O
+-	O
+fit	O
+enzymatic	O
+mechanism	O
+
+Glycoside	B-protein_type
+hydrolases	I-protein_type
+(	O
+GHs	B-protein_type
+)	O
+play	O
+fundamental	O
+roles	O
+in	O
+the	O
+decomposition	O
+of	O
+lignocellulosic	O
+biomaterials	O
+.	O
+
+Here	O
+,	O
+we	O
+report	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+structure	B-evidence
+of	O
+a	O
+cellulase	B-protein_type
+from	O
+Bacillus	B-species
+licheniformis	I-species
+(	O
+BlCel5B	B-protein
+),	O
+a	O
+member	O
+of	O
+the	O
+GH5	B-protein_type
+subfamily	I-protein_type
+4	I-protein_type
+that	O
+is	O
+entirely	O
+dependent	O
+on	O
+its	O
+two	O
+ancillary	B-structure_element
+modules	I-structure_element
+(	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+and	O
+CBM46	B-structure_element
+)	O
+for	O
+catalytic	O
+activity	O
+.	O
+
+Using	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+,	O
+small	B-experimental_method
+-	I-experimental_method
+angle	I-experimental_method
+X	I-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+scattering	I-experimental_method
+and	O
+molecular	B-experimental_method
+dynamics	I-experimental_method
+simulations	I-experimental_method
+,	O
+we	O
+propose	O
+that	O
+the	O
+C	O
+-	O
+terminal	O
+CBM46	B-structure_element
+caps	O
+the	O
+distal	O
+N	O
+-	O
+terminal	O
+catalytic	B-structure_element
+domain	I-structure_element
+(	O
+CD	B-structure_element
+)	O
+to	O
+establish	O
+a	O
+fully	B-protein_state
+functional	I-protein_state
+active	B-site
+site	I-site
+via	O
+a	O
+combination	O
+of	O
+large	O
+-	O
+scale	O
+multidomain	O
+conformational	O
+selection	O
+and	O
+induced	O
+-	O
+fit	O
+mechanisms	O
+.	O
+
+The	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+is	O
+pivoting	O
+the	O
+packing	O
+and	O
+unpacking	O
+motions	O
+of	O
+CBM46	B-structure_element
+relative	O
+to	O
+CD	B-structure_element
+in	O
+the	O
+assembly	O
+of	O
+the	O
+binding	B-site
+subsite	I-site
+.	O
+
+This	O
+is	O
+the	O
+first	O
+example	O
+of	O
+a	O
+multidomain	O
+GH	B-protein_type
+relying	O
+on	O
+large	O
+amplitude	O
+motions	O
+of	O
+the	O
+CBM46	B-structure_element
+for	O
+assembly	O
+of	O
+the	O
+catalytically	B-protein_state
+competent	I-protein_state
+form	O
+of	O
+the	O
+enzyme	O
+.	O
+
+Plant	B-taxonomy_domain
+biomass	O
+-	O
+the	O
+most	O
+abundant	O
+source	O
+of	O
+carbohydrates	B-chemical
+on	O
+Earth	O
+-	O
+is	O
+primarily	O
+composed	O
+of	O
+cellulose	B-chemical
+microfibrils	O
+surrounded	O
+by	O
+a	O
+hydrated	O
+heteropolymeric	O
+matrix	O
+of	O
+hemicellulose	B-chemical
+and	O
+lignin	B-chemical
+.	O
+
+Plant	B-taxonomy_domain
+biomass	O
+may	O
+be	O
+subjected	O
+to	O
+thermo	O
+-	O
+chemical	O
+pretreatments	O
+and	O
+enzymatic	O
+reactions	O
+to	O
+produce	O
+soluble	O
+fermentable	O
+sugars	B-chemical
+.	O
+
+The	O
+canonical	O
+model	O
+of	O
+hydrolytic	O
+degradation	O
+of	O
+cellulose	B-chemical
+requires	O
+at	O
+least	O
+three	O
+classes	O
+of	O
+enzymes	O
+.	O
+
+Cellobiohydrolases	B-protein_type
+(	O
+CBHs	B-protein_type
+)	O
+processively	O
+cleave	O
+the	O
+glycosidic	O
+bonds	O
+at	O
+the	O
+reducing	O
+and	O
+non	O
+-	O
+reducing	O
+ends	O
+of	O
+cellulose	B-chemical
+chains	O
+in	O
+crystalline	O
+regions	O
+to	O
+produce	O
+cellobiose	B-chemical
+.	O
+
+Endoglucanases	B-protein_type
+(	O
+EGs	B-protein_type
+)	O
+introduce	O
+random	O
+cuts	O
+in	O
+the	O
+amorphous	O
+regions	O
+of	O
+cellulose	B-chemical
+and	O
+create	O
+new	O
+chain	O
+extremities	O
+for	O
+CBH	B-protein_type
+attack	O
+;	O
+thus	O
+,	O
+these	O
+enzymes	O
+act	O
+synergistically	O
+.	O
+
+The	O
+released	O
+cellobiose	B-chemical
+molecules	O
+are	O
+then	O
+enzymatically	O
+converted	O
+into	O
+glucose	B-chemical
+by	O
+β	B-protein_type
+-	I-protein_type
+glucosidases	I-protein_type
+.	O
+
+The	O
+molecular	O
+architecture	O
+of	O
+glycoside	B-protein_type
+hydrolases	I-protein_type
+(	O
+GHs	B-protein_type
+)	O
+frequently	O
+consists	O
+of	O
+a	O
+catalytic	B-structure_element
+domain	I-structure_element
+(	O
+CD	B-structure_element
+),	O
+where	O
+hydrolysis	O
+occurs	O
+,	O
+and	O
+one	O
+or	O
+more	O
+ancillary	B-structure_element
+modules	I-structure_element
+(	O
+AMs	B-structure_element
+),	O
+which	O
+are	O
+usually	O
+connected	O
+by	O
+less	B-protein_state
+structured	I-protein_state
+linkers	B-structure_element
+.	O
+
+The	O
+most	O
+common	O
+type	O
+of	O
+AMs	B-structure_element
+are	O
+carbohydrate	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+modules	I-structure_element
+(	O
+CBMs	B-structure_element
+),	O
+which	O
+are	O
+able	O
+to	O
+recognize	O
+and	O
+bind	O
+specific	O
+carbohydrate	B-chemical
+chains	O
+.	O
+
+Generally	O
+distinct	O
+and	O
+independent	O
+structural	O
+domains	O
+,	O
+the	O
+CBMs	B-structure_element
+facilitate	O
+carbohydrate	B-chemical
+hydrolysis	O
+by	O
+increasing	O
+the	O
+local	O
+concentration	O
+of	O
+enzymes	O
+at	O
+the	O
+surface	O
+of	O
+insoluble	O
+substrates	O
+,	O
+thereby	O
+targeting	O
+the	O
+CD	B-structure_element
+component	O
+to	O
+its	O
+cognate	O
+ligands	O
+.	O
+
+CBMs	B-structure_element
+might	O
+also	O
+disrupt	O
+the	O
+crystalline	O
+structure	O
+of	O
+cellulose	B-chemical
+microfibrils	O
+,	O
+although	O
+the	O
+underlying	O
+mechanism	O
+remains	O
+poorly	O
+understood	O
+.	O
+
+Thus	O
+,	O
+CBMs	B-structure_element
+enhance	O
+the	O
+accessibility	O
+of	O
+CDs	B-structure_element
+to	O
+carbohydrate	B-chemical
+chains	O
+to	O
+improve	O
+enzymatic	O
+activity	O
+,	O
+making	O
+them	O
+important	O
+candidates	O
+for	O
+the	O
+development	O
+of	O
+effective	O
+biomass	O
+-	O
+degrading	O
+enzymes	O
+in	O
+industrial	O
+settings	O
+.	O
+
+Although	O
+there	O
+are	O
+examples	O
+of	O
+active	B-protein_state
+GHs	B-protein_type
+that	O
+lack	B-protein_state
+AMs	B-structure_element
+,	O
+the	O
+majority	O
+of	O
+the	O
+enzymes	O
+depend	O
+on	O
+AMs	B-structure_element
+for	O
+activity	O
+.	O
+
+In	O
+several	O
+cases	O
+,	O
+CBMs	B-structure_element
+were	O
+shown	O
+to	O
+extend	O
+and	O
+complement	O
+the	O
+CD	B-structure_element
+substrate	B-site
+-	I-site
+binding	I-site
+site	I-site
+in	O
+multimodular	O
+carbohydrate	B-protein_type
+-	I-protein_type
+active	I-protein_type
+enzymes	I-protein_type
+,	O
+such	O
+as	O
+endo	B-protein_type
+/	I-protein_type
+exocellulase	I-protein_type
+E4	B-protein
+from	O
+Thermobifida	B-species
+fusca	I-species
+,	O
+chitinase	B-protein
+B	I-protein
+from	O
+Serratia	B-species
+marcescens	I-species
+,	O
+a	O
+starch	B-protein_type
+phosphatase	I-protein_type
+from	O
+Arabidopsis	B-species
+thaliana	I-species
+and	O
+a	O
+GH5	B-protein_type
+subfamily	I-protein_type
+4	I-protein_type
+(	O
+GH5_4	B-protein_type
+)	O
+endoglucanase	B-protein_type
+from	O
+Bacillus	B-species
+halodurans	I-species
+(	O
+BhCel5B	B-protein
+).	O
+
+A	O
+pioneer	O
+work	O
+of	O
+Sakon	O
+et	O
+al	O
+.	O
+revealed	O
+that	O
+rigid	O
+structural	O
+extension	O
+of	O
+the	O
+GH9	B-protein_type
+CD	B-structure_element
+by	O
+a	O
+type	B-structure_element
+C	I-structure_element
+CBM3	I-structure_element
+imprints	O
+a	O
+processive	O
+mode	O
+of	O
+action	O
+to	O
+this	O
+endoglucanase	B-protein_type
+.	O
+
+Further	O
+publications	O
+showed	O
+that	O
+CBM	B-structure_element
+-	O
+based	O
+structural	O
+extensions	O
+of	O
+the	O
+active	B-site
+site	I-site
+are	O
+important	O
+for	O
+substrate	O
+engagement	O
+and	O
+recognition	O
+.	O
+
+Recently	O
+,	O
+Venditto	O
+et	O
+al	O
+.	O
+reported	O
+the	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+structure	I-evidence
+of	O
+the	O
+tri	B-structure_element
+-	I-structure_element
+modular	I-structure_element
+GH5_4	B-protein_type
+endoglucanase	B-protein_type
+from	O
+Bacillus	B-species
+halodurans	I-species
+(	O
+31	O
+%	O
+sequence	O
+identity	O
+to	O
+BlCel5B	B-protein
+),	O
+with	O
+the	O
+CBM46	B-structure_element
+extension	O
+of	O
+the	O
+active	B-site
+site	I-site
+appended	O
+to	O
+the	O
+CD	B-structure_element
+via	O
+an	O
+immunoglobulin	B-structure_element
+(	I-structure_element
+Ig	I-structure_element
+)-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+.	O
+
+Removal	B-experimental_method
+of	I-experimental_method
+the	O
+CBM46	B-structure_element
+caused	O
+a	O
+~	O
+60	O
+-	O
+fold	O
+reduction	O
+of	O
+the	O
+activity	O
+of	O
+the	O
+enzyme	O
+against	O
+β	B-chemical
+-	I-chemical
+glucans	I-chemical
+,	O
+but	O
+showed	O
+little	O
+or	O
+no	O
+effect	O
+against	O
+xyloglucan	B-chemical
+hydrolysis	O
+.	O
+
+Moreover	O
+,	O
+the	O
+CBM46	B-structure_element
+mediated	O
+a	O
+significant	O
+increase	O
+in	O
+the	O
+BhCel5B	B-protein
+activity	O
+in	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+settings	O
+.	O
+
+Modeling	B-experimental_method
+of	O
+cellotriose	B-chemical
+in	O
+the	O
+negative	B-site
+subsites	I-site
+of	O
+the	O
+active	B-site
+site	I-site
+of	O
+BhCel5B	B-protein
+demonstrated	O
+the	O
+structural	B-protein_state
+conservation	I-protein_state
+of	O
+the	O
+-	B-residue_number
+1	I-residue_number
+position	O
+,	O
+but	O
+provided	O
+little	O
+information	O
+about	O
+direct	O
+interactions	O
+between	O
+CBM46	B-structure_element
+and	O
+the	O
+substrate	O
+.	O
+
+It	O
+was	O
+speculated	O
+that	O
+β	O
+-	O
+1	O
+,	O
+3	O
+kink	O
+of	O
+the	O
+β	B-chemical
+-	I-chemical
+glucan	I-chemical
+might	O
+allow	O
+the	O
+ligand	O
+to	O
+reach	O
+for	O
+the	O
+CBM46	B-structure_element
+,	O
+whereas	O
+pure	O
+β	O
+-	O
+1	O
+,	O
+4	O
+linkages	O
+in	O
+the	O
+backbone	O
+of	O
+xyloglucan	B-chemical
+chains	O
+would	O
+restrict	O
+binding	O
+to	O
+the	O
+CD	B-structure_element
+,	O
+thus	O
+explaining	O
+the	O
+lack	O
+of	O
+influence	O
+of	O
+the	O
+CBM46	B-structure_element
+on	O
+the	O
+enzymatic	O
+activity	O
+of	O
+BhCel5B	B-protein
+against	O
+xyloglucans	B-chemical
+in	O
+solution	O
+.	O
+
+It	O
+was	O
+also	O
+argued	O
+that	O
+the	O
+CBM46	B-structure_element
+could	O
+potentialize	O
+the	O
+activity	O
+by	O
+driving	O
+BhCel5B	B-protein
+towards	O
+xyloglucan	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+regions	I-structure_element
+in	O
+the	O
+context	O
+of	O
+the	O
+plant	B-taxonomy_domain
+cell	O
+walls	O
+,	O
+but	O
+no	O
+large	O
+-	O
+scale	O
+conformational	O
+adjustments	O
+of	O
+the	O
+AMs	B-structure_element
+have	O
+been	O
+shown	O
+to	O
+occur	O
+or	O
+suggested	O
+to	O
+take	O
+part	O
+in	O
+the	O
+enzymatic	O
+activity	O
+.	O
+
+Although	O
+initially	O
+introduced	O
+as	O
+contradictory	O
+theories	O
+,	O
+these	O
+two	O
+limiting	O
+cases	O
+can	O
+be	O
+unified	O
+considering	O
+the	O
+flux	O
+description	O
+concept	O
+or	O
+the	O
+extended	B-protein_state
+conformational	O
+selection	O
+model	O
+.	O
+
+While	O
+local	O
+ligand	O
+-	O
+induced	O
+conformational	O
+adjustments	O
+have	O
+been	O
+reported	O
+for	O
+carbohydrate	B-protein_type
+-	I-protein_type
+active	I-protein_type
+enzymes	I-protein_type
+,	O
+cognate	O
+ligands	O
+recognition	O
+and	O
+hydrolysis	O
+mediated	O
+by	O
+a	O
+large	O
+-	O
+scale	O
+conformational	O
+mobility	O
+of	O
+distinct	O
+domains	O
+in	O
+multidomain	O
+settings	O
+is	O
+uncommon	O
+for	O
+endoglucanases	B-protein_type
+.	O
+
+Here	O
+,	O
+we	O
+report	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+a	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+GH5_4	B-protein_type
+enzyme	O
+from	O
+Bacillus	B-species
+licheniformis	I-species
+(	O
+BlCel5B	B-protein
+)	O
+that	O
+exhibits	O
+two	O
+AMs	B-structure_element
+(	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+and	O
+CBM46	B-structure_element
+)	O
+appended	O
+to	O
+the	O
+CD	B-structure_element
+.	O
+
+We	O
+structurally	B-experimental_method
+and	I-experimental_method
+functionally	I-experimental_method
+characterize	I-experimental_method
+the	O
+enzyme	O
+using	O
+a	O
+combination	O
+of	O
+protein	B-experimental_method
+crystallography	I-experimental_method
+,	O
+small	B-experimental_method
+-	I-experimental_method
+angle	I-experimental_method
+X	I-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+scattering	I-experimental_method
+(	O
+SAXS	B-experimental_method
+),	O
+molecular	B-experimental_method
+dynamics	I-experimental_method
+computer	I-experimental_method
+simulations	I-experimental_method
+and	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+,	O
+and	O
+show	O
+that	O
+the	O
+AMs	B-structure_element
+and	O
+their	O
+conformational	O
+mobility	O
+are	O
+essential	O
+for	O
+the	O
+enzymatic	O
+activity	O
+of	O
+BlCel5B	B-protein
+.	O
+
+We	O
+find	O
+that	O
+the	O
+large	O
+-	O
+scale	O
+conformational	O
+adjustments	O
+of	O
+the	O
+distal	O
+CBM46	B-structure_element
+mediated	O
+by	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+hinge	I-structure_element
+domain	I-structure_element
+are	O
+crucial	O
+in	O
+active	B-site
+-	I-site
+site	I-site
+assembly	O
+for	O
+optimal	O
+substrate	O
+binding	O
+and	O
+hydrolysis	O
+.	O
+
+We	O
+propose	O
+that	O
+the	O
+BlCel5B	B-protein
+conformational	O
+selection	O
+/	O
+induced	O
+-	O
+fit	O
+mechanism	O
+of	O
+hydrolysis	O
+represents	O
+a	O
+novel	O
+paradigm	O
+that	O
+applies	O
+to	O
+several	O
+GH5_4	B-protein_type
+members	O
+and	O
+,	O
+possibly	O
+,	O
+to	O
+a	O
+number	O
+of	O
+other	O
+multidomain	O
+GHs	B-protein_type
+.	O
+
+BlCel5B	B-protein
+Crystal	B-evidence
+Structure	I-evidence
+
+BlCel5B	B-protein
+crystals	B-evidence
+in	O
+the	O
+substrate	B-protein_state
+-	I-protein_state
+free	I-protein_state
+form	O
+and	O
+complexed	B-protein_state
+with	I-protein_state
+cellopentaose	B-chemical
+(	O
+C5	B-chemical
+)	O
+were	O
+obtained	O
+and	O
+diffracted	O
+to	O
+1	O
+.	O
+7	O
+Å	O
+and	O
+1	O
+.	O
+75	O
+Å	O
+resolutions	O
+,	O
+respectively	O
+(	O
+Supplementary	O
+Table	O
+1	O
+).	O
+
+The	O
+substrate	B-protein_state
+-	I-protein_state
+free	I-protein_state
+and	O
+complexed	B-protein_state
+structures	B-evidence
+exhibited	O
+no	O
+substantial	O
+conformational	O
+differences	O
+(	O
+with	O
+the	O
+exception	O
+of	O
+the	O
+substrate	O
+).	O
+
+Because	O
+of	O
+minor	O
+variations	O
+in	O
+the	O
+loops	B-structure_element
+located	O
+distal	O
+to	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+site	I-site
+,	O
+a	O
+root	B-evidence
+mean	I-evidence
+squared	I-evidence
+deviation	I-evidence
+(	O
+rmsd	B-evidence
+)	O
+of	O
+0	O
+.	O
+33	O
+Å	O
+between	O
+the	O
+complexed	B-protein_state
+and	O
+substrate	B-protein_state
+-	I-protein_state
+free	I-protein_state
+structures	B-evidence
+was	O
+observed	O
+.	O
+
+A	O
+single	O
+protein	O
+chain	O
+occupies	O
+the	O
+asymmetric	O
+unit	O
+,	O
+and	O
+most	O
+of	O
+the	O
+residues	O
+were	O
+built	O
+,	O
+with	O
+the	O
+exception	O
+of	O
+the	O
+first	B-residue_range
+17	I-residue_range
+residues	I-residue_range
+and	O
+those	O
+in	O
+the	O
+loop	B-structure_element
+between	O
+L398	B-residue_name_number
+and	O
+P405	B-residue_name_number
+due	O
+to	O
+weak	O
+electron	B-evidence
+density	I-evidence
+.	O
+
+The	O
+BlCel5B	B-protein
+structure	B-evidence
+comprises	O
+three	O
+distinct	O
+domains	O
+:	O
+an	O
+N	O
+-	O
+terminal	O
+CD	B-structure_element
+(	O
+residues	O
+18	B-residue_range
+to	I-residue_range
+330	I-residue_range
+),	O
+an	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+(	O
+residues	O
+335	B-residue_range
+to	I-residue_range
+428	I-residue_range
+)	O
+and	O
+a	O
+family	B-structure_element
+46	I-structure_element
+CBM	I-structure_element
+(	O
+residues	O
+432	B-residue_range
+to	I-residue_range
+533	I-residue_range
+)	O
+(	O
+Fig	O
+.	O
+1A	O
+,	O
+B	O
+).	O
+
+Similarly	O
+to	O
+other	O
+members	O
+of	O
+the	O
+GH5	B-protein_type
+family	O
+,	O
+the	O
+CD	B-structure_element
+of	O
+BlCel5B	B-protein
+has	O
+a	O
+typical	O
+TIM	B-structure_element
+barrel	I-structure_element
+fold	I-structure_element
+with	O
+eight	O
+inner	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+and	O
+eight	O
+outer	O
+α	B-structure_element
+helices	I-structure_element
+that	O
+are	O
+interconnected	O
+by	O
+loops	B-structure_element
+and	O
+three	O
+short	O
+α	B-structure_element
+helices	I-structure_element
+.	O
+
+Very	O
+short	O
+linkers	B-structure_element
+,	O
+D429	B-structure_element
+-	I-structure_element
+D430	I-structure_element
+-	I-structure_element
+P431	I-structure_element
+and	O
+V331	B-structure_element
+-	I-structure_element
+P332	I-structure_element
+-	I-structure_element
+N333	I-structure_element
+-	I-structure_element
+A334	I-structure_element
+,	O
+connect	O
+the	O
+CBM46	B-structure_element
+to	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+and	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+to	O
+the	O
+CD	B-structure_element
+,	O
+respectively	O
+.	O
+
+Both	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+and	O
+CBM46	B-structure_element
+have	O
+a	O
+β	B-structure_element
+-	I-structure_element
+sandwich	I-structure_element
+fold	I-structure_element
+composed	O
+of	O
+two	O
+β	B-structure_element
+-	I-structure_element
+sheets	I-structure_element
+of	O
+four	O
+and	O
+three	O
+antiparallel	B-structure_element
+β	I-structure_element
+-	I-structure_element
+strands	I-structure_element
+interconnected	O
+by	O
+loops	B-structure_element
+and	O
+a	O
+short	O
+α	B-structure_element
+helix	I-structure_element
+between	O
+strands	B-structure_element
+β3	B-structure_element
+and	O
+β4	B-structure_element
+(	O
+Fig	O
+.	O
+1C	O
+).	O
+
+A	O
+structural	B-experimental_method
+comparison	I-experimental_method
+between	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+and	O
+the	O
+CBM46	B-structure_element
+using	O
+the	O
+Dali	B-experimental_method
+server	I-experimental_method
+yielded	O
+an	O
+rmsd	B-evidence
+of	O
+2	O
+.	O
+3	O
+Å	O
+and	O
+a	O
+Z	B-evidence
+-	I-evidence
+score	I-evidence
+of	O
+10	O
+.	O
+2	O
+.	O
+
+A	O
+structure	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+search	I-experimental_method
+performed	O
+using	O
+the	O
+same	O
+server	O
+showed	O
+that	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+is	O
+similar	O
+to	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+from	O
+a	O
+recently	O
+solved	B-experimental_method
+crystal	B-evidence
+structure	I-evidence
+of	O
+a	O
+tri	B-structure_element
+-	I-structure_element
+modular	I-structure_element
+GH5_4	B-protein_type
+enzyme	O
+from	O
+Bacillus	B-species
+halodurans	I-species
+,	O
+BhCel5B	B-protein
+,	O
+with	O
+rmsd	B-evidence
+=	O
+1	O
+.	O
+3	O
+Å	O
+and	O
+Z	B-evidence
+-	I-evidence
+score	I-evidence
+=	O
+15	O
+.	O
+3	O
+.	O
+
+The	O
+CBM46	B-structure_element
+from	O
+BhCel5B	B-protein
+is	O
+the	O
+most	O
+structurally	O
+similar	O
+to	O
+BlCel5B	B-protein
+CBM46	B-structure_element
+,	O
+with	O
+rmsd	B-evidence
+=	O
+1	O
+.	O
+6	O
+Å	O
+and	O
+Z	B-evidence
+-	I-evidence
+score	I-evidence
+=	O
+12	O
+.	O
+4	O
+.	O
+
+The	O
+sequence	O
+identity	O
+relative	O
+to	O
+BhCel5B	B-protein
+,	O
+however	O
+,	O
+is	O
+low	O
+(	O
+28	O
+%	O
+for	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+and	O
+25	O
+%	O
+for	O
+CBM46	B-structure_element
+).	O
+
+The	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+,	O
+adjacent	O
+to	O
+the	O
+CD	B-structure_element
+,	O
+contains	O
+only	O
+one	O
+tyrosine	B-residue_name
+(	O
+Y367	B-residue_name_number
+)	O
+exposed	O
+to	O
+solvent	O
+and	O
+no	O
+tryptophan	B-residue_name
+residues	O
+.	O
+
+Because	O
+aromatic	O
+residues	O
+play	O
+a	O
+major	O
+role	O
+in	O
+glucose	B-chemical
+recognition	O
+,	O
+this	O
+observation	O
+suggests	O
+that	O
+substrate	O
+binding	O
+may	O
+not	O
+be	O
+the	O
+primary	O
+function	O
+of	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+.	O
+
+In	O
+contrast	O
+,	O
+the	O
+CBM46	B-structure_element
+has	O
+three	O
+tryptophan	B-residue_name
+residues	O
+,	O
+two	O
+of	O
+which	O
+face	O
+the	O
+CD	B-structure_element
+substrate	B-site
+binding	I-site
+site	I-site
+(	O
+Fig	O
+.	O
+1A	O
+),	O
+indicating	O
+that	O
+it	O
+may	O
+be	O
+actively	O
+engaged	O
+in	O
+the	O
+carbohydrate	B-chemical
+binding	O
+.	O
+
+Electron	B-evidence
+density	I-evidence
+maps	I-evidence
+clearly	O
+reveal	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+a	O
+cellotetraose	B-chemical
+(	O
+C4	B-chemical
+)	O
+and	O
+not	O
+a	O
+soaked	O
+cellopentaose	B-chemical
+(	O
+C5	B-chemical
+)	O
+in	O
+the	O
+CD	B-structure_element
+negative	B-site
+substrate	I-site
+-	I-site
+binding	I-site
+subsites	I-site
+(	O
+Fig	O
+.	O
+1D	O
+),	O
+indicating	O
+that	O
+BlCel5B	B-protein
+is	O
+catalytically	B-protein_state
+active	I-protein_state
+in	O
+the	O
+crystal	O
+state	O
+and	O
+able	O
+to	O
+cleave	O
+a	O
+C5	B-chemical
+molecule	O
+.	O
+
+The	O
+lack	B-evidence
+of	I-evidence
+electron	I-evidence
+density	I-evidence
+verifies	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+the	O
+fifth	B-residue_number
+glucose	B-chemical
+moiety	O
+from	O
+the	O
+soaked	O
+C5	B-chemical
+,	O
+and	O
+a	O
+closer	O
+inspection	O
+of	O
+the	O
+structure	B-evidence
+confirmed	O
+that	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+a	O
+fifth	B-residue_number
+glucose	B-chemical
+unit	O
+would	O
+be	O
+sterically	O
+hindered	O
+by	O
+the	O
+catalytic	B-site
+residues	I-site
+on	O
+the	O
+reducing	O
+end	O
+and	O
+by	O
+residue	O
+R234	B-residue_name_number
+of	O
+a	O
+symmetry	O
+-	O
+related	O
+enzyme	O
+molecule	O
+on	O
+the	O
+non	O
+-	O
+reducing	O
+end	O
+.	O
+
+The	O
+ability	O
+of	O
+BlCel5B	B-protein
+to	O
+cleave	O
+C5	B-chemical
+into	O
+glucose	B-chemical
+and	O
+C4	B-chemical
+molecules	O
+in	O
+solution	O
+was	O
+demonstrated	O
+by	O
+enzymatic	B-experimental_method
+product	I-experimental_method
+profile	I-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+analysis	O
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+
+The	O
+C4	B-chemical
+oligomer	O
+in	O
+the	O
+BlCel5B	B-protein
+binding	B-site
+site	I-site
+is	O
+coordinated	B-bond_interaction
+by	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+to	O
+residues	O
+N36	B-residue_name_number
+,	O
+H113	B-residue_name_number
+,	O
+H114	B-residue_name_number
+,	O
+N158	B-residue_name_number
+,	O
+W301	B-residue_name_number
+,	O
+and	O
+N303	B-residue_name_number
+and	O
+by	O
+a	O
+CH	B-bond_interaction
+-	I-bond_interaction
+π	I-bond_interaction
+interaction	I-bond_interaction
+with	O
+residue	O
+W47	B-residue_name_number
+(	O
+Fig	O
+.	O
+1D	O
+).	O
+
+These	O
+residues	O
+belong	O
+to	O
+the	O
+CD	B-structure_element
+and	O
+are	O
+conserved	B-protein_state
+in	O
+the	O
+GH5	B-protein_type
+family	O
+.	O
+
+BlCel5B	B-protein
+enzymatic	O
+activity	O
+
+BlCel5B	B-protein
+exhibits	O
+optimum	O
+activity	O
+toward	O
+carboxymethylcellulose	B-chemical
+(	O
+CMC	B-chemical
+;	O
+8	O
+.	O
+7	O
+U	O
+/	O
+mg	O
+)	O
+at	O
+a	O
+pH	O
+of	O
+4	O
+.	O
+0	O
+and	O
+55	O
+°	O
+C	O
+and	O
+retains	O
+approximately	O
+half	O
+of	O
+its	O
+maximum	O
+activity	O
+at	O
+80	O
+°	O
+C	O
+,	O
+demonstrating	O
+considerable	O
+thermal	O
+stability	O
+(	O
+Fig	O
+.	O
+2B	O
+,	O
+C	O
+).	O
+
+BlCel5B	B-protein
+is	O
+also	O
+active	B-protein_state
+on	O
+β	B-chemical
+-	I-chemical
+glucan	I-chemical
+(	O
+34	O
+U	O
+/	O
+mg	O
+),	O
+lichenan	B-chemical
+(	O
+17	O
+.	O
+8	O
+U	O
+/	O
+mg	O
+)	O
+and	O
+xyloglucan	B-chemical
+(	O
+15	O
+.	O
+7	O
+U	O
+/	O
+mg	O
+)	O
+substrates	O
+(	O
+Table	O
+1	O
+),	O
+whereas	O
+no	O
+activity	O
+was	O
+detected	O
+on	O
+galactomannan	B-chemical
+,	O
+rye	B-taxonomy_domain
+arabinoxylan	B-chemical
+,	O
+1	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+β	I-chemical
+-	I-chemical
+mannan	I-chemical
+or	O
+the	O
+insoluble	O
+substrate	O
+Azo	B-chemical
+-	I-chemical
+Avicel	I-chemical
+.	O
+
+Kinetic	O
+parameters	O
+were	O
+calculated	O
+assuming	O
+Michaelis	B-experimental_method
+-	I-experimental_method
+Menten	I-experimental_method
+behavior	I-experimental_method
+with	O
+CMC	B-chemical
+as	O
+substrate	O
+:	O
+KM	B-evidence
+=	O
+1	O
+.	O
+78	O
+g	O
+L	O
+−	O
+1	O
+and	O
+Vmax	B-evidence
+=	O
+1	O
+.	O
+41	O
+×	O
+10	O
+−	O
+4	O
+g	O
+s	O
+−	O
+1	O
+mg	O
+protein	O
+−	O
+1	O
+(	O
+Fig	O
+.	O
+2D	O
+).	O
+
+Although	O
+BlCel5B	B-protein
+is	O
+not	O
+a	O
+highly	O
+active	B-protein_state
+enzyme	O
+against	O
+one	O
+specific	O
+substrate	O
+as	O
+compared	O
+to	O
+others	O
+GH5_4	B-protein_type
+,	O
+it	O
+has	O
+the	O
+advantage	O
+of	O
+being	O
+active	B-protein_state
+against	O
+different	O
+substrates	O
+with	O
+β	O
+-	O
+1	O
+,	O
+3	O
+and	O
+/	O
+or	O
+β	O
+-	O
+1	O
+,	O
+4	O
+glycosidic	O
+linkages	O
+.	O
+
+To	O
+understand	O
+the	O
+importance	O
+of	O
+the	O
+ancillary	B-structure_element
+modules	I-structure_element
+for	O
+BlCel5B	B-protein
+activity	O
+,	O
+enzymatic	B-experimental_method
+assays	I-experimental_method
+were	O
+carried	O
+out	O
+using	O
+four	O
+enzyme	O
+mutants	B-protein_state
+:	O
+a	O
+CBM46	B-structure_element
+deletion	B-experimental_method
+(	O
+ΔCBM46	B-mutant
+)	O
+and	O
+an	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
++	O
+CBM46	B-structure_element
+deletion	B-experimental_method
+(	O
+ΔIg	B-mutant
+-	I-mutant
+CBM46	I-mutant
+)	O
+as	O
+well	O
+as	O
+point	B-experimental_method
+mutations	I-experimental_method
+of	O
+the	O
+CBM46	B-structure_element
+inner	O
+surface	O
+residues	O
+W479A	B-mutant
+and	O
+W481A	B-mutant
+.	O
+
+These	O
+mutants	B-protein_state
+were	O
+expressed	B-experimental_method
+and	I-experimental_method
+purified	I-experimental_method
+as	O
+described	O
+for	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+enzyme	O
+.	O
+
+Strikingly	O
+,	O
+neither	O
+of	O
+the	O
+deletion	B-protein_state
+variants	I-protein_state
+exhibited	O
+detectable	O
+activity	O
+toward	O
+any	O
+of	O
+the	O
+substrates	O
+tested	O
+using	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+BlCel5B	B-protein
+(	O
+Table	O
+1	O
+),	O
+demonstrating	O
+that	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+and	O
+the	O
+CBM46	B-structure_element
+are	O
+essential	O
+for	O
+BlCel5B	B-protein
+activity	O
+.	O
+
+Thermal	B-experimental_method
+shift	I-experimental_method
+assays	I-experimental_method
+were	O
+conducted	O
+to	O
+confirm	O
+structural	O
+stability	O
+of	O
+the	O
+mutants	B-protein_state
+(	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+).	O
+
+All	O
+of	O
+the	O
+constructs	O
+showed	O
+similar	O
+melting	B-evidence
+temperatures	I-evidence
+:	O
+62	O
+°	O
+C	O
+for	O
+BlCel5B	B-protein
+,	O
+58	O
+°	O
+C	O
+for	O
+BlCel5BΔCBM46	B-mutant
+,	O
+56	O
+°	O
+C	O
+for	O
+BlCel5BΔIg	B-mutant
+-	I-mutant
+CBM46	I-mutant
+,	O
+65	O
+°	O
+C	O
+for	O
+BlCel5BW479A	B-mutant
+and	O
+59	O
+°	O
+C	O
+for	O
+BlCel5BW479A	B-mutant
+,	O
+thus	O
+confirming	O
+their	O
+proper	O
+overall	O
+fold	O
+.	O
+
+We	O
+also	O
+examined	O
+the	O
+function	O
+of	O
+the	O
+CBM46	B-structure_element
+inner	O
+surface	B-site
+residues	O
+W479	B-residue_name_number
+and	O
+W481	B-residue_name_number
+(	O
+Fig	O
+.	O
+1A	O
+)	O
+in	O
+BlCel5B	B-protein
+activity	O
+by	O
+performing	O
+enzymatic	B-experimental_method
+assays	I-experimental_method
+with	O
+W479A	B-mutant
+and	O
+W481A	B-mutant
+mutants	B-protein_state
+.	O
+
+Both	O
+mutations	B-experimental_method
+reduced	O
+enzymatic	O
+activity	O
+toward	O
+all	O
+tested	O
+substrates	O
+(	O
+Table	O
+1	O
+),	O
+with	O
+W481A	B-mutant
+having	O
+a	O
+stronger	O
+effect	O
+than	O
+W479A	B-mutant
+(~	O
+64	O
+%	O
+vs	O
+.	O
+79	O
+%	O
+activity	O
+relative	O
+to	O
+wt	B-protein_state
+BlCel5B	B-protein
+using	O
+β	B-chemical
+-	I-chemical
+glucan	I-chemical
+and	O
+~	O
+10	O
+%	O
+vs	O
+.	O
+50	O
+%	O
+using	O
+CMC	B-chemical
+).	O
+
+This	O
+indicates	O
+that	O
+CBM46	B-structure_element
+must	O
+interact	O
+with	O
+the	O
+substrate	O
+via	O
+residues	O
+W479	B-residue_name_number
+and	O
+W481	B-residue_name_number
+.	O
+
+However	O
+,	O
+since	O
+the	O
+BlCel5B	B-protein
+crystal	B-evidence
+structure	I-evidence
+exhibits	O
+no	O
+close	B-protein_state
+contact	O
+between	O
+these	O
+residues	O
+and	O
+the	O
+substrate	O
+,	O
+these	O
+results	O
+suggest	O
+the	O
+existence	O
+of	O
+large	O
+-	O
+amplitude	O
+interdomain	O
+motions	O
+that	O
+may	O
+enable	O
+direct	O
+interactions	O
+between	O
+CBM46	B-structure_element
+and	O
+the	O
+carbohydrate	B-chemical
+.	O
+
+BlCelB5	B-protein
+dynamics	O
+and	O
+binding	B-site
+-	I-site
+site	I-site
+architecture	O
+
+Molecular	B-experimental_method
+dynamics	I-experimental_method
+(	O
+MD	B-experimental_method
+)	O
+simulations	B-experimental_method
+were	O
+performed	O
+to	O
+investigate	O
+the	O
+conformational	O
+mobility	O
+of	O
+BlCel5B	B-protein
+.	O
+
+In	O
+the	O
+simulations	B-experimental_method
+of	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+for	O
+BlCel5B	B-protein
+bound	B-protein_state
+to	I-protein_state
+C4	B-chemical
+,	O
+the	O
+substrate	O
+dissociates	O
+from	O
+the	O
+protein	O
+within	O
+the	O
+first	O
+100	O
+ns	O
+of	O
+the	O
+simulation	B-experimental_method
+time	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2A	O
+).	O
+
+This	O
+observation	O
+suggests	O
+that	O
+cellotetraose	B-chemical
+does	O
+not	O
+exhibit	O
+detectable	O
+affinity	O
+for	O
+this	O
+specific	O
+BlCel5B	B-protein
+conformation	O
+in	O
+solution	O
+,	O
+as	O
+one	O
+might	O
+otherwise	O
+expect	O
+for	O
+a	O
+reaction	O
+product	O
+.	O
+
+No	O
+changes	O
+beyond	O
+local	O
+fluctuations	O
+were	O
+observed	O
+in	O
+any	O
+of	O
+the	O
+three	O
+BlCel5B	B-protein
+domains	O
+within	O
+the	O
+time	O
+scale	O
+of	O
+these	O
+runs	O
+(	O
+400	O
+ns	O
+;	O
+Supplementary	O
+Fig	O
+.	O
+2B	O
+).	O
+
+However	O
+,	O
+the	O
+CBM46	B-structure_element
+and	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+domains	I-structure_element
+did	O
+exhibit	O
+rigid	O
+body	O
+-	O
+like	O
+motions	O
+relative	O
+to	O
+the	O
+CD	B-structure_element
+,	O
+with	O
+rmsd	B-evidence
+values	O
+around	O
+2	O
+.	O
+3	O
+Å	O
+and	O
+1	O
+.	O
+8	O
+Å	O
+,	O
+respectively	O
+,	O
+suggesting	O
+that	O
+BlCel5B	B-protein
+may	O
+execute	O
+large	O
+-	O
+amplitude	O
+interdomain	O
+motions	O
+over	O
+longer	O
+time	O
+scales	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2B	O
+,	O
+C	O
+).	O
+
+Accordingly	O
+,	O
+simulations	B-experimental_method
+were	O
+then	O
+performed	O
+using	O
+accelerated	B-experimental_method
+molecular	I-experimental_method
+dynamics	I-experimental_method
+(	O
+aMD	B-experimental_method
+)	O
+techniques	O
+to	O
+probe	O
+BlCel5B	B-protein
+interdomain	O
+motions	O
+.	O
+
+aMD	B-experimental_method
+enhances	O
+conformational	O
+sampling	O
+by	O
+raising	O
+the	O
+basins	O
+of	O
+the	O
+dihedral	B-evidence
+potential	I-evidence
+energy	I-evidence
+surface	I-evidence
+without	O
+affecting	O
+the	O
+general	O
+form	O
+of	O
+the	O
+atomistic	O
+potential	O
+,	O
+thereby	O
+increasing	O
+transition	O
+rates	O
+between	O
+different	O
+local	O
+minima	O
+.	O
+
+aMD	B-experimental_method
+trajectories	B-evidence
+corresponding	O
+to	O
+more	O
+than	O
+1	O
+.	O
+0	O
+μs	O
+of	O
+conventional	O
+MD	B-experimental_method
+runs	O
+were	O
+generated	O
+.	O
+
+During	O
+these	O
+simulations	B-experimental_method
+,	O
+we	O
+observed	O
+occlusive	O
+conformations	O
+between	O
+CBM46	B-structure_element
+and	O
+CD	B-structure_element
+that	O
+resulted	O
+in	O
+a	O
+rearrangement	O
+of	O
+the	O
+enzyme	O
+’	O
+s	O
+architecture	O
+around	O
+the	O
+active	B-site
+site	I-site
+(	O
+Video	O
+S1	O
+).	O
+
+Figure	O
+3A	O
+shows	O
+BlCel5B	B-protein
+in	O
+the	O
+crystallographic	B-experimental_method
+conformation	O
+(	O
+red	O
+)	O
+and	O
+in	O
+a	O
+selected	O
+configuration	O
+obtained	O
+with	O
+aMD	B-experimental_method
+(	O
+blue	O
+)	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+the	O
+substrate	O
+.	O
+
+Interdomain	O
+motions	O
+were	O
+gauged	O
+by	O
+the	O
+time	O
+evolution	O
+of	O
+the	O
+distance	B-evidence
+between	O
+the	O
+α	O
+carbons	O
+of	O
+residues	O
+I120	B-residue_name_number
+and	O
+E477	B-residue_name_number
+(	O
+represented	O
+as	O
+spheres	O
+in	O
+Fig	O
+.	O
+3A	O
+),	O
+belonging	O
+to	O
+the	O
+CD	B-structure_element
+and	O
+CBM46	B-structure_element
+,	O
+respectively	O
+.	O
+
+Figure	O
+3C	O
+shows	O
+that	O
+the	O
+I120	B-residue_name_number
+-	O
+E477	B-residue_name_number
+distance	B-evidence
+(	O
+red	O
+curve	O
+)	O
+gradually	O
+decreases	O
+from	O
+~	O
+35	O
+Å	O
+to	O
+~	O
+7	O
+Å	O
+within	O
+the	O
+first	O
+half	O
+of	O
+the	O
+1	O
+.	O
+0	O
+μs	O
+aMD	B-experimental_method
+trajectory	B-evidence
+,	O
+indicating	O
+a	O
+transition	O
+between	O
+the	O
+semi	B-protein_state
+-	I-protein_state
+open	I-protein_state
+(	O
+crystallographic	B-experimental_method
+)	O
+and	O
+occluded	B-protein_state
+(	O
+aMD	B-experimental_method
+sampled	O
+)	O
+configurations	O
+.	O
+
+During	O
+the	O
+second	O
+half	O
+of	O
+the	O
+aMD	B-experimental_method
+simulation	I-experimental_method
+,	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+enzyme	O
+remained	O
+in	O
+the	O
+closed	B-protein_state
+conformation	O
+,	O
+with	O
+the	O
+CBM46	B-structure_element
+covering	O
+the	O
+carbohydrate	B-site
+-	I-site
+binding	I-site
+site	I-site
+.	O
+
+These	O
+results	O
+suggest	O
+that	O
+BlCel5B	B-protein
+undergoes	O
+large	O
+-	O
+scale	O
+interdomain	O
+movements	O
+that	O
+enable	O
+interactions	O
+between	O
+CBM46	B-structure_element
+and	O
+the	O
+substrate	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+CD	B-structure_element
+.	O
+
+To	O
+study	O
+the	O
+interactions	O
+of	O
+BlCel5B	B-protein
+with	O
+a	O
+non	O
+-	O
+hydrolyzed	O
+glucan	B-chemical
+chain	O
+,	O
+we	O
+built	O
+a	O
+model	O
+structure	B-evidence
+with	O
+a	O
+cellooctaose	B-chemical
+(	O
+C8	B-chemical
+)	O
+chain	O
+spanning	O
+the	O
+entire	O
+positive	B-site
+(+	I-site
+1	I-site
+to	I-site
++	I-site
+4	I-site
+)	I-site
+and	O
+negative	B-site
+(−	I-site
+4	I-site
+to	I-site
+−	I-site
+1	I-site
+)	I-site
+subsites	B-site
+of	O
+the	O
+enzyme	O
+.	O
+
+Starting	O
+from	O
+the	O
+crystallographic	O
+BlCel5B	B-protein
+conformation	O
+,	O
+the	O
+C8	B-chemical
+molecule	O
+deviated	O
+significantly	O
+from	O
+the	O
+active	B-site
+site	I-site
+and	O
+assumed	O
+a	O
+non	O
+-	O
+productive	O
+binding	O
+mode	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2D	O
+).	O
+
+This	O
+observation	O
+suggests	O
+that	O
+the	O
+open	B-protein_state
+conformation	O
+of	O
+BlCel5B	B-protein
+is	O
+not	O
+able	O
+to	O
+hold	O
+the	O
+substrate	O
+in	O
+a	O
+position	O
+suitable	O
+for	O
+hydrolysis	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2E	O
+).	O
+
+However	O
+,	O
+after	O
+subjecting	O
+the	O
+BlCel5B	B-complex_assembly
+-	I-complex_assembly
+C8	I-complex_assembly
+complex	O
+to	O
+a	O
+0	O
+.	O
+5	O
+μs	O
+aMD	B-experimental_method
+simulation	I-experimental_method
+with	O
+harmonic	O
+restraints	O
+on	O
+the	O
+C8	B-chemical
+chain	O
+to	O
+prevent	O
+it	O
+from	O
+deviating	O
+from	O
+the	O
+productive	O
+binding	O
+mode	O
+,	O
+the	O
+CBM46	B-structure_element
+readily	O
+closed	B-protein_state
+over	O
+the	O
+CD	B-structure_element
+and	O
+trapped	O
+the	O
+C8	B-chemical
+chain	O
+in	O
+position	O
+for	O
+hydrolysis	O
+(	O
+Fig	O
+.	O
+3B	O
+).	O
+
+In	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+substrate	O
+,	O
+CBM46	B-structure_element
+adopts	O
+a	O
+final	O
+conformation	O
+intermediate	O
+between	O
+the	O
+crystallographic	B-evidence
+structure	I-evidence
+and	O
+that	O
+observed	O
+in	O
+the	O
+substrate	B-protein_state
+-	I-protein_state
+free	I-protein_state
+BlCel5B	B-protein
+aMD	B-experimental_method
+simulations	I-experimental_method
+;	O
+this	O
+is	O
+illustrated	O
+by	O
+the	O
+I120	B-residue_name_number
+-	O
+E477	B-residue_name_number
+distance	B-evidence
+,	O
+which	O
+stabilizes	O
+near	O
+20	O
+Å	O
+in	O
+the	O
+closed	B-protein_state
+configuration	O
+that	O
+traps	O
+the	O
+C8	B-chemical
+molecule	O
+(	O
+in	O
+contrast	O
+to	O
+~	O
+7	O
+Å	O
+for	O
+substrate	B-protein_state
+-	I-protein_state
+free	I-protein_state
+BlCel5B	B-protein
+)	O
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+
+This	O
+BlCel5B	B-complex_assembly
+-	I-complex_assembly
+C8	I-complex_assembly
+configuration	O
+remains	O
+stable	O
+over	O
+an	O
+additional	O
+500	O
+ns	O
+of	O
+conventional	O
+MD	B-experimental_method
+simulation	I-experimental_method
+with	O
+no	O
+restraints	O
+(	O
+Fig	O
+.	O
+3C	O
+cyan	O
+line	O
+,	O
+Supplementary	O
+Fig	O
+.	O
+2E	O
+,	O
+F	O
+).	O
+
+A	O
+closer	O
+inspection	O
+of	O
+the	O
+productive	O
+binding	O
+mode	O
+obtained	O
+from	O
+these	O
+extensive	O
+simulations	B-experimental_method
+reveals	O
+that	O
+the	O
+CBM46	B-structure_element
+tryptophan	B-residue_name
+residues	O
+W479	B-residue_name_number
+and	O
+W481	B-residue_name_number
+(	O
+along	O
+with	O
+CD	B-structure_element
+tryptophan	B-residue_name
+residues	O
+)	O
+play	O
+important	O
+roles	O
+in	O
+carbohydrate	B-chemical
+recognition	O
+and	O
+orientation	O
+by	O
+creating	O
+a	O
+tunnel	B-site
+-	O
+like	O
+topology	O
+along	O
+the	O
+BlCel5B	B-protein
+binding	B-site
+cleft	I-site
+,	O
+as	O
+depicted	O
+in	O
+Fig	O
+.	O
+3D	O
+.	O
+
+Together	O
+,	O
+these	O
+results	O
+indicate	O
+that	O
+CBM46	B-structure_element
+is	O
+a	O
+key	O
+component	O
+of	O
+the	O
+catalytic	B-protein_state
+active	I-protein_state
+complex	O
+,	O
+providing	O
+an	O
+explanation	O
+as	O
+to	O
+why	O
+CBM46	B-structure_element
+is	O
+essential	O
+for	O
+the	O
+enzymatic	O
+activity	O
+of	O
+BlCel5B	B-protein
+.	O
+
+To	O
+enable	O
+substantially	O
+longer	O
+time	O
+scales	O
+compared	O
+to	O
+atomistic	B-experimental_method
+simulations	I-experimental_method
+,	O
+we	O
+further	O
+explored	O
+the	O
+dynamics	O
+of	O
+BlCel5B	B-protein
+using	O
+coarse	B-experimental_method
+-	I-experimental_method
+grained	I-experimental_method
+MD	I-experimental_method
+(	O
+CG	B-experimental_method
+-	I-experimental_method
+MD	I-experimental_method
+)	O
+simulations	B-experimental_method
+.	O
+
+We	O
+performed	O
+three	O
+independent	O
+~	O
+120	O
+μs	O
+CG	B-experimental_method
+-	I-experimental_method
+MD	I-experimental_method
+simulations	I-experimental_method
+,	O
+for	O
+a	O
+total	O
+of	O
+approximately	O
+360	O
+μs	O
+of	O
+sampling	O
+.	O
+
+The	O
+distance	B-evidence
+between	O
+the	O
+α	O
+carbons	O
+of	O
+two	O
+residues	O
+centrally	O
+positioned	O
+in	O
+the	O
+CD	B-structure_element
+and	O
+CBM46	B-structure_element
+(	O
+Fig	O
+.	O
+4A	O
+)	O
+was	O
+monitored	O
+,	O
+and	O
+the	O
+results	O
+shown	O
+in	O
+Fig	O
+.	O
+4B	O
+indicate	O
+that	O
+the	O
+wide	O
+-	O
+amplitude	O
+events	O
+described	O
+above	O
+frequently	O
+appear	O
+in	O
+this	O
+time	O
+scale	O
+.	O
+
+The	O
+computed	B-evidence
+distance	I-evidence
+distribution	I-evidence
+depicted	O
+in	O
+Fig	O
+.	O
+4C	O
+indicates	O
+three	O
+main	O
+conformational	O
+states	O
+ranging	O
+from	O
+(	O
+I	O
+)	O
+closed	B-protein_state
+conformations	O
+similar	O
+to	O
+those	O
+encountered	O
+in	O
+the	O
+substrate	B-protein_state
+-	I-protein_state
+free	I-protein_state
+aMD	B-experimental_method
+simulations	I-experimental_method
+,	O
+in	O
+which	O
+CBM46	B-structure_element
+interacts	O
+with	O
+the	O
+CD	B-structure_element
+to	O
+shape	O
+the	O
+substrate	B-site
+binding	I-site
+site	I-site
+,	O
+to	O
+(	O
+II	O
+)	O
+semi	B-protein_state
+-	I-protein_state
+open	I-protein_state
+conformations	O
+similar	O
+to	O
+the	O
+crystallographic	B-evidence
+structure	I-evidence
+,	O
+and	O
+(	O
+III	O
+)	O
+extended	B-protein_state
+BlCel5B	B-protein
+conformations	O
+in	O
+which	O
+the	O
+CD	B-structure_element
+and	O
+CBM46	B-structure_element
+are	O
+even	O
+further	O
+apart	O
+than	O
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+.	O
+
+BlCel5B	B-protein
+conformers	O
+fit	O
+the	O
+SAXS	B-experimental_method
+envelope	B-evidence
+
+SAXS	B-experimental_method
+experiments	O
+were	O
+conducted	O
+to	O
+assess	O
+BlCel5B	B-protein
+conformational	O
+states	O
+in	O
+solution	O
+,	O
+and	O
+the	O
+results	O
+revealed	O
+the	O
+enzyme	O
+in	O
+its	O
+monomeric	B-oligomeric_state
+form	O
+,	O
+with	O
+average	O
+values	O
+of	O
+Rg	B-evidence
+=	O
+27	O
+.	O
+17	O
+Å	O
+and	O
+Dmax	B-evidence
+=	O
+87	O
+.	O
+59	O
+Å	O
+(	O
+Supplementary	O
+Table	O
+2	O
+).	O
+
+The	O
+ab	B-experimental_method
+initio	I-experimental_method
+dummy	I-experimental_method
+atom	I-experimental_method
+model	I-experimental_method
+(	O
+DAM	B-experimental_method
+)	O
+demonstrated	O
+that	O
+the	O
+SAXS	B-experimental_method
+-	O
+derived	O
+BlCel5B	B-protein
+molecular	O
+envelope	B-evidence
+could	O
+not	O
+be	O
+single	O
+-	O
+handedly	O
+filled	O
+by	O
+any	O
+of	O
+the	O
+main	O
+conformational	O
+states	O
+encountered	O
+in	O
+the	O
+simulations	B-experimental_method
+(	O
+Fig	O
+.	O
+4D	O
+).	O
+
+It	O
+is	O
+known	O
+that	O
+a	O
+Kratky	B-evidence
+plot	I-evidence
+exhibits	O
+a	O
+peak	O
+with	O
+an	O
+elevated	O
+baseline	O
+at	O
+high	O
+q	O
+for	O
+a	O
+monodisperse	O
+system	O
+composed	O
+of	O
+multi	O
+-	O
+domain	O
+particles	O
+with	O
+flexible	O
+extensions	O
+.	O
+
+Indeed	O
+,	O
+an	O
+elevation	O
+of	O
+the	O
+baseline	O
+toward	O
+a	O
+hyperbolic	O
+-	O
+like	O
+curve	O
+was	O
+observed	O
+for	O
+BlCel5B	B-protein
+,	O
+indicating	O
+a	O
+considerable	O
+degree	O
+of	O
+molecular	O
+mobility	O
+in	O
+solution	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3	O
+).	O
+
+Thus	O
+,	O
+the	O
+conformational	O
+heterogeneity	O
+of	O
+the	O
+enzyme	O
+can	O
+be	O
+decomposed	O
+in	O
+structural	O
+terms	O
+as	O
+a	O
+combination	O
+of	O
+conformational	O
+states	O
+identified	O
+in	O
+our	O
+crystallographic	B-experimental_method
+and	I-experimental_method
+MD	I-experimental_method
+studies	I-experimental_method
+.	O
+
+We	O
+found	O
+that	O
+the	O
+SAXS	B-experimental_method
+envelope	B-evidence
+can	O
+be	O
+well	O
+represented	O
+by	O
+considering	O
+the	O
+superimposition	B-experimental_method
+of	O
+three	O
+different	O
+representative	O
+molecular	O
+conformations	O
+of	O
+BlCel5B	B-protein
+(	O
+Fig	O
+.	O
+4E	O
+):	O
+a	O
+closed	B-protein_state
+or	O
+CBM46	B-structure_element
+/	O
+CD	B-structure_element
+-	O
+occluded	B-protein_state
+conformation	O
+extracted	O
+from	O
+the	O
+simulations	B-experimental_method
+with	O
+a	O
+relative	O
+weight	O
+of	O
+26	O
+%,	O
+a	O
+semi	B-protein_state
+-	I-protein_state
+open	I-protein_state
+conformation	O
+represented	O
+by	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+corresponding	O
+to	O
+40	O
+%,	O
+and	O
+an	O
+extended	B-protein_state
+conformation	O
+based	O
+on	O
+simulations	B-experimental_method
+that	O
+is	O
+responsible	O
+for	O
+34	O
+%	O
+of	O
+the	O
+SAXS	B-experimental_method
+envelope	B-evidence
+.	O
+
+The	O
+resulting	O
+average	B-evidence
+scattering	I-evidence
+curve	I-evidence
+from	O
+this	O
+model	O
+fits	O
+the	O
+experimental	O
+protein	O
+scattering	B-evidence
+intensity	I-evidence
+,	O
+with	O
+χ	B-evidence
+=	O
+1	O
+.	O
+89	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+3	O
+).	O
+
+GH5_4	B-protein_type
+phylogenetic	B-experimental_method
+analysis	I-experimental_method
+
+After	O
+the	O
+exclusion	O
+of	O
+partial	O
+sequences	O
+and	O
+the	O
+suppression	O
+of	O
+highly	O
+identical	O
+members	O
+(	O
+higher	O
+than	O
+90	O
+%	O
+identity	O
+),	O
+144	O
+sequences	O
+containing	O
+between	O
+277	B-residue_range
+and	I-residue_range
+400	I-residue_range
+residues	O
+were	O
+aligned	B-experimental_method
+and	O
+used	O
+to	O
+construct	O
+a	O
+phylogenetic	B-evidence
+tree	I-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+4A	O
+).	O
+
+According	O
+to	O
+PFAM	O
+database	O
+conserved	O
+domain	O
+classification	O
+,	O
+128	O
+GH5	B-protein_type
+enzymes	O
+have	O
+an	O
+architecture	O
+consisting	O
+of	O
+an	O
+N	O
+-	O
+terminal	O
+catalytic	B-structure_element
+module	I-structure_element
+,	O
+a	O
+CBM_X2	B-structure_element
+module	O
+and	O
+an	O
+unknown	O
+module	O
+of	O
+approximately	O
+100	O
+residues	O
+at	O
+the	O
+C	O
+-	O
+terminus	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4B	O
+).	O
+
+Of	O
+these	O
+,	O
+12	O
+enzymes	O
+have	O
+an	O
+additional	O
+CBM1	B-structure_element
+,	O
+and	O
+5	O
+have	O
+a	O
+CBM2	B-structure_element
+at	O
+the	O
+N	O
+-	O
+terminal	O
+region	O
+.	O
+
+Based	O
+on	O
+this	O
+PFAM	O
+architecture	O
+and	O
+CAZy	O
+subfamily	O
+classification	O
+,	O
+all	O
+the	O
+144	O
+enzymes	O
+(	O
+including	O
+BlCel5B	B-protein
+)	O
+belong	O
+to	O
+the	O
+GH5_4	B-protein_type
+subfamily	O
+and	O
+group	O
+together	O
+in	O
+the	O
+same	O
+branch	O
+of	O
+the	O
+phylogenetic	B-evidence
+tree	I-evidence
+,	O
+evidencing	O
+a	O
+common	O
+ancestor	O
+.	O
+
+These	O
+results	O
+support	O
+the	O
+hypothesis	O
+that	O
+the	O
+enzymes	O
+may	O
+employ	O
+the	O
+same	O
+mechanism	O
+by	O
+which	O
+ligand	O
+binding	O
+is	O
+mediated	O
+by	O
+an	O
+extensive	O
+conformational	O
+breathing	O
+of	O
+the	O
+enzyme	O
+that	O
+involves	O
+the	O
+large	O
+-	O
+scale	O
+movement	O
+of	O
+CBM46	B-structure_element
+around	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+(	O
+CBM_X2	B-structure_element
+)	O
+as	O
+a	O
+structural	B-structure_element
+hinge	I-structure_element
+.	O
+
+Here	O
+,	O
+we	O
+elucidate	O
+the	O
+trimodular	B-protein_state
+molecular	O
+architecture	O
+of	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+BlCel5B	B-protein
+,	O
+a	O
+member	O
+of	O
+the	O
+GH5_4	B-protein_type
+subfamily	O
+,	O
+for	O
+which	O
+large	O
+-	O
+scale	O
+conformational	O
+dynamics	O
+appears	O
+to	O
+play	O
+a	O
+central	O
+role	O
+in	O
+its	O
+enzymatic	O
+activity	O
+.	O
+
+Full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+BlCel5B	B-protein
+is	O
+active	B-protein_state
+on	O
+both	O
+cellulosic	B-chemical
+and	O
+hemicellulosic	B-chemical
+substrates	O
+and	O
+auxiliary	O
+modules	O
+are	O
+crucial	O
+for	O
+its	O
+activity	O
+.	O
+
+Most	O
+carbohydrate	B-protein_type
+-	I-protein_type
+active	I-protein_type
+enzymes	I-protein_type
+are	O
+modular	O
+and	O
+consist	O
+of	O
+a	O
+catalytic	B-structure_element
+domain	I-structure_element
+appended	O
+to	O
+one	O
+or	O
+more	O
+separate	O
+AMs	B-structure_element
+.	O
+
+AMs	B-structure_element
+,	O
+such	O
+as	O
+CBMs	B-structure_element
+,	O
+typically	O
+recognize	O
+carbohydrates	B-chemical
+and	O
+target	O
+their	O
+cognate	O
+catalytic	B-structure_element
+domains	I-structure_element
+toward	O
+the	O
+substrate	O
+.	O
+
+Because	O
+the	O
+structural	B-experimental_method
+analysis	I-experimental_method
+of	O
+the	O
+protein	O
+is	O
+challenging	O
+if	O
+the	O
+linkers	B-structure_element
+connecting	O
+the	O
+structural	O
+subunits	O
+of	O
+the	O
+enzyme	O
+are	O
+long	O
+and	O
+flexible	O
+,	O
+the	O
+standard	O
+approach	O
+is	O
+to	O
+study	O
+the	O
+domains	O
+separately	O
+.	O
+
+In	O
+this	O
+work	O
+,	O
+a	O
+combination	O
+of	O
+protein	B-experimental_method
+crystallography	I-experimental_method
+,	O
+computational	B-experimental_method
+molecular	I-experimental_method
+dynamics	I-experimental_method
+,	O
+and	O
+SAXS	B-experimental_method
+analyses	O
+enabled	O
+the	O
+identification	O
+of	O
+a	O
+new	O
+conformational	O
+selection	O
+-	O
+based	O
+molecular	O
+mechanism	O
+that	O
+involves	O
+GH5	B-protein_type
+catalytic	B-structure_element
+domain	I-structure_element
+and	O
+two	O
+AMs	B-structure_element
+in	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+BlCel5B	B-protein
+.	O
+
+We	O
+observed	O
+that	O
+the	O
+BlCel5B	B-protein
+distal	O
+CBM46	B-structure_element
+is	O
+directly	O
+involved	O
+in	O
+shaping	O
+the	O
+local	O
+architecture	O
+of	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+site	I-site
+.	O
+
+Although	O
+the	O
+CD	B-structure_element
+alone	B-protein_state
+appears	O
+unable	O
+to	O
+bind	O
+the	O
+substrate	O
+for	O
+catalysis	O
+,	O
+the	O
+AMs	B-structure_element
+exhibit	O
+open	B-protein_state
+-	O
+close	B-protein_state
+motions	O
+that	O
+allow	O
+the	O
+substrate	O
+to	O
+be	O
+captured	O
+in	O
+a	O
+suitable	O
+position	O
+for	O
+hydrolysis	O
+.	O
+
+Here	O
+,	O
+we	O
+advocate	O
+that	O
+large	O
+-	O
+amplitude	O
+motions	O
+of	O
+AMs	B-structure_element
+are	O
+crucial	O
+for	O
+assembling	O
+the	O
+enzyme	O
+into	O
+its	O
+active	B-protein_state
+conformation	O
+,	O
+highlighting	O
+a	O
+new	O
+function	O
+of	O
+CBMs	B-structure_element
+.	O
+
+This	O
+mechanism	O
+of	O
+substrate	O
+binding	O
+closely	O
+resembles	O
+the	O
+extended	B-protein_state
+conformational	O
+selection	O
+model	O
+,	O
+with	O
+the	O
+induced	O
+-	O
+fit	O
+mechanism	O
+of	O
+reaction	O
+as	O
+its	O
+limiting	O
+case	O
+.	O
+
+To	O
+the	O
+best	O
+of	O
+our	O
+knowledge	O
+,	O
+this	O
+enzymatic	O
+mechanism	O
+has	O
+not	O
+been	O
+proposed	O
+previously	O
+for	O
+any	O
+GH	B-protein_type
+.	O
+
+The	O
+CD	B-site
+binding	I-site
+site	I-site
+of	O
+BlCel5B	B-protein
+is	O
+open	O
+and	O
+relatively	O
+flat	O
+and	O
+is	O
+thus	O
+barely	O
+able	O
+to	O
+properly	O
+hold	O
+the	O
+substrate	O
+in	O
+position	O
+for	O
+catalysis	O
+without	O
+assistance	O
+from	O
+the	O
+CBM46	B-structure_element
+.	O
+
+In	O
+contrast	O
+,	O
+other	O
+GH5s	B-protein_type
+belonging	O
+to	O
+subfamily	O
+4	O
+listed	O
+in	O
+the	O
+Protein	O
+Data	O
+Bank	O
+exhibit	O
+a	O
+deep	O
+binding	B-site
+cleft	I-site
+or	O
+tunnel	B-site
+that	O
+can	O
+effectively	O
+entrap	O
+the	O
+substrate	O
+for	O
+catalysis	O
+(	O
+Fig	O
+.	O
+5	O
+).	O
+
+Due	O
+to	O
+the	O
+marked	O
+interdomain	O
+conformational	O
+rearrangement	O
+observed	O
+in	O
+our	O
+simulations	B-experimental_method
+,	O
+the	O
+CBM46	B-structure_element
+generates	O
+a	O
+confined	O
+binding	B-site
+site	I-site
+in	O
+BlCel5B	B-protein
+that	O
+resembles	O
+the	O
+binding	B-site
+site	I-site
+architecture	O
+of	O
+the	O
+other	O
+GH5	B-protein_type
+enzymes	O
+that	O
+lack	B-protein_state
+AMs	B-structure_element
+.	O
+
+Thus	O
+,	O
+BlCel5B	B-protein
+appears	O
+to	O
+have	O
+adopted	O
+a	O
+strategy	O
+of	O
+CBM46	B-structure_element
+-	O
+mediated	O
+interactions	O
+for	O
+proper	O
+functioning	O
+.	O
+
+Although	O
+the	O
+homologous	O
+BhCel5B	B-protein
+has	O
+the	O
+same	O
+domain	O
+architecture	O
+of	O
+BlCel5B	B-protein
+and	O
+belongs	O
+to	O
+the	O
+same	O
+subfamily	O
+(	O
+a	O
+comparison	O
+of	O
+the	O
+sequence	O
+and	O
+structure	B-evidence
+of	O
+BlCel5B	B-protein
+and	O
+BhCel5B	B-protein
+is	O
+presented	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+5	O
+),	O
+its	O
+binding	B-site
+site	I-site
+exhibits	O
+important	O
+differences	O
+that	O
+may	O
+impact	O
+the	O
+catalytic	O
+mechanism	O
+.	O
+
+The	O
+BhCel5B	B-protein
+binding	B-site
+site	I-site
+is	O
+V	B-protein_state
+-	I-protein_state
+shaped	I-protein_state
+and	O
+deeper	O
+than	O
+the	O
+BlCel5B	B-protein
+binding	B-site
+site	I-site
+(	O
+Figs	O
+5	O
+and	O
+6	O
+).	O
+
+This	O
+is	O
+due	O
+to	O
+the	O
+loop	B-structure_element
+between	O
+residues	O
+F177	B-residue_name_number
+and	O
+R185	B-residue_name_number
+from	O
+BhCel5B	B-protein
+(	O
+absent	B-protein_state
+in	O
+the	O
+BlCel5B	B-protein
+),	O
+which	O
+contains	O
+residue	O
+W181	B-residue_name_number
+that	O
+forms	O
+part	O
+of	O
+the	O
+binding	B-site
+cleft	I-site
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+Consistently	O
+,	O
+although	O
+BhCel5B	B-protein
+CBM46	B-structure_element
+is	O
+important	O
+for	O
+β	B-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+glucan	I-chemical
+hydrolysis	O
+(	O
+BhCel5B	B-protein
+is	O
+about	O
+60	O
+-	O
+fold	O
+less	O
+active	B-protein_state
+without	B-protein_state
+CBM46	B-structure_element
+),	O
+the	O
+truncated	B-protein_state
+enzyme	O
+is	O
+completely	O
+active	B-protein_state
+against	O
+xyloglucan	B-chemical
+,	O
+suggesting	O
+that	O
+the	O
+CBM46	B-structure_element
+,	O
+in	O
+this	O
+case	O
+,	O
+is	O
+necessary	O
+for	O
+the	O
+binding	O
+to	O
+specific	O
+substrates	O
+.	O
+
+A	O
+closer	O
+inspection	O
+of	O
+results	O
+of	O
+the	O
+phylogenetic	B-experimental_method
+analysis	I-experimental_method
+,	O
+more	O
+specifically	O
+of	O
+the	O
+clade	O
+composed	O
+by	O
+GH5_4	B-protein_type
+enzymes	O
+with	O
+trimodular	B-protein_state
+architecture	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4C	O
+),	O
+reveals	O
+subclades	O
+whose	O
+main	O
+characteristic	O
+is	O
+the	O
+varying	O
+length	O
+of	O
+the	O
+loop	B-structure_element
+located	O
+between	O
+residues	O
+161	B-residue_range
+and	I-residue_range
+163	I-residue_range
+(	O
+BlCel5B	B-protein
+residue	O
+numbering	O
+).	O
+
+Therefore	O
+,	O
+our	O
+results	O
+show	O
+that	O
+BlCel5B	B-protein
+represents	O
+a	O
+smaller	O
+group	O
+of	O
+enzymes	O
+that	O
+are	O
+completely	O
+dependent	O
+on	O
+its	O
+AMs	B-structure_element
+for	O
+hydrolysis	O
+of	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+polysaccharides	B-chemical
+,	O
+and	O
+that	O
+the	O
+underlying	O
+mechanism	O
+may	O
+rely	O
+on	O
+large	O
+-	O
+scale	O
+interdomain	O
+motions	O
+.	O
+
+The	O
+amino	O
+acid	O
+sequence	O
+of	O
+the	O
+BlCel5B	B-protein
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+is	O
+recognized	O
+by	O
+BLASTP	B-experimental_method
+as	O
+belonging	O
+to	O
+CBM_X2	B-structure_element
+,	O
+a	O
+poorly	O
+described	O
+group	O
+that	O
+has	O
+been	O
+compared	O
+with	O
+CBM	B-structure_element
+-	I-structure_element
+like	I-structure_element
+accessory	I-structure_element
+modules	I-structure_element
+without	O
+a	O
+defined	O
+function	O
+.	O
+
+Despite	O
+the	O
+similarity	O
+of	O
+BlCel5B	B-protein
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+to	O
+CBMs	B-structure_element
+,	O
+it	O
+lacks	O
+an	O
+identifiable	O
+aromatic	O
+residue	O
+-	O
+rich	O
+carbohydrate	B-site
+-	I-site
+binding	I-site
+site	I-site
+.	O
+
+Nonetheless	O
+,	O
+according	O
+to	O
+our	O
+results	O
+,	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+seems	O
+to	O
+play	O
+an	O
+important	O
+function	O
+as	O
+a	O
+structural	B-structure_element
+hinge	I-structure_element
+,	O
+dynamically	O
+holding	O
+the	O
+CBM46	B-structure_element
+and	O
+CD	B-structure_element
+in	O
+positions	O
+that	O
+are	O
+appropriate	O
+for	O
+enzymatic	O
+activity	O
+.	O
+
+Based	O
+on	O
+the	O
+results	O
+of	O
+our	O
+crystallographic	B-experimental_method
+,	I-experimental_method
+computer	I-experimental_method
+simulation	I-experimental_method
+,	O
+and	O
+SAXS	B-experimental_method
+structural	I-experimental_method
+analyses	I-experimental_method
+,	O
+as	O
+well	O
+as	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+and	O
+activity	B-experimental_method
+assays	I-experimental_method
+,	O
+we	O
+propose	O
+a	O
+molecular	O
+mechanism	O
+for	O
+BlCel5B	B-protein
+substrate	O
+binding	O
+,	O
+which	O
+might	O
+apply	O
+to	O
+other	O
+GH5_4	B-protein_type
+subfamily	O
+enzymes	O
+that	O
+share	O
+this	O
+tri	B-structure_element
+-	I-structure_element
+modular	I-structure_element
+architecture	O
+.	O
+
+BlCel5B	B-protein
+can	O
+be	O
+found	O
+in	O
+several	O
+different	O
+conformational	O
+states	O
+ranging	O
+from	O
+CBM46	B-structure_element
+/	O
+CD	B-structure_element
+closed	B-protein_state
+(	O
+or	O
+occluded	B-protein_state
+)	O
+to	O
+extended	B-protein_state
+conformations	O
+(	O
+Fig	O
+.	O
+7	O
+).	O
+
+In	O
+extended	B-protein_state
+configurations	O
+,	O
+the	O
+substrate	O
+may	O
+dock	O
+at	O
+the	O
+shallow	O
+substrate	B-site
+binding	I-site
+site	I-site
+of	O
+CD	B-structure_element
+in	O
+one	O
+of	O
+the	O
+semi	B-protein_state
+-	I-protein_state
+closed	I-protein_state
+conformations	O
+of	O
+the	O
+enzyme	O
+;	O
+however	O
+,	O
+its	O
+binding	O
+is	O
+properly	O
+stabilized	O
+for	O
+hydrolysis	O
+only	O
+with	O
+the	O
+aid	O
+of	O
+induced	O
+-	O
+fit	O
+repositioning	O
+mediated	O
+by	O
+CBM46	B-structure_element
+.	O
+
+After	O
+cleavage	O
+,	O
+the	O
+intrinsic	O
+dynamics	O
+of	O
+BlCel5B	B-protein
+would	O
+eventually	O
+allow	O
+the	O
+opening	O
+of	O
+the	O
+active	B-site
+site	I-site
+for	O
+product	O
+release	O
+.	O
+
+The	O
+proposed	O
+mechanism	O
+is	O
+consistent	O
+with	O
+our	O
+mutagenesis	B-experimental_method
+and	I-experimental_method
+enzymatic	I-experimental_method
+activity	I-experimental_method
+assays	I-experimental_method
+,	O
+which	O
+show	O
+that	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+module	I-structure_element
+and	O
+CBM46	B-structure_element
+are	O
+indispensable	O
+for	O
+BlCel5B	B-protein
+catalytic	O
+activity	O
+and	O
+,	O
+together	O
+with	O
+the	O
+CD	B-structure_element
+,	O
+form	O
+the	O
+unique	B-protein_state
+catalytic	B-structure_element
+domain	I-structure_element
+of	O
+the	O
+enzyme	O
+.	O
+
+These	O
+experiments	O
+reveal	O
+a	O
+novel	O
+function	O
+for	O
+CBMs	B-structure_element
+in	O
+which	O
+they	O
+are	O
+intimately	O
+involved	O
+in	O
+the	O
+assembly	O
+of	O
+the	O
+active	B-site
+site	I-site
+and	O
+catalytic	O
+process	O
+.	O
+
+Computer	B-experimental_method
+simulations	I-experimental_method
+suggest	O
+that	O
+large	O
+-	O
+scale	O
+motions	O
+of	O
+the	O
+CBM46	B-structure_element
+and	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+domains	I-structure_element
+mediate	O
+conformational	O
+selection	O
+and	O
+final	O
+induced	O
+-	O
+fit	O
+adjustments	O
+to	O
+trap	O
+the	O
+substrate	O
+at	O
+the	O
+active	B-site
+site	I-site
+and	O
+promote	O
+hydrolysis	O
+.	O
+
+SAXS	B-experimental_method
+data	O
+support	O
+the	O
+modeling	B-experimental_method
+results	O
+,	O
+providing	O
+compelling	O
+evidence	O
+for	O
+highly	B-protein_state
+mobile	I-protein_state
+domains	O
+in	O
+solution	O
+.	O
+
+Crystal	B-evidence
+models	I-evidence
+of	O
+BlCel5B	B-protein
+.	O
+
+Complete	O
+structure	B-evidence
+is	O
+shown	O
+as	O
+a	O
+cartoon	O
+illustration	O
+in	O
+(	O
+a	O
+)	O
+and	O
+a	O
+van	O
+der	O
+Waals	O
+surface	O
+in	O
+(	O
+b	O
+).	O
+
+The	O
+CD	B-structure_element
+module	O
+(	O
+red	O
+)	O
+has	O
+a	O
+typical	O
+TIM	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+fold	I-structure_element
+,	O
+and	O
+its	O
+substrate	B-site
+-	I-site
+binding	I-site
+site	I-site
+is	O
+adjacent	O
+to	O
+CBM46	B-structure_element
+(	O
+blue	O
+).	O
+
+Despite	O
+the	O
+proximity	O
+of	O
+the	O
+binding	B-site
+site	I-site
+in	O
+the	O
+crystallographic	O
+model	O
+,	O
+the	O
+CBM46	B-structure_element
+residues	O
+W479	B-residue_name_number
+and	O
+W481	B-residue_name_number
+are	O
+distant	O
+from	O
+the	O
+substrate	O
+cellotetraose	B-chemical
+(	O
+yellow	O
+).	O
+
+The	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+domain	I-structure_element
+(	O
+green	O
+)	O
+has	O
+a	O
+lateral	O
+position	O
+,	O
+serving	O
+as	O
+a	O
+connector	O
+between	O
+the	O
+CD	B-structure_element
+and	O
+CBM46	B-structure_element
+.	O
+(	O
+c	O
+)	O
+A	O
+superposition	B-experimental_method
+of	O
+the	O
+Ig	B-structure_element
+-	I-structure_element
+like	I-structure_element
+domain	I-structure_element
+and	O
+CBM46	B-structure_element
+illustrates	O
+their	O
+structural	O
+similarity	O
+,	O
+with	O
+most	O
+of	O
+the	O
+structural	O
+differences	O
+present	O
+in	O
+the	O
+loop	B-structure_element
+highlighted	O
+by	O
+a	O
+red	O
+circle	O
+.	O
+(	O
+d	O
+)	O
+Cellotetraose	B-chemical
+occupies	O
+subsites	B-site
+-	I-site
+1	I-site
+to	I-site
+-	I-site
+3	I-site
+and	O
+is	O
+primarily	O
+coordinated	B-bond_interaction
+by	O
+the	O
+residues	O
+represented	O
+in	O
+gray	O
+.	O
+
+BlCel5B	B-protein
+enzymatic	B-experimental_method
+activity	I-experimental_method
+characterization	I-experimental_method
+.	O
+
+(	O
+a	O
+)	O
+MALDI	B-experimental_method
+/	I-experimental_method
+TOF	I-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+spectra	B-evidence
+of	O
+the	O
+products	O
+released	O
+after	O
+incubation	O
+of	O
+BlCel5B	B-protein
+and	O
+its	O
+two	O
+deletion	B-experimental_method
+constructs	I-experimental_method
+(	O
+ΔCBM46	B-mutant
+and	O
+ΔIg	B-mutant
+-	I-mutant
+CBM46	I-mutant
+)	O
+with	O
+the	O
+substrate	O
+cellopentaose	B-chemical
+(	O
+C5	B-chemical
+).	O
+
+The	O
+first	O
+three	O
+spectra	B-evidence
+show	O
+the	O
+substrate	O
+,	O
+enzyme	O
+and	O
+buffer	O
+controls	O
+.	O
+
+The	O
+forth	O
+spectrum	B-evidence
+reveals	O
+that	O
+full	B-protein_state
+length	I-protein_state
+BlCel5B	B-protein
+is	O
+capable	O
+of	O
+enzymatic	O
+hydrolysis	O
+of	O
+C5	B-chemical
+into	O
+smaller	O
+oligosaccharides	B-chemical
+such	O
+as	O
+C4	B-chemical
+,	O
+C3	B-chemical
+and	O
+C2	B-chemical
+.	O
+
+The	O
+last	O
+two	O
+spectra	B-evidence
+show	O
+that	O
+the	O
+C	O
+-	O
+terminal	O
+deletions	O
+eliminate	B-protein_state
+the	I-protein_state
+enzyme	I-protein_state
+activity	I-protein_state
+.	O
+
+BlCel5B	B-protein
+activities	O
+on	O
+CMC	B-chemical
+as	O
+functions	O
+of	O
+pH	O
+and	O
+temperature	O
+are	O
+shown	O
+in	O
+(	O
+b	O
+)	O
+and	O
+(	O
+c	O
+),	O
+respectively	O
+.	O
+
+(	O
+d	O
+)	O
+Michaelis	B-evidence
+-	I-evidence
+Menten	I-evidence
+curve	I-evidence
+using	O
+CMC	B-chemical
+as	O
+a	O
+substrate	O
+.	O
+
+Open	B-protein_state
+-	O
+close	B-protein_state
+transitions	O
+of	O
+BlCel5B	B-protein
+.	O
+
+(	O
+a	O
+)	O
+BlCel5B	B-protein
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+substrate	O
+and	O
+(	O
+b	O
+)	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+cellooctaose	B-chemical
+,	O
+as	O
+observed	O
+in	O
+our	O
+aMD	B-experimental_method
+simulations	I-experimental_method
+.	O
+
+The	O
+distance	B-evidence
+between	O
+the	O
+α	O
+carbon	O
+of	O
+residues	O
+I120	B-residue_name_number
+(	O
+CD	B-structure_element
+)	O
+and	O
+E477	B-residue_name_number
+(	O
+CBM46	B-structure_element
+),	O
+illustrated	O
+as	O
+spheres	O
+in	O
+(	O
+a	O
+),	O
+is	O
+plotted	O
+in	O
+(	O
+c	O
+),	O
+revealing	O
+a	O
+transition	O
+by	O
+the	O
+decrease	O
+in	O
+the	O
+distance	B-evidence
+from	O
+40	O
+Å	O
+to	O
+7	O
+Å	O
+(	O
+substrate	B-protein_state
+-	I-protein_state
+free	I-protein_state
+)	O
+or	O
+20	O
+Å	O
+(	O
+in	O
+presence	B-protein_state
+of	I-protein_state
+cellooctaose	B-chemical
+).	O
+
+For	O
+the	O
+substrate	B-protein_state
+-	I-protein_state
+free	I-protein_state
+enzyme	O
+,	O
+the	O
+red	O
+line	O
+refers	O
+to	O
+a	O
+1	O
+μs	O
+-	O
+long	O
+aMD	B-experimental_method
+;	O
+for	O
+the	O
+BlCel5B	B-complex_assembly
+-	I-complex_assembly
+cellooctaose	I-complex_assembly
+complex	O
+,	O
+the	O
+first	O
+500	O
+ns	O
+refers	O
+to	O
+aMD	B-experimental_method
+(	O
+in	O
+blue	O
+)	O
+and	O
+the	O
+second	O
+500	O
+ns	O
+to	O
+conventional	O
+MD	B-experimental_method
+(	O
+in	O
+turquoise	O
+).	O
+
+(	O
+d	O
+)	O
+A	O
+snapshot	O
+of	O
+the	O
+BlCel5B	B-complex_assembly
+-	I-complex_assembly
+cellooctaose	I-complex_assembly
+complex	O
+,	O
+highlighting	O
+the	O
+tryptophan	B-residue_name
+residues	O
+that	O
+interact	O
+with	O
+the	O
+glucan	B-chemical
+chain	O
+in	O
+subsites	B-site
+−	I-site
+4	I-site
+to	I-site
++	I-site
+4	I-site
+.	O
+
+Residues	O
+W479	B-residue_name_number
+and	O
+W481	B-residue_name_number
+belong	O
+to	O
+CBM46	B-structure_element
+and	O
+only	O
+become	O
+available	O
+for	O
+substrate	O
+interactions	O
+in	O
+the	O
+closed	B-protein_state
+configuration	O
+of	O
+BlCel5B	B-protein
+.	O
+
+Large	O
+-	O
+scale	O
+movements	O
+of	O
+BlCel5B	B-protein
+modules	O
+and	O
+superposition	B-experimental_method
+of	O
+their	O
+representative	O
+conformations	O
+with	O
+the	O
+SAXS	B-experimental_method
+envelope	B-evidence
+.	O
+
+(	O
+a	O
+)	O
+BlCel5B	B-protein
+structure	B-evidence
+showing	O
+the	O
+distance	B-evidence
+between	O
+the	O
+backbone	O
+beads	O
+of	O
+residues	O
+I120	B-residue_name_number
+and	O
+E477	B-residue_name_number
+,	O
+which	O
+are	O
+centrally	O
+located	O
+in	O
+CD	B-structure_element
+and	O
+CBM46	B-structure_element
+,	O
+respectively	O
+,	O
+as	O
+a	O
+metric	O
+for	O
+the	O
+relative	O
+disposition	O
+between	O
+the	O
+two	O
+domains	O
+.	O
+(	O
+b	O
+)	O
+Time	O
+history	O
+of	O
+the	O
+I120	B-residue_name_number
+-	O
+E477	B-residue_name_number
+distance	B-evidence
+computed	O
+using	O
+CG	B-experimental_method
+-	I-experimental_method
+MD	I-experimental_method
+simulations	I-experimental_method
+.	O
+
+Different	O
+colors	O
+separated	O
+by	O
+vertical	O
+lines	O
+correspond	O
+to	O
+independent	O
+simulations	B-experimental_method
+of	O
+approximately	O
+120	O
+μs	O
+.	O
+(	O
+c	O
+)	O
+The	O
+distance	B-evidence
+distribution	I-evidence
+indicates	O
+three	O
+major	O
+peaks	O
+:	O
+closed	B-protein_state
+or	O
+occluded	B-protein_state
+CBM46	B-structure_element
+/	O
+CD	B-structure_element
+conformations	O
+(	O
+I	O
+);	O
+semi	B-protein_state
+-	I-protein_state
+open	I-protein_state
+(	O
+II	O
+),	O
+which	O
+is	O
+similar	O
+to	O
+the	O
+crystallographic	B-evidence
+structure	I-evidence
+;	O
+and	O
+extended	B-protein_state
+conformers	O
+(	O
+III	O
+).	O
+
+(	O
+d	O
+)	O
+Superimposition	B-experimental_method
+of	O
+the	O
+three	O
+representative	O
+molecular	O
+conformations	O
+of	O
+BlCel5B	B-protein
+with	O
+the	O
+SAXS	B-experimental_method
+model	B-evidence
+.	O
+(	O
+e	O
+)	O
+Average	O
+structures	B-evidence
+obtained	O
+from	O
+the	O
+simulation	B-experimental_method
+segments	O
+corresponding	O
+to	O
+population	O
+groups	O
+I	O
+-	O
+III	O
+,	O
+which	O
+are	O
+individually	O
+superposed	B-experimental_method
+on	O
+the	O
+SAXS	B-experimental_method
+envelope	B-evidence
+.	O
+
+Comparison	B-experimental_method
+of	O
+the	O
+binding	B-site
+site	I-site
+shape	O
+of	O
+GH5_4	B-protein_type
+enzymes	O
+available	O
+on	O
+the	O
+Protein	O
+Data	O
+Bank	O
+.	O
+
+(	O
+a	O
+)	O
+BlCel5B	B-protein
+in	O
+the	O
+crystallographic	B-experimental_method
+and	O
+closed	B-protein_state
+configuration	O
+;	O
+(	O
+b	O
+)	O
+Bacillus	B-species
+halodurans	I-species
+Cel5B	B-protein
+(	O
+BhCel5B	B-protein
+)	O
+(	O
+PDB	O
+id	O
+:	O
+4V2X	O
+)	O
+(	O
+c	O
+)	O
+Piromyces	B-species
+rhizinflata	I-species
+GH5	B-protein_type
+endoglucanase	B-protein_type
+(	O
+PDB	O
+id	O
+:	O
+3AYR	O
+);	O
+(	O
+d	O
+)	O
+Clostridium	B-species
+cellulolyticum	I-species
+GH5	B-protein_type
+endoglucanase	B-protein_type
+(	O
+PDB	O
+id	O
+:	O
+1EDG	O
+);	O
+(	O
+e	O
+)	O
+Clostridium	B-species
+cellulovorans	I-species
+GH5	B-protein_type
+endoglucanase	B-protein_type
+(	O
+PDB	O
+id	O
+:	O
+3NDY	O
+);	O
+(	O
+f	O
+)	O
+Bacteroides	B-species
+ovatus	I-species
+GH5	B-protein_type
+xyloglucanase	B-protein_type
+(	O
+PDB	O
+id	O
+:	O
+3ZMR	O
+);	O
+(	O
+g	O
+)	O
+Paenibacillus	B-species
+pabuli	I-species
+GH5	B-protein_type
+xyloglucanase	B-protein_type
+(	O
+PDB	O
+id	O
+:	O
+2JEP	O
+);	O
+(	O
+h	O
+)	O
+Prevotella	B-species
+bryantii	I-species
+GH5	B-protein_type
+endoglucanase	B-protein_type
+(	O
+PDB	O
+id	O
+:	O
+3VDH	O
+);	O
+(	O
+i	O
+)	O
+Ruminiclostridium	B-species
+thermocellum	I-species
+multifunctional	O
+GH5	B-protein_type
+cellulase	B-protein_type
+,	O
+xylanase	B-protein_type
+and	O
+mannase	B-protein_type
+(	O
+PDB	O
+id	O
+:	O
+4IM4	O
+);	O
+(	O
+j	O
+)	O
+Bacteroidetes	B-taxonomy_domain
+bacterium	I-taxonomy_domain
+AC2a	B-protein_type
+endocellulase	B-protein_type
+(	O
+PDB	O
+id	O
+:	O
+4YHE	O
+).	O
+
+Comparison	B-experimental_method
+of	O
+the	O
+binding	B-site
+cleft	I-site
+of	O
+the	O
+BlCel5B	B-protein
+and	O
+BhCel5B	B-protein
+.	O
+
+The	O
+main	O
+difference	O
+between	O
+BlCel5B	B-protein
+and	O
+BhCel5B	B-protein
+is	O
+that	O
+the	O
+latter	O
+exhibits	O
+a	O
+deeper	O
+cleft	B-site
+due	O
+to	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+residue	O
+W181	B-residue_name_number
+in	O
+the	O
+loop	B-structure_element
+between	O
+F177	B-residue_name_number
+and	O
+R185	B-residue_name_number
+.	O
+
+We	O
+conjecture	O
+that	O
+this	O
+difference	O
+in	O
+the	O
+binding	B-site
+site	I-site
+architecture	O
+relates	O
+to	O
+the	O
+importance	O
+that	O
+the	O
+CBM46	B-structure_element
+plays	O
+in	O
+the	O
+BlCel5B	B-protein
+enzymatic	O
+mechanism	O
+.	O
+
+Proposed	O
+molecular	O
+mechanism	O
+of	O
+BlCel5B	B-protein
+conformational	O
+selection	O
+.	O
+
+As	O
+suggested	O
+by	O
+the	O
+simulations	B-experimental_method
+and	O
+SAXS	B-experimental_method
+data	O
+,	O
+BlCel5B	B-protein
+spans	O
+multiple	O
+conformations	O
+ranging	O
+from	O
+closed	B-protein_state
+to	O
+extended	B-protein_state
+CBM46	B-structure_element
+/	O
+CD	B-structure_element
+states	O
+.	O
+
+In	O
+a	O
+given	O
+open	B-protein_state
+state	O
+,	O
+the	O
+substrate	O
+may	O
+reach	O
+the	O
+active	B-site
+site	I-site
+and	O
+become	O
+entrapped	O
+by	O
+the	O
+capping	O
+of	O
+CBM46	B-structure_element
+onto	O
+CD	B-structure_element
+and	O
+induced	O
+-	O
+fit	O
+conformational	O
+adjustments	O
+.	O
+
+After	O
+hydrolysis	O
+,	O
+the	O
+reaction	O
+product	O
+is	O
+released	O
+to	O
+yield	O
+apo	B-protein_state
+-	O
+BlCel5B	B-protein
+,	O
+which	O
+becomes	O
+ready	O
+for	O
+a	O
+new	O
+cycle	O
+.	O
+
+Activity	O
+of	O
+BlCel5B	B-protein
+constructs	O
+against	O
+tested	O
+substrates	O
+.	O
+
+Substrate	O
+(	O
+1	O
+%)	O
+Relative	O
+Activity	O
+(%)	O
+WT	B-protein_state
+*	O
+W479A	B-mutant
+W481A	B-mutant
+ΔCBM46	B-mutant
+ΔIg	B-mutant
+-	I-mutant
+CBM46	I-mutant
+β	B-chemical
+-	I-chemical
+glucan	I-chemical
+100	O
+79	O
+.	O
+1	O
+63	O
+.	O
+6	O
+nd	O
+nd	O
+CMC	B-chemical
+25	O
+.	O
+5	O
+12	O
+.	O
+2	O
+2	O
+.	O
+4	O
+nd	O
+nd	O
+Lichenan	B-chemical
+52	O
+.	O
+4	O
+41	O
+28	O
+.	O
+6	O
+nd	O
+nd	O
+Xyloglucan	B-chemical
+45	O
+.	O
+2	O
+41	O
+.	O
+2	O
+30	O
+.	O
+8	O
+nd	O
+nd	O
+Azo	B-chemical
+-	I-chemical
+Avicel	I-chemical
+nd	O
+**	O
+nd	O
+nd	O
+nd	O
+nd	O
+Arabinoxylan	B-chemical
+nd	O
+nd	O
+nd	O
+nd	O
+nd	O
+Galactomannan	B-chemical
+nd	O
+nd	O
+nd	O
+nd	O
+nd	O
+1	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+β	I-chemical
+-	I-chemical
+mannan	I-chemical
+nd	O
+nd	O
+nd	O
+nd	O
+nd	O
+
+*	O
+WT	B-protein_state
+=	O
+wild	B-protein_state
+type	I-protein_state
+.	O
+
diff --git a/annotation_IOB/PMC4980666.tsv b/annotation_IOB/PMC4980666.tsv
new file mode 100644
index 0000000000000000000000000000000000000000..458d0bef702fe3dc2ad0d3da89cf1b800158e300
--- /dev/null
+++ b/annotation_IOB/PMC4980666.tsv
@@ -0,0 +1,5903 @@
+N	B-chemical
+-	I-chemical
+acylhydrazone	I-chemical
+inhibitors	O
+of	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+PA	B-protein
+endonuclease	B-protein_type
+with	O
+versatile	O
+metal	O
+binding	O
+modes	O
+
+Influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+PA	B-protein
+endonuclease	B-protein_type
+has	O
+recently	O
+emerged	O
+as	O
+an	O
+attractive	O
+target	O
+for	O
+the	O
+development	O
+of	O
+novel	O
+antiviral	O
+therapeutics	O
+.	O
+
+This	O
+is	O
+an	O
+enzyme	O
+with	O
+divalent	O
+metal	O
+ion	O
+(	O
+s	O
+)	O
+(	O
+Mg2	B-chemical
++	I-chemical
+or	O
+Mn2	B-chemical
++)	I-chemical
+in	O
+its	O
+catalytic	B-site
+site	I-site
+:	O
+chelation	B-bond_interaction
+of	O
+these	O
+metal	O
+cofactors	O
+is	O
+an	O
+attractive	O
+strategy	O
+to	O
+inhibit	O
+enzymatic	O
+activity	O
+.	O
+
+Here	O
+we	O
+report	O
+the	O
+activity	O
+of	O
+a	O
+series	O
+of	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+in	O
+an	O
+enzymatic	B-experimental_method
+assay	I-experimental_method
+with	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+endonuclease	B-protein_type
+,	O
+as	O
+well	O
+as	O
+in	O
+cell	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+influenza	I-experimental_method
+vRNP	I-experimental_method
+reconstitution	I-experimental_method
+and	O
+virus	B-experimental_method
+yield	I-experimental_method
+assays	I-experimental_method
+.	O
+
+Several	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+were	O
+found	O
+to	O
+have	O
+promising	O
+anti	O
+-	O
+influenza	B-taxonomy_domain
+activity	O
+in	O
+the	O
+low	O
+micromolar	O
+concentration	O
+range	O
+and	O
+good	O
+selectivity	O
+.	O
+
+Computational	B-experimental_method
+docking	I-experimental_method
+studies	I-experimental_method
+are	O
+carried	O
+on	O
+to	O
+investigate	O
+the	O
+key	O
+features	O
+that	O
+determine	O
+inhibition	O
+of	O
+the	O
+endonuclease	B-protein_type
+enzyme	O
+by	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+.	O
+
+Moreover	O
+,	O
+we	O
+here	O
+describe	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+one	O
+of	O
+the	O
+most	O
+active	O
+inhibitors	O
+,	O
+revealing	O
+its	O
+interactions	O
+within	O
+the	O
+protein	O
+’	O
+s	O
+active	B-site
+site	I-site
+.	O
+
+Influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+is	O
+an	O
+enveloped	B-taxonomy_domain
+virus	I-taxonomy_domain
+with	O
+a	O
+segmented	O
+negative	B-chemical
+-	I-chemical
+oriented	I-chemical
+single	I-chemical
+-	I-chemical
+stranded	I-chemical
+RNA	I-chemical
+genome	O
+,	O
+belonging	O
+to	O
+the	O
+Orthomyxoviridae	B-taxonomy_domain
+.	O
+
+Seasonal	O
+influenza	B-taxonomy_domain
+A	I-taxonomy_domain
+and	O
+B	B-taxonomy_domain
+viruses	B-taxonomy_domain
+affect	O
+each	O
+year	O
+approximately	O
+5	O
+–	O
+10	O
+%	O
+of	O
+the	O
+adult	O
+and	O
+20	O
+–	O
+30	O
+%	O
+of	O
+the	O
+paediatric	O
+population	O
+,	O
+and	O
+there	O
+is	O
+a	O
+permanent	O
+risk	O
+of	O
+sudden	O
+influenza	B-taxonomy_domain
+pandemics	O
+,	O
+such	O
+as	O
+the	O
+notorious	O
+‘	O
+Spanish	O
+flu	O
+’	O
+in	O
+1918	O
+and	O
+the	O
+swine	O
+-	O
+origin	O
+H1N1	B-species
+pandemic	O
+in	O
+2009	O
+.	O
+
+Two	O
+classes	O
+of	O
+anti	O
+-	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+drugs	O
+are	O
+available	O
+,	O
+acting	O
+on	O
+the	O
+viral	B-taxonomy_domain
+M2	B-protein_type
+ion	I-protein_type
+-	I-protein_type
+channel	I-protein_type
+(	O
+amantadine	B-chemical
+and	O
+rimantadine	B-chemical
+)	O
+or	O
+on	O
+the	O
+viral	B-taxonomy_domain
+neuraminidase	B-protein_type
+(	O
+zanamivir	B-chemical
+and	O
+oseltamivir	B-chemical
+).	O
+
+The	O
+M2	B-protein_type
+inhibitors	O
+have	O
+limited	O
+clinical	O
+utility	O
+due	O
+to	O
+their	O
+central	O
+nervous	O
+system	O
+side	O
+effects	O
+and	O
+widespread	O
+resistance	O
+,	O
+as	O
+in	O
+the	O
+case	O
+of	O
+the	O
+2009	O
+pandemic	O
+H1N1	B-species
+virus	B-taxonomy_domain
+;	O
+resistance	O
+is	O
+also	O
+a	O
+growing	O
+concern	O
+for	O
+oseltamivir	B-chemical
+.	O
+
+The	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+polymerase	B-protein_type
+complex	O
+is	O
+composed	O
+of	O
+three	O
+subunits	O
+:	O
+PB1	B-protein
+,	O
+PB2	B-protein
+and	O
+PA	B-protein
+.	O
+
+The	O
+PA	B-protein
+subunit	B-structure_element
+performs	O
+the	O
+‘	O
+cap	O
+-	O
+snatching	O
+’	O
+endonuclease	B-protein_type
+reaction	O
+,	O
+the	O
+PB2	B-protein
+subunit	B-structure_element
+is	O
+responsible	O
+for	O
+initial	O
+binding	O
+of	O
+the	O
+capped	B-chemical
+RNAs	I-chemical
+,	O
+while	O
+the	O
+actual	O
+RNA	B-chemical
+synthesis	O
+is	O
+performed	O
+by	O
+the	O
+PB1	B-protein
+protein	O
+.	O
+
+Given	O
+its	O
+crucial	O
+role	O
+in	O
+the	O
+viral	B-taxonomy_domain
+life	O
+cycle	O
+,	O
+the	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+polymerase	B-protein_type
+is	O
+widely	O
+recognized	O
+as	O
+a	O
+superior	O
+target	O
+for	O
+antiviral	O
+drug	O
+development	O
+and	O
+,	O
+in	O
+particular	O
+,	O
+inhibition	O
+of	O
+the	O
+PA	B-protein
+endonuclease	B-protein_type
+has	O
+deserved	O
+much	O
+attention	O
+in	O
+recent	O
+years	O
+.	O
+
+The	O
+endonuclease	B-protein_type
+catalytic	B-site
+site	I-site
+resides	O
+in	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+of	O
+PA	B-protein
+(	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+;	O
+residues	O
+1	B-residue_range
+~	I-residue_range
+195	I-residue_range
+).	O
+
+It	O
+comprises	O
+a	O
+histidine	B-residue_name
+(	O
+His41	B-residue_name_number
+)	O
+and	O
+a	O
+cluster	O
+of	O
+three	O
+strictly	B-protein_state
+conserved	I-protein_state
+acidic	B-protein_state
+residues	O
+(	O
+Glu80	B-residue_name_number
+,	O
+Asp108	B-residue_name_number
+,	O
+Glu119	B-residue_name_number
+),	O
+which	O
+coordinate	B-bond_interaction
+(	O
+together	O
+with	O
+Ile120	B-residue_name_number
+)	O
+one	O
+,	O
+two	O
+,	O
+or	O
+three	O
+manganese	B-chemical
+or	O
+magnesium	B-chemical
+ions	O
+.	O
+
+Since	O
+the	O
+intracellular	O
+concentration	O
+of	O
+Mg2	B-chemical
++	I-chemical
+is	O
+at	O
+least	O
+1000	O
+-	O
+fold	O
+higher	O
+than	O
+that	O
+of	O
+Mn2	B-chemical
++,	I-chemical
+magnesium	B-chemical
+may	O
+be	O
+more	O
+biologically	O
+relevant	O
+.	O
+
+A	O
+controversy	O
+about	O
+number	O
+and	O
+type	O
+of	O
+metal	O
+ions	O
+exists	O
+also	O
+for	O
+the	O
+active	B-site
+site	I-site
+of	O
+HIV	B-species
+-	I-species
+1	I-species
+integrase	B-protein_type
+.	O
+
+HIV	B-species
+-	I-species
+1	I-species
+integrase	B-protein_type
+inhibitors	O
+are	O
+a	O
+paradigm	O
+for	O
+the	O
+innovative	O
+drug	O
+concept	O
+that	O
+is	O
+based	O
+on	O
+coordination	O
+with	O
+the	O
+metal	B-chemical
+cofactor	O
+(	O
+s	O
+)	O
+of	O
+viral	B-taxonomy_domain
+enzymes	O
+:	O
+similarly	O
+,	O
+several	O
+PA	B-protein
+-	O
+binding	O
+agents	O
+with	O
+metal	O
+-	O
+chelating	O
+properties	O
+have	O
+been	O
+identified	O
+as	O
+influenza	B-taxonomy_domain
+endonuclease	B-protein_type
+inhibitors	O
+(	O
+Fig	O
+.	O
+1	O
+),	O
+including	O
+2	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+dioxobutanoic	I-chemical
+acid	I-chemical
+derivatives	O
+,	O
+flutimide	B-chemical
+and	O
+its	O
+derivatives	O
+,	O
+2	B-chemical
+-	I-chemical
+hydroxyphenyl	I-chemical
+amide	I-chemical
+derivatives	O
+,	O
+as	O
+well	O
+as	O
+tetramic	B-chemical
+acids	I-chemical
+,	O
+5	B-chemical
+-	I-chemical
+hydroxypyrimidin	I-chemical
+-	I-chemical
+4	I-chemical
+-	I-chemical
+one	I-chemical
+derivatives	O
+,	O
+marchantins	B-chemical
+and	O
+green	B-taxonomy_domain
+tea	I-taxonomy_domain
+catechins	B-chemical
+,	O
+like	O
+epigallocatechin	B-chemical
+-	I-chemical
+3	I-chemical
+-	I-chemical
+gallate	I-chemical
+(	O
+EGCG	B-chemical
+,	O
+Fig	O
+.	O
+1	O
+).	O
+
+In	O
+recent	O
+years	O
+,	O
+we	O
+focused	O
+our	O
+research	O
+on	O
+chemical	O
+scaffolds	O
+that	O
+are	O
+able	O
+to	O
+chelate	O
+metal	O
+ions	O
+of	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+,	O
+resulting	O
+in	O
+inhibition	O
+of	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+replication	O
+.	O
+
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+represent	O
+an	O
+appealing	O
+class	O
+of	O
+chelating	O
+ligands	O
+with	O
+a	O
+broad	O
+spectrum	B-evidence
+of	O
+biological	O
+activities	O
+,	O
+such	O
+as	O
+activity	O
+against	O
+HIV	B-taxonomy_domain
+,	O
+hepatitis	B-taxonomy_domain
+A	I-taxonomy_domain
+,	O
+vaccinia	B-taxonomy_domain
+and	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+.	O
+
+In	O
+the	O
+present	O
+work	O
+,	O
+we	O
+report	O
+the	O
+biological	O
+activity	O
+of	O
+a	O
+series	O
+of	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+(	O
+Fig	O
+.	O
+2	O
+),	O
+as	O
+determined	O
+in	O
+an	O
+enzymatic	B-experimental_method
+assay	I-experimental_method
+with	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+endonuclease	B-protein_type
+as	O
+well	O
+as	O
+in	O
+cell	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+influenza	I-experimental_method
+viral	I-experimental_method
+ribonucleoprotein	I-experimental_method
+(	I-experimental_method
+vRNP	I-experimental_method
+)	I-experimental_method
+reconstitution	I-experimental_method
+and	O
+virus	B-experimental_method
+yield	I-experimental_method
+assays	I-experimental_method
+.	O
+
+Several	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+were	O
+found	O
+to	O
+have	O
+promising	O
+anti	O
+-	O
+influenza	B-taxonomy_domain
+activity	O
+with	O
+50	B-evidence
+%	I-evidence
+effective	I-evidence
+concentration	I-evidence
+values	O
+(	O
+EC50	B-evidence
+)	O
+in	O
+the	O
+range	O
+of	O
+3	O
+–	O
+20	O
+μM	O
+and	O
+good	O
+selectivity	O
+(	O
+Table	O
+1	O
+and	O
+Fig	O
+.	O
+3	O
+).	O
+
+Computational	B-experimental_method
+docking	I-experimental_method
+studies	I-experimental_method
+of	O
+two	O
+candidate	O
+ligands	O
+in	O
+the	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+active	B-site
+site	I-site
+gave	O
+information	O
+about	O
+the	O
+features	O
+that	O
+could	O
+determine	O
+inhibition	O
+of	O
+endonuclease	B-protein_type
+activity	O
+.	O
+
+Moreover	O
+,	O
+we	O
+describe	O
+the	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+crystal	I-evidence
+structure	I-evidence
+of	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+one	O
+of	O
+the	O
+most	O
+active	O
+inhibitors	O
+.	O
+
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+1	B-chemical
+–	I-chemical
+27	I-chemical
+(	O
+Fig	O
+.	O
+2	O
+)	O
+were	O
+prepared	O
+in	O
+high	O
+yields	O
+by	O
+following	O
+literature	O
+methods	O
+(	O
+Fig	O
+.	O
+2A	O
+);	O
+they	O
+were	O
+characterized	O
+by	O
+spectroscopic	O
+tools	O
+,	O
+mass	B-experimental_method
+spectrometry	I-experimental_method
+and	O
+elemental	B-experimental_method
+analysis	I-experimental_method
+.	O
+
+Even	O
+if	O
+isomerism	O
+around	O
+the	O
+C	O
+=	O
+N	O
+bond	O
+is	O
+possible	O
+,	O
+1	B-chemical
+–	I-chemical
+27	I-chemical
+are	O
+present	O
+in	O
+the	O
+E	O
+form	O
+in	O
+solution	O
+,	O
+as	O
+evidenced	O
+by	O
+the	O
+chemical	O
+shift	O
+values	O
+of	O
+the	O
+HC	O
+=	O
+N	O
+and	O
+NH	O
+protons	O
+in	O
+the	O
+1H	B-experimental_method
+-	I-experimental_method
+NMR	I-experimental_method
+spectrum	B-evidence
+.	O
+
+Exceptions	O
+are	O
+represented	O
+by	O
+the	O
+alkyl	O
+-	O
+derivatives	O
+3	B-chemical
+and	O
+4	B-chemical
+(	O
+2	O
+:	O
+1	O
+and	O
+5	O
+:	O
+3	O
+E	O
+:	O
+Z	O
+ratio	O
+,	O
+respectively	O
+).	O
+
+If	O
+R	O
+’	O
+(	O
+Fig	O
+.	O
+2A	O
+)	O
+is	O
+a	O
+2	O
+-	O
+hydroxy	O
+substituted	O
+phenyl	O
+ring	O
+,	O
+the	O
+corresponding	O
+acylhydrazones	B-chemical
+can	O
+coordinate	B-bond_interaction
+one	O
+or	O
+,	O
+depending	O
+on	O
+denticity	O
+,	O
+two	O
+metal	O
+centers	O
+(	O
+modes	O
+A	O
+and	O
+B	O
+in	O
+Fig	O
+.	O
+4	O
+).	O
+
+Starting	O
+from	O
+N	B-chemical
+’-(	I-chemical
+2	I-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+dihydroxybenzylidene	I-chemical
+)-	I-chemical
+semicarbazide	I-chemical
+(	O
+1	B-chemical
+)	O
+and	O
+its	O
+methoxy	O
+-	O
+analogue	O
+(	O
+2	B-chemical
+),	O
+we	O
+modified	O
+the	O
+acylhydrazonic	O
+substituent	O
+R	O
+”	O
+(	O
+3	B-chemical
+–	I-chemical
+8	I-chemical
+,	O
+18	B-chemical
+,	O
+19	B-chemical
+,	O
+Fig	O
+.	O
+2A	O
+).	O
+
+In	O
+18	B-chemical
+and	O
+19	B-chemical
+,	O
+also	O
+the	O
+gallic	B-chemical
+moiety	O
+can	O
+be	O
+involved	O
+in	O
+the	O
+chelation	B-bond_interaction
+of	O
+the	O
+metal	O
+cofactors	O
+(	O
+mode	O
+C	O
+,	O
+Fig	O
+.	O
+4	O
+).	O
+
+In	O
+order	O
+to	O
+investigate	O
+the	O
+role	O
+of	O
+hydroxyl	O
+substituents	O
+9	B-chemical
+–	I-chemical
+11	I-chemical
+,	O
+13	B-chemical
+–	I-chemical
+17	I-chemical
+,	O
+20	B-chemical
+–	I-chemical
+23	I-chemical
+and	O
+27	B-chemical
+were	O
+also	O
+synthesized	O
+.	O
+
+Compound	O
+12	B-chemical
+was	O
+synthesized	O
+in	O
+order	O
+to	O
+confirm	O
+the	O
+crucial	O
+influence	O
+of	O
+the	O
+gallic	B-chemical
+moiety	O
+.	O
+
+Finally	O
+,	O
+26	B-chemical
+was	O
+here	O
+considered	O
+,	O
+because	O
+it	O
+is	O
+an	O
+inhibitor	O
+of	O
+HIV	B-taxonomy_domain
+RNase	B-protein
+H	I-protein
+,	O
+another	O
+enzyme	O
+with	O
+two	O
+magnesium	B-chemical
+ions	O
+in	O
+its	O
+active	B-site
+site	I-site
+.	O
+
+Since	O
+the	O
+inhibitory	O
+activity	O
+of	O
+the	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+could	O
+be	O
+related	O
+to	O
+chelation	B-bond_interaction
+of	O
+the	O
+divalent	O
+metal	B-chemical
+cofactor	O
+(	O
+s	O
+)	O
+in	O
+the	O
+influenza	B-taxonomy_domain
+PA	B-protein
+-	O
+Nter	B-structure_element
+active	B-site
+site	I-site
+,	O
+we	O
+investigated	O
+the	O
+coordination	O
+properties	O
+of	O
+one	O
+model	O
+ligand	O
+(	O
+i	O
+.	O
+e	O
+.	O
+19	B-chemical
+,	O
+H2L	B-chemical
+)	O
+towards	O
+Mg2	B-chemical
++.	I-chemical
+
+Different	O
+reaction	O
+conditions	O
+were	O
+used	O
+(	O
+1	O
+:	O
+1	O
+and	O
+1	O
+:	O
+2	O
+metal	O
+to	O
+ligand	O
+ratio	O
+,	O
+up	O
+to	O
+4	O
+equivalents	O
+of	O
+triethylamine	B-chemical
+),	O
+but	O
+in	O
+any	O
+case	O
+the	O
+same	O
+chemical	O
+species	O
+Mg	B-chemical
+(	I-chemical
+HL	I-chemical
+)	I-chemical
+2	I-chemical
+∙	I-chemical
+4H2O	I-chemical
+was	O
+recovered	O
+and	O
+conveniently	O
+characterized	O
+.	O
+
+The	O
+use	O
+of	O
+a	O
+coordinating	O
+solvent	O
+as	O
+d6	B-chemical
+-	I-chemical
+DMSO	I-chemical
+causes	O
+partial	O
+decoordination	O
+of	O
+the	O
+ligand	O
+,	O
+but	O
+the	O
+1H	B-experimental_method
+-	I-experimental_method
+NMR	I-experimental_method
+spectrum	B-evidence
+in	O
+MeOD	O
+,	O
+instead	O
+,	O
+shows	O
+only	O
+the	O
+signals	O
+attributable	O
+to	O
+the	O
+complex	O
+.	O
+
+In	O
+the	O
+13C	B-experimental_method
+-	I-experimental_method
+NMR	I-experimental_method
+spectrum	B-evidence
+,	O
+the	O
+signal	O
+of	O
+the	O
+C	O
+=	O
+O	O
+quaternary	O
+carbon	O
+is	O
+practically	O
+unaffected	O
+by	O
+complexation	O
+,	O
+suggesting	O
+that	O
+the	O
+C	O
+=	O
+O	O
+group	O
+is	O
+weakly	O
+involved	O
+in	O
+the	O
+coordination	O
+to	O
+the	O
+metal	O
+ion	O
+.	O
+
+This	O
+is	O
+confirmed	O
+,	O
+in	O
+the	O
+IR	B-experimental_method
+spectrum	B-evidence
+,	O
+by	O
+the	O
+shift	O
+of	O
+about	O
+20	O
+cm	O
+−	O
+1	O
+of	O
+the	O
+C	O
+=	O
+O	O
+absorption	O
+,	O
+while	O
+a	O
+shift	O
+of	O
+30	O
+–	O
+50	O
+cm	O
+−	O
+1	O
+is	O
+expected	O
+when	O
+the	O
+carbonylic	O
+oxygen	O
+is	O
+tightly	O
+bound	O
+to	O
+the	O
+metal	O
+ion	O
+.	O
+
+ESI	B-experimental_method
+-	I-experimental_method
+mass	I-experimental_method
+spectra	B-evidence
+and	O
+elemental	B-experimental_method
+analysis	I-experimental_method
+confirmed	O
+the	O
+formula	O
+Mg	B-chemical
+(	I-chemical
+HL	I-chemical
+)	I-chemical
+2	I-chemical
+∙	I-chemical
+4H2O	I-chemical
+.	O
+
+The	O
+interaction	O
+between	O
+the	O
+N	B-chemical
+-	I-chemical
+acylhydrazone	I-chemical
+ligands	O
+and	O
+the	O
+magnesium	B-chemical
+cation	O
+was	O
+investigated	O
+also	O
+by	O
+means	O
+of	O
+UV	B-experimental_method
+-	I-experimental_method
+visible	I-experimental_method
+spectroscopy	I-experimental_method
+(	O
+UV	B-experimental_method
+-	I-experimental_method
+visible	I-experimental_method
+titrations	I-experimental_method
+of	O
+23	B-chemical
+and	O
+19	B-chemical
+with	O
+increasing	B-experimental_method
+amount	I-experimental_method
+of	O
+Mg	B-chemical
+(	I-chemical
+CH3COO	I-chemical
+)	I-chemical
+2	I-chemical
+are	O
+shown	O
+in	O
+Figure	O
+S1	O
+).	O
+
+The	O
+spectrum	B-evidence
+of	O
+19	B-chemical
+includes	O
+a	O
+band	O
+at	O
+313	O
+nm	O
+assignable	O
+to	O
+n	O
+-	O
+π	O
+*	O
+transitions	O
+of	O
+the	O
+C	O
+=	O
+N	O
+and	O
+C	O
+=	O
+O	O
+groups	O
+.	O
+
+By	O
+adding	O
+increasing	O
+equivalents	O
+of	O
+Mg	B-chemical
+(	I-chemical
+CH3COO	I-chemical
+)	I-chemical
+2	I-chemical
+,	O
+the	O
+absorption	O
+around	O
+400	O
+nm	O
+increases	O
+,	O
+and	O
+a	O
+new	O
+band	O
+appears	O
+with	O
+a	O
+maximum	O
+at	O
+397	O
+nm	O
+.	O
+
+When	O
+the	O
+same	O
+experiment	O
+was	O
+performed	O
+with	O
+23	B-chemical
+,	O
+a	O
+different	O
+behavior	O
+was	O
+observed	O
+.	O
+
+Increasing	O
+concentration	O
+of	O
+Mg2	B-chemical
++,	I-chemical
+in	O
+fact	O
+,	O
+caused	O
+a	O
+diminution	O
+in	O
+the	O
+maximum	O
+absorption	O
+,	O
+an	O
+isosbestic	O
+point	O
+is	O
+visible	O
+at	O
+about	O
+345	O
+nm	O
+,	O
+but	O
+a	O
+new	O
+band	O
+at	O
+400	O
+nm	O
+does	O
+not	O
+appear	O
+.	O
+
+Ligands	O
+19	B-chemical
+and	O
+23	B-chemical
+coordinate	B-bond_interaction
+the	O
+Mg2	B-chemical
++	I-chemical
+ions	O
+in	O
+different	O
+ways	O
+:	O
+19	B-chemical
+chelates	O
+the	O
+metal	O
+ion	O
+by	O
+using	O
+the	O
+deprotonated	O
+salicyl	O
+oxygen	O
+and	O
+the	O
+iminic	O
+nitrogen	O
+,	O
+while	O
+for	O
+23	B-chemical
+,	O
+the	O
+gallic	O
+moiety	O
+is	O
+supposed	O
+to	O
+be	O
+involved	O
+(	O
+Fig	O
+.	O
+4A	O
+,	O
+B	O
+versus	O
+C	O
+),	O
+leading	O
+to	O
+different	O
+,	O
+less	O
+extensive	O
+,	O
+modifications	O
+of	O
+the	O
+UV	B-experimental_method
+spectrum	B-evidence
+.	O
+
+Inhibition	O
+of	O
+the	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+enzyme	O
+
+All	O
+the	O
+compounds	O
+were	O
+tested	O
+for	O
+their	O
+ability	O
+to	O
+inhibit	O
+the	O
+influenza	B-taxonomy_domain
+endonuclease	B-protein_type
+in	O
+an	O
+enzymatic	B-experimental_method
+plasmid	I-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+assay	I-experimental_method
+with	O
+recombinant	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+,	O
+as	O
+well	O
+as	O
+in	O
+cell	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+influenza	I-experimental_method
+methods	I-experimental_method
+(	O
+i	O
+.	O
+e	O
+.	O
+virus	B-experimental_method
+yield	I-experimental_method
+and	I-experimental_method
+vRNP	I-experimental_method
+reconstitution	I-experimental_method
+assays	I-experimental_method
+).	O
+
+The	O
+results	O
+are	O
+shown	O
+in	O
+Table	O
+1	O
+and	O
+summarized	O
+in	O
+Fig	O
+.	O
+3	O
+to	O
+visualize	O
+the	O
+structure	O
+-	O
+activity	O
+relationships	O
+;	O
+Figure	O
+S2	O
+shows	O
+the	O
+dose	B-evidence
+-	I-evidence
+response	I-evidence
+curves	I-evidence
+for	O
+three	O
+representative	O
+compounds	O
+(	O
+i	O
+.	O
+e	O
+.	O
+10	B-chemical
+,	O
+13	B-chemical
+and	O
+23	B-chemical
+)	O
+in	O
+either	O
+the	O
+PA	B-experimental_method
+-	I-experimental_method
+enzyme	I-experimental_method
+or	I-experimental_method
+vRNP	I-experimental_method
+reconstitution	I-experimental_method
+assay	I-experimental_method
+.	O
+
+The	O
+moderate	O
+activity	O
+(	O
+IC50	B-evidence
+=	O
+24	O
+μM	O
+)	O
+of	O
+N	B-chemical
+’-	I-chemical
+2	I-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+dihydroxybenzylidene	I-chemical
+semicarbazide	I-chemical
+(	O
+1	B-chemical
+)	O
+was	O
+completely	O
+lost	O
+when	O
+the	O
+NH2	O
+moiety	O
+was	O
+replaced	O
+by	O
+a	O
+hydrophobic	O
+heptyl	O
+chain	O
+(	O
+3	B-chemical
+),	O
+but	O
+it	O
+is	O
+less	O
+affected	O
+when	O
+a	O
+phenyl	O
+or	O
+a	O
+2	O
+-	O
+hydroxyphenyl	O
+is	O
+present	O
+(	O
+5	B-chemical
+and	O
+7	B-chemical
+,	O
+IC50	B-evidence
+=	O
+84	O
+and	O
+54	O
+μM	O
+,	O
+respectively	O
+).	O
+
+When	O
+the	O
+hydroxyl	O
+in	O
+position	O
+3	O
+on	O
+R1	O
+(	O
+2	B-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+dihydroxybenzylidene	I-chemical
+)	O
+was	O
+replaced	O
+by	O
+a	O
+methoxy	O
+group	O
+(	O
+2	B-chemical
+-	I-chemical
+hydroxy	I-chemical
+-	I-chemical
+3	I-chemical
+-	I-chemical
+methoxybenzylidene	I-chemical
+),	O
+the	O
+activity	O
+disappeared	O
+(	O
+compounds	O
+2	B-chemical
+,	O
+4	B-chemical
+,	O
+6	B-chemical
+and	O
+8	B-chemical
+).	O
+
+The	O
+activity	O
+is	O
+unaffected	O
+(	O
+IC50	B-evidence
+values	O
+ranging	O
+from	O
+45	O
+to	O
+75	O
+μM	O
+)	O
+when	O
+going	O
+from	O
+two	O
+hydroxyls	O
+in	O
+R1	O
+(	O
+7	B-chemical
+)	O
+to	O
+compounds	O
+with	O
+three	O
+hydroxyls	O
+(	O
+i	O
+.	O
+e	O
+.	O
+9	B-chemical
+,	O
+10	B-chemical
+and	O
+11	B-chemical
+).	O
+
+Similarly	O
+,	O
+11	B-chemical
+(	O
+R1	O
+=	O
+3	O
+,	O
+4	O
+,	O
+5	O
+-	O
+trihydroxyphenyl	O
+,	O
+R2	O
+=	O
+2	O
+-	O
+hydroxyphenyl	O
+)	O
+had	O
+comparable	O
+activity	O
+as	O
+27	B-chemical
+(	O
+R1	O
+=	O
+3	O
+,	O
+4	O
+,	O
+5	O
+-	O
+trihydroxyphenyl	O
+,	O
+R2	O
+=	O
+NH2	O
+).	O
+
+Within	O
+the	O
+series	O
+carrying	O
+a	O
+2	O
+-	O
+hydroxyphenyl	O
+R2	O
+group	O
+,	O
+the	O
+activity	O
+of	O
+11	B-chemical
+is	O
+particularly	O
+intriguing	O
+.	O
+
+11	B-chemical
+does	O
+not	O
+have	O
+the	O
+possibility	O
+to	O
+chelate	O
+in	O
+a	O
+tridentate	O
+ONO	O
+fashion	O
+(	O
+mode	O
+A	O
+in	O
+Fig	O
+.	O
+4	O
+),	O
+but	O
+it	O
+can	O
+coordinate	B-bond_interaction
+two	O
+cations	O
+by	O
+means	O
+of	O
+its	O
+three	O
+OH	O
+groups	O
+in	O
+R1	O
+(	O
+mode	O
+C	O
+,	O
+Fig	O
+.	O
+4	O
+).	O
+
+Note	O
+that	O
+a	O
+similar	O
+chelating	O
+mode	O
+was	O
+observed	O
+in	O
+a	O
+crystal	B-evidence
+structure	I-evidence
+,	O
+solved	O
+by	O
+Cusack	O
+and	O
+coworkers	O
+,	O
+of	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+endonuclease	B-protein_type
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+inhibitor	O
+EGCG	B-chemical
+.	O
+
+The	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+inhibitory	O
+activity	O
+strongly	O
+depends	O
+on	O
+the	O
+number	O
+and	O
+position	O
+of	O
+hydroxyl	O
+substituents	O
+in	O
+R1	O
+and	O
+R2	O
+:	O
+this	O
+is	O
+clearly	O
+highlighted	O
+by	O
+the	O
+data	O
+obtained	O
+with	O
+compounds	O
+13	B-chemical
+–	I-chemical
+23	I-chemical
+,	O
+in	O
+which	O
+R2	O
+is	O
+a	O
+3	O
+,	O
+4	O
+,	O
+5	O
+-	O
+trihydroxyphenyl	O
+(	O
+gallic	O
+)	O
+group	O
+,	O
+the	O
+most	O
+active	O
+scaffold	O
+in	O
+our	O
+series	O
+.	O
+
+The	O
+analogue	O
+carrying	O
+an	O
+unsubstituted	O
+aromatic	O
+ring	O
+as	O
+R1	O
+(	O
+compound	O
+13	B-chemical
+)	O
+had	O
+moderate	O
+activity	O
+(	O
+IC50	B-evidence
+=	O
+69	O
+μM	O
+).	O
+
+When	O
+one	O
+OH	O
+was	O
+added	O
+at	O
+position	O
+2	O
+of	O
+the	O
+R1	O
+ring	O
+(	O
+14	B-chemical
+),	O
+the	O
+activity	O
+was	O
+lost	O
+.	O
+
+Adding	O
+a	O
+second	O
+OH	O
+substituent	O
+at	O
+position	O
+5	O
+resulted	O
+in	O
+strong	O
+activity	O
+(	O
+compound	O
+15	B-chemical
+,	O
+IC50	B-evidence
+=	O
+9	O
+μM	O
+);	O
+medium	O
+activity	O
+for	O
+a	O
+3	O
+-	O
+OH	O
+(	O
+18	B-chemical
+;	O
+IC50	B-evidence
+=	O
+83	O
+μM	O
+),	O
+and	O
+marginal	O
+activity	O
+when	O
+the	O
+second	O
+OH	O
+is	O
+at	O
+position	O
+4	O
+(	O
+17	B-chemical
+,	O
+IC50	B-evidence
+≥	O
+370	O
+μM	O
+).	O
+
+The	O
+addition	O
+of	O
+a	O
+3	O
+-	O
+methoxy	O
+group	O
+(	O
+19	B-chemical
+)	O
+abolished	O
+all	O
+inhibitory	O
+activity	O
+.	O
+
+This	O
+cannot	O
+be	O
+related	O
+to	O
+variations	O
+in	O
+the	O
+chelating	O
+features	O
+displayed	O
+by	O
+the	O
+R1	O
+moiety	O
+,	O
+since	O
+compounds	O
+14	B-chemical
+–	I-chemical
+19	I-chemical
+all	O
+have	O
+,	O
+in	O
+theory	O
+,	O
+the	O
+capacity	O
+to	O
+chelate	O
+one	O
+metal	O
+ion	O
+through	O
+the	O
+ortho	O
+-	O
+OH	O
+and	O
+iminic	O
+nitrogen	O
+(	O
+mode	O
+A	O
+in	O
+Fig	O
+.	O
+4	O
+).	O
+
+Moreover	O
+,	O
+compound	O
+18	B-chemical
+can	O
+,	O
+in	O
+principle	O
+,	O
+chelate	O
+the	O
+two	O
+M2	B-chemical
++	I-chemical
+ions	O
+in	O
+the	O
+active	B-site
+site	I-site
+according	O
+to	O
+mode	O
+B	O
+(	O
+Fig	O
+.	O
+4	O
+),	O
+yet	O
+it	O
+(	O
+IC50	B-evidence
+=	O
+83	O
+μM	O
+)	O
+has	O
+nine	O
+-	O
+fold	O
+lower	O
+activity	O
+than	O
+15	B-chemical
+,	O
+that	O
+does	O
+not	O
+possess	O
+this	O
+two	O
+-	O
+metal	O
+chelating	O
+feature	O
+.	O
+
+Therefore	O
+,	O
+we	O
+hypothesized	O
+that	O
+the	O
+inhibitory	O
+activity	O
+of	O
+the	O
+series	O
+containing	O
+the	O
+gallic	O
+moiety	O
+is	O
+determined	O
+by	O
+:	O
+(	O
+i	O
+)	O
+the	O
+capacity	O
+of	O
+the	O
+moiety	O
+R2	O
+to	O
+chelate	O
+two	O
+metal	O
+ions	O
+in	O
+the	O
+active	B-site
+site	I-site
+of	O
+the	O
+enzyme	O
+,	O
+according	O
+to	O
+mode	O
+C	O
+(	O
+Fig	O
+.	O
+4	O
+);	O
+and	O
+(	O
+ii	O
+)	O
+the	O
+presence	O
+and	O
+position	O
+of	O
+one	O
+or	O
+more	O
+hydroxyl	O
+substituents	O
+in	O
+R1	O
+,	O
+which	O
+may	O
+possibly	O
+result	O
+in	O
+ligand	O
+-	O
+protein	O
+interactions	O
+(	O
+e	O
+.	O
+g	O
+.	O
+through	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+).	O
+
+This	O
+assumption	O
+was	O
+supported	O
+by	O
+molecular	B-experimental_method
+docking	I-experimental_method
+calculations	I-experimental_method
+and	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+analysis	I-experimental_method
+of	O
+inhibitor	O
+23	B-chemical
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+PA	B-protein
+-	O
+Nter	B-structure_element
+(	O
+vide	O
+infra	O
+).	O
+
+Substitution	O
+of	O
+the	O
+5	O
+-	O
+hydroxyl	O
+in	O
+15	B-chemical
+by	O
+a	O
+methoxy	O
+group	O
+(	O
+16	B-chemical
+)	O
+causes	O
+a	O
+dramatic	O
+drop	O
+in	O
+activity	O
+(	O
+IC50	B-evidence
+=	O
+9	O
+and	O
+454	O
+μM	O
+for	O
+15	B-chemical
+and	O
+16	B-chemical
+,	O
+respectively	O
+).	O
+
+In	O
+particular	O
+,	O
+all	O
+the	O
+compounds	O
+with	O
+a	O
+trihydroxylated	O
+phenyl	O
+group	O
+as	O
+R1	O
+(	O
+i	O
+.	O
+e	O
+.	O
+20	B-chemical
+,	O
+21	B-chemical
+,	O
+22	B-chemical
+and	O
+23	B-chemical
+)	O
+were	O
+able	O
+to	O
+inhibit	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+quite	O
+potently	O
+.	O
+
+The	O
+lowest	O
+IC50	B-evidence
+values	O
+were	O
+obtained	O
+for	O
+21	B-chemical
+and	O
+23	B-chemical
+(	O
+IC50	B-evidence
+=	O
+13	O
+and	O
+7	O
+μM	O
+,	O
+respectively	O
+),	O
+which	O
+both	O
+have	O
+one	O
+of	O
+their	O
+three	O
+hydroxyl	O
+groups	O
+at	O
+position	O
+5	O
+.	O
+
+The	O
+most	O
+active	O
+compound	O
+in	O
+this	O
+series	O
+was	O
+23	B-chemical
+,	O
+which	O
+lacks	O
+the	O
+hydroxyl	O
+group	O
+at	O
+position	O
+2	O
+of	O
+R1	O
+,	O
+further	O
+confirming	O
+that	O
+this	O
+function	O
+is	O
+undesirable	O
+or	O
+even	O
+detrimental	O
+for	O
+inhibitory	O
+activity	O
+against	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+,	O
+as	O
+already	O
+noticed	O
+above	O
+for	O
+14	B-chemical
+.	O
+
+Consistent	O
+with	O
+a	O
+crucial	O
+role	O
+of	O
+the	O
+R2	O
+gallic	O
+moiety	O
+in	O
+metal	O
+chelation	B-bond_interaction
+,	O
+the	O
+strong	O
+activity	O
+of	O
+15	B-chemical
+was	O
+completely	O
+lost	O
+in	O
+its	O
+3	O
+,	O
+4	O
+,	O
+5	O
+-	O
+trimethoxy	O
+analogue	O
+12	B-chemical
+.	O
+
+On	O
+the	O
+other	O
+hand	O
+,	O
+the	O
+R2	O
+gallic	O
+containing	O
+compounds	O
+displayed	O
+moderate	O
+activity	O
+(	O
+IC50	B-evidence
+values	O
+around	O
+40	O
+μM	O
+)	O
+when	O
+R1	O
+was	O
+absent	O
+(	O
+i	O
+.	O
+e	O
+.	O
+the	O
+3	B-chemical
+,	I-chemical
+4	I-chemical
+,	I-chemical
+5	I-chemical
+-	I-chemical
+trihydroxybenzohydrazide	I-chemical
+28	B-chemical
+,	O
+Fig	O
+.	O
+2	O
+),	O
+or	O
+composed	O
+of	O
+an	O
+extended	O
+ring	O
+system	O
+(	O
+26	B-chemical
+)	O
+or	O
+a	O
+pyrrole	O
+ring	O
+(	O
+25	B-chemical
+).	O
+
+Still	O
+lower	O
+activity	O
+was	O
+seen	O
+with	O
+the	O
+pyridine	O
+analogue	O
+24	B-chemical
+.	O
+
+Evidently	O
+,	O
+the	O
+3	O
+,	O
+4	O
+,	O
+5	O
+-	O
+trihydroxybenzyl	O
+moiety	O
+at	O
+R2	O
+is	O
+fundamental	O
+but	O
+not	O
+sufficient	O
+to	O
+ensure	O
+potent	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+endonuclease	B-protein_type
+inhibition	O
+,	O
+since	O
+the	O
+interactions	O
+of	O
+R1	O
+with	O
+the	O
+amino	O
+acid	O
+side	O
+chains	O
+of	O
+the	O
+protein	O
+appear	O
+crucial	O
+in	O
+modulating	O
+activity	O
+.	O
+
+Inhibition	O
+of	O
+vRNP	B-complex_assembly
+activity	O
+or	O
+virus	B-taxonomy_domain
+replication	O
+in	O
+cells	O
+
+To	O
+determine	O
+the	O
+anti	O
+-	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+activity	O
+of	O
+compounds	O
+1	B-chemical
+–	I-chemical
+28	I-chemical
+in	O
+cell	O
+culture	O
+,	O
+we	O
+performed	O
+an	O
+influenza	B-experimental_method
+vRNP	I-experimental_method
+reconstitution	I-experimental_method
+assay	I-experimental_method
+in	O
+human	B-species
+embryonic	O
+kidney	O
+293	O
+T	O
+(	O
+HEK293T	O
+)	O
+cells	O
+,	O
+then	O
+subjected	O
+the	O
+active	O
+compounds	O
+(	O
+i	O
+.	O
+e	O
+.	O
+EC50	B-evidence
+<	O
+100	O
+μM	O
+)	O
+to	O
+a	O
+virus	B-experimental_method
+yield	I-experimental_method
+assay	I-experimental_method
+in	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+-	O
+infected	O
+Madin	O
+-	O
+Darby	O
+canine	O
+kidney	O
+(	O
+MDCK	O
+)	O
+cells	O
+(	O
+Table	O
+1	O
+and	O
+Fig	O
+.	O
+3	O
+).	O
+
+For	O
+some	O
+N	B-chemical
+-	I-chemical
+acylhydrazone	I-chemical
+compounds	O
+,	O
+we	O
+observed	O
+quite	O
+potent	O
+and	O
+selective	O
+activity	O
+in	O
+the	O
+vRNP	B-experimental_method
+reconstitution	I-experimental_method
+assay	I-experimental_method
+.	O
+
+This	O
+indicates	O
+that	O
+they	O
+are	O
+able	O
+to	O
+inhibit	O
+viral	B-taxonomy_domain
+RNA	B-chemical
+synthesis	O
+and	O
+suggests	O
+that	O
+they	O
+could	O
+be	O
+classified	O
+as	O
+original	O
+PA	B-protein
+inhibitors	O
+.	O
+
+Values	O
+for	O
+EC50	B-evidence
+(	O
+vRNP	B-complex_assembly
+)	O
+or	O
+EC90	B-evidence
+(	O
+virus	B-taxonomy_domain
+yield	O
+)	O
+in	O
+the	O
+range	O
+of	O
+0	O
+.	O
+4	O
+–	O
+18	O
+μM	O
+were	O
+obtained	O
+for	O
+compounds	O
+15	B-chemical
+and	O
+20	B-chemical
+–	I-chemical
+23	I-chemical
+,	O
+which	O
+all	O
+carry	O
+a	O
+3	O
+,	O
+4	O
+,	O
+5	O
+-	O
+trihydroxyphenyl	O
+as	O
+R2	O
+,	O
+and	O
+possess	O
+either	O
+two	O
+(	O
+15	B-chemical
+)	O
+or	O
+three	O
+(	O
+20	B-chemical
+–	O
+23	B-chemical
+)	O
+hydroxyl	O
+substituents	O
+in	O
+the	O
+R1	O
+moiety	O
+.	O
+
+As	O
+in	O
+the	O
+enzymatic	B-experimental_method
+PA	I-experimental_method
+-	I-experimental_method
+Nter	I-experimental_method
+assays	I-experimental_method
+,	O
+the	O
+compounds	O
+having	O
+R2	O
+as	O
+a	O
+gallic	O
+moiety	O
+(	O
+Fig	O
+.	O
+3	O
+:	O
+21	B-chemical
+,	O
+22	B-chemical
+and	O
+23	B-chemical
+)	O
+showed	O
+slightly	O
+higher	O
+activity	O
+than	O
+the	O
+compounds	O
+carrying	O
+a	O
+2	O
+-	O
+hydroxyl	O
+R2	O
+group	O
+(	O
+9	B-chemical
+,	O
+10	B-chemical
+and	O
+11	B-chemical
+);	O
+10	B-chemical
+and	O
+22	B-chemical
+have	O
+substantially	O
+the	O
+same	O
+EC50	B-evidence
+in	O
+the	O
+vRNP	B-experimental_method
+reconstitution	I-experimental_method
+assay	I-experimental_method
+in	O
+HEK293T	O
+cells	O
+.	O
+
+The	O
+hydrazide	B-chemical
+28	B-chemical
+displayed	O
+weak	O
+(	O
+virus	B-taxonomy_domain
+yield	O
+)	O
+to	O
+moderate	O
+(	O
+vRNP	B-experimental_method
+reconstitution	I-experimental_method
+)	O
+activity	O
+,	O
+albeit	O
+less	O
+than	O
+the	O
+most	O
+active	O
+molecules	O
+in	O
+the	O
+3	O
+,	O
+4	O
+,	O
+5	O
+-	O
+trihydroxyphenyl	O
+series	O
+(	O
+i	O
+.	O
+e	O
+.	O
+18	B-chemical
+and	O
+21	B-chemical
+–	I-chemical
+23	I-chemical
+).	O
+
+Even	O
+if	O
+there	O
+are	O
+no	O
+data	O
+indicating	O
+that	O
+the	O
+compounds	O
+reported	O
+in	O
+the	O
+paper	O
+are	O
+subject	O
+to	O
+hydrolysis	O
+,	O
+the	O
+activity	O
+of	O
+28	B-chemical
+could	O
+raise	O
+the	O
+concern	O
+that	O
+for	O
+some	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+the	O
+antiviral	O
+activity	O
+in	O
+cell	O
+culture	O
+may	O
+be	O
+related	O
+to	O
+their	O
+intracellular	O
+hydrolysis	O
+.	O
+
+However	O
+,	O
+this	O
+is	O
+unlikely	O
+,	O
+since	O
+the	O
+antiviral	O
+potency	O
+showed	O
+large	O
+differences	O
+(	O
+i	O
+.	O
+e	O
+.	O
+EC50	B-evidence
+values	O
+between	O
+0	O
+.	O
+42	O
+and	O
+29	O
+μM	O
+)	O
+for	O
+compounds	O
+with	O
+the	O
+same	O
+R2	O
+but	O
+different	O
+R1	O
+groups	O
+,	O
+meaning	O
+that	O
+R1	O
+does	O
+play	O
+a	O
+role	O
+in	O
+modulating	O
+the	O
+antiviral	O
+effect	O
+.	O
+
+Most	O
+compounds	O
+carrying	O
+as	O
+R1	O
+a	O
+2	B-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+dihydroxybenzylidene	I-chemical
+(	O
+i	O
+.	O
+e	O
+.	O
+3	B-chemical
+,	O
+5	B-chemical
+and	O
+7	B-chemical
+)	O
+or	O
+2	B-chemical
+-	I-chemical
+hydroxy	I-chemical
+-	I-chemical
+3	I-chemical
+-	I-chemical
+methoxybenzylidene	I-chemical
+moiety	O
+(	O
+i	O
+.	O
+e	O
+.	O
+4	B-chemical
+,	O
+6	B-chemical
+and	O
+8	B-chemical
+)	O
+showed	O
+relatively	O
+high	O
+cytotoxicity	O
+in	O
+the	O
+vRNP	B-experimental_method
+assay	I-experimental_method
+,	O
+with	O
+CC50	B-evidence
+values	O
+below	O
+50	O
+μM	O
+and	O
+a	O
+selectivity	B-evidence
+index	I-evidence
+(	O
+ratio	O
+of	O
+CC50	B-evidence
+to	O
+EC50	B-evidence
+)	O
+below	O
+8	O
+.	O
+
+Two	O
+notable	O
+exceptions	O
+are	O
+18	B-chemical
+and	O
+19	B-chemical
+(	O
+containing	O
+a	O
+2	B-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+dihydroxybenzylidene	I-chemical
+or	O
+2	B-chemical
+-	I-chemical
+hydroxy	I-chemical
+-	I-chemical
+3	I-chemical
+-	I-chemical
+methoxybenzylidene	I-chemical
+R1	O
+,	O
+respectively	O
+)	O
+which	O
+were	O
+not	O
+cytotoxic	O
+at	O
+200	O
+μM	O
+and	O
+displayed	O
+favorable	O
+antiviral	O
+selectivity	O
+.	O
+
+Some	O
+N	B-chemical
+-	I-chemical
+acylhydrazone	I-chemical
+compounds	O
+were	O
+devoid	O
+of	O
+activity	O
+in	O
+the	O
+enzymatic	B-experimental_method
+assay	I-experimental_method
+,	O
+yet	O
+showed	O
+good	O
+to	O
+moderate	O
+efficacy	O
+in	O
+cell	O
+culture	O
+(	O
+e	O
+.	O
+g	O
+.	O
+14	B-chemical
+and	O
+19	B-chemical
+,	O
+having	O
+EC50	B-evidence
+values	O
+of	O
+2	O
+.	O
+2	O
+and	O
+7	O
+.	O
+1	O
+μM	O
+,	O
+respectively	O
+).	O
+
+For	O
+most	O
+of	O
+the	O
+active	O
+compounds	O
+(	O
+i	O
+.	O
+e	O
+.	O
+9	B-chemical
+,	O
+11	B-chemical
+,	O
+13	B-chemical
+,	O
+15	B-chemical
+–	I-chemical
+21	I-chemical
+,	O
+23	B-chemical
+,	O
+24	B-chemical
+and	O
+26	B-chemical
+)	O
+a	O
+fair	O
+correlation	O
+was	O
+seen	O
+for	O
+the	O
+two	O
+cell	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+assays	I-experimental_method
+,	O
+since	O
+the	O
+EC50	B-evidence
+values	O
+obtained	O
+in	O
+the	O
+vRNP	B-experimental_method
+assay	I-experimental_method
+were	O
+maximum	O
+5	O
+-	O
+fold	O
+different	O
+from	O
+the	O
+EC90	B-evidence
+values	O
+in	O
+the	O
+virus	B-experimental_method
+yield	I-experimental_method
+assay	I-experimental_method
+.	O
+
+On	O
+the	O
+other	O
+hand	O
+,	O
+this	O
+difference	O
+was	O
+8	O
+-	O
+fold	O
+or	O
+more	O
+for	O
+7	B-chemical
+,	O
+10	B-chemical
+,	O
+14	B-chemical
+,	O
+22	B-chemical
+,	O
+25	B-chemical
+and	O
+28	B-chemical
+.	O
+
+Some	O
+N	B-chemical
+-	I-chemical
+acylhydrazone	I-chemical
+compounds	O
+showed	O
+good	O
+to	O
+moderate	O
+efficacy	O
+in	O
+the	O
+vRNP	B-experimental_method
+assay	I-experimental_method
+(	O
+e	O
+.	O
+g	O
+.	O
+14	B-chemical
+and	O
+19	B-chemical
+,	O
+having	O
+EC50	B-evidence
+values	O
+of	O
+2	O
+.	O
+3	O
+and	O
+5	O
+.	O
+7	O
+μM	O
+,	O
+respectively	O
+),	O
+yet	O
+were	O
+devoid	O
+of	O
+activity	O
+in	O
+the	O
+enzymatic	B-experimental_method
+assay	I-experimental_method
+.	O
+
+This	O
+observation	O
+suggests	O
+that	O
+they	O
+may	O
+inhibit	O
+the	O
+viral	B-taxonomy_domain
+polymerase	B-protein_type
+in	O
+an	O
+endonuclease	B-protein_type
+-	O
+independent	O
+manner	O
+.	O
+
+To	O
+achieve	O
+a	O
+clear	O
+insight	O
+into	O
+the	O
+antiviral	O
+profile	O
+of	O
+the	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+,	O
+specific	O
+mechanistic	O
+experiments	O
+are	O
+currently	O
+ongoing	O
+in	O
+our	O
+laboratory	O
+,	O
+in	O
+which	O
+we	O
+are	O
+analyzing	O
+in	O
+full	O
+depth	O
+their	O
+effects	O
+on	O
+virus	B-taxonomy_domain
+entry	O
+,	O
+polymerase	B-protein_type
+-	O
+dependent	O
+RNA	B-chemical
+synthesis	O
+or	O
+the	O
+late	O
+stage	O
+(	O
+maturation	O
+and	O
+release	O
+)	O
+of	O
+the	O
+virus	B-taxonomy_domain
+replication	O
+cycle	O
+.	O
+
+Docking	B-experimental_method
+studies	I-experimental_method
+
+In	O
+order	O
+to	O
+explore	O
+the	O
+possible	O
+binding	O
+mode	O
+of	O
+the	O
+synthesized	O
+compounds	O
+,	O
+docking	B-experimental_method
+simulations	I-experimental_method
+by	O
+GOLD	B-experimental_method
+program	I-experimental_method
+were	O
+performed	O
+by	O
+using	O
+the	O
+structural	O
+coordinates	O
+(	O
+PDB	O
+code	O
+4AWM	O
+)	O
+for	O
+the	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+endonuclease	B-protein_type
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+EGCG	B-chemical
+.	O
+
+Considering	O
+that	O
+the	O
+position	O
+of	O
+the	O
+side	O
+-	O
+chains	O
+of	O
+some	O
+residues	O
+changes	O
+depending	O
+on	O
+which	O
+pocket	O
+the	O
+ligand	O
+is	O
+occupying	O
+,	O
+we	O
+superimposed	B-experimental_method
+some	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+structures	I-evidence
+of	O
+complexes	O
+between	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+endonuclease	B-protein_type
+and	O
+known	O
+active	O
+ligands	O
+.	O
+
+It	O
+was	O
+observed	O
+that	O
+the	O
+side	O
+-	O
+chain	O
+of	O
+amino	O
+acid	O
+Tyr24	B-residue_name_number
+shows	O
+greater	O
+movement	O
+than	O
+the	O
+other	O
+residues	O
+and	O
+for	O
+this	O
+reason	O
+we	O
+considered	O
+it	O
+as	O
+a	O
+flexible	B-protein_state
+residue	O
+during	O
+the	O
+docking	B-experimental_method
+procedure	I-experimental_method
+.	O
+
+First	O
+,	O
+test	B-experimental_method
+docking	I-experimental_method
+calculations	I-experimental_method
+,	O
+using	O
+EGCG	B-chemical
+,	O
+L	B-chemical
+-	I-chemical
+742	I-chemical
+,	I-chemical
+001	I-chemical
+and	O
+2	B-chemical
+-(	I-chemical
+4	I-chemical
+-(	I-chemical
+1H	I-chemical
+-	I-chemical
+tetrazol	I-chemical
+-	I-chemical
+5	I-chemical
+-	I-chemical
+yl	I-chemical
+)	I-chemical
+phenyl	I-chemical
+)-	I-chemical
+5	I-chemical
+-	I-chemical
+hydroxypyrimidin	I-chemical
+-	I-chemical
+4	I-chemical
+(	I-chemical
+3H	I-chemical
+)-	I-chemical
+one	I-chemical
+(	O
+Fig	O
+.	O
+1	O
+),	O
+were	O
+carried	O
+out	O
+to	O
+compare	O
+experimental	O
+and	O
+predicted	O
+binding	O
+modes	O
+and	O
+validate	O
+docking	B-experimental_method
+procedure	I-experimental_method
+.	O
+
+Their	O
+best	O
+docking	O
+poses	O
+agreed	O
+well	O
+with	O
+the	O
+experimental	O
+binding	O
+modes	O
+(	O
+rmsd	B-evidence
+values	O
+of	O
+0	O
+.	O
+8	O
+,	O
+1	O
+.	O
+2	O
+and	O
+0	O
+.	O
+7	O
+,	O
+respectively	O
+).	O
+
+Next	O
+,	O
+docking	B-experimental_method
+of	O
+several	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+was	O
+performed	O
+and	O
+this	O
+generated	O
+a	O
+number	O
+of	O
+possible	O
+binding	O
+conformations	O
+,	O
+highlighting	O
+that	O
+the	O
+active	B-site
+site	I-site
+cavity	I-site
+of	O
+the	O
+PA	B-protein
+endonuclease	B-protein_type
+is	O
+quite	O
+spacious	O
+,	O
+as	O
+already	O
+demonstrated	O
+by	O
+crystallographic	B-experimental_method
+studies	I-experimental_method
+,	O
+and	O
+confirming	O
+the	O
+ability	O
+of	O
+this	O
+scaffold	O
+to	O
+chelate	O
+the	O
+two	O
+M2	B-chemical
++	I-chemical
+ions	O
+in	O
+different	O
+ways	O
+(	O
+Mode	O
+A	O
+-	O
+C	O
+in	O
+Fig	O
+.	O
+4	O
+).	O
+
+Figure	O
+5	O
+displays	O
+the	O
+first	O
+(	O
+panel	O
+A	O
+)	O
+and	O
+second	O
+(	O
+panel	O
+B	O
+)	O
+GOLD	B-experimental_method
+cluster	I-experimental_method
+docked	I-experimental_method
+solutions	O
+for	O
+compound	O
+23	B-chemical
+.	O
+
+These	O
+two	O
+complex	O
+structures	B-evidence
+represent	O
+the	O
+largest	O
+clusters	O
+with	O
+similar	O
+fitness	O
+values	O
+(	O
+59	O
+.	O
+20	O
+and	O
+58	O
+.	O
+65	O
+,	O
+respectively	O
+).	O
+
+In	O
+both	O
+cases	O
+,	O
+23	B-chemical
+appears	O
+able	O
+to	O
+coordinate	B-bond_interaction
+the	O
+two	O
+M2	B-chemical
++	I-chemical
+ions	O
+in	O
+the	O
+active	B-site
+site	I-site
+through	O
+the	O
+three	O
+contiguous	O
+OH	O
+groups	O
+(	O
+Fig	O
+.	O
+5	O
+).	O
+
+In	O
+addition	O
+,	O
+23	B-chemical
+was	O
+predicted	O
+to	O
+form	O
+two	O
+hydrogen	B-bond_interaction
+bonding	I-bond_interaction
+interactions	I-bond_interaction
+,	O
+i	O
+.	O
+e	O
+.	O
+with	O
+the	O
+catalytic	B-protein_state
+Lys134	B-residue_name_number
+on	O
+the	O
+one	O
+side	O
+and	O
+Glu26	B-residue_name_number
+on	O
+the	O
+other	O
+side	O
+.	O
+
+Furthermore	O
+,	O
+in	O
+these	O
+two	O
+different	O
+binding	O
+modes	O
+,	O
+23	B-chemical
+forms	O
+π	B-bond_interaction
+–	I-bond_interaction
+π	I-bond_interaction
+interactions	I-bond_interaction
+with	O
+the	O
+aromatic	O
+ring	O
+of	O
+Tyr24	B-residue_name_number
+,	O
+in	O
+a	O
+fashion	O
+similar	O
+to	O
+that	O
+described	O
+for	O
+other	O
+endonuclease	B-protein_type
+inhibitors	O
+,	O
+i	O
+.	O
+e	O
+.	O
+EGCG	B-chemical
+and	O
+L	B-chemical
+-	I-chemical
+742	I-chemical
+,	I-chemical
+001	I-chemical
+.	O
+
+The	O
+best	O
+docked	O
+conformation	O
+for	O
+compound	O
+15	B-chemical
+(	O
+Fig	O
+.	O
+6	O
+,	O
+fitness	B-evidence
+value	I-evidence
+68	O
+.	O
+56	O
+),	O
+which	O
+has	O
+an	O
+activity	O
+slightly	O
+lower	O
+than	O
+23	O
+,	O
+reveals	O
+a	O
+different	O
+role	O
+for	O
+the	O
+gallic	O
+moiety	O
+.	O
+
+The	O
+ligand	O
+seems	O
+to	O
+form	O
+two	O
+hydrogen	B-bond_interaction
+bonding	I-bond_interaction
+interactions	I-bond_interaction
+with	O
+Tyr130	B-residue_name_number
+as	O
+well	O
+as	O
+a	O
+cation	B-bond_interaction
+–	I-bond_interaction
+π	I-bond_interaction
+interaction	I-bond_interaction
+with	O
+Lys134	B-residue_name_number
+.	O
+
+Tyr130	B-residue_name_number
+lies	O
+in	O
+a	O
+pocket	B-site
+that	O
+also	O
+contains	O
+Arg124	B-residue_name_number
+,	O
+a	O
+residue	O
+that	O
+was	O
+proposed	O
+to	O
+have	O
+a	O
+crucial	O
+role	O
+in	O
+binding	O
+of	O
+the	O
+RNA	B-chemical
+substrate	O
+.	O
+
+Compound	O
+15	B-chemical
+appears	O
+further	O
+stabilized	O
+by	O
+hydrogen	B-bond_interaction
+bonding	I-bond_interaction
+interactions	I-bond_interaction
+between	O
+two	O
+hydroxyl	O
+groups	O
+and	O
+Arg82	B-residue_name_number
+and	O
+Asp108	B-residue_name_number
+.	O
+
+In	O
+this	O
+case	O
+,	O
+chelation	B-bond_interaction
+of	O
+the	O
+two	O
+M2	B-chemical
++	I-chemical
+ions	O
+is	O
+carried	O
+out	O
+by	O
+involving	O
+the	O
+imine	O
+group	O
+(	O
+mode	O
+A	O
+in	O
+Fig	O
+.	O
+4	O
+).	O
+
+It	O
+is	O
+important	O
+to	O
+highlight	O
+that	O
+compounds	O
+23	B-chemical
+and	O
+15	B-chemical
+,	O
+although	O
+in	O
+different	O
+ways	O
+,	O
+both	O
+are	O
+able	O
+to	O
+chelate	O
+the	O
+metal	O
+cofactors	O
+and	O
+to	O
+establish	O
+interactions	O
+with	O
+highly	B-protein_state
+conserved	I-protein_state
+aminoacids	O
+(	O
+Tyr24	B-residue_name_number
+,	O
+Glu26	B-residue_name_number
+,	O
+Arg124	B-residue_name_number
+,	O
+Tyr130	B-residue_name_number
+and	O
+Lys134	B-residue_name_number
+)	O
+that	O
+are	O
+very	O
+important	O
+for	O
+both	O
+endonuclease	B-protein_type
+activity	O
+and	O
+transcription	O
+in	O
+vitro	O
+.	O
+
+The	O
+crucial	O
+role	O
+of	O
+such	O
+interactions	O
+is	O
+underlined	O
+by	O
+the	O
+differences	O
+in	O
+activity	O
+between	O
+15	B-chemical
+(	O
+IC50	B-evidence
+=	O
+9	O
+.	O
+0	O
+μM	O
+)	O
+and	O
+19	B-chemical
+(>	O
+500	O
+μM	O
+):	O
+their	O
+coordinating	O
+features	O
+are	O
+similar	O
+,	O
+since	O
+both	O
+coordinate	B-bond_interaction
+to	O
+the	O
+divalent	O
+metal	O
+ion	O
+through	O
+the	O
+phenolic	O
+oxygen	O
+,	O
+the	O
+iminic	O
+nitrogen	O
+and	O
+the	O
+carbonylic	O
+oxygen	O
+(	O
+mode	O
+A	O
+in	O
+Fig	O
+.	O
+4	O
+),	O
+but	O
+the	O
+biological	O
+activity	O
+could	O
+be	O
+related	O
+to	O
+their	O
+different	O
+ability	O
+to	O
+engage	O
+interactions	O
+with	O
+the	O
+protein	O
+environment	O
+.	O
+
+Crystallographic	B-experimental_method
+Studies	I-experimental_method
+
+Attempts	O
+were	O
+made	O
+to	O
+co	B-experimental_method
+-	I-experimental_method
+crystallize	I-experimental_method
+PA	B-protein
+-	O
+Nter	B-structure_element
+with	O
+15	B-chemical
+,	O
+20	B-chemical
+,	O
+21	B-chemical
+and	O
+23	B-chemical
+in	O
+one	O
+to	O
+four	O
+molar	O
+excess	O
+.	O
+
+While	O
+crystals	B-evidence
+appeared	O
+and	O
+diffracted	O
+well	O
+,	O
+upon	O
+data	O
+processing	O
+,	O
+no	O
+or	O
+very	O
+little	O
+electron	B-evidence
+density	I-evidence
+for	O
+the	O
+inhibitors	O
+was	O
+observed	O
+.	O
+
+Attempts	O
+to	O
+soak	O
+apo	B-protein_state
+crystals	B-evidence
+in	O
+crystallization	O
+solution	O
+containing	O
+5	O
+mM	O
+inhibitor	O
+overnight	O
+also	O
+did	O
+not	O
+result	O
+in	O
+substantial	O
+electron	B-evidence
+density	I-evidence
+for	O
+the	O
+inhibitor	O
+.	O
+
+As	O
+a	O
+last	O
+resort	O
+,	O
+dry	O
+powder	O
+of	O
+the	O
+inhibitor	O
+was	O
+sprinkled	O
+over	O
+the	O
+crystallization	O
+drop	O
+containing	O
+apo	B-protein_state
+crystals	B-evidence
+and	O
+left	O
+over	O
+night	O
+.	O
+
+This	O
+experiment	O
+was	O
+successful	O
+for	O
+compound	O
+23	B-chemical
+,	O
+the	O
+crystals	B-evidence
+diffracted	O
+to	O
+2	O
+.	O
+15	O
+Å	O
+and	O
+diffraction	O
+data	O
+were	O
+collected	O
+(	O
+PDB	O
+ID	O
+5EGA	O
+).	O
+
+The	O
+refined	O
+structure	B-evidence
+shows	O
+unambiguous	O
+electron	B-evidence
+density	I-evidence
+for	O
+the	O
+inhibitor	O
+(	O
+Table	O
+S1	O
+and	O
+Fig	O
+.	O
+7	O
+).	O
+
+The	O
+complex	B-evidence
+structure	I-evidence
+confirms	O
+one	O
+of	O
+the	O
+two	O
+binding	O
+modes	O
+predicted	O
+by	O
+the	O
+docking	B-experimental_method
+simulations	I-experimental_method
+(	O
+Fig	O
+.	O
+5	O
+,	O
+panel	O
+B	O
+).	O
+
+The	O
+galloyl	O
+moiety	O
+chelates	O
+the	O
+manganese	B-chemical
+ions	O
+,	O
+while	O
+the	O
+trihydroxyphenyl	O
+group	O
+stacks	O
+against	O
+the	O
+Tyr24	B-residue_name_number
+side	O
+chain	O
+.	O
+
+It	O
+is	O
+interesting	O
+to	O
+note	O
+that	O
+two	O
+of	O
+these	O
+hydroxyl	O
+groups	O
+are	O
+in	O
+position	O
+to	O
+form	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+the	O
+side	O
+chain	O
+of	O
+Glu26	B-residue_name_number
+and	O
+Lys34	B-residue_name_number
+(	O
+Fig	O
+.	O
+7	O
+).	O
+
+These	O
+interactions	O
+suggest	O
+that	O
+other	O
+functional	O
+groups	O
+,	O
+e	O
+.	O
+g	O
+.	O
+halogens	O
+,	O
+could	O
+be	O
+used	O
+in	O
+place	O
+of	O
+the	O
+hydroxyl	O
+groups	O
+for	O
+better	O
+interactions	O
+with	O
+Glu26	B-residue_name_number
+and	O
+Lys34	B-residue_name_number
+side	O
+chains	O
+,	O
+and	O
+the	O
+inhibitory	O
+potency	O
+of	O
+these	O
+compounds	O
+could	O
+be	O
+further	O
+improved	O
+.	O
+
+Chemical	O
+structures	O
+of	O
+some	O
+prototype	O
+inhibitors	O
+of	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+endonuclease	B-protein_type
+.	O
+
+Inhibitor	O
+activity	O
+in	O
+enzymatic	B-experimental_method
+assays	I-experimental_method
+(	O
+IC50	B-evidence
+,	O
+μM	O
+)	O
+as	O
+reported	O
+in	O
+:	O
+aref	O
+.,	O
+bref	O
+.,	O
+cref	O
+.,	O
+dref	O
+..	O
+
+General	O
+synthesis	O
+for	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+1	B-chemical
+–	I-chemical
+27	I-chemical
+and	O
+hydrazides	B-chemical
+28	B-chemical
+and	O
+29	B-chemical
+(	O
+A	O
+).	O
+
+Chemical	O
+structures	O
+of	O
+compounds	O
+1	B-chemical
+–	I-chemical
+27	I-chemical
+(	O
+B	O
+).	O
+
+Overview	O
+of	O
+the	O
+structure	O
+-	O
+activity	O
+relationship	O
+for	O
+compounds	O
+1	B-chemical
+–	I-chemical
+27	I-chemical
+.	O
+
+Scheme	O
+of	O
+possible	O
+binding	O
+modes	O
+of	O
+the	O
+studied	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+.	O
+
+First	O
+(	O
+A	O
+)	O
+and	O
+second	O
+(	O
+B	O
+)	O
+GOLD	B-experimental_method
+cluster	I-experimental_method
+docked	I-experimental_method
+solutions	O
+of	O
+compound	O
+23	B-chemical
+(	O
+orange	O
+and	O
+cyan	O
+,	O
+respectively	O
+)	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+PA	B-protein
+endonuclease	B-protein_type
+.	O
+
+Key	O
+residues	O
+of	O
+the	O
+pocket	B-site
+are	O
+presented	O
+using	O
+PyMOL	O
+[	O
+http	O
+://	O
+www	O
+.	O
+pymol	O
+.	O
+org	O
+]	O
+and	O
+LIGPLUS	B-experimental_method
+[	O
+Laskowski	O
+,	O
+R	O
+.	O
+A	O
+.;	O
+Swindells	O
+,	O
+M	O
+.	O
+B	O
+.	O
+Journal	O
+of	O
+chemical	O
+information	O
+and	O
+modeling	O
+2011	O
+,	O
+51	O
+,	O
+2778	O
+].	O
+
+Hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+are	O
+illustrated	O
+by	O
+dotted	O
+lines	O
+,	O
+while	O
+the	O
+divalent	O
+metal	O
+ions	O
+are	O
+shown	O
+as	O
+purple	O
+spheres	O
+.	O
+
+Schematic	O
+drawings	O
+of	O
+the	O
+interactions	O
+of	O
+the	O
+first	O
+(	O
+C	O
+)	O
+and	O
+second	O
+(	O
+D	O
+)	O
+GOLD	B-experimental_method
+cluster	I-experimental_method
+docked	I-experimental_method
+solutions	O
+generated	O
+using	O
+LIGPLUS	B-experimental_method
+.	O
+
+Dashed	O
+lines	O
+are	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+and	O
+‘	O
+eyelashes	O
+’	O
+show	O
+residues	O
+involved	O
+in	O
+hydrophobic	B-bond_interaction
+interactions	I-bond_interaction
+.	O
+
+(	O
+A	O
+)	O
+Binding	O
+mode	O
+of	O
+compound	O
+15	B-chemical
+(	O
+orange	O
+)	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+PA	B-protein
+endonuclease	B-protein_type
+.	O
+
+Hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+are	O
+illustrated	O
+by	O
+dotted	O
+lines	O
+while	O
+the	O
+divalent	O
+metal	O
+ions	O
+are	O
+shown	O
+as	O
+purple	O
+spheres	O
+.	O
+
+(	O
+B	O
+)	O
+Schematic	O
+drawing	O
+of	O
+the	O
+interactions	O
+of	O
+compound	O
+15	B-chemical
+generated	O
+using	O
+LIGPLUS	B-experimental_method
+.	O
+
+Crystal	B-evidence
+structure	I-evidence
+of	O
+PANΔLoop	B-mutant
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+compound	O
+23	B-chemical
+.	O
+
+Active	B-site
+site	I-site
+residues	O
+are	O
+shown	O
+in	O
+sticks	O
+with	O
+green	O
+carbons	O
+,	O
+manganese	B-chemical
+atoms	O
+are	O
+shown	O
+as	O
+purple	O
+spheres	O
+and	O
+water	B-chemical
+molecules	O
+as	O
+red	O
+spheres	O
+.	O
+
+Compound	O
+23	B-chemical
+is	O
+shown	O
+in	O
+sticks	O
+with	O
+yellow	O
+carbons	O
+.	O
+
+2Fo	B-evidence
+-	I-evidence
+Fc	I-evidence
+electron	I-evidence
+density	I-evidence
+map	I-evidence
+contoured	O
+at	O
+1σ	O
+is	O
+shown	O
+as	O
+blue	O
+mesh	O
+.	O
+
+Hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+and	O
+metal	B-bond_interaction
+coordination	I-bond_interaction
+are	O
+shown	O
+with	O
+dotted	O
+lines	O
+.	O
+
+The	O
+H	B-bond_interaction
+-	I-bond_interaction
+bond	I-bond_interaction
+distances	O
+from	O
+the	O
+side	O
+chain	O
+carboxyl	O
+group	O
+of	O
+Glu26	B-residue_name_number
+to	O
+p	O
+-	O
+OH	O
+and	O
+m	O
+-	O
+OH	O
+of	O
+the	O
+trihydroxyphenyl	O
+group	O
+of	O
+the	O
+inhibitor	O
+are	O
+2	O
+.	O
+7	O
+Å	O
+and	O
+3	O
+.	O
+0	O
+Å	O
+,	O
+respectively	O
+.	O
+
+The	O
+H	B-bond_interaction
+-	I-bond_interaction
+bond	I-bond_interaction
+distance	O
+from	O
+the	O
+side	O
+chain	O
+of	O
+Lys34	B-residue_name_number
+to	O
+p	O
+-	O
+OH	O
+of	O
+the	O
+trihydroxyphenyl	O
+group	O
+is	O
+3	O
+.	O
+6	O
+Å	O
+.	O
+The	O
+H	B-bond_interaction
+-	I-bond_interaction
+bond	I-bond_interaction
+distance	O
+to	O
+the	O
+water	B-chemical
+molecule	O
+from	O
+m	O
+-	O
+OH	O
+of	O
+the	O
+galloyl	O
+moiety	O
+is	O
+3	O
+.	O
+0	O
+Å	O
+,	O
+which	O
+in	O
+turn	O
+is	O
+H	B-bond_interaction
+-	I-bond_interaction
+bonded	I-bond_interaction
+to	O
+the	O
+side	O
+chain	O
+of	O
+Tyr130	B-residue_name_number
+with	O
+a	O
+distance	O
+of	O
+2	O
+.	O
+7	O
+Å	O
+.	O
+Crystal	B-evidence
+structure	I-evidence
+has	O
+been	O
+deposited	O
+in	O
+the	O
+RCSB	O
+Protein	O
+Data	O
+Bank	O
+with	O
+PDB	O
+ID	O
+:	O
+5EGA	O
+.	O
+
+Inhibitory	O
+activity	O
+of	O
+the	O
+N	B-chemical
+-	I-chemical
+acylhydrazones	I-chemical
+1	B-chemical
+–	I-chemical
+27	I-chemical
+and	O
+hydrazide	B-chemical
+28	B-chemical
+in	O
+the	O
+enzymatic	B-experimental_method
+assay	I-experimental_method
+with	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+PA	B-protein
+-	O
+Nter	B-structure_element
+endonuclease	B-protein_type
+,	O
+or	O
+in	O
+cellular	B-experimental_method
+influenza	I-experimental_method
+virus	I-experimental_method
+assays	I-experimental_method
+.	O
+
+Compound	B-experimental_method
+Enzyme	I-experimental_method
+assay	I-experimental_method
+with	O
+PA	B-protein
+-	O
+Ntera	O
+Virus	B-experimental_method
+yield	I-experimental_method
+assay	I-experimental_method
+in	O
+influenza	B-taxonomy_domain
+virus	B-taxonomy_domain
+-	O
+infected	O
+MDCK	O
+cellsb	O
+vRNP	B-experimental_method
+reconstitution	I-experimental_method
+assay	I-experimental_method
+in	O
+HEK293T	O
+cellsc	O
+Antiviral	O
+activity	O
+Cytotoxicity	O
+SId	O
+Activity	O
+Cytotoxicity	O
+IC50	B-evidence
+EC99	B-evidence
+EC90	B-evidence
+CC50	B-evidence
+EC50	B-evidence
+CC50	B-evidence
+(	O
+1	O
+)	O
+24	O
+NDf	O
+ND	O
+ND	O
+107	O
+>	O
+200	O
+(	O
+2	O
+)	O
+>	O
+500	O
+ND	O
+ND	O
+ND	O
+>	O
+100	O
+>	O
+200	O
+(	O
+3	O
+)	O
+>	O
+500	O
+ND	O
+ND	O
+>	O
+200	O
+5	O
+.	O
+9	O
+48	O
+(	O
+4	O
+)	O
+>	O
+500	O
+ND	O
+ND	O
+>	O
+200	O
+6	O
+.	O
+3	O
+33	O
+(	O
+5	O
+)	O
+67	O
+>	O
+25	O
+>	O
+25	O
+≥	O
+146	O
+2	O
+.	O
+6	O
+10	O
+(	O
+6	O
+)	O
+>	O
+500	O
+>	O
+50	O
+>	O
+50	O
+>	O
+200	O
+15	O
+14	O
+(	O
+7	O
+)	O
+54	O
+172	O
+100	O
+>	O
+200	O
+>	O
+2	O
+.	O
+0	O
+3	O
+.	O
+2	O
+8	O
+.	O
+9	O
+(	O
+8	O
+)	O
+>	O
+500	O
+>	O
+12	O
+.	O
+5	O
+>	O
+12	O
+.	O
+5	O
+>	O
+200	O
+1	O
+.	O
+9	O
+15	O
+(	O
+9	O
+)	O
+34	O
+16	O
+5	O
+.	O
+3	O
+>	O
+200	O
+>	O
+38	O
+5	O
+.	O
+5	O
+>	O
+200	O
+(	O
+10	O
+)	O
+68	O
+14	O
+8	O
+.	O
+5	O
+111	O
+>	O
+13	O
+0	O
+.	O
+40	O
+132	O
+(	O
+11	O
+)	O
+45	O
+30	O
+12	O
+>	O
+200	O
+>	O
+17	O
+5	O
+.	O
+6	O
+>	O
+200	O
+(	O
+12	O
+)	O
+>	O
+500	O
+>	O
+12	O
+.	O
+5	O
+>	O
+12	O
+.	O
+5	O
+>	O
+200	O
+20	O
+39	O
+(	O
+13	O
+)	O
+69	O
+71	O
+34	O
+>	O
+200	O
+>	O
+5	O
+.	O
+9	O
+6	O
+.	O
+3	O
+>	O
+200	O
+(	O
+14	O
+)	O
+>	O
+500	O
+63	O
+37	O
+>	O
+200	O
+>	O
+5	O
+.	O
+4	O
+2	O
+.	O
+3	O
+>	O
+200	O
+(	O
+15	O
+)	O
+8	O
+.	O
+9	O
+18	O
+7	O
+.	O
+5	O
+≥	O
+172	O
+≥	O
+23	O
+14	O
+>	O
+200	O
+(	O
+16	O
+)	O
+454	O
+67	O
+28	O
+>	O
+200	O
+>	O
+7	O
+.	O
+1	O
+5	O
+.	O
+2	O
+>	O
+200	O
+(	O
+17	O
+)	O
+482	O
+21	O
+8	O
+.	O
+1	O
+>	O
+200	O
+>	O
+25	O
+7	O
+.	O
+1	O
+>	O
+200	O
+(	O
+18	O
+)	O
+83	O
+6	O
+.	O
+2	O
+2	O
+.	O
+2	O
+>	O
+200	O
+>	O
+91	O
+3	O
+.	O
+3	O
+>	O
+200	O
+(	O
+19	O
+)	O
+>	O
+500	O
+53	O
+26	O
+>	O
+200	O
+>	O
+7	O
+.	O
+7	O
+5	O
+.	O
+7	O
+>	O
+200	O
+(	O
+20	O
+)	O
+18	O
+35	O
+11	O
+>	O
+200	O
+>	O
+18	O
+2	O
+.	O
+2	O
+>	O
+200	O
+(	O
+21	O
+)	O
+13	O
+8	O
+.	O
+3	O
+3	O
+.	O
+6	O
+>	O
+200	O
+>	O
+56	O
+2	O
+.	O
+5	O
+>	O
+200	O
+(	O
+22	O
+)	O
+75	O
+7	O
+.	O
+4	O
+3	O
+.	O
+4	O
+>	O
+200	O
+>	O
+59	O
+0	O
+.	O
+42	O
+>	O
+200	O
+(	O
+23	O
+)	O
+8	O
+.	O
+7	O
+11	O
+3	O
+.	O
+5	O
+>	O
+200	O
+>	O
+57	O
+3	O
+.	O
+1	O
+>	O
+200	O
+(	O
+24	O
+)	O
+131	O
+58	O
+26	O
+>	O
+200	O
+>	O
+7	O
+.	O
+7	O
+25	O
+>	O
+200	O
+(	O
+25	O
+)	O
+40	O
+132	O
+70	O
+>	O
+200	O
+>	O
+2	O
+.	O
+9	O
+4	O
+.	O
+1	O
+>	O
+200	O
+(	O
+26	O
+)	O
+30	O
+36	O
+13	O
+>	O
+200	O
+>	O
+15	O
+5	O
+.	O
+5	O
+>	O
+200	O
+(	O
+27	O
+)	O
+36	O
+ND	O
+ND	O
+ND	O
+21	O
+>	O
+200	O
+(	O
+28	O
+)	O
+40	O
+158	O
+85	O
+>	O
+200	O
+>	O
+2	O
+.	O
+4	O
+7	O
+.	O
+2	O
+>	O
+200	O
+DPBAe	O
+5	O
+.	O
+3	O
+ND	O
+ND	O
+ND	O
+ND	O
+ND	O
+Ribavirin	O
+ND	O
+13	O
+8	O
+.	O
+5	O
+>	O
+200	O
+>	O
+24	O
+9	O
+.	O
+4	O
+>	O
+200	O
+
+aRecombinant	O
+PA	B-protein
+-	O
+Nter	B-structure_element
+was	O
+incubated	B-experimental_method
+with	O
+the	O
+ssDNA	B-chemical
+plasmid	O
+substrate	O
+,	O
+a	O
+Mn2	B-chemical
++-	I-chemical
+containing	O
+buffer	O
+and	O
+test	O
+compounds	O
+.	O
+
+The	O
+IC50	B-evidence
+represents	O
+the	O
+compound	O
+concentration	O
+(	O
+in	O
+μM	O
+)	O
+required	O
+to	O
+obtain	O
+50	O
+%	O
+inhibition	O
+of	O
+cleavage	O
+,	O
+calculated	O
+by	O
+nonlinear	B-experimental_method
+least	I-experimental_method
+-	I-experimental_method
+squares	I-experimental_method
+regression	I-experimental_method
+analysis	I-experimental_method
+(	O
+using	O
+GraphPad	O
+Prism	O
+software	O
+)	O
+of	O
+the	O
+results	O
+from	O
+2	O
+–	O
+4	O
+independent	O
+experiments	O
+.	O
+
+bMDCK	O
+cells	O
+were	O
+infected	O
+with	O
+influenza	B-taxonomy_domain
+A	I-taxonomy_domain
+virus	B-taxonomy_domain
+(	O
+strain	O
+A	O
+/	O
+PR	O
+/	O
+8	O
+/	O
+34	O
+)	O
+and	O
+incubated	O
+with	O
+the	O
+compounds	O
+during	O
+24	O
+h	O
+.	O
+The	O
+virus	B-taxonomy_domain
+yield	O
+in	O
+the	O
+supernatant	O
+was	O
+assessed	O
+by	O
+real	B-experimental_method
+-	I-experimental_method
+time	I-experimental_method
+qPCR	I-experimental_method
+.	O
+
+The	O
+EC99	B-evidence
+and	O
+EC90	B-evidence
+values	O
+represent	O
+the	O
+compound	O
+concentrations	O
+(	O
+in	O
+μM	O
+)	O
+producing	O
+a	O
+2	O
+-	O
+log10	O
+or	O
+1	O
+-	O
+log10	O
+reduction	O
+in	O
+virus	B-taxonomy_domain
+titer	O
+,	O
+respectively	O
+,	O
+determined	O
+in	O
+2	O
+–	O
+3	O
+independent	O
+experiments	O
+.	O
+
+The	O
+cytotoxicity	O
+,	O
+assessed	O
+in	O
+uninfected	O
+MDCK	O
+cells	O
+,	O
+was	O
+expressed	O
+as	O
+the	O
+CC50	B-evidence
+value	O
+(	O
+50	O
+%	O
+cytotoxic	O
+concentration	O
+,	O
+determined	O
+with	O
+the	O
+MTS	B-experimental_method
+cell	I-experimental_method
+viability	I-experimental_method
+assay	I-experimental_method
+,	O
+in	O
+μM	O
+).	O
+
+cHEK293T	O
+cells	O
+were	O
+co	B-experimental_method
+-	I-experimental_method
+transfected	I-experimental_method
+with	O
+the	O
+four	O
+vRNP	B-complex_assembly
+-	O
+reconstituting	O
+plasmids	O
+and	O
+the	O
+luciferase	O
+reporter	O
+plasmid	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+test	O
+compounds	O
+.	O
+
+The	O
+EC50	B-evidence
+represents	O
+the	O
+compound	O
+concentration	O
+(	O
+in	O
+μM	O
+)	O
+producing	O
+50	O
+%	O
+reduction	O
+in	O
+vRNP	B-complex_assembly
+-	O
+driven	O
+firefly	O
+reporter	O
+signal	O
+,	O
+estimated	O
+at	O
+24	O
+h	O
+after	O
+transfection	O
+.	O
+
+The	O
+EC50	B-evidence
+value	O
+was	O
+derived	O
+from	O
+data	O
+from	O
+2	O
+–	O
+4	O
+independent	O
+experiments	O
+,	O
+by	O
+nonlinear	B-experimental_method
+least	I-experimental_method
+-	I-experimental_method
+squares	I-experimental_method
+regression	I-experimental_method
+analysis	I-experimental_method
+(	O
+using	O
+GraphPad	O
+Prism	O
+software	O
+).	O
+
+The	O
+CC50	B-evidence
+(	O
+in	O
+μM	O
+),	O
+i	O
+.	O
+e	O
+.	O
+the	O
+50	O
+%	O
+cytotoxic	O
+concentration	O
+,	O
+was	O
+determined	O
+in	O
+untransfected	O
+HEK293T	O
+cells	O
+by	O
+MTS	B-experimental_method
+cell	I-experimental_method
+viability	I-experimental_method
+assay	I-experimental_method
+.	O
+
+dSI	B-evidence
+,	O
+selectivity	B-evidence
+index	I-evidence
+,	O
+defined	O
+as	O
+the	O
+ratio	O
+between	O
+the	O
+CC50	B-evidence
+and	O
+EC90	B-evidence
+.	O
+
+eDPBA	B-chemical
+,	O
+2	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+dioxo	I-chemical
+-	I-chemical
+4	I-chemical
+-	I-chemical
+phenylbutanoic	I-chemical
+acid	I-chemical
+.	O
+
diff --git a/annotation_IOB/PMC4981400.tsv b/annotation_IOB/PMC4981400.tsv
new file mode 100644
index 0000000000000000000000000000000000000000..7e645bf8f882b3350490ea301e8fd320b87cebfb
--- /dev/null
+++ b/annotation_IOB/PMC4981400.tsv
@@ -0,0 +1,3348 @@
+Crystal	B-evidence
+Structure	I-evidence
+of	O
+the	O
+SPOC	B-structure_element
+Domain	O
+of	O
+the	O
+Arabidopsis	B-taxonomy_domain
+Flowering	B-protein_type
+Regulator	I-protein_type
+FPA	B-protein
+
+The	O
+Arabidopsis	B-taxonomy_domain
+protein	O
+FPA	B-protein
+controls	O
+flowering	O
+time	O
+by	O
+regulating	O
+the	O
+alternative	O
+3	O
+′-	O
+end	O
+processing	O
+of	O
+the	O
+FLOWERING	B-gene
+LOCUS	I-gene
+(	O
+FLC	B-gene
+)	O
+antisense	B-chemical
+RNA	I-chemical
+.	O
+
+FPA	B-protein
+belongs	O
+to	O
+the	O
+split	B-protein_type
+ends	I-protein_type
+(	O
+SPEN	B-protein_type
+)	O
+family	O
+of	O
+proteins	O
+,	O
+which	O
+contain	O
+N	O
+-	O
+terminal	O
+RNA	B-structure_element
+recognition	I-structure_element
+motifs	I-structure_element
+(	O
+RRMs	B-structure_element
+)	O
+and	O
+a	O
+SPEN	B-structure_element
+paralog	I-structure_element
+and	I-structure_element
+ortholog	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+(	O
+SPOC	B-structure_element
+)	O
+domain	O
+.	O
+
+The	O
+SPOC	B-structure_element
+domain	O
+is	O
+highly	B-protein_state
+conserved	I-protein_state
+among	O
+FPA	B-protein
+homologs	O
+in	O
+plants	B-taxonomy_domain
+,	O
+but	O
+the	O
+conservation	O
+with	O
+the	O
+domain	O
+in	O
+other	O
+SPEN	B-protein_type
+proteins	O
+is	O
+much	O
+lower	O
+.	O
+
+We	O
+have	O
+determined	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+Arabidopsis	B-species
+thaliana	I-species
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+at	O
+2	O
+.	O
+7	O
+Å	O
+resolution	O
+.	O
+
+The	O
+overall	O
+structure	B-evidence
+is	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+SPOC	B-structure_element
+domain	O
+in	O
+human	B-species
+SMRT	B-protein
+/	I-protein
+HDAC1	I-protein
+Associated	I-protein
+Repressor	I-protein
+Protein	I-protein
+(	O
+SHARP	B-protein
+),	O
+although	O
+there	O
+are	O
+also	O
+substantial	O
+conformational	O
+differences	O
+between	O
+them	O
+.	O
+
+Structural	B-experimental_method
+and	I-experimental_method
+sequence	I-experimental_method
+analyses	I-experimental_method
+identify	O
+a	O
+surface	B-site
+patch	I-site
+that	O
+is	O
+conserved	B-protein_state
+among	O
+plant	B-taxonomy_domain
+FPA	B-protein
+homologs	O
+.	O
+
+Mutations	B-experimental_method
+of	O
+two	O
+residues	O
+in	O
+this	O
+surface	B-site
+patch	I-site
+did	O
+not	O
+disrupt	O
+FPA	B-protein
+functions	O
+,	O
+suggesting	O
+that	O
+either	O
+the	O
+SPOC	B-structure_element
+domain	O
+is	O
+not	O
+required	O
+for	O
+the	O
+role	O
+of	O
+FPA	B-protein
+in	O
+regulating	O
+RNA	B-chemical
+3	O
+′-	O
+end	O
+formation	O
+or	O
+the	O
+functions	O
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+cannot	O
+be	O
+disrupted	O
+by	O
+the	O
+combination	O
+of	O
+mutations	O
+,	O
+in	O
+contrast	O
+to	O
+observations	O
+with	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+.	O
+
+Eukaryotic	B-taxonomy_domain
+messenger	B-chemical
+RNAs	I-chemical
+(	O
+mRNAs	B-chemical
+)	O
+are	O
+made	O
+as	O
+precursors	O
+through	O
+transcription	O
+by	O
+RNA	B-complex_assembly
+polymerase	I-complex_assembly
+II	I-complex_assembly
+(	O
+Pol	B-complex_assembly
+II	I-complex_assembly
+),	O
+and	O
+these	O
+primary	O
+transcripts	O
+undergo	O
+extensive	O
+processing	O
+,	O
+including	O
+3	O
+′-	O
+end	O
+cleavage	O
+and	O
+polyadenylation	O
+.	O
+
+In	O
+addition	O
+,	O
+alternative	O
+3	O
+′-	O
+end	O
+cleavage	O
+and	O
+polyadenylation	O
+is	O
+an	O
+essential	O
+and	O
+ubiquitous	O
+process	O
+in	O
+eukaryotes	B-taxonomy_domain
+.	O
+
+Recently	O
+,	O
+the	O
+split	B-protein_type
+ends	I-protein_type
+(	O
+SPEN	B-protein_type
+)	O
+family	O
+of	O
+proteins	O
+was	O
+identified	O
+as	O
+RNA	B-protein_type
+binding	I-protein_type
+proteins	I-protein_type
+that	O
+regulate	O
+alternative	O
+3	O
+′-	O
+end	O
+cleavage	O
+and	O
+polyadenylation	O
+.	O
+
+They	O
+are	O
+characterized	O
+by	O
+possessing	O
+N	O
+-	O
+terminal	O
+RNA	B-structure_element
+recognition	I-structure_element
+motifs	I-structure_element
+(	O
+RRMs	B-structure_element
+)	O
+and	O
+a	O
+conserved	B-protein_state
+SPEN	B-structure_element
+paralog	I-structure_element
+and	I-structure_element
+ortholog	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+(	O
+SPOC	B-structure_element
+)	O
+domain	O
+(	O
+Fig	O
+1A	O
+).	O
+
+The	O
+SPOC	B-structure_element
+domain	O
+is	O
+believed	O
+to	O
+mediate	O
+protein	O
+-	O
+protein	O
+interactions	O
+and	O
+has	O
+diverse	O
+functions	O
+among	O
+SPEN	B-protein_type
+family	O
+proteins	O
+,	O
+but	O
+the	O
+molecular	O
+mechanism	O
+of	O
+these	O
+functions	O
+is	O
+not	O
+well	O
+understood	O
+.	O
+
+Sequence	B-evidence
+conservation	I-evidence
+of	O
+SPOC	B-structure_element
+domains	O
+.	O
+
+Domain	O
+organization	O
+of	O
+A	B-species
+.	I-species
+thaliana	I-species
+FPA	B-protein
+.	O
+(	O
+B	O
+).	O
+
+Sequence	B-experimental_method
+alignment	I-experimental_method
+of	O
+the	O
+SPOC	B-structure_element
+domains	O
+of	O
+Arabidopsis	B-species
+thaliana	I-species
+FPA	B-protein
+,	O
+human	B-species
+RBM15	B-protein
+,	O
+Drosophila	B-taxonomy_domain
+SPEN	B-protein_type
+,	O
+mouse	B-taxonomy_domain
+MINT	B-protein
+,	O
+and	O
+human	B-species
+SHARP	B-protein
+.	O
+
+Residues	O
+in	O
+surface	B-site
+patch	I-site
+1	I-site
+are	O
+indicated	O
+with	O
+the	O
+orange	O
+dots	O
+,	O
+and	O
+those	O
+in	O
+surface	B-site
+patch	I-site
+2	I-site
+with	O
+the	O
+green	O
+dots	O
+.	O
+
+The	O
+secondary	O
+structure	O
+elements	O
+in	O
+the	O
+structure	B-evidence
+of	O
+FPA	B-protein
+SPOC	B-structure_element
+are	O
+labeled	O
+.	O
+
+Residues	O
+that	O
+are	O
+strictly	B-protein_state
+conserved	I-protein_state
+among	O
+the	O
+five	O
+proteins	O
+are	O
+shown	O
+in	O
+white	O
+with	O
+a	O
+red	O
+background	O
+,	O
+and	O
+those	O
+that	O
+are	O
+mostly	B-protein_state
+conserved	I-protein_state
+in	O
+red	O
+.	O
+
+FPA	B-protein
+,	O
+a	O
+SPEN	B-protein_type
+family	O
+protein	O
+in	O
+Arabidopsis	B-species
+thaliana	I-species
+and	O
+other	O
+plants	B-taxonomy_domain
+,	O
+was	O
+found	O
+to	O
+regulate	O
+the	O
+3	O
+′-	O
+end	O
+alternative	O
+cleavage	O
+and	O
+polyadenylation	O
+of	O
+the	O
+antisense	B-chemical
+RNAs	I-chemical
+of	O
+FLOWERING	B-gene
+LOCUS	I-gene
+(	O
+FLC	B-gene
+),	O
+a	O
+flowering	O
+repressor	O
+gene	O
+.	O
+
+FPA	B-protein
+promotes	O
+the	O
+3	O
+′-	O
+end	O
+processing	O
+of	O
+class	O
+I	O
+FLC	B-gene
+antisense	B-chemical
+RNAs	I-chemical
+,	O
+which	O
+includes	O
+the	O
+proximal	O
+polyadenylation	B-site
+site	I-site
+.	O
+
+This	O
+is	O
+associated	O
+with	O
+histone	B-protein_type
+demethylase	I-protein_type
+activity	O
+and	O
+down	O
+-	O
+regulation	O
+of	O
+FLC	B-gene
+transcription	O
+.	O
+
+Although	O
+a	O
+SPOC	B-structure_element
+domain	O
+is	O
+found	O
+in	O
+all	O
+the	O
+SPEN	B-protein_type
+family	O
+proteins	O
+,	O
+its	O
+sequence	O
+conservation	O
+is	O
+rather	O
+low	O
+.	O
+
+For	O
+example	O
+,	O
+the	O
+sequence	O
+identity	O
+between	O
+the	O
+SPOC	B-structure_element
+domains	O
+of	O
+A	B-species
+.	I-species
+thaliana	I-species
+FPA	B-protein
+and	O
+human	B-species
+SMRT	B-protein
+/	I-protein
+HDAC1	I-protein
+Associated	I-protein
+Repressor	I-protein
+Protein	I-protein
+(	O
+SHARP	B-protein
+)	O
+is	O
+only	O
+19	O
+%	O
+(	O
+Fig	O
+1B	O
+).	O
+
+Currently	O
+,	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+is	O
+the	O
+only	O
+one	O
+with	O
+structural	O
+information	O
+.	O
+
+As	O
+a	O
+first	O
+step	O
+toward	O
+understanding	O
+the	O
+molecular	O
+basis	O
+for	O
+the	O
+regulation	O
+of	O
+alternative	O
+3	O
+′-	O
+end	O
+processing	O
+and	O
+flowering	O
+by	O
+FPA	B-protein
+,	O
+we	O
+have	O
+determined	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+SPOC	B-structure_element
+domain	O
+of	O
+A	B-species
+.	I-species
+thaliana	I-species
+FPA	B-protein
+at	O
+2	O
+.	O
+7	O
+Å	O
+resolution	O
+.	O
+
+The	O
+overall	O
+structure	B-evidence
+is	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+,	O
+although	O
+there	O
+are	O
+also	O
+substantial	O
+conformational	O
+differences	O
+between	O
+them	O
+.	O
+
+The	O
+structure	B-evidence
+reveals	O
+a	O
+surface	B-site
+patch	I-site
+that	O
+is	O
+conserved	B-protein_state
+among	O
+FPA	B-protein
+homologs	O
+.	O
+
+Structure	B-evidence
+of	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+SPOC	B-structure_element
+domain	O
+of	O
+A	B-species
+.	I-species
+thaliana	I-species
+FPA	B-protein
+has	O
+been	O
+determined	O
+at	O
+2	O
+.	O
+7	O
+Å	O
+resolution	O
+using	O
+the	O
+selenomethionyl	B-experimental_method
+single	I-experimental_method
+-	I-experimental_method
+wavelength	I-experimental_method
+anomalous	I-experimental_method
+dispersion	I-experimental_method
+method	I-experimental_method
+.	O
+
+The	O
+expression	O
+construct	O
+contained	O
+residues	O
+433	B-residue_range
+–	I-residue_range
+565	I-residue_range
+of	O
+FPA	B-protein
+,	O
+but	O
+only	O
+residues	O
+439	B-residue_range
+–	I-residue_range
+460	I-residue_range
+and	O
+465	B-residue_range
+–	I-residue_range
+565	I-residue_range
+are	O
+ordered	O
+in	O
+the	O
+crystal	B-evidence
+.	O
+
+The	O
+atomic	B-evidence
+model	I-evidence
+has	O
+good	O
+agreement	O
+with	O
+the	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+diffraction	I-evidence
+data	I-evidence
+and	O
+the	O
+expected	O
+bond	O
+lengths	O
+,	O
+bond	O
+angles	O
+and	O
+other	O
+geometric	O
+parameters	O
+(	O
+Table	O
+1	O
+).	O
+
+All	O
+the	O
+residues	O
+are	O
+located	O
+in	O
+the	O
+favored	O
+regions	O
+of	O
+the	O
+Ramachandran	B-evidence
+plot	I-evidence
+(	O
+data	O
+not	O
+shown	O
+).	O
+
+The	O
+structure	B-evidence
+has	O
+been	O
+deposited	O
+in	O
+the	O
+Protein	O
+Data	O
+Bank	O
+,	O
+with	O
+accession	O
+code	O
+5KXF	O
+.	O
+
+Resolution	O
+range	O
+(	O
+Å	O
+)	O
+1	O
+50	O
+–	O
+2	O
+.	O
+7	O
+(	O
+2	O
+.	O
+8	O
+–	O
+2	O
+.	O
+7	O
+)	O
+Number	O
+of	O
+observations	O
+78	O
+,	O
+008	O
+Rmerge	O
+(%)	O
+10	O
+.	O
+5	O
+(	O
+45	O
+.	O
+3	O
+)	O
+I	O
+/	O
+σI	O
+24	O
+.	O
+1	O
+(	O
+6	O
+.	O
+3	O
+)	O
+Redundancy	O
+Completeness	O
+(%)	O
+100	O
+(	O
+100	O
+)	O
+R	B-evidence
+factor	I-evidence
+(%)	O
+19	O
+.	O
+2	O
+(	O
+25	O
+.	O
+0	O
+)	O
+Free	B-evidence
+R	I-evidence
+factor	I-evidence
+(%)	O
+25	O
+.	O
+4	O
+(	O
+35	O
+.	O
+4	O
+)	O
+Rms	O
+deviation	O
+in	O
+bond	O
+lengths	O
+(	O
+Å	O
+)	O
+0	O
+.	O
+017	O
+Rms	O
+deviation	O
+in	O
+bond	O
+angles	O
+(°)	O
+1	O
+.	O
+9	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+contains	O
+a	O
+seven	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+,	I-structure_element
+mostly	I-structure_element
+anti	I-structure_element
+-	I-structure_element
+parallel	I-structure_element
+β	I-structure_element
+-	I-structure_element
+barrel	I-structure_element
+(	O
+β1	B-structure_element
+-	I-structure_element
+β7	I-structure_element
+)	O
+and	O
+three	O
+helices	B-structure_element
+(	O
+αA	B-structure_element
+-	I-structure_element
+αC	I-structure_element
+)	O
+(	O
+Fig	O
+2A	O
+).	O
+
+Only	O
+two	O
+of	O
+the	O
+neighboring	O
+strands	B-structure_element
+,	O
+β1	B-structure_element
+and	O
+β3	B-structure_element
+,	O
+are	O
+parallel	O
+to	O
+each	O
+other	O
+.	O
+
+Helix	B-structure_element
+αB	B-structure_element
+covers	O
+one	O
+end	O
+of	O
+the	O
+barrel	B-structure_element
+,	O
+while	O
+helices	B-structure_element
+αA	B-structure_element
+and	O
+αC	B-structure_element
+are	O
+located	O
+next	O
+to	O
+each	O
+other	O
+at	O
+one	O
+side	O
+of	O
+the	O
+barrel	B-structure_element
+(	O
+Fig	O
+2B	O
+).	O
+
+The	O
+other	O
+end	O
+of	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+is	O
+covered	O
+by	O
+the	O
+loop	B-structure_element
+connecting	O
+strands	B-structure_element
+β2	B-structure_element
+and	O
+β3	B-structure_element
+,	O
+which	O
+contains	O
+the	O
+disordered	B-protein_state
+461	B-residue_range
+–	I-residue_range
+464	I-residue_range
+segment	O
+.	O
+
+The	O
+center	O
+of	O
+the	O
+barrel	B-structure_element
+is	O
+filled	O
+with	O
+hydrophobic	O
+side	O
+chains	O
+and	O
+is	O
+not	O
+accessible	O
+to	O
+the	O
+solvent	O
+.	O
+
+Crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+SPOC	B-structure_element
+domain	O
+of	O
+A	B-species
+.	I-species
+thaliana	I-species
+FPA	B-protein
+.	O
+
+Schematic	O
+drawing	O
+of	O
+the	O
+structure	B-evidence
+of	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+,	O
+colored	O
+from	O
+blue	O
+at	O
+the	O
+N	O
+terminus	O
+to	O
+red	O
+at	O
+the	O
+C	O
+terminus	O
+.	O
+
+The	O
+view	O
+is	O
+from	O
+the	O
+side	O
+of	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+.	O
+
+The	O
+disordered	B-protein_state
+segment	O
+(	O
+residues	O
+460	B-residue_range
+–	I-residue_range
+465	I-residue_range
+)	O
+is	O
+indicated	O
+with	O
+the	O
+dotted	O
+line	O
+.	O
+
+Structure	B-evidence
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+,	O
+viewed	O
+from	O
+the	O
+end	O
+of	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+,	O
+after	O
+90	O
+°	O
+rotation	O
+around	O
+the	O
+horizontal	O
+axis	O
+from	O
+panel	O
+A	O
+.	O
+All	O
+structure	O
+figures	O
+were	O
+produced	O
+with	O
+PyMOL	O
+(	O
+www	O
+.	O
+pymol	O
+.	O
+org	O
+).	O
+
+Comparisons	B-experimental_method
+to	I-experimental_method
+structural	I-experimental_method
+homologs	I-experimental_method
+of	O
+the	O
+SPOC	B-structure_element
+domain	O
+
+Only	O
+five	O
+structural	O
+homologs	O
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+were	O
+found	O
+in	O
+the	O
+Protein	O
+Data	O
+Bank	O
+with	O
+the	O
+DaliLite	B-experimental_method
+server	I-experimental_method
+,	O
+suggesting	O
+that	O
+the	O
+SPOC	B-structure_element
+domain	O
+structure	B-evidence
+is	O
+relatively	O
+unique	O
+.	O
+
+The	O
+top	O
+hit	O
+is	O
+the	O
+SPOC	B-structure_element
+domain	O
+of	O
+human	B-species
+SHARP	B-protein
+(	O
+Fig	O
+3A	O
+),	O
+with	O
+a	O
+Z	B-evidence
+score	I-evidence
+of	O
+12	O
+.	O
+3	O
+.	O
+
+The	O
+other	O
+four	O
+structural	O
+homologs	O
+include	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+domain	O
+of	O
+the	O
+proteins	O
+Ku70	B-protein
+and	O
+Ku80	B-protein
+(	O
+Z	B-evidence
+score	I-evidence
+11	O
+.	O
+4	O
+)	O
+(	O
+Fig	O
+3B	O
+),	O
+a	O
+domain	O
+in	O
+the	O
+chromodomain	B-protein_type
+protein	I-protein_type
+Chp1	B-protein
+(	O
+Z	B-evidence
+score	I-evidence
+10	O
+.	O
+8	O
+)	O
+(	O
+Fig	O
+3C	O
+),	O
+and	O
+the	O
+activator	B-structure_element
+interacting	I-structure_element
+domain	I-structure_element
+(	O
+ACID	B-structure_element
+)	O
+of	O
+the	O
+Med25	B-protein
+subunit	O
+of	O
+the	O
+Mediator	O
+complex	O
+(	O
+Z	B-evidence
+score	I-evidence
+8	O
+.	O
+5	O
+)	O
+(	O
+Fig	O
+3D	O
+).	O
+
+The	O
+next	O
+structural	O
+homolog	O
+has	O
+a	O
+Z	B-evidence
+score	I-evidence
+of	O
+3	O
+.	O
+0	O
+.	O
+
+Structural	O
+homologs	O
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+.	O
+
+Overlay	B-experimental_method
+of	O
+the	O
+structures	B-evidence
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+(	O
+cyan	O
+)	O
+and	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+(	O
+gray	O
+).	O
+
+The	O
+bound	O
+position	O
+of	O
+a	O
+doubly	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+peptide	B-chemical
+from	O
+SMRT	B-protein
+is	O
+shown	O
+in	O
+magenta	O
+.	O
+
+Overlay	B-experimental_method
+of	O
+the	O
+structures	B-evidence
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+(	O
+cyan	O
+)	O
+and	O
+the	O
+Ku70	B-protein
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+domain	O
+(	O
+gray	O
+).	O
+
+Ku80	B-protein
+contains	O
+a	O
+homologous	O
+domain	O
+(	O
+green	O
+),	O
+which	O
+forms	O
+a	O
+hetero	B-oligomeric_state
+-	I-oligomeric_state
+dimer	I-oligomeric_state
+with	O
+that	O
+in	O
+Ku70	B-protein
+.	O
+
+The	O
+two	O
+domains	O
+,	O
+and	O
+inserted	O
+segments	O
+on	O
+them	O
+,	O
+mediate	O
+the	O
+binding	O
+of	O
+dsDNA	B-chemical
+(	O
+orange	O
+).	O
+
+The	O
+red	O
+rectangle	O
+highlights	O
+the	O
+region	O
+of	O
+contact	O
+between	O
+the	O
+two	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+domains	O
+.	O
+
+Overlay	B-experimental_method
+of	O
+the	O
+structures	B-evidence
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+(	O
+cyan	O
+)	O
+and	O
+the	O
+homologous	O
+domain	O
+in	O
+Chp1	B-protein
+(	O
+gray	O
+).	O
+
+The	O
+binding	O
+partner	O
+of	O
+Chp1	B-protein
+,	O
+Tas3	B-protein
+,	O
+is	O
+shown	O
+in	O
+green	O
+.	O
+
+The	O
+red	O
+rectangle	O
+indicates	O
+the	O
+region	O
+equivalent	O
+to	O
+the	O
+binding	B-site
+site	I-site
+of	O
+the	O
+SMART	B-protein
+phosphopeptide	B-ptm
+in	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+,	O
+where	O
+a	O
+loop	B-structure_element
+of	O
+Tas3	B-protein
+is	O
+also	O
+located	O
+.	O
+(	O
+D	O
+).	O
+
+Overlay	B-experimental_method
+of	O
+the	O
+structures	B-evidence
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+(	O
+cyan	O
+)	O
+and	O
+the	O
+Med25	B-protein
+ACID	B-structure_element
+(	O
+gray	O
+).	O
+
+SHARP	B-protein
+is	O
+a	O
+transcriptional	B-protein_type
+co	I-protein_type
+-	I-protein_type
+repressor	I-protein_type
+in	O
+the	O
+nuclear	B-protein_type
+receptor	I-protein_type
+and	O
+Notch	B-protein
+/	O
+RBP	B-protein
+-	I-protein
+Jκ	I-protein
+signaling	O
+pathways	O
+.	O
+
+The	O
+SPOC	B-structure_element
+domain	O
+of	O
+SHARP	B-protein
+interacts	O
+directly	O
+with	O
+silencing	B-protein
+mediator	I-protein
+for	I-protein
+retinoid	I-protein
+and	I-protein
+thyroid	I-protein
+receptor	I-protein
+(	O
+SMRT	B-protein
+),	O
+nuclear	B-protein_type
+receptor	I-protein_type
+co	I-protein_type
+-	I-protein_type
+repressor	I-protein_type
+(	O
+N	B-protein_type
+-	I-protein_type
+CoR	I-protein_type
+),	O
+HDAC	B-protein
+,	O
+and	O
+other	O
+components	O
+to	O
+represses	O
+transcription	O
+.	O
+
+While	O
+the	O
+overall	O
+structure	B-evidence
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+is	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+,	O
+there	O
+are	O
+noticeable	O
+differences	O
+in	O
+the	O
+positioning	O
+of	O
+the	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+and	O
+the	O
+helices	B-structure_element
+,	O
+and	O
+most	O
+of	O
+the	O
+loops	B-structure_element
+have	O
+substantially	O
+different	O
+conformations	O
+as	O
+well	O
+(	O
+Fig	O
+3A	O
+).	O
+
+In	O
+addition	O
+,	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+has	O
+three	O
+extra	O
+helices	B-structure_element
+.	O
+
+One	O
+of	O
+them	O
+covers	O
+the	O
+other	O
+end	O
+of	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+,	O
+and	O
+the	O
+other	O
+two	O
+shield	O
+an	O
+additional	O
+surface	O
+of	O
+the	O
+side	O
+of	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+from	O
+solvent	O
+.	O
+
+A	O
+doubly	B-protein_state
+-	I-protein_state
+phosphorylated	I-protein_state
+peptide	B-chemical
+from	O
+SMRT	B-protein
+is	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+side	O
+of	O
+the	O
+barrel	B-structure_element
+,	O
+near	O
+strands	B-structure_element
+β1	B-structure_element
+and	O
+β3	B-structure_element
+(	O
+Fig	O
+3A	O
+).	O
+
+Such	O
+a	O
+binding	O
+mode	O
+probably	O
+would	O
+not	O
+be	O
+possible	O
+in	O
+FPA	B-protein
+,	O
+as	O
+the	O
+peptide	B-chemical
+would	O
+clash	O
+with	O
+the	O
+β1	B-structure_element
+-	I-structure_element
+β2	I-structure_element
+loop	I-structure_element
+.	O
+
+The	O
+Ku70	B-complex_assembly
+-	I-complex_assembly
+Ku80	I-complex_assembly
+hetero	B-oligomeric_state
+-	I-oligomeric_state
+dimer	I-oligomeric_state
+is	O
+involved	O
+in	O
+DNA	O
+double	O
+-	O
+strand	O
+break	O
+repair	O
+and	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+domain	O
+contributes	O
+to	O
+DNA	B-chemical
+binding	O
+.	O
+
+In	O
+fact	O
+,	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+domains	O
+of	O
+Ku70	B-protein
+and	O
+Ku80	B-protein
+form	O
+a	O
+hetero	B-oligomeric_state
+-	I-oligomeric_state
+dimer	I-oligomeric_state
+,	O
+primarily	O
+through	O
+interactions	O
+between	O
+the	O
+loops	B-structure_element
+connecting	O
+the	O
+third	B-structure_element
+and	I-structure_element
+fourth	I-structure_element
+strands	I-structure_element
+of	O
+the	O
+barrel	B-structure_element
+(	O
+Fig	O
+3B	O
+).	O
+
+The	O
+open	O
+ends	O
+of	O
+the	O
+two	O
+β	B-structure_element
+-	I-structure_element
+barrels	I-structure_element
+face	O
+the	O
+DNA	B-site
+binding	I-site
+sites	I-site
+,	O
+and	O
+contact	O
+the	O
+phosphodiester	O
+backbone	O
+of	O
+the	O
+dsDNA	B-chemical
+.	O
+
+In	O
+addition	O
+,	O
+a	O
+long	B-structure_element
+insert	I-structure_element
+connecting	O
+strands	B-structure_element
+β2	B-structure_element
+and	O
+β3	B-structure_element
+in	O
+the	O
+two	O
+domains	O
+form	O
+an	O
+arch	B-structure_element
+-	I-structure_element
+like	I-structure_element
+structure	I-structure_element
+,	O
+encircling	O
+the	O
+dsDNA	B-chemical
+.	O
+
+Chp1	B-protein
+is	O
+a	O
+subunit	O
+of	O
+the	O
+RNA	B-complex_assembly
+-	I-complex_assembly
+induced	I-complex_assembly
+initiation	I-complex_assembly
+of	I-complex_assembly
+transcriptional	I-complex_assembly
+gene	I-complex_assembly
+silencing	I-complex_assembly
+(	O
+RITS	B-complex_assembly
+)	O
+complex	O
+.	O
+
+The	O
+partner	O
+of	O
+Chp1	B-protein
+,	O
+Tas3	B-protein
+,	O
+is	O
+bound	O
+between	O
+the	O
+barrel	B-structure_element
+domain	I-structure_element
+and	O
+the	O
+second	B-structure_element
+domain	I-structure_element
+of	O
+Chp1	B-protein
+,	O
+and	O
+the	O
+linker	B-structure_element
+between	O
+the	O
+two	O
+domains	O
+is	O
+also	O
+crucial	O
+for	O
+this	O
+interaction	O
+(	O
+Fig	O
+3C	O
+).	O
+
+It	O
+is	O
+probably	O
+unlikely	O
+that	O
+the	O
+β	B-structure_element
+-	I-structure_element
+barrel	I-structure_element
+itself	O
+is	O
+sufficient	O
+to	O
+bind	O
+Tas3	B-protein
+.	O
+
+Interestingly	O
+,	O
+a	O
+loop	B-structure_element
+in	O
+Tas3	B-protein
+contacts	O
+strand	B-structure_element
+β3	B-structure_element
+of	O
+the	O
+barrel	B-structure_element
+domain	I-structure_element
+,	O
+at	O
+a	O
+location	O
+somewhat	O
+similar	O
+to	O
+that	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+segment	O
+of	O
+the	O
+SMRT	B-protein
+peptide	B-chemical
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+(	O
+Fig	O
+3A	O
+).	O
+
+Mediator	B-protein_type
+is	O
+a	O
+coactivator	O
+complex	O
+that	O
+promotes	O
+transcription	O
+by	O
+Pol	B-complex_assembly
+II	I-complex_assembly
+.	O
+
+The	O
+Med25	B-protein
+subunit	O
+ACID	B-structure_element
+is	O
+the	O
+target	O
+of	O
+the	O
+potent	O
+activator	O
+VP16	B-protein
+of	O
+the	O
+herpes	B-species
+simplex	I-species
+virus	I-species
+.	O
+
+The	O
+structure	B-evidence
+of	O
+ACID	B-structure_element
+contains	O
+a	O
+helix	B-structure_element
+at	O
+the	O
+C	O
+-	O
+terminus	O
+as	O
+well	O
+as	O
+an	O
+extended	O
+β1	B-structure_element
+-	I-structure_element
+β2	I-structure_element
+loop	I-structure_element
+.	O
+
+Nonetheless	O
+,	O
+the	O
+binding	B-site
+site	I-site
+for	O
+VP16	B-protein
+has	O
+been	O
+mapped	O
+to	O
+roughly	O
+the	O
+same	O
+surface	B-site
+patch	I-site
+,	O
+near	O
+strands	B-structure_element
+β1	B-structure_element
+and	O
+β3	B-structure_element
+,	O
+that	O
+is	O
+used	O
+by	O
+the	O
+SHARP	B-protein
+and	O
+Tas3	B-protein
+SPOC	B-structure_element
+domains	O
+for	O
+binding	O
+their	O
+partners	O
+.	O
+
+A	O
+conserved	B-protein_state
+surface	B-site
+patch	I-site
+in	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+
+An	O
+analysis	O
+of	O
+the	O
+SPOC	B-structure_element
+domain	O
+indicates	O
+a	O
+large	O
+surface	B-site
+patch	I-site
+near	O
+strands	B-structure_element
+β1	B-structure_element
+,	O
+β3	B-structure_element
+,	O
+β5	B-structure_element
+and	O
+β6	B-structure_element
+that	O
+is	O
+conserved	B-protein_state
+among	O
+plant	B-taxonomy_domain
+FPA	B-protein
+homologs	O
+(	O
+Fig	O
+4A	O
+).	O
+
+This	O
+surface	B-site
+patch	I-site
+can	O
+be	O
+broken	O
+into	O
+two	O
+sub	B-site
+-	I-site
+patches	I-site
+,	O
+with	O
+residues	O
+Lys447	B-residue_name_number
+(	O
+in	O
+strand	B-structure_element
+β1	B-structure_element
+),	O
+Arg477	B-residue_name_number
+(	O
+β3	B-structure_element
+),	O
+Tyr515	B-residue_name_number
+(	O
+αB	B-structure_element
+)	O
+and	O
+Arg521	B-residue_name_number
+(	O
+β5	B-structure_element
+)	O
+in	O
+one	O
+sub	B-site
+-	I-site
+patch	I-site
+,	O
+and	O
+residues	O
+His486	B-residue_name_number
+(	O
+αA	B-structure_element
+),	O
+Thr478	B-residue_name_number
+(	O
+β3	B-structure_element
+),	O
+Val524	B-residue_name_number
+(	O
+β5	B-structure_element
+)	O
+and	O
+Phe534	B-residue_name_number
+(	O
+β6	B-structure_element
+)	O
+in	O
+the	O
+other	O
+sub	B-site
+-	I-site
+patch	I-site
+(	O
+Fig	O
+4B	O
+).	O
+
+The	O
+first	B-site
+surface	I-site
+patch	I-site
+is	O
+electropositive	B-protein_state
+in	O
+nature	O
+(	O
+Fig	O
+4C	O
+),	O
+and	O
+residues	O
+Arg477	B-residue_name_number
+and	O
+Tyr515	B-residue_name_number
+are	O
+also	O
+conserved	B-protein_state
+in	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+(	O
+Fig	O
+1B	O
+).	O
+
+In	O
+fact	O
+,	O
+one	O
+of	O
+the	O
+phosphorylated	B-protein_state
+residues	O
+of	O
+the	O
+SMRT	B-protein
+peptide	B-chemical
+interacts	O
+with	O
+this	O
+surface	B-site
+patch	I-site
+(	O
+Fig	O
+3A	O
+),	O
+suggesting	O
+that	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+might	O
+also	O
+interact	O
+with	O
+a	O
+phosphorylated	B-protein_state
+segment	O
+here	O
+.	O
+
+In	O
+comparison	O
+,	O
+the	O
+second	B-site
+surface	I-site
+patch	I-site
+is	O
+more	O
+hydrophobic	B-protein_state
+in	O
+nature	O
+(	O
+Fig	O
+4C	O
+).	O
+
+A	O
+conserved	B-protein_state
+surface	B-site
+patch	I-site
+of	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+.	O
+
+Two	O
+views	O
+of	O
+the	O
+molecular	O
+surface	O
+of	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+colored	O
+based	O
+on	O
+sequence	O
+conservation	O
+among	O
+plant	B-taxonomy_domain
+FPA	B-protein
+homologs	O
+.	O
+
+Residues	O
+in	O
+the	O
+conserved	B-protein_state
+surface	B-site
+patch	I-site
+of	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+.	O
+
+The	O
+side	O
+chains	O
+of	O
+the	O
+residues	O
+are	O
+shown	O
+in	O
+stick	O
+models	O
+,	O
+colored	O
+orange	O
+in	O
+the	O
+first	B-site
+sub	I-site
+-	I-site
+patch	I-site
+and	O
+green	O
+in	O
+the	O
+second	O
+.	O
+(	O
+C	O
+).	O
+
+Molecular	O
+surface	O
+of	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+colored	O
+based	O
+on	O
+electrostatic	O
+potential	O
+.	O
+
+Testing	O
+the	O
+requirement	O
+of	O
+specific	O
+conserved	O
+amino	O
+acids	O
+for	O
+FPA	B-protein
+functions	O
+
+We	O
+next	O
+examined	O
+the	O
+potential	O
+impact	O
+of	O
+the	O
+conserved	B-protein_state
+surface	B-site
+patch	I-site
+on	O
+FPA	B-protein
+function	O
+in	O
+vivo	O
+.	O
+
+We	O
+mutated	B-experimental_method
+two	O
+residues	O
+,	O
+Arg477	B-residue_name_number
+and	O
+Tyr515	B-residue_name_number
+,	O
+of	O
+the	O
+surface	B-site
+patch	I-site
+,	O
+which	O
+are	O
+also	O
+conserved	B-protein_state
+in	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+(	O
+Fig	O
+1B	O
+)	O
+and	O
+were	O
+found	O
+to	O
+be	O
+functionally	O
+important	O
+.	O
+
+The	O
+mutations	B-experimental_method
+were	O
+introduced	B-experimental_method
+into	O
+a	O
+transgene	O
+designed	O
+to	O
+express	O
+FPA	B-protein
+from	O
+its	O
+native	O
+control	O
+elements	O
+(	O
+promoter	O
+,	O
+introns	O
+and	O
+3	O
+′	O
+UTR	O
+).	O
+
+The	O
+resulting	O
+transgenes	O
+were	O
+then	O
+stably	B-experimental_method
+transformed	I-experimental_method
+into	O
+an	O
+fpa	B-gene
+-	I-gene
+8	I-gene
+mutant	B-protein_state
+background	O
+so	O
+that	O
+the	O
+impact	O
+of	O
+the	O
+mutations	B-experimental_method
+on	O
+FPA	B-protein
+function	O
+could	O
+be	O
+assessed	O
+.	O
+
+Control	O
+transformation	O
+of	O
+the	O
+same	O
+expression	B-experimental_method
+constructs	I-experimental_method
+into	O
+fpa	B-gene
+-	I-gene
+8	I-gene
+designed	O
+to	O
+express	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+FPA	B-protein
+protein	O
+restored	O
+FPA	B-protein
+protein	O
+expression	B-evidence
+levels	I-evidence
+to	O
+near	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+levels	O
+(	O
+panel	O
+A	O
+in	O
+S1	O
+Fig	O
+)	O
+and	O
+rescued	O
+the	O
+function	O
+of	O
+FPA	B-protein
+in	O
+controlling	O
+RNA	B-chemical
+3	O
+′-	O
+end	O
+formation	O
+,	O
+for	O
+example	O
+in	O
+FPA	B-protein
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+(	O
+panel	O
+B	O
+in	O
+S1	O
+Fig	O
+).	O
+
+We	O
+examined	O
+independent	O
+transgenic	O
+lines	O
+expressing	O
+each	O
+R477A	B-mutant
+and	O
+Y515A	B-mutant
+mutation	B-experimental_method
+.	O
+
+In	O
+each	O
+case	O
+,	O
+we	O
+confirmed	O
+that	O
+detectable	O
+levels	O
+of	O
+FPA	B-protein
+protein	O
+expression	O
+were	O
+restored	O
+close	O
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+levels	O
+in	O
+protein	B-experimental_method
+blot	I-experimental_method
+analyses	O
+using	O
+antibodies	O
+that	O
+specifically	O
+recognize	O
+FPA	B-protein
+(	O
+S2	O
+Fig	O
+).	O
+
+We	O
+then	O
+examined	O
+the	O
+impact	O
+of	O
+the	O
+surface	B-site
+patch	I-site
+mutations	B-experimental_method
+on	O
+FPA	B-protein
+’	O
+s	O
+function	O
+in	O
+controlling	O
+RNA	O
+3	O
+′-	O
+end	O
+formation	O
+by	O
+determining	O
+whether	O
+the	O
+mutant	B-protein_state
+proteins	O
+functioned	O
+in	O
+FPA	B-protein
+autoregulation	O
+and	O
+the	O
+repression	O
+of	O
+FLC	B-gene
+expression	O
+.	O
+
+FPA	B-protein
+autoregulates	O
+its	O
+expression	O
+by	O
+promoting	O
+cleavage	O
+and	O
+polyadenylation	O
+within	O
+intron	O
+1	O
+of	O
+its	O
+own	O
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+,	O
+resulting	O
+in	O
+a	O
+truncated	O
+transcript	O
+that	O
+does	O
+not	O
+encode	O
+functional	O
+protein	O
+.	O
+
+We	O
+used	O
+RNA	B-experimental_method
+gel	I-experimental_method
+blot	I-experimental_method
+analyses	I-experimental_method
+to	O
+reveal	O
+that	O
+in	O
+each	O
+of	O
+three	O
+independent	O
+transgenic	O
+lines	O
+for	O
+each	O
+single	O
+mutant	B-protein_state
+,	O
+rescue	O
+of	O
+proximally	O
+polyadenylated	O
+FPA	B-protein
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+can	O
+be	O
+detected	O
+(	O
+Fig	O
+5A	O
+and	O
+5B	O
+).	O
+
+We	O
+therefore	O
+conclude	O
+that	O
+neither	O
+of	O
+these	O
+mutations	O
+disrupted	O
+the	O
+ability	O
+of	O
+FPA	B-protein
+to	O
+promote	O
+RNA	O
+3	O
+′-	O
+end	O
+formation	O
+in	O
+its	O
+own	O
+transcript	O
+.	O
+
+Impact	O
+of	O
+individual	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+mutations	B-experimental_method
+on	O
+alternative	O
+polyadenylation	O
+of	O
+FPA	B-protein
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+.	O
+
+RNA	B-experimental_method
+gel	I-experimental_method
+blot	I-experimental_method
+analysis	O
+of	O
+WT	B-protein_state
+A	B-species
+.	I-species
+thaliana	I-species
+accession	O
+Columbia	O
+(	O
+Col	O
+-	O
+0	O
+)	O
+plants	B-taxonomy_domain
+fpa	B-gene
+-	I-gene
+8	I-gene
+and	O
+fpa	B-gene
+-	I-gene
+8	I-gene
+mutants	B-protein_state
+expressing	O
+either	O
+FPA	B-protein
+::	O
+FPA	B-mutant
+R477A	I-mutant
+(	O
+A	O
+),	O
+or	O
+FPA	B-protein
+::	O
+FPA	B-mutant
+Y515A	I-mutant
+(	O
+B	O
+)	O
+using	O
+poly	O
+(	O
+A	O
+)+	O
+purified	O
+mRNAs	B-chemical
+.	O
+
+A	O
+probe	O
+corresponding	O
+to	O
+the	O
+5	O
+’	O
+UTR	O
+region	O
+of	O
+FPA	B-protein
+mRNA	B-chemical
+was	O
+used	O
+to	O
+detect	O
+FPA	B-protein
+specific	O
+mRNAs	B-chemical
+.	O
+
+Proximally	O
+and	O
+distally	O
+polyadenylated	O
+FPA	B-protein
+transcripts	O
+are	O
+marked	O
+with	O
+arrows	O
+.	O
+
+The	O
+ratio	O
+of	O
+distal	O
+:	O
+proximal	O
+polyadenylated	O
+forms	O
+is	O
+given	O
+under	O
+each	O
+lane	O
+.	O
+(	O
+C	O
+,	O
+D	O
+)	O
+Impact	O
+of	O
+individual	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+mutations	B-experimental_method
+on	O
+FLC	B-gene
+transcript	O
+levels	O
+.	O
+
+qRT	B-experimental_method
+-	I-experimental_method
+PCR	I-experimental_method
+analysis	O
+was	O
+performed	O
+with	O
+total	O
+RNA	B-chemical
+purified	O
+from	O
+Col	O
+-	O
+0	O
+,	O
+fpa	B-gene
+-	I-gene
+8	I-gene
+,	O
+35S	O
+::	O
+FPA	B-protein
+:	O
+YFP	B-experimental_method
+and	O
+FPA	B-protein
+::	O
+FPA	B-mutant
+R477A	I-mutant
+(	O
+C	O
+),	O
+FPA	B-protein
+::	O
+FPA	B-mutant
+Y515A	I-mutant
+(	O
+D	O
+)	O
+plants	B-taxonomy_domain
+.	O
+
+Histograms	B-evidence
+show	O
+mean	O
+values	O
+±	O
+SE	O
+for	O
+three	O
+independent	O
+PCR	B-experimental_method
+amplifications	O
+of	O
+three	O
+biological	O
+replicates	O
+.	O
+
+We	O
+next	O
+examined	O
+whether	O
+the	O
+corresponding	O
+mutations	O
+disrupted	O
+the	O
+ability	O
+of	O
+FPA	B-protein
+to	O
+control	O
+FLC	B-gene
+expression	O
+.	O
+
+We	O
+used	O
+RT	B-experimental_method
+-	I-experimental_method
+qPCR	I-experimental_method
+to	O
+measure	O
+the	O
+expression	O
+of	O
+FLC	B-gene
+mRNA	B-chemical
+and	O
+found	O
+that	O
+in	O
+each	O
+independent	O
+transgenic	O
+line	O
+encoding	O
+each	O
+mutated	B-protein_state
+FPA	B-protein
+protein	O
+,	O
+the	O
+elevated	O
+levels	O
+of	O
+FLC	B-gene
+detected	O
+in	O
+fpa	B-gene
+-	I-gene
+8	I-gene
+mutants	B-protein_state
+were	O
+restored	O
+to	O
+near	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+levels	O
+by	O
+expression	O
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+conserved	B-protein_state
+patch	B-site
+mutant	B-protein_state
+proteins	O
+(	O
+Fig	O
+5C	O
+and	O
+5D	O
+).	O
+
+Since	O
+each	O
+surface	B-site
+patch	I-site
+mutation	B-experimental_method
+appeared	O
+to	O
+be	O
+insufficient	O
+to	O
+disrupt	O
+FPA	B-protein
+functions	O
+on	O
+its	O
+own	O
+,	O
+we	O
+combined	O
+both	O
+mutations	O
+into	O
+the	O
+same	O
+transgene	O
+.	O
+
+We	O
+could	O
+again	O
+confirm	O
+that	O
+near	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+levels	O
+of	O
+FPA	B-protein
+protein	O
+were	O
+expressed	O
+from	O
+three	O
+independent	O
+transgenic	O
+lines	O
+expressing	O
+the	O
+FPA	B-mutant
+R477A	I-mutant
+;	I-mutant
+Y515A	I-mutant
+doubly	B-protein_state
+mutated	I-protein_state
+protein	O
+in	O
+an	O
+fpa	B-gene
+-	I-gene
+8	I-gene
+mutant	B-protein_state
+background	O
+(	O
+S3	O
+Fig	O
+).	O
+
+We	O
+found	O
+that	O
+FPA	B-mutant
+R477A	I-mutant
+;	I-mutant
+Y515A	I-mutant
+protein	O
+functioned	O
+like	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+FPA	B-protein
+to	O
+restore	O
+FPA	B-protein
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+proximal	O
+polyadenylation	O
+(	O
+Fig	O
+6A	O
+)	O
+and	O
+FLC	B-gene
+expression	O
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+levels	O
+(	O
+Fig	O
+6B	O
+).	O
+
+Impact	O
+of	O
+double	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+mutations	B-experimental_method
+on	O
+alternative	O
+polyadenylation	O
+of	O
+FPA	B-protein
+pre	B-chemical
+-	I-chemical
+mRNA	I-chemical
+and	O
+FLC	B-gene
+expression	O
+.	O
+
+(	O
+A	O
+)	O
+RNA	B-experimental_method
+gel	I-experimental_method
+blot	I-experimental_method
+analysis	O
+of	O
+WT	B-protein_state
+A	B-species
+.	I-species
+thaliana	I-species
+accession	O
+Columbia	O
+(	O
+Col	O
+-	O
+0	O
+)	O
+plants	B-taxonomy_domain
+fpa	B-gene
+-	I-gene
+8	I-gene
+and	O
+fpa	B-gene
+-	I-gene
+8	I-gene
+mutants	B-protein_state
+expressing	O
+FPA	B-protein
+::	O
+FPA	B-mutant
+R477A	I-mutant
+;	I-mutant
+Y515A	I-mutant
+using	O
+poly	O
+(	O
+A	O
+)+	O
+purified	O
+mRNAs	B-chemical
+.	O
+
+Black	O
+arrows	O
+indicate	O
+the	O
+proximally	O
+and	O
+distally	O
+polyadenylated	O
+FPA	B-protein
+mRNAs	B-chemical
+.	O
+
+qRT	B-experimental_method
+-	I-experimental_method
+PCR	I-experimental_method
+analysis	O
+was	O
+performed	O
+with	O
+total	O
+RNA	B-chemical
+purified	O
+from	O
+Col	O
+-	O
+0	O
+,	O
+fpa	B-gene
+-	I-gene
+8	I-gene
+,	O
+and	O
+FPA	B-protein
+::	O
+FPA	B-mutant
+R477A	I-mutant
+;	I-mutant
+Y515A	I-mutant
+plants	B-taxonomy_domain
+.	O
+
+Together	O
+our	O
+findings	O
+suggest	O
+that	O
+either	O
+the	O
+SPOC	B-structure_element
+domain	O
+is	O
+not	O
+required	O
+for	O
+the	O
+role	O
+of	O
+FPA	B-protein
+in	O
+regulating	O
+RNA	B-chemical
+3	O
+′-	O
+end	O
+formation	O
+,	O
+or	O
+that	O
+this	O
+combination	O
+of	O
+mutations	B-experimental_method
+is	O
+not	O
+sufficient	O
+to	O
+critically	O
+disrupt	O
+the	O
+function	O
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+.	O
+
+Since	O
+the	O
+corresponding	O
+mutations	B-experimental_method
+in	O
+the	O
+SHARP	B-protein
+SPOC	B-structure_element
+domain	O
+do	O
+disrupt	O
+its	O
+recognition	O
+of	O
+unphosphorylated	B-protein_state
+SMRT	B-protein
+peptides	B-chemical
+,	O
+these	O
+observations	O
+may	O
+reinforce	O
+the	O
+idea	O
+that	O
+the	O
+features	O
+and	O
+functions	O
+of	O
+the	O
+FPA	B-protein
+SPOC	B-structure_element
+domain	O
+differ	O
+from	O
+those	O
+of	O
+the	O
+only	O
+other	O
+well	O
+-	O
+characterized	O
+SPOC	B-structure_element
+domain	O
+.	O
+
diff --git a/annotation_IOB/PMC4993997.tsv b/annotation_IOB/PMC4993997.tsv
new file mode 100644
index 0000000000000000000000000000000000000000..bfc61706f2019d616c75dd611715754f233a858b
--- /dev/null
+++ b/annotation_IOB/PMC4993997.tsv
@@ -0,0 +1,7856 @@
+Structure	O
+and	O
+function	O
+of	O
+human	B-species
+Naa60	B-protein
+(	O
+NatF	B-complex_assembly
+),	O
+a	O
+Golgi	O
+-	O
+localized	O
+bi	O
+-	O
+functional	O
+acetyltransferase	B-protein_type
+
+N	B-ptm
+-	I-ptm
+terminal	I-ptm
+acetylation	I-ptm
+(	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+),	O
+carried	O
+out	O
+by	O
+N	B-protein_type
+-	I-protein_type
+terminal	I-protein_type
+acetyltransferases	I-protein_type
+(	O
+NATs	B-protein_type
+),	O
+is	O
+a	O
+conserved	O
+and	O
+primary	O
+modification	O
+of	O
+nascent	O
+peptide	B-chemical
+chains	O
+.	O
+
+Naa60	B-protein
+(	O
+also	O
+named	O
+NatF	B-complex_assembly
+)	O
+is	O
+a	O
+recently	O
+identified	O
+NAT	B-protein_type
+found	O
+only	O
+in	O
+multicellular	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+.	O
+
+This	O
+protein	O
+was	O
+shown	O
+to	O
+locate	O
+on	O
+the	O
+Golgi	O
+apparatus	O
+and	O
+mainly	O
+catalyze	O
+the	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+of	O
+transmembrane	O
+proteins	O
+,	O
+and	O
+it	O
+also	O
+harbors	O
+lysine	B-protein_type
+Nε	I-protein_type
+-	I-protein_type
+acetyltransferase	I-protein_type
+(	O
+KAT	B-protein_type
+)	O
+activity	O
+to	O
+catalyze	O
+the	O
+acetylation	B-ptm
+of	O
+lysine	B-residue_name
+ε	O
+-	O
+amine	O
+.	O
+
+Here	O
+,	O
+we	O
+report	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+human	B-species
+Naa60	B-protein
+(	O
+hNaa60	B-protein
+)	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+Acetyl	B-chemical
+-	I-chemical
+Coenzyme	I-chemical
+A	I-chemical
+(	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+)	O
+or	O
+Coenzyme	B-chemical
+A	I-chemical
+(	O
+CoA	B-chemical
+).	O
+
+The	O
+hNaa60	B-protein
+protein	O
+contains	O
+an	O
+amphipathic	B-structure_element
+helix	I-structure_element
+following	O
+its	O
+GNAT	B-structure_element
+domain	I-structure_element
+that	O
+may	O
+contribute	O
+to	O
+Golgi	O
+localization	O
+of	O
+hNaa60	B-protein
+,	O
+and	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+adopted	O
+different	O
+conformations	O
+in	O
+the	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+and	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+crystal	B-evidence
+structures	I-evidence
+.	O
+
+Remarkably	O
+,	O
+we	O
+found	O
+that	O
+the	O
+side	O
+-	O
+chain	O
+of	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+can	O
+influence	O
+the	O
+position	O
+of	O
+the	O
+coenzyme	B-chemical
+,	O
+indicating	O
+a	O
+new	O
+regulatory	O
+mechanism	O
+involving	O
+enzyme	O
+,	O
+co	O
+-	O
+factor	O
+and	O
+substrates	O
+interactions	O
+.	O
+
+Moreover	O
+,	O
+structural	B-experimental_method
+comparison	I-experimental_method
+and	I-experimental_method
+biochemical	I-experimental_method
+studies	I-experimental_method
+indicated	O
+that	O
+Tyr	B-residue_name_number
+97	I-residue_name_number
+and	O
+His	B-residue_name_number
+138	I-residue_name_number
+are	O
+key	O
+residues	O
+for	O
+catalytic	O
+reaction	O
+and	O
+that	O
+a	O
+non	B-protein_state
+-	I-protein_state
+conserved	I-protein_state
+β3	B-structure_element
+-	I-structure_element
+β4	I-structure_element
+long	I-structure_element
+loop	I-structure_element
+participates	O
+in	O
+the	O
+regulation	O
+of	O
+hNaa60	B-protein
+activity	O
+.	O
+
+Acetylation	B-ptm
+is	O
+one	O
+of	O
+the	O
+most	O
+ubiquitous	O
+modifications	O
+that	O
+plays	O
+a	O
+vital	O
+role	O
+in	O
+many	O
+biological	O
+processes	O
+,	O
+such	O
+as	O
+transcriptional	O
+regulation	O
+,	O
+protein	O
+-	O
+protein	O
+interaction	O
+,	O
+enzyme	O
+activity	O
+,	O
+protein	O
+stability	O
+,	O
+antibiotic	O
+resistance	O
+,	O
+biological	O
+rhythm	O
+and	O
+so	O
+on	O
+.	O
+
+Protein	O
+acetylation	B-ptm
+can	O
+be	O
+grouped	O
+into	O
+lysine	B-ptm
+Nε	I-ptm
+-	I-ptm
+acetylation	I-ptm
+and	O
+peptide	B-chemical
+N	B-ptm
+-	I-ptm
+terminal	I-ptm
+acetylation	I-ptm
+(	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+).	O
+
+Generally	O
+,	O
+Nε	B-ptm
+-	I-ptm
+acetylation	I-ptm
+refers	O
+to	O
+the	O
+transfer	O
+of	O
+an	O
+acetyl	B-chemical
+group	O
+from	O
+an	O
+acetyl	B-chemical
+coenzyme	I-chemical
+A	I-chemical
+(	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+)	O
+to	O
+the	O
+ε	O
+-	O
+amino	O
+group	O
+of	O
+lysine	B-residue_name
+.	O
+
+This	O
+kind	O
+of	O
+modification	O
+is	O
+catalyzed	O
+by	O
+lysine	B-protein_type
+acetyltransferases	I-protein_type
+(	O
+KATs	B-protein_type
+),	O
+some	O
+of	O
+which	O
+are	O
+named	O
+histone	B-protein_type
+acetyltransferases	I-protein_type
+(	O
+HATs	B-protein_type
+)	O
+because	O
+early	O
+studies	O
+focused	O
+mostly	O
+on	O
+the	O
+post	O
+-	O
+transcriptional	O
+acetylation	B-ptm
+of	O
+histones	B-protein_type
+.	O
+
+Despite	O
+the	O
+prominent	O
+accomplishments	O
+in	O
+the	O
+field	O
+regarding	O
+Nε	B-ptm
+-	I-ptm
+acetylation	I-ptm
+by	O
+KATs	B-protein_type
+for	O
+over	O
+50	O
+years	O
+,	O
+the	O
+significance	O
+of	O
+the	O
+more	O
+evolutionarily	O
+conserved	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+is	O
+still	O
+inconclusive	O
+.	O
+
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+is	O
+an	O
+abundant	O
+and	O
+evolutionarily	O
+conserved	O
+modification	O
+occurring	O
+in	O
+bacteria	B-taxonomy_domain
+,	O
+archaea	B-taxonomy_domain
+and	O
+eukaryotes	B-taxonomy_domain
+.	O
+
+It	O
+is	O
+estimated	O
+that	O
+about	O
+80	O
+–	O
+90	O
+%	O
+of	O
+soluble	O
+human	B-species
+proteins	O
+and	O
+50	O
+–	O
+70	O
+%	O
+of	O
+yeast	B-taxonomy_domain
+proteins	O
+are	O
+subjected	O
+to	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+,	O
+where	O
+an	O
+acetyl	B-chemical
+moiety	O
+is	O
+transferred	O
+from	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+to	O
+the	O
+α	O
+-	O
+amino	O
+group	O
+of	O
+the	O
+first	O
+residue	O
+.	O
+
+Recently	O
+Nt	O
+-	O
+acetylome	O
+expands	O
+the	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+to	O
+transmembrane	O
+proteins	O
+.	O
+
+Unlike	O
+Nε	B-ptm
+-	I-ptm
+acetylation	I-ptm
+that	O
+can	O
+be	O
+eliminated	O
+by	O
+deacetylases	B-protein_type
+,	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+is	O
+considered	O
+irreversible	B-protein_state
+since	O
+no	O
+corresponding	O
+deacetylase	B-protein_type
+is	O
+found	O
+to	O
+date	O
+.	O
+
+Although	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+has	O
+been	O
+regarded	O
+as	O
+a	O
+co	O
+-	O
+translational	O
+modification	O
+traditionally	O
+,	O
+there	O
+is	O
+evidence	O
+that	O
+post	O
+-	O
+translational	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+exists	O
+.	O
+
+During	O
+the	O
+past	O
+decades	O
+,	O
+a	O
+large	O
+number	O
+of	O
+Nt	O
+-	O
+acetylome	O
+researches	O
+have	O
+shed	O
+light	O
+on	O
+the	O
+functional	O
+roles	O
+of	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+,	O
+including	O
+protein	O
+degradation	O
+,	O
+subcellular	O
+localization	O
+,	O
+protein	O
+-	O
+protein	O
+interaction	O
+,	O
+protein	O
+-	O
+membrane	O
+interaction	O
+,	O
+plant	B-taxonomy_domain
+development	O
+,	O
+stress	O
+-	O
+response	O
+and	O
+protein	O
+stability	O
+.	O
+
+The	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+is	O
+carried	O
+out	O
+by	O
+N	B-protein_type
+-	I-protein_type
+terminal	I-protein_type
+acetyltransferases	I-protein_type
+(	O
+NATs	B-protein_type
+)	O
+that	O
+belong	O
+to	O
+the	O
+GNAT	B-protein_type
+superfamily	I-protein_type
+.	O
+
+To	O
+date	O
+,	O
+six	O
+NATs	B-protein_type
+(	O
+NatA	B-complex_assembly
+/	O
+B	B-complex_assembly
+/	O
+C	B-complex_assembly
+/	O
+D	B-complex_assembly
+/	O
+E	B-complex_assembly
+/	O
+F	B-complex_assembly
+)	O
+have	O
+been	O
+identified	O
+in	O
+eukaryotes	B-taxonomy_domain
+.	O
+
+About	O
+40	O
+percent	O
+of	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+of	O
+soluble	O
+proteins	O
+in	O
+cells	O
+is	O
+catalyzed	O
+by	O
+NatA	B-complex_assembly
+complex	O
+which	O
+is	O
+composed	O
+of	O
+the	O
+catalytic	O
+subunit	O
+Naa10p	B-protein
+and	O
+the	O
+auxiliary	O
+subunit	O
+Naa15p	B-protein
+.	O
+
+NatE	B-complex_assembly
+was	O
+found	O
+to	O
+physically	O
+interact	O
+with	O
+the	O
+NatA	B-complex_assembly
+complex	O
+without	O
+any	O
+observation	O
+of	O
+impact	O
+on	O
+NatA	B-complex_assembly
+-	O
+activity	O
+.	O
+
+Two	O
+other	O
+multimeric	O
+complexes	O
+of	O
+NATs	B-protein_type
+are	O
+NatB	B-complex_assembly
+and	O
+NatC	B-complex_assembly
+which	O
+contain	O
+the	O
+catalytic	O
+subunits	O
+Naa20	B-protein
+and	O
+Naa30	B-protein
+and	O
+the	O
+auxiliary	O
+subunits	O
+Naa25	B-protein
+and	O
+Naa35	B-protein
+/	O
+Naa38	B-protein
+,	O
+respectively	O
+.	O
+
+Furthermore	O
+,	O
+only	O
+the	O
+catalytic	O
+subunits	O
+Naa40	B-protein
+and	O
+Naa60	B-protein
+were	O
+found	O
+for	O
+NatD	B-complex_assembly
+and	O
+NatF	B-complex_assembly
+,	O
+respectively	O
+.	O
+
+Besides	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+,	O
+accumulating	O
+reports	O
+have	O
+proposed	O
+Nε	B-ptm
+-	I-ptm
+acetylation	I-ptm
+carried	O
+out	O
+by	O
+NATs	B-protein_type
+.	O
+
+There	O
+is	O
+an	O
+evolutionary	O
+increasing	O
+in	O
+the	O
+degree	O
+of	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+between	O
+yeast	B-taxonomy_domain
+and	O
+human	B-species
+which	O
+could	O
+partly	O
+be	O
+explained	O
+by	O
+the	O
+contribution	O
+of	O
+NatF	B-complex_assembly
+.	O
+As	O
+the	O
+first	O
+N	B-protein_type
+-	I-protein_type
+terminal	I-protein_type
+acetyltransferase	I-protein_type
+discovered	O
+on	O
+an	O
+organelle	O
+,	O
+NatF	B-complex_assembly
+,	O
+encoded	O
+by	O
+NAA60	B-protein
+and	O
+also	O
+named	O
+as	O
+Histone	B-protein
+acetyltransferase	I-protein
+type	I-protein
+B	I-protein
+protein	I-protein
+4	I-protein
+(	O
+HAT4	B-protein
+),	O
+Naa60	B-protein
+or	O
+N	B-protein
+-	I-protein
+acetyltransferase	I-protein
+15	I-protein
+(	O
+NAT15	B-protein
+),	O
+is	O
+the	O
+youngest	O
+member	O
+of	O
+the	O
+NAT	B-protein_type
+family	O
+.	O
+
+Unlike	O
+other	O
+NATs	B-protein_type
+that	O
+are	O
+highly	B-protein_state
+conserved	I-protein_state
+among	O
+lower	B-taxonomy_domain
+and	O
+higher	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+,	O
+NatF	B-complex_assembly
+only	O
+exists	O
+in	O
+higher	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+.	O
+
+Subsequent	O
+researches	O
+indicated	O
+that	O
+NatF	B-complex_assembly
+displays	O
+its	O
+catalytic	O
+ability	O
+with	O
+both	O
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+and	O
+lysine	B-ptm
+Nε	I-ptm
+-	I-ptm
+acetylation	I-ptm
+.	O
+
+As	O
+an	O
+N	B-protein_type
+-	I-protein_type
+terminal	I-protein_type
+acetyltransferase	I-protein_type
+,	O
+NatF	B-complex_assembly
+can	O
+specifically	O
+catalyze	O
+acetylation	B-ptm
+of	O
+the	O
+N	O
+-	O
+terminal	O
+α	O
+-	O
+amine	O
+of	O
+most	O
+transmembrane	O
+proteins	O
+and	O
+has	O
+substrate	O
+preference	O
+towards	O
+proteins	O
+with	O
+Met	B-structure_element
+-	I-structure_element
+Lys	I-structure_element
+-,	I-structure_element
+Met	B-structure_element
+-	I-structure_element
+Val	I-structure_element
+-,	I-structure_element
+Met	B-structure_element
+-	I-structure_element
+Ala	I-structure_element
+-	I-structure_element
+and	O
+Met	B-structure_element
+-	I-structure_element
+Met	I-structure_element
+-	I-structure_element
+N	O
+-	O
+termini	O
+,	O
+thus	O
+partially	O
+overlapping	O
+substrate	O
+selectivity	O
+with	O
+NatC	B-complex_assembly
+and	O
+NatE	B-complex_assembly
+.	O
+On	O
+the	O
+other	O
+hand	O
+,	O
+NatF	B-complex_assembly
+,	O
+with	O
+its	O
+lysine	B-protein_type
+acetyltransferase	I-protein_type
+activity	O
+,	O
+mediates	O
+the	O
+lysine	B-ptm
+acetylation	I-ptm
+of	O
+free	O
+histone	B-protein_type
+H4	B-protein_type
+,	O
+including	O
+H4K20	B-protein_type
+,	O
+H4K79	B-protein_type
+and	O
+H4K91	B-protein_type
+.	O
+
+Another	O
+important	O
+feature	O
+of	O
+NatF	B-complex_assembly
+is	O
+that	O
+this	O
+protein	O
+is	O
+anchored	O
+on	O
+the	O
+Golgi	O
+apparatus	O
+through	O
+its	O
+C	O
+-	O
+terminal	O
+membrane	B-structure_element
+-	I-structure_element
+integrating	I-structure_element
+region	I-structure_element
+and	O
+takes	O
+part	O
+in	O
+the	O
+maintaining	O
+of	O
+Golgi	O
+integrity	O
+.	O
+
+With	O
+its	O
+unique	O
+intracellular	O
+organellar	O
+localization	O
+and	O
+substrate	O
+selectivity	O
+,	O
+NatF	B-complex_assembly
+appears	O
+to	O
+provide	O
+more	O
+evolutionary	O
+information	O
+among	O
+the	O
+NAT	B-protein_type
+family	O
+members	O
+.	O
+
+It	O
+was	O
+recently	O
+found	O
+that	O
+NatF	B-complex_assembly
+facilitates	O
+nucleosomes	B-complex_assembly
+assembly	O
+and	O
+that	O
+NAA60	B-protein
+knockdown	O
+in	O
+MCF7	O
+-	O
+cell	O
+inhibits	O
+cell	O
+proliferation	O
+,	O
+sensitizes	O
+cells	O
+to	O
+DNA	O
+damage	O
+and	O
+induces	O
+cell	O
+apoptosis	O
+.	O
+
+In	O
+Drosophila	B-taxonomy_domain
+cells	O
+,	O
+NAA60	B-protein
+knockdown	O
+induces	O
+chromosomal	O
+segregation	O
+defects	O
+during	O
+anaphase	O
+including	O
+lagging	O
+chromosomes	O
+and	O
+chromosomal	O
+bridges	O
+.	O
+
+Much	O
+recent	O
+attention	O
+has	O
+also	O
+been	O
+focused	O
+on	O
+the	O
+requirement	O
+of	O
+NatF	B-complex_assembly
+for	O
+regulation	O
+of	O
+organellar	O
+structure	O
+.	O
+
+In	O
+HeLa	O
+cells	O
+,	O
+NAA60	B-protein
+knockdown	O
+causes	O
+Golgi	O
+apparatus	O
+fragmentation	O
+which	O
+can	O
+be	O
+rescued	O
+by	O
+overexpression	B-experimental_method
+Naa60	B-protein
+.	O
+
+The	O
+systematic	O
+investigation	O
+of	O
+publicly	O
+available	O
+microarray	O
+data	O
+showed	O
+that	O
+NATs	B-protein_type
+share	O
+distinct	O
+tissue	O
+-	O
+specific	O
+expression	O
+patterns	O
+in	O
+Drosophila	B-taxonomy_domain
+and	O
+NatF	B-complex_assembly
+shows	O
+a	O
+higher	O
+expression	O
+level	O
+in	O
+central	O
+nervous	O
+system	O
+of	O
+Drosophila	B-taxonomy_domain
+.	O
+
+In	O
+this	O
+study	O
+,	O
+we	O
+solved	B-experimental_method
+the	O
+structures	B-evidence
+of	O
+human	B-species
+Naa60	B-protein
+(	O
+NatF	B-complex_assembly
+)	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+coenzyme	B-chemical
+.	O
+
+The	O
+hNaa60	B-protein
+protein	O
+contains	O
+a	O
+unique	O
+amphipathic	B-structure_element
+α	I-structure_element
+-	I-structure_element
+helix	I-structure_element
+(	O
+α5	B-structure_element
+)	O
+following	O
+its	O
+GNAT	B-structure_element
+domain	I-structure_element
+that	O
+might	O
+account	O
+for	O
+the	O
+Golgi	O
+localization	O
+of	O
+this	O
+protein	O
+.	O
+
+Crystal	B-evidence
+structures	I-evidence
+showed	O
+that	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+rotated	O
+about	O
+50	O
+degrees	O
+upon	O
+removing	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+the	O
+protein	O
+and	O
+this	O
+movement	O
+substantially	O
+changed	O
+the	O
+geometry	O
+of	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+.	O
+
+Remarkably	O
+,	O
+we	O
+find	O
+that	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+may	O
+participate	O
+in	O
+the	O
+proper	O
+positioning	O
+of	O
+the	O
+coenzyme	B-chemical
+for	O
+the	O
+transfer	O
+reaction	O
+to	O
+occur	O
+.	O
+
+Further	O
+structure	B-experimental_method
+comparison	I-experimental_method
+and	O
+biochemical	B-experimental_method
+studies	I-experimental_method
+also	O
+identified	O
+other	O
+key	O
+structural	O
+elements	O
+essential	O
+for	O
+the	O
+enzyme	O
+activity	O
+of	O
+Naa60	B-protein
+.	O
+
+Overall	O
+structure	B-evidence
+of	O
+hNaa60	B-protein
+
+In	O
+the	O
+effort	O
+to	O
+prepare	O
+the	O
+protein	O
+for	O
+structural	O
+studies	O
+,	O
+we	O
+tried	O
+a	O
+large	O
+number	O
+of	O
+hNaa60	B-protein
+constructs	O
+but	O
+all	O
+failed	O
+due	O
+to	O
+heavy	O
+precipitation	O
+or	O
+aggregation	O
+.	O
+
+Sequence	B-experimental_method
+alignment	I-experimental_method
+of	O
+Naa60	B-protein
+from	O
+different	O
+species	O
+revealed	O
+a	O
+Glu	B-structure_element
+-	I-structure_element
+Glu	I-structure_element
+-	I-structure_element
+Arg	I-structure_element
+(	O
+EER	B-structure_element
+)	O
+versus	O
+Val	B-structure_element
+-	I-structure_element
+Val	I-structure_element
+-	I-structure_element
+Pro	I-structure_element
+(	O
+VVP	B-structure_element
+)	O
+sequence	O
+difference	O
+near	O
+the	O
+N	O
+-	O
+terminus	O
+of	O
+the	O
+protein	O
+in	O
+Xenopus	B-species
+Laevis	I-species
+versus	O
+Homo	B-species
+sapiens	I-species
+(	O
+Fig	O
+.	O
+1A	O
+).	O
+
+Considering	O
+that	O
+terminal	O
+residues	O
+may	O
+lack	O
+higher	O
+-	O
+order	O
+structure	O
+and	O
+hydrophobic	O
+residues	O
+in	O
+this	O
+region	O
+may	O
+expose	O
+to	O
+solvent	O
+and	O
+hence	O
+cause	O
+protein	O
+aggregation	O
+,	O
+we	O
+mutated	B-experimental_method
+residues	O
+4	B-residue_range
+–	I-residue_range
+6	I-residue_range
+from	O
+VVP	B-mutant
+to	I-mutant
+EER	I-mutant
+for	O
+the	O
+purpose	O
+of	O
+improving	O
+solubility	O
+of	O
+this	O
+protein	O
+.	O
+
+According	O
+to	O
+previous	O
+studies	O
+,	O
+this	O
+N	O
+-	O
+terminal	O
+region	O
+should	O
+not	O
+interfere	O
+with	O
+hNaa60	B-protein
+’	O
+s	O
+Golgi	O
+localization	O
+.	O
+
+We	O
+tried	O
+many	O
+hNaa60	B-protein
+constructs	O
+with	O
+the	O
+three	O
+-	O
+residues	O
+mutation	B-experimental_method
+but	O
+only	O
+the	O
+truncated	B-protein_state
+variant	O
+1	B-residue_range
+-	I-residue_range
+199	I-residue_range
+and	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+protein	O
+behaved	O
+well	O
+.	O
+
+We	O
+obtained	O
+the	O
+crystal	B-evidence
+of	O
+the	O
+truncated	B-protein_state
+variant	O
+1	B-residue_range
+-	I-residue_range
+199	I-residue_range
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+CoA	B-chemical
+first	O
+,	O
+and	O
+after	O
+extensive	O
+trials	O
+we	O
+got	O
+the	O
+crystal	B-evidence
+of	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+protein	O
+(	O
+spanning	O
+residues	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+(	O
+Fig	O
+.	O
+1B	O
+,	O
+C	O
+).	O
+
+Hereafter	O
+,	O
+all	O
+deletions	O
+or	O
+point	O
+mutants	B-protein_state
+of	O
+hNaa60	B-protein
+we	O
+describe	O
+here	O
+are	O
+with	O
+the	O
+EER	B-structure_element
+mutation	B-experimental_method
+.	O
+
+The	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+and	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+were	O
+determined	O
+by	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+and	O
+refined	O
+to	O
+1	O
+.	O
+38	O
+Å	O
+and	O
+1	O
+.	O
+60	O
+Å	O
+resolution	O
+,	O
+respectively	O
+(	O
+Table	O
+1	O
+).	O
+
+The	O
+electron	B-evidence
+density	I-evidence
+maps	I-evidence
+were	O
+of	O
+sufficient	O
+quality	O
+to	O
+trace	O
+residues	O
+1	B-residue_range
+-	I-residue_range
+211	I-residue_range
+of	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+and	O
+residues	O
+5	B-residue_range
+-	I-residue_range
+199	I-residue_range
+of	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+).	I-mutant
+
+The	O
+structure	B-evidence
+of	O
+hNaa60	B-protein
+protein	O
+contains	O
+a	O
+central	B-structure_element
+domain	I-structure_element
+exhibiting	O
+a	O
+classic	O
+GCN5	B-protein_type
+-	I-protein_type
+related	I-protein_type
+N	I-protein_type
+-	I-protein_type
+acetyltransferase	I-protein_type
+(	O
+GNAT	B-protein_type
+)	O
+folding	O
+,	O
+along	O
+with	O
+the	O
+extended	B-protein_state
+N	B-structure_element
+-	I-structure_element
+and	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+regions	I-structure_element
+(	O
+Fig	O
+.	O
+1B	O
+,	O
+C	O
+).	O
+
+The	O
+central	B-structure_element
+domain	I-structure_element
+includes	O
+nine	O
+β	B-structure_element
+strands	I-structure_element
+(	O
+β1	B-structure_element
+-	I-structure_element
+β9	I-structure_element
+)	O
+and	O
+four	O
+α	B-structure_element
+-	I-structure_element
+helixes	I-structure_element
+(	O
+α1	B-structure_element
+-	I-structure_element
+α4	I-structure_element
+)	O
+and	O
+is	O
+highly	B-protein_state
+similar	I-protein_state
+to	O
+the	O
+known	O
+hNaa50p	B-protein
+and	O
+other	O
+reported	O
+NATs	B-protein_type
+(	O
+Fig	O
+.	O
+1D	O
+).	O
+
+However	O
+,	O
+in	O
+hNaa60	B-protein
+,	O
+there	O
+is	O
+an	O
+extra	B-structure_element
+20	I-structure_element
+-	I-structure_element
+residue	I-structure_element
+loop	I-structure_element
+between	O
+β3	B-structure_element
+and	O
+β4	B-structure_element
+that	O
+forms	O
+a	O
+small	B-structure_element
+subdomain	I-structure_element
+with	O
+well	O
+-	O
+defined	O
+3D	O
+structure	O
+(	O
+Fig	O
+.	O
+1B	O
+–	O
+D	O
+).	O
+
+Furthermore	O
+,	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+strands	I-structure_element
+form	O
+an	O
+approximately	B-structure_element
+antiparallel	I-structure_element
+β	I-structure_element
+-	I-structure_element
+hairpin	I-structure_element
+structure	I-structure_element
+remarkably	O
+different	O
+from	O
+that	O
+in	O
+hNaa50p	B-protein
+(	O
+Fig	O
+.	O
+1D	O
+).	O
+
+The	O
+N	B-structure_element
+-	I-structure_element
+and	I-structure_element
+C	I-structure_element
+-	I-structure_element
+terminal	I-structure_element
+regions	I-structure_element
+form	O
+helical	B-structure_element
+structures	I-structure_element
+(	O
+α0	B-structure_element
+and	O
+α5	B-structure_element
+)	O
+stretching	O
+out	O
+from	O
+the	O
+central	O
+GCN5	B-structure_element
+-	I-structure_element
+domain	I-structure_element
+(	O
+Fig	O
+.	O
+1C	O
+).	O
+
+Interestingly	O
+,	O
+we	O
+found	O
+that	O
+the	O
+catalytic	O
+activity	O
+of	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+is	O
+much	O
+lower	O
+than	O
+that	O
+of	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+(	O
+Figure	O
+S1	O
+),	O
+indicating	O
+that	O
+residues	O
+200	B-residue_range
+–	I-residue_range
+242	I-residue_range
+may	O
+have	O
+some	O
+auto	O
+-	O
+inhibitory	O
+effect	O
+on	O
+the	O
+activity	O
+of	O
+the	O
+enzyme	O
+.	O
+
+However	O
+,	O
+since	O
+this	O
+region	O
+was	O
+not	O
+visible	O
+in	O
+the	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+crystal	B-evidence
+structure	I-evidence
+,	O
+we	O
+do	O
+not	O
+yet	O
+understand	O
+how	O
+this	O
+happens	O
+.	O
+
+Another	O
+possibility	O
+is	O
+that	O
+since	O
+hNaa60	B-protein
+is	O
+localized	O
+on	O
+Golgi	O
+apparatus	O
+,	O
+the	O
+observed	O
+low	O
+activity	O
+of	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+hNaa60	B-protein
+might	O
+be	O
+related	O
+to	O
+lack	O
+of	O
+Golgi	O
+localization	O
+of	O
+the	O
+enzyme	O
+in	O
+our	O
+in	O
+vitro	O
+studies	O
+.	O
+
+For	O
+the	O
+convenience	O
+of	O
+studying	O
+the	O
+kinetics	O
+of	O
+mutants	B-protein_state
+,	O
+the	O
+mutagenesis	B-experimental_method
+studies	I-experimental_method
+described	O
+hereafter	O
+were	O
+all	O
+based	O
+on	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+).	I-mutant
+
+An	O
+amphipathic	B-structure_element
+α	I-structure_element
+-	I-structure_element
+helix	I-structure_element
+in	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+may	O
+contribute	O
+to	O
+Golgi	O
+localization	O
+of	O
+hNaa60	B-protein
+
+There	O
+is	O
+one	O
+hNaa60	B-protein
+molecule	O
+in	O
+the	O
+asymmetric	O
+unit	O
+in	O
+the	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+structure	B-evidence
+.	O
+
+The	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+extended	O
+from	O
+the	O
+GCN5	B-structure_element
+-	I-structure_element
+domain	I-structure_element
+forms	O
+an	O
+amphipathic	B-structure_element
+helix	I-structure_element
+(	O
+α5	B-structure_element
+)	O
+and	O
+interacts	O
+with	O
+a	O
+molecule	O
+in	O
+a	O
+neighbor	O
+asymmetric	O
+unit	O
+through	O
+hydrophobic	B-bond_interaction
+interactions	I-bond_interaction
+between	O
+α5	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+and	O
+a	O
+hydrophobic	B-site
+groove	I-site
+between	O
+the	O
+N	O
+-	O
+terminal	O
+β1	B-structure_element
+and	O
+β3	B-structure_element
+strands	I-structure_element
+of	O
+the	O
+neighbor	O
+molecule	O
+(	O
+Fig	O
+.	O
+2A	O
+).	O
+
+The	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+extension	I-structure_element
+following	O
+α5	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+forms	O
+a	O
+β	B-structure_element
+-	I-structure_element
+turn	I-structure_element
+that	O
+wraps	O
+around	O
+and	O
+interacts	O
+with	O
+the	O
+neighbor	O
+protein	O
+molecule	O
+through	O
+hydrophobic	B-bond_interaction
+interactions	I-bond_interaction
+,	O
+too	O
+.	O
+
+In	O
+the	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+structure	B-evidence
+,	O
+a	O
+part	O
+of	O
+the	O
+α5	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+is	O
+deleted	O
+due	O
+to	O
+truncation	O
+of	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+(	O
+Fig	O
+.	O
+1B	O
+).	O
+
+Interestingly	O
+,	O
+the	O
+remaining	O
+residues	O
+in	O
+α5	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+still	O
+form	O
+an	O
+amphipathic	B-structure_element
+helix	I-structure_element
+although	O
+the	O
+hydrophobic	B-bond_interaction
+interaction	I-bond_interaction
+with	O
+the	O
+N	O
+-	O
+terminal	O
+hydrophobic	B-site
+groove	I-site
+of	O
+a	O
+neighbor	O
+molecule	O
+is	O
+abolished	O
+and	O
+the	O
+helix	B-structure_element
+is	O
+largely	O
+exposed	O
+in	O
+solvent	O
+due	O
+to	O
+different	O
+crystal	B-evidence
+packing	I-evidence
+(	O
+Fig	O
+.	O
+2B	O
+).	O
+
+A	O
+recent	O
+research	O
+showed	O
+that	O
+residues	O
+182	B-residue_range
+–	I-residue_range
+216	I-residue_range
+are	O
+important	O
+for	O
+the	O
+localization	O
+of	O
+hNaa60	B-protein
+on	O
+Golgi	O
+.	O
+According	O
+to	O
+our	O
+structure	B-evidence
+,	O
+the	O
+solvent	B-protein_state
+-	I-protein_state
+exposed	I-protein_state
+amphipathic	B-structure_element
+helix	I-structure_element
+(	O
+α5	B-structure_element
+)	O
+formed	O
+by	O
+residues	O
+190	B-residue_range
+-	I-residue_range
+202	I-residue_range
+with	O
+an	O
+array	O
+of	O
+hydrophobic	O
+residues	O
+located	O
+on	O
+one	O
+side	O
+(	O
+Ile	B-residue_name_number
+190	I-residue_name_number
+,	O
+Leu	B-residue_name_number
+191	I-residue_name_number
+,	O
+Ile	B-residue_name_number
+194	I-residue_name_number
+,	O
+Leu	B-residue_name_number
+197	I-residue_name_number
+and	O
+Leu	B-residue_name_number
+201	I-residue_name_number
+)	O
+and	O
+hydrophilic	O
+residues	O
+on	O
+the	O
+other	O
+side	O
+(	O
+Fig	O
+.	O
+S2	O
+)	O
+might	O
+account	O
+for	O
+interaction	O
+between	O
+hNaa60	B-protein
+and	O
+Golgi	O
+membrane	O
+,	O
+as	O
+it	O
+is	O
+a	O
+typical	O
+structure	O
+accounting	O
+for	O
+membrane	O
+association	O
+through	O
+immersing	O
+into	O
+the	O
+lipid	O
+bi	O
+-	O
+layer	O
+with	O
+its	O
+hydrophobic	O
+side	O
+as	O
+was	O
+observed	O
+with	O
+KalSec14	B-protein
+,	O
+Atg3	B-protein
+,	O
+PB1	B-protein
+-	I-protein
+F2	I-protein
+etc	O
+.	O
+
+The	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+showed	O
+alternative	O
+conformations	O
+in	O
+the	O
+hNaa60	B-protein
+crystal	B-evidence
+structures	I-evidence
+
+Superposition	B-experimental_method
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+,	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+and	O
+hNaa50	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+(	O
+PDB	O
+3TFY	O
+)	O
+revealed	O
+considerable	O
+difference	O
+in	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+region	O
+despite	O
+the	O
+overall	O
+stability	O
+and	O
+similarity	O
+of	O
+the	O
+GNAT	B-structure_element
+domain	I-structure_element
+(	O
+Fig	O
+.	O
+1D	O
+).	O
+
+In	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+),	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+is	O
+located	O
+in	O
+close	O
+proximity	O
+to	O
+the	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+,	O
+creating	O
+a	O
+more	O
+compact	O
+substrate	B-site
+binding	I-site
+site	I-site
+than	O
+that	O
+in	O
+hNaa50	B-protein
+,	O
+where	O
+this	O
+region	O
+adopts	O
+a	O
+more	O
+flexible	B-protein_state
+loop	B-structure_element
+conformation	O
+(	O
+β6	B-structure_element
+-	I-structure_element
+β7	I-structure_element
+loop	I-structure_element
+).	O
+
+Upon	O
+removing	B-experimental_method
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+hNaa60	B-protein
+,	O
+we	O
+observed	O
+that	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+molecules	O
+pack	O
+in	O
+a	O
+different	O
+way	O
+involving	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+in	O
+the	O
+crystal	B-evidence
+,	O
+leading	O
+to	O
+about	O
+50	O
+degree	O
+rotation	O
+of	O
+the	O
+hairpin	B-structure_element
+which	O
+moves	O
+away	O
+from	O
+the	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+(	O
+Figs	O
+1D	O
+and	O
+2C	O
+).	O
+
+This	O
+conformational	O
+change	O
+substantially	O
+altered	O
+the	O
+geometry	O
+of	O
+the	O
+substrate	B-site
+binding	I-site
+site	I-site
+,	O
+which	O
+could	O
+potentially	O
+change	O
+the	O
+way	O
+in	O
+which	O
+the	O
+substrate	O
+accesses	O
+the	O
+active	B-site
+site	I-site
+of	O
+the	O
+enzyme	O
+.	O
+
+In	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+),	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+covers	O
+the	O
+active	B-site
+site	I-site
+in	O
+a	O
+way	O
+similar	O
+to	O
+that	O
+observed	O
+in	O
+hNaa50	B-protein
+,	O
+presumably	O
+leaving	O
+only	O
+one	O
+way	O
+for	O
+the	O
+substrate	O
+to	O
+access	O
+the	O
+active	B-site
+site	I-site
+,	O
+i	O
+.	O
+e	O
+.	O
+to	O
+enter	O
+from	O
+the	O
+opposite	O
+end	O
+into	O
+the	O
+same	O
+tunnel	B-site
+where	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+/	O
+CoA	B-chemical
+binds	O
+(	O
+Fig	O
+.	O
+2D	O
+),	O
+which	O
+may	O
+accommodate	O
+access	O
+of	O
+a	O
+NAT	B-protein_type
+substrate	O
+only	O
+.	O
+
+KAT	B-protein_type
+activity	O
+of	O
+hNaa60	B-protein
+toward	O
+histone	B-protein_type
+H4	B-protein_type
+has	O
+been	O
+noted	O
+in	O
+previous	O
+study	O
+,	O
+and	O
+our	O
+enzyme	B-evidence
+kinetic	I-evidence
+data	I-evidence
+also	O
+indicated	O
+that	O
+hNaa60	B-protein
+can	O
+acetylate	O
+H3	B-complex_assembly
+-	I-complex_assembly
+H4	I-complex_assembly
+tetramer	B-oligomeric_state
+in	O
+vitro	O
+(	O
+Figure	O
+S3	O
+).	O
+
+Furthermore	O
+,	O
+we	O
+analyzed	O
+the	O
+acetylation	B-ptm
+status	O
+of	O
+histone	B-protein_type
+H3	B-complex_assembly
+-	I-complex_assembly
+H4	I-complex_assembly
+tetramer	B-oligomeric_state
+using	O
+mass	B-experimental_method
+spectrometry	I-experimental_method
+and	O
+observed	O
+that	O
+multiple	O
+lysine	B-residue_name
+residues	O
+in	O
+the	O
+protein	O
+showed	O
+significantly	O
+increased	O
+acetylation	B-ptm
+level	O
+and	O
+changed	O
+acetylation	B-ptm
+profile	O
+upon	O
+treatment	O
+with	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+(	O
+Figure	O
+S4	O
+).	O
+
+We	O
+also	O
+conducted	O
+liquid	B-experimental_method
+chromatography	I-experimental_method
+-	I-experimental_method
+tandem	I-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+(	O
+LC	B-experimental_method
+/	I-experimental_method
+MS	I-experimental_method
+/	I-experimental_method
+MS	I-experimental_method
+)	O
+analysis	O
+on	O
+a	O
+synthetic	O
+peptide	B-chemical
+(	O
+NH2	B-chemical
+-	I-chemical
+MKGKEEKEGGAR	I-chemical
+-	I-chemical
+COOH	I-chemical
+)	O
+after	O
+treatment	O
+with	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+),	I-mutant
+and	O
+the	O
+data	O
+confirmed	O
+that	O
+both	O
+the	O
+N	O
+-	O
+terminal	O
+α	O
+-	O
+amine	O
+and	O
+lysine	B-residue_name
+side	O
+-	O
+chain	O
+ε	O
+-	O
+amine	O
+were	O
+robustly	O
+acetylated	B-protein_state
+after	O
+the	O
+treatment	O
+(	O
+Table	O
+S1	O
+).	O
+
+Recent	O
+structural	B-experimental_method
+investigation	I-experimental_method
+of	O
+other	O
+NATs	B-protein_type
+proposed	O
+that	O
+the	O
+β6	B-structure_element
+-	I-structure_element
+β7	I-structure_element
+loop	I-structure_element
+,	O
+corresponding	O
+to	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+in	O
+hNaa60	B-protein
+,	O
+and	O
+the	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+flanking	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+site	I-site
+of	O
+NATs	B-protein_type
+,	O
+prevent	O
+the	O
+lysine	B-residue_name
+side	O
+-	O
+chain	O
+of	O
+the	O
+KAT	B-protein_type
+substrates	O
+from	O
+inserting	O
+into	O
+the	O
+active	B-site
+site	I-site
+.	O
+
+Indeed	O
+,	O
+superposition	B-experimental_method
+of	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+structure	B-evidence
+on	O
+that	O
+of	O
+Hat1p	B-protein
+,	O
+a	O
+typical	O
+KAT	B-protein_type
+,	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+a	O
+histone	B-protein_type
+H4	B-protein_type
+peptide	B-chemical
+revealed	O
+obvious	O
+overlapping	O
+/	O
+clashing	O
+of	O
+the	O
+H4	B-protein_type
+peptide	B-chemical
+(	O
+a	O
+KAT	B-protein_type
+substrate	O
+)	O
+with	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+of	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+(	O
+Fig	O
+.	O
+2D	O
+).	O
+
+Interestingly	O
+,	O
+in	O
+the	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+crystal	B-evidence
+structure	I-evidence
+,	O
+the	O
+displaced	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+opened	O
+a	O
+second	O
+way	O
+for	O
+the	O
+substrate	O
+to	O
+access	O
+the	O
+active	B-site
+center	I-site
+that	O
+would	O
+readily	O
+accommodate	O
+the	O
+binding	O
+of	O
+the	O
+H4	B-protein_type
+peptide	B-chemical
+(	O
+Fig	O
+.	O
+2E	O
+),	O
+thus	O
+implied	O
+a	O
+potential	O
+explanation	O
+for	O
+KAT	B-protein_type
+activity	O
+of	O
+this	O
+enzyme	O
+from	O
+a	O
+structural	O
+biological	O
+view	O
+.	O
+
+However	O
+,	O
+since	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+and	O
+hNaa60	B-protein
+(	O
+1	O
+-	O
+199	O
+)	O
+were	O
+crystallized	B-experimental_method
+in	O
+different	O
+crystal	B-evidence
+forms	I-evidence
+,	O
+the	O
+observed	O
+conformational	O
+change	O
+of	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+may	O
+simply	O
+be	O
+an	O
+artifact	O
+related	O
+to	O
+the	O
+different	O
+crystal	B-evidence
+packing	I-evidence
+.	O
+
+Whether	O
+the	O
+KAT	B-protein_type
+substrates	O
+bind	O
+to	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+displaced	O
+conformation	O
+of	O
+the	O
+enzyme	O
+needs	O
+to	O
+be	O
+verified	O
+by	O
+further	O
+structural	B-experimental_method
+and	I-experimental_method
+functional	I-experimental_method
+studies	I-experimental_method
+.	O
+
+Phe	B-residue_name_number
+34	I-residue_name_number
+facilitates	O
+proper	O
+positioning	O
+of	O
+the	O
+cofactor	O
+for	O
+acetyl	B-chemical
+-	O
+transfer	O
+
+The	O
+electron	B-evidence
+density	I-evidence
+of	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+side	O
+-	O
+chain	O
+is	O
+well	O
+defined	O
+in	O
+the	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+structure	B-evidence
+,	O
+but	O
+becomes	O
+invisible	O
+in	O
+the	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+structure	B-evidence
+,	O
+indicating	O
+displacement	O
+of	O
+the	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+side	O
+-	O
+chain	O
+in	O
+the	O
+latter	O
+(	O
+Fig	O
+.	O
+3A	O
+,	O
+B	O
+).	O
+
+A	O
+solvent	O
+-	O
+derived	O
+malonate	B-chemical
+molecule	O
+is	O
+found	O
+beside	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+and	O
+the	O
+ethanethioate	B-chemical
+moiety	O
+of	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+in	O
+the	O
+high	O
+-	O
+resolution	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+structure	B-evidence
+(	O
+Fig	O
+.	O
+3A	O
+).	O
+
+Superposition	B-experimental_method
+of	O
+this	O
+structure	B-evidence
+on	O
+that	O
+of	O
+hNaa50p	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+shows	O
+that	O
+the	O
+malonate	B-chemical
+molecule	O
+overlaps	O
+well	O
+on	O
+the	O
+N	O
+-	O
+terminal	O
+methionine	B-residue_name
+of	O
+the	O
+substrate	O
+peptide	B-chemical
+and	O
+residue	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+in	O
+hNaa60	B-protein
+overlaps	O
+well	O
+on	O
+Phe	B-residue_name_number
+27	I-residue_name_number
+in	O
+hNaa50	B-protein
+(	O
+Fig	O
+.	O
+4A	O
+).	O
+
+Interestingly	O
+,	O
+in	O
+the	O
+structure	B-evidence
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+,	O
+the	O
+terminal	O
+thiol	O
+of	O
+CoA	B-chemical
+adopts	O
+alternative	O
+conformations	O
+.	O
+
+One	O
+is	O
+to	O
+approach	O
+the	O
+substrate	O
+amine	B-chemical
+(	O
+as	O
+indicated	O
+by	O
+the	O
+superimposed	B-experimental_method
+hNaa50	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+structure	B-evidence
+),	O
+similar	O
+to	O
+the	O
+terminal	O
+ethanethioate	B-chemical
+of	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+in	O
+the	O
+structure	B-evidence
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+;	O
+the	O
+other	O
+is	O
+to	O
+approach	O
+the	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+and	O
+away	O
+from	O
+the	O
+substrate	O
+amine	O
+(	O
+Fig	O
+.	O
+3B	O
+).	O
+
+To	O
+rule	O
+out	O
+the	O
+possibility	O
+that	O
+the	O
+electron	B-evidence
+density	I-evidence
+we	O
+define	O
+as	O
+the	O
+alternative	O
+conformation	O
+of	O
+the	O
+thiol	O
+terminus	O
+is	O
+residual	O
+electron	B-evidence
+density	I-evidence
+of	O
+the	O
+displaced	O
+side	O
+-	O
+chain	O
+of	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+,	O
+we	O
+solved	B-experimental_method
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)	I-complex_assembly
+F34A	I-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+.	O
+The	O
+structure	B-evidence
+of	O
+this	O
+mutant	B-protein_state
+is	O
+highly	O
+similar	O
+to	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+and	O
+there	O
+is	O
+essentially	O
+the	O
+same	O
+electron	B-evidence
+density	I-evidence
+corresponding	O
+to	O
+the	O
+alternative	O
+conformation	O
+of	O
+the	O
+thiol	O
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+
+Phe	B-residue_name_number
+27	I-residue_name_number
+in	O
+hNaa50p	B-protein
+(	O
+equivalent	O
+to	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+in	O
+hNaa60	B-protein
+)	O
+has	O
+been	O
+implicated	O
+to	O
+facilitate	O
+the	O
+binding	O
+of	O
+N	O
+-	O
+terminal	O
+methionine	B-residue_name
+of	O
+the	O
+substrate	O
+peptide	B-chemical
+through	O
+hydrophobic	B-bond_interaction
+interaction	I-bond_interaction
+.	O
+
+However	O
+,	O
+in	O
+the	O
+hNaa60	B-complex_assembly
+/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+structure	B-evidence
+,	O
+a	O
+hydrophilic	O
+malonate	B-chemical
+molecule	O
+is	O
+found	O
+at	O
+the	O
+same	O
+location	O
+where	O
+the	O
+N	O
+-	O
+terminal	O
+methionine	B-residue_name
+should	O
+bind	O
+as	O
+is	O
+indicated	O
+by	O
+the	O
+superposition	B-experimental_method
+(	O
+Fig	O
+.	O
+3A	O
+),	O
+suggesting	O
+that	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+may	O
+accommodate	O
+binding	O
+of	O
+hydrophilic	O
+substrate	O
+,	O
+too	O
+.	O
+
+Moreover	O
+,	O
+orientation	O
+of	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+side	O
+-	O
+chain	O
+seems	O
+to	O
+be	O
+co	O
+-	O
+related	O
+to	O
+positioning	O
+of	O
+the	O
+terminus	O
+of	O
+the	O
+co	O
+-	O
+enzyme	O
+and	O
+important	O
+for	O
+placing	O
+it	O
+at	O
+a	O
+location	O
+in	O
+close	O
+proximity	O
+to	O
+the	O
+substrate	O
+amine	O
+.	O
+
+We	O
+hypothesize	O
+that	O
+if	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+only	O
+works	O
+to	O
+facilitate	O
+the	O
+binding	O
+of	O
+the	O
+hydrophobic	O
+N	O
+-	O
+terminal	O
+Met	B-residue_name
+residue	O
+,	O
+to	O
+mutate	B-experimental_method
+it	O
+from	O
+Phe	B-residue_name
+to	O
+Ala	B-residue_name
+would	O
+not	O
+abolish	O
+the	O
+catalytic	O
+activity	O
+of	O
+this	O
+enzyme	O
+,	O
+while	O
+if	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+also	O
+plays	O
+an	O
+essential	O
+role	O
+to	O
+position	O
+the	O
+ethanethioate	B-chemical
+moiety	O
+of	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+,	O
+the	O
+mutation	B-experimental_method
+would	O
+be	O
+expected	O
+to	O
+abrogate	O
+the	O
+activity	O
+of	O
+the	O
+enzyme	O
+.	O
+
+Indeed	O
+,	O
+our	O
+enzyme	B-evidence
+kinetic	I-evidence
+data	I-evidence
+showed	O
+that	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+F34A	B-mutant
+mutant	B-protein_state
+showed	O
+no	O
+detectable	O
+activity	O
+(	O
+Fig	O
+.	O
+5A	O
+).	O
+
+In	O
+order	O
+to	O
+rule	O
+out	O
+the	O
+possibility	O
+that	O
+the	O
+observed	O
+loss	O
+of	O
+activity	O
+may	O
+be	O
+related	O
+to	O
+bad	O
+folding	O
+of	O
+the	O
+mutant	B-protein_state
+protein	O
+,	O
+we	O
+studied	O
+the	O
+circular	B-experimental_method
+dichroism	I-experimental_method
+(	O
+CD	B-experimental_method
+)	O
+spectrum	B-evidence
+of	O
+the	O
+protein	O
+(	O
+Fig	O
+.	O
+5B	O
+)	O
+and	O
+determined	O
+its	O
+crystal	B-evidence
+structure	I-evidence
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+
+Both	O
+studies	O
+proved	O
+that	O
+the	O
+F34A	B-mutant
+mutant	B-protein_state
+protein	O
+is	O
+well	B-protein_state
+-	I-protein_state
+folded	I-protein_state
+.	O
+
+Many	O
+studies	O
+have	O
+addressed	O
+the	O
+crucial	O
+effect	O
+of	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+on	O
+catalysis	O
+,	O
+showing	O
+that	O
+some	O
+residues	O
+located	O
+in	O
+this	O
+area	O
+are	O
+involved	O
+in	O
+the	O
+binding	O
+of	O
+substrates	O
+.	O
+
+We	O
+propose	O
+that	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+may	O
+play	O
+a	O
+dual	O
+role	O
+both	O
+in	O
+interacting	O
+with	O
+the	O
+peptide	B-chemical
+substrate	O
+(	O
+recognition	O
+)	O
+and	O
+in	O
+positioning	O
+of	O
+the	O
+ethanethioate	B-chemical
+moiety	O
+of	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+to	O
+the	O
+right	O
+location	O
+to	O
+facilitate	O
+acetyl	B-chemical
+-	O
+transfer	O
+.	O
+
+Structural	O
+basis	O
+for	O
+hNaa60	B-protein
+substrate	O
+binding	O
+
+Several	O
+studies	O
+have	O
+demonstrated	O
+that	O
+the	O
+substrate	O
+specificities	O
+of	O
+hNaa60	B-protein
+and	O
+hNaa50	B-protein
+are	O
+highly	O
+overlapped	O
+.	O
+
+The	O
+structure	B-evidence
+of	O
+hNaa50p	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+provides	O
+detailed	O
+information	O
+about	O
+the	O
+position	O
+of	O
+substrate	O
+N	O
+-	O
+terminal	O
+residues	O
+in	O
+the	O
+active	B-site
+site	I-site
+of	O
+hNaa50	B-protein
+.	O
+
+Comparing	O
+the	O
+active	B-site
+site	I-site
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+with	O
+hNaa50p	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+revealed	O
+that	O
+key	O
+catalytic	B-site
+and	I-site
+substrate	I-site
+binding	I-site
+residues	I-site
+are	O
+highly	B-protein_state
+conserved	I-protein_state
+in	O
+both	O
+proteins	O
+(	O
+Fig	O
+.	O
+4A	O
+).	O
+
+With	O
+respect	O
+to	O
+catalysis	O
+,	O
+hNaa50p	B-protein
+has	O
+been	O
+shown	O
+to	O
+employ	O
+residues	O
+Tyr	B-residue_name_number
+73	I-residue_name_number
+and	O
+His	B-residue_name_number
+112	I-residue_name_number
+to	O
+abstract	O
+proton	O
+from	O
+the	O
+α	O
+-	O
+amino	O
+group	O
+from	O
+the	O
+substrate	O
+’	O
+s	O
+first	O
+residue	O
+through	O
+a	O
+well	B-protein_state
+-	I-protein_state
+ordered	I-protein_state
+water	B-chemical
+.	O
+
+A	O
+well	B-protein_state
+-	I-protein_state
+ordered	I-protein_state
+water	B-chemical
+was	O
+also	O
+found	O
+between	O
+Tyr	B-residue_name_number
+97	I-residue_name_number
+and	O
+His	B-residue_name_number
+138	I-residue_name_number
+in	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+and	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+(	O
+Fig	O
+.	O
+4B	O
+).	O
+
+To	O
+determine	O
+the	O
+function	O
+of	O
+Tyr	B-residue_name_number
+97	I-residue_name_number
+and	O
+His	B-residue_name_number
+138	I-residue_name_number
+in	O
+hNaa60	B-protein
+catalysis	O
+,	O
+we	O
+mutated	B-experimental_method
+these	O
+residues	O
+to	O
+alanine	B-residue_name
+and	O
+phenylalanine	B-residue_name
+,	O
+respectively	O
+,	O
+and	O
+confirmed	O
+that	O
+all	O
+these	O
+mutants	B-protein_state
+used	O
+in	O
+our	O
+kinetic	B-experimental_method
+assays	I-experimental_method
+are	O
+well	B-protein_state
+-	I-protein_state
+folded	I-protein_state
+by	O
+CD	B-experimental_method
+spectra	B-evidence
+(	O
+Fig	O
+.	O
+5B	O
+).	O
+
+Purity	O
+of	O
+all	O
+proteins	O
+were	O
+also	O
+analyzed	O
+by	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+(	O
+Figure	O
+S5	O
+).	O
+
+As	O
+show	O
+in	O
+Fig	O
+.	O
+5A	O
+,	O
+the	O
+mutants	B-protein_state
+Y97A	B-mutant
+,	O
+Y97F	B-mutant
+,	O
+H138A	B-mutant
+and	O
+H138F	B-mutant
+abolished	B-protein_state
+the	I-protein_state
+activity	I-protein_state
+of	O
+hNaa60	B-protein
+.	O
+
+In	O
+contrast	O
+,	O
+to	O
+mutate	B-experimental_method
+the	O
+nearby	O
+solvent	B-protein_state
+exposed	I-protein_state
+residue	O
+Glu	B-residue_name_number
+37	I-residue_name_number
+to	O
+Ala	B-residue_name
+(	O
+E37A	B-mutant
+)	O
+has	O
+little	O
+impact	O
+on	O
+the	O
+activity	O
+of	O
+hNaa60	B-protein
+(	O
+Figs	O
+4B	O
+and	O
+5A	O
+).	O
+
+In	O
+conclusion	O
+,	O
+the	O
+structural	B-experimental_method
+and	I-experimental_method
+functional	I-experimental_method
+studies	I-experimental_method
+indicate	O
+that	O
+hNaa60	B-protein
+applies	O
+the	O
+same	O
+two	O
+base	O
+mechanism	O
+through	O
+Tyr	B-residue_name_number
+97	I-residue_name_number
+,	O
+His	B-residue_name_number
+138	I-residue_name_number
+and	O
+a	O
+well	B-protein_state
+-	I-protein_state
+ordered	I-protein_state
+water	B-chemical
+as	O
+was	O
+described	O
+for	O
+hNaa50	B-protein
+.	O
+
+The	O
+malonate	B-chemical
+molecule	O
+observed	O
+in	O
+the	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+crystal	B-evidence
+structure	I-evidence
+may	O
+be	O
+indicative	O
+of	O
+the	O
+substrate	O
+binding	O
+position	O
+of	O
+hNaa60	B-protein
+since	O
+it	O
+is	O
+located	O
+in	O
+the	O
+active	B-site
+site	I-site
+and	O
+overlaps	O
+the	O
+N	O
+-	O
+terminal	O
+Met	B-residue_name
+of	O
+the	O
+substrate	O
+peptide	B-chemical
+in	O
+the	O
+superposition	B-experimental_method
+with	O
+the	O
+hNaa50p	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+structure	B-evidence
+(	O
+Fig	O
+.	O
+4A	O
+).	O
+
+Residues	O
+Tyr	B-residue_name_number
+38	I-residue_name_number
+,	O
+Asn	B-residue_name_number
+143	I-residue_name_number
+and	O
+Tyr	B-residue_name_number
+165	I-residue_name_number
+are	O
+located	O
+around	O
+the	O
+malonate	B-chemical
+and	O
+interact	O
+with	O
+it	O
+through	O
+direct	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+or	O
+water	B-bond_interaction
+bridge	I-bond_interaction
+(	O
+Fig	O
+.	O
+4C	O
+).	O
+
+Although	O
+malonate	B-chemical
+is	O
+negatively	O
+charged	O
+,	O
+which	O
+is	O
+different	O
+from	O
+that	O
+of	O
+lysine	B-residue_name
+ε	O
+-	O
+amine	O
+or	O
+peptide	B-chemical
+N	O
+-	O
+terminal	O
+amine	O
+,	O
+similar	O
+hydrophilic	B-bond_interaction
+interactions	I-bond_interaction
+may	O
+take	O
+place	O
+when	O
+substrate	O
+amine	O
+presents	O
+in	O
+the	O
+same	O
+position	O
+,	O
+since	O
+Tyr	B-residue_name_number
+38	I-residue_name_number
+,	O
+Asn	B-residue_name_number
+143	I-residue_name_number
+and	O
+Tyr	B-residue_name_number
+165	I-residue_name_number
+are	O
+not	O
+positively	O
+or	O
+negatively	O
+charged	O
+.	O
+
+In	O
+agreement	O
+with	O
+this	O
+hypothesis	O
+,	O
+it	O
+was	O
+found	O
+that	O
+the	O
+Y38A	B-mutant
+,	O
+N143A	B-mutant
+and	O
+Y165A	B-mutant
+mutants	B-protein_state
+all	O
+showed	O
+remarkably	O
+reduced	O
+activities	O
+as	O
+compared	O
+to	O
+WT	B-protein_state
+,	O
+implying	O
+that	O
+these	O
+residues	O
+may	O
+be	O
+critical	O
+for	O
+substrate	O
+binding	O
+(	O
+Figs	O
+4C	O
+and	O
+5A	O
+).	O
+
+The	O
+β3	B-structure_element
+-	I-structure_element
+β4	I-structure_element
+loop	I-structure_element
+participates	O
+in	O
+the	O
+regulation	O
+of	O
+hNaa60	B-protein
+-	O
+activity	O
+
+Residues	O
+between	O
+β3	B-structure_element
+and	O
+β4	B-structure_element
+of	O
+hNaa60	B-protein
+form	O
+a	O
+unique	O
+20	B-structure_element
+-	I-structure_element
+residue	I-structure_element
+long	I-structure_element
+loop	I-structure_element
+(	O
+residues	O
+73	B-residue_range
+–	I-residue_range
+92	I-residue_range
+)	O
+that	O
+is	O
+a	O
+short	B-structure_element
+turn	I-structure_element
+in	O
+many	O
+other	O
+NAT	B-protein_type
+members	O
+(	O
+Fig	O
+.	O
+1D	O
+).	O
+
+Previous	O
+study	O
+indicated	O
+that	O
+auto	B-ptm
+-	I-ptm
+acetylation	I-ptm
+of	O
+hNaa60K79	B-protein
+could	O
+influence	O
+the	O
+activity	O
+of	O
+hNaa60	B-protein
+;	O
+however	O
+,	O
+we	O
+were	O
+not	O
+able	O
+to	O
+determine	O
+if	O
+Lys	B-residue_name_number
+79	I-residue_name_number
+is	O
+acetylated	B-protein_state
+in	O
+our	O
+crystal	B-evidence
+structures	I-evidence
+due	O
+to	O
+poor	O
+quality	O
+of	O
+the	O
+electron	B-evidence
+density	I-evidence
+of	O
+Lys	B-residue_name_number
+79	I-residue_name_number
+side	O
+-	O
+chain	O
+.	O
+
+We	O
+therefore	O
+used	O
+mass	B-experimental_method
+spectrometry	I-experimental_method
+to	O
+analyze	O
+if	O
+Lys	B-residue_name_number
+79	I-residue_name_number
+was	O
+acetylated	B-protein_state
+in	O
+our	O
+bacterially	O
+purified	O
+proteins	O
+,	O
+and	O
+observed	O
+no	O
+modification	O
+on	O
+this	O
+residue	O
+(	O
+Figure	O
+S6	O
+).	O
+
+To	O
+assess	O
+the	O
+impact	O
+of	O
+hNaa60K79	B-protein
+auto	B-ptm
+-	I-ptm
+acetylation	I-ptm
+,	O
+we	O
+studied	O
+the	O
+kinetics	O
+of	O
+K79R	B-mutant
+and	O
+K79Q	B-mutant
+mutants	B-protein_state
+which	O
+mimic	O
+the	O
+un	B-protein_state
+-	I-protein_state
+acetylated	I-protein_state
+and	O
+acetylated	B-protein_state
+form	O
+of	O
+Lys	B-residue_name_number
+79	I-residue_name_number
+,	O
+respectively	O
+.	O
+
+Interestingly	O
+,	O
+both	O
+K79R	B-mutant
+and	O
+K79Q	B-mutant
+mutants	B-protein_state
+led	O
+to	O
+an	O
+increase	O
+in	O
+the	O
+catalytic	O
+activity	O
+of	O
+hNaa60	B-protein
+,	O
+while	O
+K79A	B-mutant
+mutant	B-protein_state
+led	O
+to	O
+modest	O
+decrease	O
+of	O
+the	O
+activity	O
+(	O
+Fig	O
+.	O
+5A	O
+).	O
+
+These	O
+data	O
+indicate	O
+that	O
+the	O
+acetylation	B-ptm
+of	O
+Lys	B-residue_name_number
+79	I-residue_name_number
+is	O
+not	O
+required	O
+for	O
+optimal	O
+catalytic	O
+activity	O
+of	O
+hNaa60	B-protein
+in	O
+vitro	O
+.	O
+
+It	O
+is	O
+noted	O
+that	O
+the	O
+β3	B-structure_element
+-	I-structure_element
+β4	I-structure_element
+loop	I-structure_element
+of	O
+hNaa60	B-protein
+acts	O
+like	O
+a	O
+door	O
+leaf	O
+to	O
+partly	O
+cover	O
+the	O
+substrate	B-site
+-	I-site
+binding	I-site
+pathway	I-site
+.	O
+
+We	O
+hence	O
+hypothesize	O
+that	O
+the	O
+β3	B-structure_element
+-	I-structure_element
+β4	I-structure_element
+loop	I-structure_element
+may	O
+interfere	O
+with	O
+the	O
+access	O
+of	O
+the	O
+peptide	B-chemical
+substrates	O
+and	O
+that	O
+the	O
+solvent	B-protein_state
+-	I-protein_state
+exposing	I-protein_state
+Lys	B-residue_name_number
+79	I-residue_name_number
+may	O
+play	O
+a	O
+potential	O
+role	O
+to	O
+remove	O
+the	O
+door	O
+leaf	O
+when	O
+it	O
+hovers	O
+in	O
+solvent	O
+(	O
+Fig	O
+.	O
+4D	O
+).	O
+
+Acidic	O
+residues	O
+Glu	B-residue_name_number
+80	I-residue_name_number
+,	O
+Asp	B-residue_name_number
+81	I-residue_name_number
+and	O
+Asp	B-residue_name_number
+83	I-residue_name_number
+interact	O
+with	O
+His	B-residue_name_number
+138	I-residue_name_number
+,	O
+His	B-residue_name_number
+159	I-residue_name_number
+and	O
+His	B-residue_name_number
+158	I-residue_name_number
+to	O
+maintain	O
+the	O
+conformation	O
+of	O
+the	O
+β3	B-structure_element
+-	I-structure_element
+β4	I-structure_element
+loop	I-structure_element
+,	O
+thus	O
+contribute	O
+to	O
+control	O
+the	O
+substrate	O
+binding	O
+(	O
+Fig	O
+.	O
+4D	O
+).	O
+
+To	O
+verify	O
+this	O
+hypothesis	O
+,	O
+we	O
+mutated	B-experimental_method
+Glu	B-residue_name_number
+80	I-residue_name_number
+,	O
+Asp	B-residue_name_number
+81	I-residue_name_number
+and	O
+Asp	B-residue_name_number
+83	I-residue_name_number
+to	O
+Ala	B-residue_name
+respectively	O
+.	O
+
+In	O
+line	O
+with	O
+our	O
+hypothesis	O
+,	O
+E80A	B-mutant
+,	O
+D81A	B-mutant
+and	O
+D83A	B-mutant
+mutants	B-protein_state
+exhibit	O
+at	O
+least	O
+2	O
+-	O
+fold	O
+increase	O
+in	O
+hNaa60	B-protein
+-	O
+activity	O
+(	O
+Fig	O
+.	O
+5A	O
+).	O
+
+Interestingly	O
+,	O
+the	O
+structure	B-evidence
+of	O
+an	O
+ancestral	O
+NAT	B-protein_type
+from	O
+S	B-species
+.	I-species
+solfataricus	I-species
+also	O
+exhibits	O
+a	O
+10	B-structure_element
+-	I-structure_element
+residue	I-structure_element
+long	I-structure_element
+extension	I-structure_element
+between	O
+β3	B-structure_element
+and	O
+β4	B-structure_element
+,	O
+and	O
+the	O
+structure	B-experimental_method
+and	I-experimental_method
+biochemical	I-experimental_method
+studies	I-experimental_method
+showed	O
+that	O
+the	O
+extension	B-structure_element
+of	O
+SsNat	B-protein
+has	O
+the	O
+ability	O
+to	O
+stabilize	O
+structure	O
+of	O
+the	O
+active	B-site
+site	I-site
+and	O
+potentiate	O
+SsNat	B-protein
+-	O
+activity	O
+.	O
+
+Nt	B-ptm
+-	I-ptm
+acetylation	I-ptm
+,	O
+which	O
+is	O
+carried	O
+out	O
+by	O
+the	O
+NAT	B-protein_type
+family	I-protein_type
+acetyltransferases	I-protein_type
+,	O
+is	O
+an	O
+ancient	O
+and	O
+essential	O
+modification	O
+of	O
+proteins	O
+.	O
+
+Although	O
+many	O
+NATs	B-protein_type
+are	O
+highly	B-protein_state
+conserved	I-protein_state
+from	O
+lower	B-taxonomy_domain
+to	O
+higher	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+and	O
+the	O
+substrate	O
+bias	O
+of	O
+them	O
+appears	O
+to	O
+be	O
+partially	O
+overlapped	O
+,	O
+there	O
+is	O
+a	O
+significant	O
+increase	O
+in	O
+the	O
+overall	O
+level	O
+of	O
+N	B-ptm
+-	I-ptm
+terminal	I-ptm
+acetylation	I-ptm
+from	O
+lower	B-taxonomy_domain
+to	O
+higher	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+.	O
+
+In	O
+this	O
+study	O
+we	O
+provide	O
+structural	O
+insights	O
+into	O
+Naa60	B-protein
+found	O
+only	O
+in	O
+multicellular	B-taxonomy_domain
+eukaryotes	I-taxonomy_domain
+.	O
+
+The	O
+N	O
+-	O
+terminus	O
+of	O
+hNaa60	B-protein
+harbors	O
+three	O
+hydrophobic	O
+residues	O
+(	O
+VVP	B-structure_element
+)	O
+that	O
+makes	O
+it	O
+very	O
+difficult	O
+to	O
+express	O
+and	O
+purify	O
+the	O
+protein	O
+.	O
+
+This	O
+problem	O
+was	O
+solved	O
+by	O
+replacing	B-experimental_method
+residues	O
+4	B-residue_range
+–	I-residue_range
+6	I-residue_range
+from	O
+VVP	B-structure_element
+to	O
+EER	B-structure_element
+that	O
+are	O
+found	O
+in	O
+Naa60	B-protein
+from	O
+Xenopus	B-species
+Laevis	I-species
+.	O
+
+Since	O
+Naa60	B-protein
+from	O
+human	B-species
+and	O
+from	O
+Xenopus	B-species
+Laevis	I-species
+are	O
+highly	B-protein_state
+homologous	I-protein_state
+(	O
+Fig	O
+.	O
+1A	O
+),	O
+we	O
+speculate	O
+that	O
+these	O
+two	O
+proteins	O
+should	O
+have	O
+the	O
+same	O
+biological	O
+function	O
+.	O
+
+Therefore	O
+it	O
+is	O
+deduced	O
+that	O
+the	O
+VVP	B-mutant
+to	I-mutant
+EER	I-mutant
+replacement	B-experimental_method
+on	O
+the	O
+N	O
+-	O
+terminus	O
+of	O
+hNaa60	B-protein
+may	O
+not	O
+interfere	O
+with	O
+its	O
+function	O
+.	O
+
+However	O
+,	O
+in	O
+the	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+structure	B-evidence
+the	O
+N	O
+-	O
+terminus	O
+adopts	O
+an	O
+α	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+structure	I-structure_element
+which	O
+will	O
+probably	O
+be	O
+kinked	O
+if	O
+residue	O
+6	B-residue_number
+is	O
+proline	B-residue_name
+(	O
+Fig	O
+.	O
+1C	O
+),	O
+and	O
+in	O
+the	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+structure	B-evidence
+the	O
+N	O
+-	O
+terminus	O
+adopts	O
+a	O
+different	O
+semi	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+structure	I-structure_element
+(	O
+Fig	O
+.	O
+1B	O
+)	O
+likely	O
+due	O
+to	O
+different	O
+crystal	B-evidence
+packing	I-evidence
+.	O
+
+Hence	O
+it	O
+is	O
+not	O
+clear	O
+if	O
+the	O
+N	O
+-	O
+terminal	O
+end	O
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+hNaa60	B-protein
+is	O
+an	O
+α	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+,	O
+and	O
+what	O
+roles	O
+the	O
+hydrophobic	O
+residues	O
+4	B-residue_range
+–	I-residue_range
+6	I-residue_range
+play	O
+in	O
+structure	O
+and	O
+function	O
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+hNaa60	B-protein
+.	O
+
+In	O
+addition	O
+to	O
+the	O
+three	O
+-	O
+residue	O
+mutation	B-experimental_method
+(	O
+VVP	B-structure_element
+to	O
+EER	B-structure_element
+),	O
+we	O
+also	O
+tried	O
+many	O
+other	O
+hNaa60	B-protein
+constructs	O
+,	O
+but	O
+only	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+protein	O
+and	O
+the	O
+truncated	B-protein_state
+variant	O
+1	B-residue_range
+-	I-residue_range
+199	I-residue_range
+behaved	O
+well	O
+.	O
+
+The	O
+finding	O
+that	O
+the	O
+catalytic	O
+activity	O
+of	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+is	O
+much	O
+lower	O
+than	O
+that	O
+of	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+is	O
+intriguing	O
+.	O
+
+We	O
+speculate	O
+that	O
+low	O
+activity	O
+of	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+hNaa60	B-protein
+might	O
+be	O
+related	O
+to	O
+lack	O
+of	O
+Golgi	O
+localization	O
+of	O
+the	O
+enzyme	O
+in	O
+our	O
+in	O
+vitro	O
+studies	O
+or	O
+there	O
+remains	O
+some	O
+undiscovered	O
+auto	O
+-	O
+inhibitory	O
+regulation	O
+in	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+protein	O
+.	O
+
+The	O
+hNaa60	B-protein
+protein	O
+was	O
+proven	O
+to	O
+be	O
+localized	O
+on	O
+Golgi	O
+apparatus	O
+.	O
+
+Aksnes	O
+and	O
+colleagues	O
+predicted	O
+putative	O
+transmembrane	B-structure_element
+domains	I-structure_element
+and	O
+two	O
+putative	O
+sites	O
+of	O
+S	B-ptm
+-	I-ptm
+palmitoylation	I-ptm
+,	O
+by	O
+bioinformatics	O
+means	O
+,	O
+to	O
+account	O
+for	O
+Golgi	O
+localization	O
+of	O
+the	O
+protein	O
+.	O
+
+They	O
+then	O
+mutated	B-experimental_method
+all	O
+five	O
+cysteine	B-residue_name
+residues	O
+of	O
+hNaa60	B-protein
+’	O
+s	O
+to	O
+serine	B-residue_name
+,	O
+including	O
+the	O
+two	O
+putative	O
+S	B-site
+-	I-site
+palmitoylation	I-site
+sites	I-site
+.	O
+
+However	O
+,	O
+these	O
+mutations	B-experimental_method
+did	O
+not	O
+abolish	O
+Naa60	B-protein
+membrane	O
+localization	O
+,	O
+indicating	O
+that	O
+S	B-ptm
+-	I-ptm
+palmitoylation	I-ptm
+is	O
+unlikely	O
+to	O
+(	O
+solely	O
+)	O
+account	O
+for	O
+targeting	O
+hNaa60	B-protein
+on	O
+Golgi	O
+.	O
+
+Furthermore	O
+,	O
+adding	B-experimental_method
+residues	O
+217	B-residue_range
+–	I-residue_range
+242	I-residue_range
+of	O
+hNaa60	B-protein
+(	O
+containing	O
+residues	O
+217	B-residue_range
+–	I-residue_range
+236	I-residue_range
+,	O
+one	O
+of	O
+the	O
+putative	O
+transmembrane	B-structure_element
+domains	I-structure_element
+)	O
+to	O
+the	O
+C	O
+terminus	O
+of	O
+eGFP	B-experimental_method
+were	O
+not	O
+sufficient	O
+to	O
+localize	O
+the	O
+protein	O
+on	O
+Golgi	O
+apparatus	O
+,	O
+while	O
+eGFP	B-experimental_method
+-	O
+hNaa60182	B-mutant
+-	I-mutant
+242	I-mutant
+was	O
+sufficient	O
+to	O
+,	O
+suggesting	O
+that	O
+residues	O
+182	B-residue_range
+–	I-residue_range
+216	I-residue_range
+are	O
+important	O
+for	O
+Golgi	O
+localization	O
+of	O
+hNaa60	B-protein
+.	O
+
+We	O
+found	O
+that	O
+residues	O
+190	B-residue_range
+–	I-residue_range
+202	I-residue_range
+formed	O
+an	O
+amphipathic	B-structure_element
+helix	I-structure_element
+with	O
+an	O
+array	O
+of	O
+hydrophobic	O
+residues	O
+located	O
+on	O
+one	O
+side	O
+.	O
+
+This	O
+observation	O
+is	O
+reminiscent	O
+of	O
+the	O
+protein	O
+/	O
+membrane	O
+interaction	O
+through	O
+amphipathic	B-structure_element
+helices	I-structure_element
+in	O
+the	O
+cases	O
+of	O
+KalSec14	B-protein
+,	O
+Atg3	B-protein
+,	O
+PB1	B-protein
+-	I-protein
+F2	I-protein
+etc	O
+.	O
+
+In	O
+this	O
+model	O
+an	O
+amphipathic	B-structure_element
+helix	I-structure_element
+can	O
+immerse	O
+its	O
+hydrophobic	O
+side	O
+into	O
+the	O
+lipid	O
+bilayer	O
+through	O
+hydrophobic	B-bond_interaction
+interactions	I-bond_interaction
+.	O
+
+Therefore	O
+we	O
+propose	O
+that	O
+the	O
+amphipathic	B-structure_element
+helix	I-structure_element
+α5	B-structure_element
+may	O
+contribute	O
+to	O
+Golgi	O
+localization	O
+of	O
+hNaa60	B-protein
+.	O
+
+Previous	O
+studies	O
+indicated	O
+that	O
+members	O
+of	O
+NAT	B-protein_type
+family	O
+are	O
+bi	O
+-	O
+functional	O
+NAT	B-protein_type
+and	O
+KAT	B-protein_type
+enzymes	O
+.	O
+
+However	O
+,	O
+known	O
+structures	B-evidence
+of	O
+NATs	B-protein_type
+do	O
+not	O
+well	O
+support	O
+this	O
+hypothesis	O
+,	O
+since	O
+the	O
+β6	B-structure_element
+-	I-structure_element
+β7	I-structure_element
+hairpin	I-structure_element
+/	O
+loop	B-structure_element
+of	O
+most	O
+of	O
+NATs	B-protein_type
+is	O
+involved	O
+in	O
+the	O
+formation	O
+of	O
+a	O
+tunnel	B-site
+-	I-site
+like	I-site
+substrate	I-site
+-	I-site
+binding	I-site
+site	I-site
+with	O
+the	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+,	O
+which	O
+would	O
+be	O
+good	O
+for	O
+the	O
+NAT	B-protein_type
+but	O
+not	O
+KAT	B-protein_type
+activity	O
+of	O
+the	O
+enzyme	O
+.	O
+
+Kinetic	B-experimental_method
+studies	I-experimental_method
+have	O
+been	O
+conducted	O
+to	O
+compare	O
+the	O
+NAT	B-protein_type
+and	O
+KAT	B-protein_type
+activity	O
+of	O
+hNaa50	B-protein
+in	O
+vitro	O
+,	O
+and	O
+indicate	O
+that	O
+the	O
+NAT	B-protein_type
+activity	O
+of	O
+Naa50	B-protein
+is	O
+much	O
+higher	O
+than	O
+KAT	B-protein_type
+activity	O
+.	O
+
+However	O
+,	O
+the	O
+substrate	O
+used	O
+in	O
+this	O
+study	O
+for	O
+assessing	O
+KAT	B-protein_type
+activity	O
+was	O
+a	O
+small	O
+peptide	B-chemical
+which	O
+could	O
+not	O
+really	O
+mimic	O
+the	O
+3D	B-evidence
+structure	I-evidence
+of	O
+a	O
+folded	B-protein_state
+protein	O
+substrate	O
+in	O
+vivo	O
+.	O
+
+Our	O
+mass	B-experimental_method
+spectrometry	I-experimental_method
+data	B-evidence
+indicated	O
+that	O
+there	O
+were	O
+robust	O
+acetylation	B-ptm
+of	O
+histone	B-protein_type
+H3	B-complex_assembly
+-	I-complex_assembly
+H4	I-complex_assembly
+tetramer	B-oligomeric_state
+lysines	B-residue_name
+and	O
+both	O
+N	B-ptm
+-	I-ptm
+terminal	I-ptm
+acetylation	I-ptm
+and	O
+lysine	B-ptm
+acetylation	I-ptm
+of	O
+the	O
+peptide	B-chemical
+used	O
+in	O
+the	O
+activity	B-experimental_method
+assay	I-experimental_method
+,	O
+thus	O
+confirmed	O
+the	O
+KAT	B-protein_type
+activity	O
+of	O
+this	O
+enzyme	O
+in	O
+vitro	O
+.	O
+
+Conformational	O
+change	O
+of	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+(	O
+corresponding	O
+to	O
+the	O
+β6	B-structure_element
+-	I-structure_element
+β7	I-structure_element
+loop	I-structure_element
+of	O
+other	O
+NATs	B-protein_type
+)	O
+is	O
+noted	O
+in	O
+our	O
+structures	B-evidence
+(	O
+Figs	O
+1D	O
+and	O
+2C	O
+),	O
+which	O
+might	O
+provide	O
+an	O
+explanation	O
+to	O
+the	O
+NAT	B-protein_type
+/	O
+KAT	B-protein_type
+dual	O
+-	O
+activity	O
+in	O
+a	O
+structural	O
+biological	O
+view	O
+,	O
+but	O
+we	O
+were	O
+unable	O
+to	O
+rule	O
+out	O
+the	O
+possibility	O
+that	O
+the	O
+observed	O
+conformational	O
+change	O
+of	O
+this	O
+hairpin	B-structure_element
+might	O
+be	O
+an	O
+artifact	O
+related	O
+to	O
+crystal	B-evidence
+packing	I-evidence
+or	O
+truncation	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+end	O
+of	O
+the	O
+protein	O
+.	O
+
+Further	O
+studies	O
+are	O
+therefore	O
+needed	O
+to	O
+reveal	O
+the	O
+mechanism	O
+for	O
+the	O
+KAT	B-protein_type
+activity	O
+of	O
+this	O
+enzyme	O
+.	O
+
+In	O
+early	O
+years	O
+,	O
+researchers	O
+found	O
+adjustment	O
+of	O
+GCN5	B-protein_type
+histone	I-protein_type
+acetyltransferase	I-protein_type
+structure	B-evidence
+when	O
+it	O
+binds	O
+CoA	B-chemical
+molecule	O
+.	O
+
+The	O
+complexed	B-protein_state
+form	O
+of	O
+NatA	B-complex_assembly
+is	O
+more	O
+suitable	O
+for	O
+catalytic	O
+activation	O
+,	O
+since	O
+the	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+undergoes	O
+a	O
+conformation	O
+change	O
+to	O
+participate	O
+in	O
+the	O
+formation	O
+of	O
+substrate	B-site
+-	I-site
+binding	I-site
+site	I-site
+when	O
+the	O
+auxiliary	O
+subunit	O
+Naa15	B-protein
+interacts	O
+with	O
+Naa10	B-protein
+(	O
+the	O
+catalytic	B-protein_state
+subunit	B-structure_element
+of	O
+NatA	B-complex_assembly
+).	O
+
+In	O
+the	O
+structure	B-evidence
+of	O
+hNaa50	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+,	O
+Phe	B-residue_name_number
+27	I-residue_name_number
+in	O
+the	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+appears	O
+to	O
+make	O
+hydrophobic	B-bond_interaction
+interaction	I-bond_interaction
+with	O
+the	O
+N	O
+-	O
+terminal	O
+Met	B-residue_name
+of	O
+substrate	O
+peptide	B-chemical
+.	O
+
+However	O
+,	O
+the	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+crystal	B-evidence
+structure	I-evidence
+indicated	O
+that	O
+its	O
+counterpart	O
+in	O
+hNaa60	B-protein
+,	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+,	O
+could	O
+also	O
+accommodate	O
+the	O
+binding	O
+of	O
+a	O
+hydrophilic	O
+malonate	B-chemical
+that	O
+occupied	O
+the	O
+substrate	B-site
+binding	I-site
+site	I-site
+although	O
+it	O
+maintained	O
+the	O
+same	O
+conformation	O
+as	O
+that	O
+observed	O
+in	O
+hNaa50	B-protein
+.	O
+
+Interestingly	O
+,	O
+the	O
+terminal	O
+thiol	B-chemical
+of	O
+CoA	B-chemical
+adopted	O
+alternative	O
+conformations	O
+in	O
+the	O
+structure	B-evidence
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+.	O
+One	O
+was	O
+to	O
+approach	O
+the	O
+substrate	O
+amine	O
+;	O
+the	O
+other	O
+was	O
+to	O
+approach	O
+the	O
+α1	B-structure_element
+-	I-structure_element
+α2	I-structure_element
+loop	I-structure_element
+and	O
+away	O
+from	O
+the	O
+substrate	O
+amine	O
+.	O
+
+Same	O
+alternative	O
+conformations	O
+of	O
+CoA	B-chemical
+were	O
+observed	O
+in	O
+the	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)(	I-mutant
+F34A	I-mutant
+)	I-mutant
+crystal	B-evidence
+structure	I-evidence
+,	O
+and	O
+our	O
+kinetic	B-evidence
+data	I-evidence
+showed	O
+that	O
+the	O
+F34A	B-mutant
+mutation	B-experimental_method
+abolished	O
+the	O
+activity	O
+of	O
+the	O
+enzyme	O
+.	O
+
+Taken	O
+together	O
+,	O
+our	O
+data	O
+indicated	O
+that	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+in	O
+hNaa60	B-protein
+may	O
+play	O
+a	O
+role	O
+in	O
+placing	O
+co	O
+-	O
+enzyme	O
+at	O
+the	O
+right	O
+location	O
+to	O
+facilitate	O
+the	O
+acetyl	B-chemical
+-	O
+transfer	O
+.	O
+
+However	O
+,	O
+these	O
+data	O
+did	O
+not	O
+rule	O
+out	O
+that	O
+possibility	O
+that	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+may	O
+coordinate	O
+the	O
+binding	O
+of	O
+the	O
+N	O
+-	O
+terminal	O
+Met	B-residue_name
+through	O
+hydrophobic	B-bond_interaction
+interaction	I-bond_interaction
+as	O
+was	O
+proposed	O
+by	O
+previous	O
+studies	O
+.	O
+
+Furthermore	O
+,	O
+we	O
+showed	O
+that	O
+hNaa60	B-protein
+adopts	O
+the	O
+classical	O
+two	O
+base	O
+mechanism	O
+to	O
+catalyze	O
+acetyl	B-chemical
+-	O
+transfer	O
+.	O
+
+Although	O
+sequence	O
+identity	O
+between	O
+hNaa60	B-protein
+and	O
+hNaa50	B-protein
+is	O
+low	O
+,	O
+key	O
+residues	O
+in	O
+the	O
+active	B-site
+site	I-site
+of	O
+both	O
+enzymes	O
+are	O
+highly	B-protein_state
+conserved	I-protein_state
+.	O
+
+This	O
+can	O
+reasonably	O
+explain	O
+the	O
+high	O
+overlapping	O
+substrates	O
+specificities	O
+between	O
+hNaa60	B-protein
+and	O
+hNaa50	B-protein
+.	O
+
+Another	O
+structural	O
+feature	O
+of	O
+hNaa60	B-protein
+that	O
+distinguishes	O
+it	O
+from	O
+other	O
+NATs	B-protein_type
+is	O
+the	O
+β3	B-structure_element
+-	I-structure_element
+β4	I-structure_element
+long	I-structure_element
+loop	I-structure_element
+which	O
+appears	O
+to	O
+inhibit	O
+the	O
+catalytic	O
+activity	O
+of	O
+hNaa60	B-protein
+.	O
+
+However	O
+,	O
+this	O
+loop	B-structure_element
+also	O
+seems	O
+to	O
+stabilize	O
+the	O
+whole	O
+hNaa60	B-protein
+structure	B-evidence
+,	O
+because	O
+deletion	B-experimental_method
+mutations	I-experimental_method
+of	O
+this	O
+region	O
+led	O
+to	O
+protein	O
+precipitation	O
+and	O
+aggregation	O
+(	O
+Figure	O
+S7	O
+).	O
+
+A	O
+previous	O
+study	O
+suggested	O
+that	O
+the	O
+auto	B-ptm
+-	I-ptm
+acetylation	I-ptm
+of	O
+Lys	B-residue_name_number
+79	I-residue_name_number
+was	O
+important	O
+for	O
+hNaa60	B-protein
+-	O
+activity	O
+,	O
+whereas	O
+the	O
+point	B-experimental_method
+mutation	I-experimental_method
+K79R	B-mutant
+did	O
+not	O
+decrease	O
+the	O
+activity	O
+of	O
+hNaa60	B-protein
+in	O
+our	O
+study	O
+.	O
+
+Meanwhile	O
+,	O
+no	O
+electron	B-evidence
+density	I-evidence
+of	O
+acetyl	B-chemical
+group	O
+was	O
+found	O
+on	O
+Lys	B-residue_name_number
+79	I-residue_name_number
+in	O
+our	O
+structures	B-evidence
+and	O
+mass	B-experimental_method
+spectrometry	I-experimental_method
+analysis	O
+.	O
+
+Hence	O
+,	O
+it	O
+appears	O
+that	O
+the	O
+auto	B-ptm
+-	I-ptm
+acetylation	I-ptm
+of	O
+hNaa60	B-protein
+is	O
+not	O
+an	O
+essential	O
+modification	O
+for	O
+its	O
+activity	O
+for	O
+the	O
+protein	O
+we	O
+used	O
+here	O
+.	O
+
+As	O
+for	O
+the	O
+reason	O
+why	O
+K79R	B-mutant
+in	O
+Yang	O
+’	O
+s	O
+previous	O
+studies	O
+reduced	O
+the	O
+activity	O
+of	O
+the	O
+enzyme	O
+,	O
+but	O
+in	O
+our	O
+studies	O
+it	O
+didn	O
+’	O
+t	O
+,	O
+we	O
+suspect	O
+that	O
+the	O
+stability	O
+of	O
+this	O
+mutant	B-protein_state
+may	O
+play	O
+some	O
+role	O
+.	O
+
+K79R	B-mutant
+is	O
+less	O
+stable	B-protein_state
+than	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+enzyme	O
+as	O
+was	O
+judged	O
+by	O
+its	O
+poorer	O
+gel	B-experimental_method
+-	I-experimental_method
+filtration	I-experimental_method
+behavior	O
+and	O
+tendency	O
+to	O
+precipitate	O
+.	O
+
+In	O
+our	O
+studies	O
+we	O
+have	O
+paid	O
+special	O
+attention	O
+and	O
+carefully	O
+handled	O
+this	O
+protein	O
+to	O
+ensure	O
+that	O
+we	O
+did	O
+get	O
+enough	O
+of	O
+the	O
+protein	O
+in	O
+good	O
+condition	O
+for	O
+kinetic	B-experimental_method
+assays	I-experimental_method
+.	O
+
+The	O
+intracellular	O
+environment	O
+is	O
+more	O
+complicated	O
+than	O
+our	O
+in	O
+vitro	O
+assay	O
+and	O
+the	O
+substrate	O
+specificity	O
+of	O
+hNaa60	B-protein
+most	O
+focuses	O
+on	O
+transmembrane	O
+proteins	O
+.	O
+
+The	O
+interaction	O
+between	O
+hNaa60	B-protein
+and	O
+its	O
+substrates	O
+may	O
+involve	O
+the	O
+protein	O
+-	O
+membrane	O
+interaction	O
+which	O
+would	O
+further	O
+increase	O
+the	O
+complexity	O
+.	O
+
+It	O
+is	O
+not	O
+clear	O
+if	O
+the	O
+structure	B-evidence
+of	O
+hNaa60	B-protein
+is	O
+different	O
+in	O
+vivo	O
+or	O
+if	O
+other	O
+potential	O
+partner	O
+proteins	O
+may	O
+help	O
+to	O
+regulate	O
+its	O
+activity	O
+.	O
+
+Nevertheless	O
+,	O
+our	O
+study	O
+may	O
+be	O
+an	O
+inspiration	O
+for	O
+further	O
+studies	O
+on	O
+the	O
+functions	O
+and	O
+regulation	O
+of	O
+this	O
+youngest	O
+member	O
+of	O
+the	O
+NAT	B-protein_type
+family	O
+.	O
+
+Overall	O
+structure	B-evidence
+of	O
+Naa60	B-protein
+.	O
+
+(	O
+A	O
+)	O
+Sequence	B-experimental_method
+alignment	I-experimental_method
+of	O
+Naa60	B-protein
+(	O
+NatF	B-complex_assembly
+,	O
+HAT4	B-protein
+)	O
+from	O
+different	O
+species	O
+including	O
+Homo	B-species
+sapiens	I-species
+(	O
+Homo	B-species
+),	O
+Bos	B-species
+mutus	I-species
+(	O
+Bos	B-species
+),	O
+Salmo	B-species
+salar	I-species
+(	O
+Salmo	B-species
+)	O
+and	O
+Xenopus	B-species
+(	O
+Silurana	B-species
+)	O
+tropicalis	B-species
+(	O
+Xenopus	B-species
+).	O
+
+Alignment	B-experimental_method
+was	O
+generated	O
+using	O
+NPS	O
+@	O
+and	O
+ESPript	O
+.	O
+3	O
+.	O
+0	O
+(	O
+http	O
+://	O
+espript	O
+.	O
+ibcp	O
+.	O
+fr	O
+/	O
+ESPript	O
+/	O
+ESPript	O
+/).	O
+
+Residues	O
+4	B-residue_range
+–	I-residue_range
+6	I-residue_range
+are	O
+highlighted	O
+in	O
+red	O
+box	O
+.	O
+
+(	O
+B	O
+)	O
+The	O
+structure	B-evidence
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+complex	O
+is	O
+shown	O
+as	O
+a	O
+yellow	O
+cartoon	O
+model	O
+.	O
+
+The	O
+CoA	B-chemical
+molecule	O
+is	O
+shown	O
+as	O
+sticks	O
+.	O
+(	O
+C	O
+)	O
+The	O
+structure	B-evidence
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+complex	O
+is	O
+presented	O
+as	O
+a	O
+cartoon	O
+model	O
+in	O
+cyan	O
+.	O
+
+The	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+and	O
+malonate	B-chemical
+molecules	O
+are	O
+shown	O
+as	O
+cyan	O
+and	O
+purple	O
+sticks	O
+,	O
+respectively	O
+.	O
+
+The	O
+secondary	O
+structures	O
+are	O
+labeled	O
+starting	O
+with	O
+α0	B-structure_element
+.	O
+(	O
+D	O
+)	O
+Superposition	B-experimental_method
+of	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+(	O
+cyan	O
+),	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+(	O
+yellow	O
+)	O
+and	O
+hNaa50	B-protein
+(	O
+pink	O
+,	O
+PDB	O
+3TFY	O
+).	O
+
+The	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+of	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+complex	O
+is	O
+represented	O
+as	O
+cyan	O
+sticks	O
+.	O
+
+Amphipathicity	B-protein_state
+of	O
+the	O
+α5	B-structure_element
+helix	I-structure_element
+and	O
+alternative	O
+conformations	O
+of	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+.	O
+
+(	O
+A	O
+)	O
+The	O
+α5	B-structure_element
+helix	I-structure_element
+of	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+in	O
+one	O
+asymmetric	O
+unit	O
+(	O
+slate	O
+)	O
+interacts	O
+with	O
+another	O
+hNaa60	B-protein
+molecule	O
+in	O
+a	O
+neighboring	O
+asymmetric	O
+unit	O
+(	O
+cyan	O
+).	O
+
+Side	O
+-	O
+chains	O
+of	O
+hydrophobic	O
+residues	O
+on	O
+α5	B-structure_element
+helix	I-structure_element
+and	O
+the	O
+neighboring	O
+molecule	O
+participating	O
+in	O
+the	O
+interaction	O
+are	O
+shown	O
+as	O
+yellow	O
+and	O
+green	O
+sticks	O
+,	O
+respectively	O
+.	O
+(	O
+B	O
+)	O
+The	O
+α5	B-structure_element
+helix	I-structure_element
+of	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+in	O
+one	O
+asymmetric	O
+unit	O
+(	O
+yellow	O
+)	O
+interacts	O
+with	O
+another	O
+hNaa60	B-protein
+molecule	O
+in	O
+the	O
+neighboring	O
+asymmetric	O
+units	O
+(	O
+green	O
+).	O
+
+Side	O
+-	O
+chains	O
+of	O
+hydrophobic	O
+residues	O
+on	O
+α5	B-structure_element
+helix	I-structure_element
+and	O
+the	O
+neighboring	O
+molecule	O
+(	O
+green	O
+)	O
+participating	O
+in	O
+the	O
+interaction	O
+are	O
+shown	O
+as	O
+yellow	O
+and	O
+green	O
+sticks	O
+,	O
+respectively	O
+.	O
+
+The	O
+third	O
+molecule	O
+(	O
+pink	O
+)	O
+does	O
+not	O
+directly	O
+interact	O
+with	O
+the	O
+α5	B-structure_element
+helix	I-structure_element
+.	O
+
+(	O
+C	O
+)	O
+Superposition	B-experimental_method
+of	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+(	O
+yellow	O
+)	O
+and	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+(	O
+cyan	O
+)	O
+showing	O
+conformational	O
+change	O
+of	O
+the	O
+β7	B-structure_element
+-	I-structure_element
+β8	I-structure_element
+hairpin	I-structure_element
+in	O
+these	O
+two	O
+structures	B-evidence
+.	O
+(	O
+D	O
+,	O
+E	O
+)	O
+Superposition	B-experimental_method
+of	O
+Hat1p	B-protein
+/	O
+H4	B-protein_type
+(	O
+gray	O
+,	O
+drawn	O
+from	O
+PDB	O
+4PSW	O
+)	O
+with	O
+hNaa60	B-protein
+(	O
+1	B-residue_range
+-	I-residue_range
+242	I-residue_range
+)	O
+(	O
+cyan	O
+,	O
+D	O
+)	O
+or	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+(	O
+yellow	O
+,	O
+E	O
+).	O
+
+The	O
+histone	B-protein_type
+H4	B-protein_type
+peptide	B-chemical
+(	O
+a	O
+KAT	B-protein_type
+substrate	O
+)	O
+bound	B-protein_state
+to	I-protein_state
+Hat1p	B-protein
+is	O
+shown	O
+in	O
+purple	O
+(	O
+D	O
+,	O
+E	O
+),	O
+while	O
+the	O
+peptide	B-chemical
+bound	B-protein_state
+to	I-protein_state
+hNaa50	B-protein
+(	O
+a	O
+NAT	B-protein_type
+substrate	O
+,	O
+drawn	O
+from	O
+PDB	O
+3TFY	O
+)	O
+is	O
+shown	O
+in	O
+orange	O
+(	O
+Nt	B-chemical
+-	I-chemical
+peptide	I-chemical
+)	O
+after	O
+superimposing	B-experimental_method
+hNaa50	B-protein
+(	O
+not	O
+shown	O
+in	O
+figure	O
+)	O
+on	O
+hNaa60	B-protein
+(	O
+D	O
+).	O
+
+The	O
+α	O
+-	O
+amine	O
+of	O
+the	O
+NAT	B-protein_type
+substrate	O
+and	O
+ε	O
+-	O
+amine	O
+of	O
+the	O
+KAT	B-protein_type
+substrate	O
+(	O
+along	O
+with	O
+the	O
+lysine	B-residue_name
+side	O
+-	O
+chain	O
+)	O
+subject	O
+to	O
+acetylation	B-ptm
+are	O
+shown	O
+as	O
+sticks	O
+.	O
+
+Electron	B-evidence
+density	I-evidence
+map	I-evidence
+of	O
+the	O
+active	B-site
+site	I-site
+.	O
+
+The	O
+2Fo	B-evidence
+-	I-evidence
+Fc	I-evidence
+maps	I-evidence
+contoured	O
+at	O
+1	O
+.	O
+0σ	O
+are	O
+shown	O
+for	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+(	O
+A	O
+),	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+(	O
+B	O
+)	O
+and	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)	I-complex_assembly
+F34A	I-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+(	O
+C	O
+).	O
+
+The	O
+putative	O
+substrate	B-site
+peptide	I-site
+binding	I-site
+site	I-site
+is	O
+indicated	O
+by	O
+the	O
+peptide	B-chemical
+(	O
+shown	O
+as	O
+pink	O
+sticks	O
+)	O
+from	O
+the	O
+hNaa50	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+complex	O
+structure	B-evidence
+after	O
+superimposing	B-experimental_method
+hNaa50	B-protein
+on	O
+the	O
+hNaa60	B-protein
+structures	B-evidence
+determined	O
+in	O
+this	O
+study	O
+.	O
+
+The	O
+black	O
+arrow	O
+indicates	O
+the	O
+α	O
+-	O
+amine	O
+of	O
+the	O
+first	B-residue_name_number
+Met	I-residue_name_number
+(	O
+M1	B-residue_name_number
+)	O
+(	O
+all	O
+panels	O
+).	O
+
+The	O
+purple	O
+arrow	O
+indicates	O
+the	O
+acetyl	B-chemical
+moiety	O
+of	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+(	O
+A	O
+).	O
+
+The	O
+red	O
+arrow	O
+indicates	O
+the	O
+alternative	O
+conformation	O
+of	O
+the	O
+thiol	O
+moiety	O
+of	O
+the	O
+co	O
+-	O
+enzyme	O
+when	O
+Phe	B-residue_name_number
+34	I-residue_name_number
+side	O
+-	O
+chain	O
+is	O
+displaced	O
+(	O
+B	O
+)	O
+or	O
+mutated	B-experimental_method
+to	O
+Ala	B-residue_name
+(	O
+C	O
+).	O
+
+Structural	O
+basis	O
+for	O
+hNaa60	B-protein
+catalytic	O
+activity	O
+.	O
+
+(	O
+A	O
+)	O
+Superposition	B-experimental_method
+of	O
+hNaa60	B-protein
+active	B-site
+site	I-site
+(	O
+cyan	O
+)	O
+on	O
+that	O
+of	O
+hNaa50	B-protein
+(	O
+pink	O
+,	O
+PDB	O
+3TFY	O
+).	O
+
+Side	O
+-	O
+chains	O
+of	O
+key	O
+catalytic	B-site
+and	I-site
+substrate	I-site
+-	I-site
+binding	I-site
+residues	I-site
+are	O
+highlighted	O
+as	O
+sticks	O
+.	O
+
+The	O
+malonate	B-chemical
+molecule	O
+in	O
+the	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+structure	B-evidence
+and	O
+the	O
+peptide	B-chemical
+in	O
+the	O
+hNaa50	B-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+/	I-complex_assembly
+peptide	I-complex_assembly
+structure	B-evidence
+are	O
+shown	O
+as	O
+purple	O
+and	O
+yellow	O
+sticks	O
+respectively	O
+.	O
+(	O
+B	O
+)	O
+A	O
+close	O
+view	O
+of	O
+the	O
+active	B-site
+site	I-site
+of	O
+hNaa60	B-protein
+.	O
+
+Residues	O
+Glu	B-residue_name_number
+37	I-residue_name_number
+,	O
+Tyr	B-residue_name_number
+97	I-residue_name_number
+and	O
+His	B-residue_name_number
+138	I-residue_name_number
+in	O
+hNaa60	B-protein
+(	O
+cyan	O
+)	O
+and	O
+corresponding	O
+residues	O
+(	O
+Tyr	B-residue_name_number
+73	I-residue_name_number
+and	O
+His	B-residue_name_number
+112	I-residue_name_number
+)	O
+in	O
+hNaa50	B-protein
+(	O
+pink	O
+)	O
+as	O
+well	O
+as	O
+the	O
+side	O
+-	O
+chain	O
+of	O
+corresponding	O
+residues	O
+(	O
+Glu	B-residue_name_number
+24	I-residue_name_number
+,	O
+His	B-residue_name_number
+72	I-residue_name_number
+and	O
+His	B-residue_name_number
+111	I-residue_name_number
+)	O
+in	O
+complexed	B-protein_state
+formed	O
+hNaa10p	B-protein
+(	O
+warmpink	O
+)	O
+are	O
+highlighted	O
+as	O
+sticks	O
+.	O
+
+The	O
+water	B-chemical
+molecules	O
+participating	O
+in	O
+catalysis	O
+in	O
+the	O
+hNaa60	B-protein
+and	O
+hNaa50	B-protein
+structures	B-evidence
+are	O
+showed	O
+as	O
+green	O
+and	O
+red	O
+spheres	O
+,	O
+separately	O
+.	O
+(	O
+C	O
+)	O
+The	O
+interaction	O
+between	O
+the	O
+malonate	B-chemical
+molecule	O
+and	O
+surrounding	O
+residues	O
+observed	O
+in	O
+the	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+structure	B-evidence
+.	O
+
+The	O
+yellow	O
+dotted	O
+lines	O
+indicate	O
+the	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+.	O
+(	O
+D	O
+)	O
+A	O
+zoomed	O
+view	O
+of	O
+β3	B-structure_element
+-	I-structure_element
+β4	I-structure_element
+loop	I-structure_element
+of	O
+hNaa60	B-protein
+.	O
+
+Key	O
+residues	O
+discussed	O
+in	O
+the	O
+text	O
+(	O
+cyan	O
+),	O
+the	O
+malonate	B-chemical
+(	O
+purple	O
+)	O
+and	O
+Ac	B-chemical
+-	I-chemical
+CoA	I-chemical
+(	O
+gray	O
+)	O
+are	O
+shown	O
+as	O
+sticks	O
+.	O
+
+The	O
+yellow	O
+dotted	O
+lines	O
+indicate	O
+the	O
+salt	B-bond_interaction
+bridges	I-bond_interaction
+.	O
+
+Catalytic	O
+activity	O
+of	O
+hNaa60	B-protein
+and	O
+mutant	B-protein_state
+proteins	O
+.	O
+
+(	O
+A	O
+)	O
+Catalytic	B-evidence
+efficiency	I-evidence
+(	O
+shown	O
+as	O
+kcat	B-evidence
+/	O
+Km	B-evidence
+values	O
+)	O
+of	O
+hNaa60	B-mutant
+(	I-mutant
+1	I-mutant
+-	I-mutant
+199	I-mutant
+)	I-mutant
+WT	B-protein_state
+and	O
+mutants	B-protein_state
+.	O
+
+(	O
+B	O
+)	O
+CD	B-experimental_method
+spectra	B-evidence
+of	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+mutant	B-protein_state
+proteins	O
+from	O
+250	O
+nm	O
+to	O
+190	O
+nm	O
+.	O
+
+The	O
+sample	O
+concentration	O
+was	O
+4	O
+.	O
+5	O
+μM	O
+in	O
+20	O
+mM	O
+Tris	O
+,	O
+pH	O
+8	O
+.	O
+0	O
+,	O
+150	O
+mM	O
+NaCl	O
+,	O
+1	O
+%	O
+glycerol	O
+and	O
+1	O
+mM	O
+TCEP	B-chemical
+at	O
+room	O
+temperature	O
+.	O
+
+Data	B-evidence
+collection	I-evidence
+and	I-evidence
+refinement	I-evidence
+statistics	I-evidence
+.	O
+
+Structure	O
+and	O
+PDB	O
+ID	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+242	I-complex_assembly
+)/	I-complex_assembly
+Ac	I-complex_assembly
+-	I-complex_assembly
+CoA	I-complex_assembly
+5HGZ	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)/	I-complex_assembly
+CoA	I-complex_assembly
+5HH0	O
+hNaa60	B-complex_assembly
+(	I-complex_assembly
+1	I-complex_assembly
+-	I-complex_assembly
+199	I-complex_assembly
+)	I-complex_assembly
+F34A	I-complex_assembly
+/	I-complex_assembly
+CoA	I-complex_assembly
+5HH1	O
+Data	O
+collection	O
+*	O
+Space	O
+group	O
+P212121	O
+P21212	O
+P21212	O
+Cell	O
+dimensions	O
+a	O
+,	O
+b	O
+,	O
+c	O
+(	O
+Å	O
+)	O
+53	O
+.	O
+3	O
+,	O
+57	O
+.	O
+4	O
+,	O
+68	O
+.	O
+8	O
+67	O
+.	O
+8	O
+,	O
+73	O
+.	O
+8	O
+,	O
+43	O
+.	O
+2	O
+66	O
+.	O
+7	O
+,	O
+74	O
+.	O
+0	O
+,	O
+43	O
+.	O
+5	O
+α	O
+,	O
+β	O
+,	O
+γ	O
+(°)	O
+90	O
+.	O
+0	O
+,	O
+90	O
+.	O
+0	O
+,	O
+90	O
+.	O
+0	O
+90	O
+.	O
+0	O
+,	O
+90	O
+.	O
+0	O
+,	O
+90	O
+.	O
+0	O
+90	O
+.	O
+0	O
+,	O
+90	O
+.	O
+0	O
+,	O
+90	O
+.	O
+0	O
+Resolution	O
+(	O
+Å	O
+)	O
+50	O
+–	O
+1	O
+.	O
+38	O
+(	O
+1	O
+.	O
+42	O
+–	O
+1	O
+.	O
+38	O
+)	O
+50	O
+–	O
+1	O
+.	O
+60	O
+(	O
+1	O
+.	O
+66	O
+–	O
+1	O
+.	O
+60	O
+)	O
+50	O
+–	O
+1	O
+.	O
+80	O
+(	O
+1	O
+.	O
+86	O
+–	O
+1	O
+.	O
+80	O
+)	O
+Rp	O
+.	O
+i	O
+.	O
+m	O
+.(%)**	O
+3	O
+.	O
+0	O
+(	O
+34	O
+.	O
+4	O
+)	O
+2	O
+.	O
+1	O
+(	O
+32	O
+.	O
+5	O
+)	O
+2	O
+.	O
+6	O
+(	O
+47	O
+.	O
+8	O
+)	O
+I	O
+/	O
+σ	O
+21	O
+.	O
+5	O
+(	O
+2	O
+.	O
+0	O
+)	O
+31	O
+.	O
+8	O
+(	O
+2	O
+.	O
+0	O
+)	O
+28	O
+.	O
+0	O
+(	O
+2	O
+.	O
+4	O
+)	O
+Completeness	O
+(%)	O
+99	O
+.	O
+8	O
+(	O
+99	O
+.	O
+1	O
+)	O
+99	O
+.	O
+6	O
+(	O
+98	O
+.	O
+5	O
+)	O
+99	O
+.	O
+9	O
+(	O
+99	O
+.	O
+7	O
+)	O
+Redundancy	O
+6	O
+.	O
+9	O
+(	O
+5	O
+.	O
+0	O
+)	O
+6	O
+.	O
+9	O
+(	O
+6	O
+.	O
+2	O
+)	O
+6	O
+.	O
+3	O
+(	O
+5	O
+.	O
+9	O
+)	O
+Refinement	O
+Resolution	O
+(	O
+Å	O
+)	O
+25	O
+.	O
+81	O
+–	O
+1	O
+.	O
+38	O
+33	O
+.	O
+55	O
+–	O
+1	O
+.	O
+60	O
+43	O
+.	O
+52	O
+–	O
+1	O
+.	O
+80	O
+No	O
+.	O
+reflections	O
+43660	O
+28588	O
+20490	O
+Rwork	O
+/	O
+Rfree	O
+0	O
+.	O
+182	O
+/	O
+0	O
+.	O
+192	O
+0	O
+.	O
+181	O
+/	O
+0	O
+.	O
+184	O
+0	O
+.	O
+189	O
+/	O
+0	O
+.	O
+209	O
+No	O
+.	O
+atoms	O
+Protein	O
+1717	O
+1576	O
+1566	O
+Ligand	O
+/	O
+ion	O
+116	O
+96	O
+96	O
+Water	B-chemical
+289	O
+258	O
+168	O
+B	O
+-	O
+factors	O
+Protein	O
+23	O
+.	O
+8	O
+32	O
+.	O
+0	O
+37	O
+.	O
+4	O
+Ligand	O
+/	O
+ion	O
+22	O
+.	O
+2	O
+34	O
+.	O
+6	O
+43	O
+.	O
+7	O
+Water	B-chemical
+35	O
+.	O
+1	O
+46	O
+.	O
+4	O
+49	O
+.	O
+1	O
+R	O
+.	O
+m	O
+.	O
+s	O
+.	O
+
+One	O
+crystal	B-evidence
+was	O
+used	O
+for	O
+each	O
+data	O
+set	O
+.	O
+
+**	O
+Rp	O
+.	O
+i	O
+.	O
+m	O
+.,	O
+a	O
+redundancy	O
+-	O
+independent	O
+R	B-evidence
+factor	I-evidence
+was	O
+used	O
+to	O
+evaluate	O
+the	O
+diffraction	B-evidence
+data	I-evidence
+quality	O
+as	O
+was	O
+proposed	O
+by	O
+Evans	O
+.	O
+
diff --git a/annotation_IOB/PMC5012862.tsv b/annotation_IOB/PMC5012862.tsv
new file mode 100644
index 0000000000000000000000000000000000000000..64a84710091547b2986a54daa768c8253a7a0c89
--- /dev/null
+++ b/annotation_IOB/PMC5012862.tsv
@@ -0,0 +1,15440 @@
+Structural	B-experimental_method
+characterization	I-experimental_method
+of	O
+encapsulated	B-protein_state
+ferritin	B-protein_type
+provides	O
+insight	O
+into	O
+iron	B-chemical
+storage	O
+in	O
+bacterial	B-taxonomy_domain
+nanocompartments	B-complex_assembly
+
+Ferritins	B-protein_type
+are	O
+ubiquitous	O
+proteins	O
+that	O
+oxidise	O
+and	O
+store	O
+iron	B-chemical
+within	O
+a	O
+protein	O
+shell	B-structure_element
+to	O
+protect	O
+cells	O
+from	O
+oxidative	O
+damage	O
+.	O
+
+We	O
+have	O
+characterized	O
+the	O
+structure	B-evidence
+and	O
+function	O
+of	O
+a	O
+new	O
+member	O
+of	O
+the	O
+ferritin	B-protein_type
+superfamily	O
+that	O
+is	O
+sequestered	O
+within	O
+an	O
+encapsulin	B-protein
+capsid	O
+.	O
+
+We	O
+show	O
+that	O
+this	O
+encapsulated	B-protein_state
+ferritin	B-protein_type
+(	O
+EncFtn	B-protein
+)	O
+has	O
+two	O
+main	B-structure_element
+alpha	I-structure_element
+helices	I-structure_element
+,	O
+which	O
+assemble	O
+in	O
+a	O
+metal	B-protein_state
+dependent	I-protein_state
+manner	O
+to	O
+form	O
+a	O
+ferroxidase	B-site
+center	I-site
+at	O
+a	O
+dimer	B-site
+interface	I-site
+.	O
+
+EncFtn	B-protein
+adopts	O
+an	O
+open	B-protein_state
+decameric	B-oligomeric_state
+structure	B-evidence
+that	O
+is	O
+topologically	O
+distinct	O
+from	O
+other	O
+ferritins	B-protein_type
+.	O
+
+While	O
+EncFtn	B-protein
+acts	O
+as	O
+a	O
+ferroxidase	B-protein_type
+,	O
+it	O
+cannot	O
+mineralize	O
+iron	B-chemical
+.	O
+
+Conversely	O
+,	O
+the	O
+encapsulin	B-protein
+shell	B-structure_element
+associates	O
+with	O
+iron	B-chemical
+,	O
+but	O
+is	O
+not	B-protein_state
+enzymatically	I-protein_state
+active	I-protein_state
+,	O
+and	O
+we	O
+demonstrate	O
+that	O
+EncFtn	B-protein
+must	O
+be	O
+housed	O
+within	O
+the	O
+encapsulin	B-protein
+for	O
+iron	B-chemical
+storage	O
+.	O
+
+This	O
+encapsulin	B-protein
+nanocompartment	B-complex_assembly
+is	O
+widely	O
+distributed	O
+in	O
+bacteria	B-taxonomy_domain
+and	O
+archaea	B-taxonomy_domain
+and	O
+represents	O
+a	O
+distinct	O
+class	O
+of	O
+iron	B-chemical
+storage	O
+system	O
+,	O
+where	O
+the	O
+oxidation	O
+and	O
+mineralization	O
+of	O
+iron	B-chemical
+are	O
+distributed	O
+between	O
+two	O
+proteins	O
+.	O
+
+Iron	B-chemical
+is	O
+essential	O
+for	O
+life	O
+as	O
+it	O
+is	O
+a	O
+key	O
+component	O
+of	O
+many	O
+different	O
+enzymes	O
+that	O
+participate	O
+in	O
+processes	O
+such	O
+as	O
+energy	O
+production	O
+and	O
+metabolism	O
+.	O
+
+However	O
+,	O
+iron	B-chemical
+can	O
+also	O
+be	O
+highly	O
+toxic	O
+to	O
+cells	O
+because	O
+it	O
+readily	O
+reacts	O
+with	O
+oxygen	B-chemical
+.	O
+
+To	O
+balance	O
+the	O
+cell	O
+’	O
+s	O
+need	O
+for	O
+iron	B-chemical
+against	O
+its	O
+potential	O
+damaging	O
+effects	O
+,	O
+organisms	O
+have	O
+evolved	O
+iron	B-protein_type
+storage	I-protein_type
+proteins	I-protein_type
+known	O
+as	O
+ferritins	B-protein_type
+that	O
+form	O
+cage	B-structure_element
+-	I-structure_element
+like	I-structure_element
+structures	I-structure_element
+.	O
+
+The	O
+ferritins	B-protein_type
+convert	O
+iron	B-chemical
+into	O
+a	O
+less	O
+reactive	O
+form	O
+that	O
+is	O
+mineralised	O
+and	O
+safely	O
+stored	O
+in	O
+the	O
+central	B-site
+cavity	I-site
+of	O
+the	O
+ferritin	B-protein_type
+cage	O
+and	O
+is	O
+available	O
+for	O
+cells	O
+when	O
+they	O
+need	O
+it	O
+.	O
+
+Recently	O
+,	O
+a	O
+new	O
+family	O
+of	O
+ferritins	B-protein_type
+known	O
+as	O
+encapsulated	B-protein_state
+ferritins	B-protein_type
+have	O
+been	O
+found	O
+in	O
+some	O
+microorganisms	B-taxonomy_domain
+.	O
+
+These	O
+ferritins	B-protein_type
+are	O
+found	O
+in	O
+bacterial	B-taxonomy_domain
+genomes	O
+with	O
+a	O
+gene	O
+that	O
+codes	O
+for	O
+a	O
+protein	O
+cage	O
+called	O
+an	O
+encapsulin	B-protein
+.	O
+
+Although	O
+the	O
+structure	B-evidence
+of	O
+the	O
+encapsulin	B-protein
+cage	O
+is	O
+known	O
+to	O
+look	O
+like	O
+the	O
+shell	B-structure_element
+of	O
+a	O
+virus	B-taxonomy_domain
+,	O
+the	O
+structure	B-evidence
+that	O
+the	O
+encapsulated	B-protein_state
+ferritin	B-protein_type
+itself	O
+forms	O
+is	O
+not	O
+known	O
+.	O
+
+It	O
+is	O
+also	O
+not	O
+clear	O
+how	O
+encapsulin	B-protein
+and	O
+the	O
+encapsulated	B-protein_state
+ferritin	B-protein_type
+work	O
+together	O
+to	O
+store	O
+iron	B-chemical
+.	O
+
+He	O
+et	O
+al	O
+.	O
+have	O
+now	O
+used	O
+the	O
+techniques	O
+of	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+and	O
+mass	B-experimental_method
+spectrometry	I-experimental_method
+to	O
+determine	O
+the	O
+structure	B-evidence
+of	O
+the	O
+encapsulated	B-protein_state
+ferritin	B-protein_type
+found	O
+in	O
+some	O
+bacteria	B-taxonomy_domain
+.	O
+
+The	O
+encapsulated	B-protein_state
+ferritin	B-protein_type
+forms	O
+a	O
+ring	B-structure_element
+-	I-structure_element
+shaped	I-structure_element
+doughnut	B-structure_element
+in	O
+which	O
+ten	O
+subunits	B-structure_element
+of	O
+ferritin	B-protein_type
+are	O
+arranged	O
+in	O
+a	O
+ring	B-structure_element
+;	O
+this	O
+is	O
+totally	O
+different	O
+from	O
+the	O
+enclosed	O
+cages	B-structure_element
+that	O
+other	O
+ferritins	B-protein_type
+form	O
+.	O
+
+Biochemical	B-experimental_method
+studies	I-experimental_method
+revealed	O
+that	O
+the	O
+encapsulated	B-protein_state
+ferritin	B-protein_type
+is	O
+able	O
+to	O
+convert	O
+iron	B-chemical
+into	O
+a	O
+less	O
+reactive	O
+form	O
+,	O
+but	O
+it	O
+cannot	O
+store	O
+iron	B-chemical
+on	O
+its	O
+own	O
+since	O
+it	O
+does	O
+not	O
+form	O
+a	O
+cage	O
+.	O
+
+Thus	O
+,	O
+the	O
+encapsulated	B-protein_state
+ferritin	B-protein_type
+needs	O
+to	O
+be	O
+housed	O
+within	O
+the	O
+encapsulin	B-protein
+cage	O
+to	O
+store	O
+iron	B-chemical
+.	O
+
+Further	O
+work	O
+is	O
+needed	O
+to	O
+investigate	O
+how	O
+iron	B-chemical
+moves	O
+into	O
+the	O
+encapsulin	B-protein
+cage	O
+to	O
+reach	O
+the	O
+ferritin	B-protein_type
+proteins	O
+.	O
+
+Some	O
+organisms	O
+have	O
+both	O
+standard	O
+ferritin	B-protein_type
+cages	O
+and	O
+encapsulated	B-protein_state
+ferritins	B-protein_type
+;	O
+why	O
+this	O
+is	O
+the	O
+case	O
+also	O
+remains	O
+to	O
+be	O
+discovered	O
+.	O
+
+Encapsulin	B-protein_type
+nanocompartments	B-complex_assembly
+are	O
+a	O
+family	O
+of	O
+proteinaceous	O
+metabolic	O
+compartments	O
+that	O
+are	O
+widely	O
+distributed	O
+in	O
+bacteria	B-taxonomy_domain
+and	O
+archaea	B-taxonomy_domain
+.	O
+
+They	O
+share	O
+a	O
+common	O
+architecture	O
+,	O
+comprising	O
+an	O
+icosahedral	B-protein_state
+shell	B-structure_element
+formed	O
+by	O
+the	O
+oligomeric	O
+assembly	O
+of	O
+a	O
+protein	O
+,	O
+encapsulin	B-protein_type
+,	O
+that	O
+is	O
+structurally	O
+related	O
+to	O
+the	O
+HK97	B-taxonomy_domain
+bacteriophage	I-taxonomy_domain
+capsid	O
+protein	O
+gp5	B-protein
+.	O
+
+Gp5	B-protein
+is	O
+known	O
+to	O
+assemble	O
+as	O
+a	O
+66	O
+nm	O
+diameter	O
+icosahedral	B-protein_state
+shell	B-structure_element
+of	O
+420	O
+subunits	B-structure_element
+.	O
+
+In	O
+contrast	O
+,	O
+both	O
+the	O
+Pyrococcus	B-species
+furiosus	I-species
+and	O
+Myxococcus	B-species
+xanthus	I-species
+encapsulin	B-protein
+shell	B-structure_element
+-	O
+proteins	O
+form	O
+32	O
+nm	O
+icosahedra	B-structure_element
+with	O
+180	O
+subunits	B-structure_element
+;	O
+while	O
+the	O
+Thermotoga	B-species
+maritima	I-species
+encapsulin	B-protein
+is	O
+smaller	O
+still	O
+with	O
+a	O
+25	O
+nm	O
+,	O
+60	O
+-	O
+subunit	O
+icosahedron	B-structure_element
+.	O
+
+The	O
+high	O
+structural	O
+similarity	O
+of	O
+the	O
+encapsulin	B-protein_type
+shell	B-structure_element
+-	O
+proteins	O
+to	O
+gp5	B-protein
+suggests	O
+a	O
+common	O
+evolutionary	O
+origin	O
+for	O
+these	O
+proteins	O
+.	O
+
+The	O
+genes	O
+encoding	O
+encapsulin	B-protein_type
+proteins	O
+are	O
+found	O
+downstream	O
+of	O
+genes	O
+for	O
+dye	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+peroxidase	I-protein_type
+(	O
+DyP	B-protein_type
+)	O
+family	O
+enzymes	O
+,	O
+or	O
+encapsulin	B-protein_type
+-	I-protein_type
+associated	I-protein_type
+ferritins	I-protein_type
+(	O
+EncFtn	B-protein_type
+).	O
+
+Enzymes	O
+in	O
+the	O
+DyP	B-protein_type
+family	I-protein_type
+are	O
+active	O
+against	O
+polyphenolic	O
+compounds	O
+such	O
+as	O
+azo	O
+dyes	O
+and	O
+lignin	O
+breakdown	O
+products	O
+;	O
+although	O
+their	O
+physiological	O
+function	O
+and	O
+natural	O
+substrates	O
+are	O
+not	O
+known	O
+.	O
+
+Ferritin	B-protein_type
+family	O
+proteins	O
+are	O
+found	O
+in	O
+all	O
+kingdoms	B-taxonomy_domain
+and	O
+have	O
+a	O
+wide	O
+range	O
+of	O
+activities	O
+,	O
+including	O
+ribonucleotide	B-protein_type
+reductase	I-protein_type
+,	O
+protecting	O
+DNA	O
+from	O
+oxidative	O
+damage	O
+,	O
+and	O
+iron	B-chemical
+storage	O
+.	O
+
+The	O
+classical	B-protein_state
+iron	B-complex_assembly
+storage	I-complex_assembly
+ferritin	I-complex_assembly
+nanocages	I-complex_assembly
+are	O
+found	O
+in	O
+all	O
+kingdoms	B-taxonomy_domain
+and	O
+are	O
+essential	O
+in	O
+eukaryotes	B-taxonomy_domain
+;	O
+they	O
+play	O
+a	O
+central	O
+role	O
+in	O
+iron	B-chemical
+homeostasis	O
+,	O
+where	O
+they	O
+protect	O
+the	O
+cell	O
+from	O
+toxic	O
+free	O
+Fe2	B-chemical
++	I-chemical
+by	O
+oxidizing	O
+it	O
+and	O
+storing	O
+the	O
+resulting	O
+Fe3	B-chemical
++	I-chemical
+as	O
+ferrihydrite	B-chemical
+minerals	O
+within	O
+their	O
+central	B-site
+cavity	I-site
+.	O
+
+The	O
+encapsulin	B-protein_type
+-	O
+associated	O
+enzymes	O
+are	O
+sequestered	O
+within	O
+the	O
+icosahedral	B-protein_state
+shell	B-structure_element
+through	O
+interactions	O
+between	O
+the	O
+shell	B-structure_element
+’	O
+s	O
+inner	O
+surface	O
+and	O
+a	O
+short	B-structure_element
+localization	I-structure_element
+sequence	I-structure_element
+(	O
+Gly	B-structure_element
+-	I-structure_element
+Ser	I-structure_element
+-	I-structure_element
+Leu	I-structure_element
+-	I-structure_element
+Lys	I-structure_element
+)	O
+appended	O
+to	O
+their	O
+C	O
+-	O
+termini	O
+.	O
+
+This	B-structure_element
+motif	I-structure_element
+is	O
+well	B-protein_state
+-	I-protein_state
+conserved	I-protein_state
+,	O
+and	O
+the	O
+addition	O
+of	O
+this	O
+sequence	O
+to	O
+heterologous	O
+proteins	O
+is	O
+sufficient	O
+to	O
+direct	O
+them	O
+to	O
+the	O
+interior	O
+of	O
+encapsulins	B-protein_type
+.	O
+
+A	O
+recent	O
+study	O
+of	O
+the	O
+Myxococcus	B-species
+xanthus	I-species
+encapsulin	B-protein
+showed	O
+that	O
+it	O
+sequesters	O
+a	O
+number	O
+of	O
+different	O
+EncFtn	B-protein_type
+proteins	O
+and	O
+acts	O
+as	O
+an	O
+‘	O
+iron	B-chemical
+-	O
+megastore	O
+’	O
+to	O
+protect	O
+these	O
+bacteria	B-taxonomy_domain
+from	O
+oxidative	O
+stress	O
+.	O
+
+At	O
+32	O
+nm	O
+in	O
+diameter	O
+,	O
+it	O
+is	O
+much	O
+larger	O
+than	O
+other	O
+members	O
+of	O
+the	O
+ferritin	B-protein_type
+superfamily	O
+,	O
+such	O
+as	O
+the	O
+12	O
+nm	O
+24	O
+-	O
+subunit	O
+classical	B-protein_state
+ferritin	B-protein_type
+nanocage	B-complex_assembly
+and	O
+the	O
+8	O
+nm	O
+12	O
+-	O
+subunit	O
+Dps	B-protein_type
+(	O
+DNA	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+protein	I-protein_type
+from	O
+starved	O
+cells	O
+)	O
+complex	O
+;	O
+and	O
+is	O
+thus	O
+capable	O
+of	O
+sequestering	O
+up	O
+to	O
+ten	O
+times	O
+more	O
+iron	B-chemical
+than	O
+these	O
+ferritins	B-protein_type
+.	O
+
+The	O
+primary	O
+sequences	O
+of	O
+EncFtn	B-protein_type
+proteins	O
+have	O
+Glu	B-structure_element
+-	I-structure_element
+X	I-structure_element
+-	I-structure_element
+X	I-structure_element
+-	I-structure_element
+His	I-structure_element
+metal	B-site
+coordination	I-site
+sites	I-site
+,	O
+which	O
+are	O
+shared	O
+features	O
+of	O
+the	O
+ferritin	B-protein_type
+family	O
+proteins	O
+.	O
+
+Secondary	B-experimental_method
+structure	I-experimental_method
+prediction	I-experimental_method
+identifies	O
+two	O
+major	B-structure_element
+α	I-structure_element
+-	I-structure_element
+helical	I-structure_element
+regions	I-structure_element
+in	O
+these	O
+proteins	O
+;	O
+this	O
+is	O
+in	O
+contrast	O
+to	O
+other	O
+members	O
+of	O
+the	O
+ferritin	B-protein_type
+superfamily	O
+,	O
+which	O
+have	O
+four	O
+major	B-structure_element
+α	I-structure_element
+-	I-structure_element
+helices	I-structure_element
+(	O
+Supplementary	O
+file	O
+1	O
+).	O
+
+The	O
+‘	O
+half	O
+-	O
+ferritin	B-protein_type
+’	O
+primary	O
+sequence	O
+of	O
+the	O
+EncFtn	B-protein_type
+family	O
+and	O
+their	O
+association	O
+with	O
+encapsulin	B-protein
+nanocompartments	B-complex_assembly
+suggests	O
+a	O
+distinct	O
+biochemical	O
+and	O
+structural	O
+organization	O
+to	O
+other	O
+ferritin	B-protein_type
+family	O
+proteins	O
+.	O
+
+The	O
+Rhodospirillum	B-species
+rubrum	I-species
+EncFtn	B-protein
+protein	O
+(	O
+Rru_A0973	B-gene
+)	O
+shares	O
+33	O
+%	O
+protein	O
+sequence	O
+identity	O
+with	O
+the	O
+M	B-species
+.	I-species
+xanthus	I-species
+(	O
+MXAN_4464	B-gene
+),	O
+53	O
+%	O
+with	O
+the	O
+T	B-species
+.	I-species
+maritima	I-species
+(	O
+Tmari_0787	B-gene
+),	O
+and	O
+29	O
+%	O
+with	O
+the	O
+P	B-species
+.	I-species
+furiosus	I-species
+(	O
+PF1192	B-gene
+)	O
+homologues	O
+.	O
+
+The	O
+GXXH	B-structure_element
+motifs	O
+are	O
+strictly	B-protein_state
+conserved	I-protein_state
+in	O
+each	O
+of	O
+these	O
+species	O
+(	O
+Supplementary	O
+file	O
+1	O
+).	O
+
+Here	O
+we	O
+investigate	O
+the	O
+structure	B-evidence
+and	O
+biochemistry	O
+of	O
+EncFtn	B-protein
+in	O
+order	O
+to	O
+understand	O
+iron	B-chemical
+storage	O
+within	O
+the	O
+encapsulin	B-protein
+nanocompartment	B-complex_assembly
+.	O
+
+We	O
+have	O
+produced	O
+recombinant	O
+encapsulin	B-protein
+(	O
+Enc	B-protein
+)	O
+and	O
+EncFtn	B-protein
+from	O
+the	O
+aquatic	B-taxonomy_domain
+purple	B-taxonomy_domain
+-	I-taxonomy_domain
+sulfur	I-taxonomy_domain
+bacterium	I-taxonomy_domain
+R	B-species
+.	I-species
+rubrum	I-species
+,	O
+which	O
+serves	O
+as	O
+a	O
+model	O
+organism	O
+for	O
+the	O
+study	O
+of	O
+the	O
+control	O
+of	O
+the	O
+bacterial	B-taxonomy_domain
+nitrogen	O
+fixation	O
+machinery	O
+,	O
+in	O
+Escherichia	B-species
+coli	I-species
+.	O
+
+Analysis	O
+by	O
+transmission	B-experimental_method
+electron	I-experimental_method
+microscopy	I-experimental_method
+(	O
+TEM	B-experimental_method
+)	O
+indicates	O
+that	O
+their	O
+co	B-experimental_method
+-	I-experimental_method
+expression	I-experimental_method
+leads	O
+to	O
+the	O
+production	O
+of	O
+an	O
+icosahedral	B-protein_state
+nanocompartment	B-complex_assembly
+with	O
+encapsulated	B-protein_state
+EncFtn	B-protein
+.	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+a	O
+truncated	B-protein_state
+hexahistidine	B-protein_state
+-	I-protein_state
+tagged	I-protein_state
+variant	O
+of	O
+the	O
+EncFtn	B-protein
+protein	O
+(	O
+EncFtnsH	B-protein
+)	O
+shows	O
+that	O
+it	O
+forms	O
+a	O
+decameric	B-oligomeric_state
+structure	B-evidence
+with	O
+an	O
+annular	O
+‘	O
+ring	B-structure_element
+-	I-structure_element
+doughnut	I-structure_element
+’	O
+topology	O
+,	O
+which	O
+is	O
+distinct	O
+from	O
+the	O
+four	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+bundles	I-structure_element
+of	O
+the	O
+24meric	B-oligomeric_state
+ferritins	B-protein_type
+and	O
+dodecahedral	B-oligomeric_state
+DPS	B-protein_type
+proteins	O
+.	O
+
+We	O
+identify	O
+a	O
+symmetrical	O
+iron	B-protein_state
+bound	I-protein_state
+ferroxidase	B-site
+center	I-site
+(	O
+FOC	B-site
+)	O
+formed	O
+between	O
+subunits	B-structure_element
+in	O
+the	O
+decamer	B-oligomeric_state
+and	O
+additional	O
+metal	B-site
+-	I-site
+binding	I-site
+sites	I-site
+close	O
+to	O
+the	O
+center	O
+of	O
+the	O
+ring	B-structure_element
+and	O
+on	O
+the	O
+outer	O
+surface	O
+.	O
+
+We	O
+also	O
+demonstrate	O
+the	O
+metal	O
+-	O
+dependent	O
+assembly	O
+of	O
+EncFtn	B-protein
+decamers	B-oligomeric_state
+using	O
+native	B-experimental_method
+PAGE	I-experimental_method
+,	O
+analytical	B-experimental_method
+gel	I-experimental_method
+-	I-experimental_method
+filtration	I-experimental_method
+,	O
+and	O
+native	B-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+.	O
+
+Biochemical	B-experimental_method
+assays	I-experimental_method
+show	O
+that	O
+EncFtn	B-protein
+is	O
+active	B-protein_state
+as	O
+a	O
+ferroxidase	B-protein_type
+enzyme	O
+.	O
+
+Through	O
+site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+we	O
+show	O
+that	O
+the	O
+conserved	B-protein_state
+glutamic	B-residue_name
+acid	I-residue_name
+and	O
+histidine	B-residue_name
+residues	O
+in	O
+the	O
+FOC	B-site
+influence	O
+protein	O
+assembly	O
+and	O
+activity	O
+.	O
+
+We	O
+use	O
+our	O
+combined	O
+structural	B-evidence
+and	I-evidence
+biochemical	I-evidence
+data	I-evidence
+to	O
+propose	O
+a	O
+model	O
+for	O
+the	O
+EncFtn	B-protein
+-	O
+catalyzed	O
+sequestration	O
+of	O
+iron	B-chemical
+within	O
+the	O
+encapsulin	B-protein
+shell	B-structure_element
+.	O
+
+Assembly	O
+of	O
+R	B-species
+.	I-species
+rubrum	I-species
+EncFtn	B-protein
+encapsulin	B-protein
+nanocompartments	B-complex_assembly
+in	O
+E	B-species
+.	I-species
+coli	I-species
+
+Full	B-evidence
+-	I-evidence
+frame	I-evidence
+transmission	I-evidence
+electron	I-evidence
+micrographs	I-evidence
+of	O
+R	B-species
+.	I-species
+rubrum	I-species
+nanocompartments	B-complex_assembly
+.	O
+
+(	O
+A	O
+/	O
+B	O
+)	O
+Negative	B-experimental_method
+stain	I-experimental_method
+TEM	I-experimental_method
+image	B-evidence
+of	O
+recombinant	O
+R	B-species
+.	I-species
+rubrum	I-species
+encapsulin	B-protein
+and	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+nanocompartments	B-complex_assembly
+.	O
+
+All	O
+samples	O
+were	O
+imaged	O
+at	O
+143	O
+,	O
+000	O
+x	O
+magnification	O
+;	O
+the	O
+scale	O
+bar	O
+length	O
+corresponds	O
+to	O
+50	O
+nm	O
+.	O
+(	O
+C	O
+)	O
+Histogram	B-evidence
+showing	O
+the	O
+distribution	O
+of	O
+nanocompartment	B-complex_assembly
+diameters	O
+.	O
+
+A	O
+model	O
+Gaussian	B-experimental_method
+nonlinear	I-experimental_method
+least	I-experimental_method
+square	I-experimental_method
+function	I-experimental_method
+was	O
+fitted	O
+to	O
+the	O
+data	O
+to	O
+obtain	O
+a	O
+mean	O
+diameter	O
+of	O
+24	O
+.	O
+6	O
+nm	O
+with	O
+a	O
+standard	O
+deviation	O
+of	O
+2	O
+.	O
+0	O
+nm	O
+for	O
+encapsulin	B-protein
+(	O
+grey	O
+)	O
+and	O
+a	O
+mean	O
+value	O
+of	O
+23	O
+.	O
+9	O
+nm	O
+with	O
+a	O
+standard	O
+deviation	O
+of	O
+2	O
+.	O
+2	O
+nm	O
+for	O
+co	B-experimental_method
+-	I-experimental_method
+expressed	I-experimental_method
+EncFtn	B-protein
+and	O
+encapsulin	B-protein
+(	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+,	O
+black	O
+).	O
+
+Purification	O
+of	O
+recombinant	O
+R	B-species
+.	I-species
+rubrum	I-species
+encapsulin	B-protein
+nanocompartments	B-complex_assembly
+.	O
+
+(	O
+A	O
+)	O
+Recombinantly	B-experimental_method
+expressed	I-experimental_method
+encapsulin	B-protein
+(	O
+Enc	B-protein
+)	O
+and	O
+co	B-experimental_method
+-	I-experimental_method
+expressed	I-experimental_method
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+were	O
+purified	O
+by	O
+sucrose	B-experimental_method
+gradient	I-experimental_method
+ultracentrifugation	I-experimental_method
+from	O
+E	B-species
+.	I-species
+coli	I-species
+B834	O
+(	O
+DE3	O
+)	O
+grown	O
+in	O
+SeMet	B-chemical
+medium	O
+.	O
+
+Samples	O
+were	O
+resolved	O
+by	O
+18	O
+%	O
+acrylamide	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+;	O
+the	O
+position	O
+of	O
+the	O
+proteins	O
+found	O
+in	O
+the	O
+complexes	O
+as	O
+resolved	O
+on	O
+the	O
+gel	O
+are	O
+shown	O
+with	O
+arrows	O
+.	O
+
+(	O
+B	O
+/	O
+C	O
+)	O
+Negative	B-experimental_method
+stain	I-experimental_method
+TEM	I-experimental_method
+image	O
+of	O
+recombinant	O
+encapsulin	B-protein
+and	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+nanocompartments	B-complex_assembly
+.	O
+
+Representative	O
+encapsulin	B-protein
+and	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+complexes	O
+are	O
+indicated	O
+with	O
+red	O
+arrows	O
+.	O
+
+We	O
+produced	O
+recombinant	O
+R	B-species
+.	I-species
+rubrum	I-species
+encapsulin	B-protein
+nanocompartments	B-complex_assembly
+in	O
+E	B-species
+.	I-species
+coli	I-species
+by	O
+co	B-experimental_method
+-	I-experimental_method
+expression	I-experimental_method
+of	O
+the	O
+encapsulin	B-protein
+(	O
+Rru_A0974	B-gene
+)	O
+and	O
+EncFtn	B-protein
+(	O
+Rru_A0973	B-gene
+)	O
+proteins	O
+,	O
+and	O
+purified	O
+these	O
+by	O
+sucrose	B-experimental_method
+gradient	I-experimental_method
+ultra	I-experimental_method
+-	I-experimental_method
+centrifugation	I-experimental_method
+(	O
+Figure	O
+1A	O
+).	O
+
+TEM	B-experimental_method
+imaging	O
+of	O
+uranyl	O
+acetate	O
+-	O
+stained	O
+samples	O
+revealed	O
+that	O
+,	O
+when	O
+expressed	B-experimental_method
+in	I-experimental_method
+isolation	I-experimental_method
+,	O
+the	O
+encapsulin	B-protein
+protein	O
+forms	O
+empty	B-protein_state
+compartments	B-complex_assembly
+with	O
+an	O
+average	O
+diameter	O
+of	O
+24	O
+nm	O
+(	O
+Figure	O
+1B	O
+and	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+1A	O
+/	O
+C	O
+),	O
+consistent	O
+with	O
+the	O
+appearance	O
+and	O
+size	O
+of	O
+the	O
+T	B-species
+.	I-species
+maritima	I-species
+encapsulin	B-protein
+.	O
+
+We	O
+were	O
+not	O
+able	O
+to	O
+resolve	O
+any	O
+higher	O
+-	O
+order	O
+structures	O
+of	O
+EncFtn	B-protein
+by	O
+TEM	B-experimental_method
+.	O
+
+Protein	O
+purified	O
+from	O
+co	B-experimental_method
+-	I-experimental_method
+expression	I-experimental_method
+of	O
+the	O
+encapsulin	B-protein
+and	O
+EncFtn	B-protein
+resulted	O
+in	O
+24	O
+nm	O
+compartments	O
+with	O
+regions	O
+in	O
+the	O
+center	O
+that	O
+exclude	O
+stain	O
+,	O
+consistent	O
+with	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+EncFtn	B-protein
+within	O
+the	O
+encapsulin	B-protein
+shell	B-structure_element
+(	O
+Figure	O
+1C	O
+and	O
+Figure	O
+1	O
+—	O
+figure	O
+supplement	O
+1B	O
+/	O
+C	O
+).	O
+
+R	B-species
+.	I-species
+rubrum	I-species
+EncFtn	B-protein
+forms	O
+a	O
+metal	O
+-	O
+ion	O
+stabilized	O
+decamer	B-oligomeric_state
+in	O
+solution	O
+
+Purification	B-experimental_method
+of	I-experimental_method
+recombinant	I-experimental_method
+R	B-species
+.	I-species
+rubrum	I-species
+EncFtnsH	B-protein
+.	O
+
+(	O
+A	O
+)	O
+Recombinant	O
+SeMet	B-protein_state
+-	I-protein_state
+labeled	I-protein_state
+EncFtnsH	B-protein
+produced	O
+with	O
+1	O
+mM	O
+Fe	B-chemical
+(	I-chemical
+NH4	I-chemical
+)	I-chemical
+2	I-chemical
+(	I-chemical
+SO4	I-chemical
+)	I-chemical
+2	I-chemical
+in	O
+the	O
+growth	O
+medium	O
+was	O
+purified	O
+by	O
+nickel	B-experimental_method
+affinity	I-experimental_method
+chromatography	I-experimental_method
+and	O
+size	B-experimental_method
+-	I-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+using	O
+a	O
+Superdex	O
+200	O
+16	O
+/	O
+60	O
+column	O
+(	O
+GE	O
+Healthcare	O
+).	O
+
+Chromatogram	B-evidence
+traces	O
+measured	O
+at	O
+280	O
+nm	O
+and	O
+315	O
+nm	O
+are	O
+shown	O
+with	O
+the	O
+results	O
+from	O
+ICP	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+analysis	O
+of	O
+the	O
+iron	B-chemical
+content	O
+of	O
+the	O
+fractions	O
+collected	O
+during	O
+the	O
+experiment	O
+.	O
+
+The	O
+peak	O
+around	O
+73	O
+ml	O
+corresponds	O
+to	O
+a	O
+molecular	B-evidence
+weight	I-evidence
+of	O
+around	O
+130	O
+kDa	O
+when	O
+compared	O
+to	O
+calibration	O
+standards	O
+;	O
+this	O
+is	O
+consistent	O
+with	O
+a	O
+decamer	B-oligomeric_state
+of	O
+EncFtnsH	B-protein
+.	O
+The	O
+small	O
+peak	O
+at	O
+85	O
+ml	O
+corresponds	O
+to	O
+the	O
+13	O
+kDa	O
+monomer	B-oligomeric_state
+compared	O
+to	O
+the	O
+standards	O
+.	O
+
+Only	O
+the	O
+decamer	B-oligomeric_state
+peak	O
+contains	O
+significant	O
+amounts	O
+of	O
+iron	B-chemical
+as	O
+indicated	O
+by	O
+the	O
+ICP	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+analysis	O
+.	O
+
+(	O
+B	O
+)	O
+Peak	O
+fractions	O
+from	O
+the	O
+gel	B-experimental_method
+filtration	I-experimental_method
+run	O
+were	O
+resolved	O
+by	O
+15	O
+%	O
+acrylamide	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+and	O
+stained	O
+with	O
+Coomassie	O
+blue	O
+stain	O
+.	O
+
+The	O
+bands	O
+around	O
+13	O
+kDa	O
+and	O
+26	O
+kDa	O
+correspond	O
+to	O
+EncFtnsH	B-protein
+,	O
+as	O
+identified	O
+by	O
+MALDI	B-experimental_method
+peptide	I-experimental_method
+mass	I-experimental_method
+fingerprinting	I-experimental_method
+.	O
+
+The	O
+band	O
+at	O
+13	O
+kDa	O
+is	O
+consistent	O
+with	O
+the	O
+monomer	B-oligomeric_state
+mass	O
+,	O
+while	O
+the	O
+band	O
+at	O
+26	O
+kDa	O
+is	O
+consistent	O
+with	O
+a	O
+dimer	B-oligomeric_state
+of	O
+EncFtnsH	B-protein
+.	O
+The	O
+dimer	B-oligomeric_state
+species	O
+only	O
+appears	O
+in	O
+the	O
+decamer	B-oligomeric_state
+fractions	O
+.	O
+
+(	O
+C	O
+)	O
+SEC	B-experimental_method
+-	I-experimental_method
+MALLS	I-experimental_method
+analysis	O
+of	O
+EncFtnsH	B-protein
+from	O
+decamer	B-oligomeric_state
+fractions	O
+and	O
+monomer	B-oligomeric_state
+fractions	O
+allows	O
+assignment	O
+of	O
+an	O
+average	O
+mass	O
+of	O
+132	O
+kDa	O
+to	O
+decamer	B-oligomeric_state
+fractions	O
+and	O
+13	O
+kDa	O
+to	O
+monomer	B-oligomeric_state
+fractions	O
+,	O
+consistent	O
+with	O
+decamer	B-oligomeric_state
+and	O
+monomer	B-oligomeric_state
+species	O
+(	O
+Table	O
+2	O
+).	O
+
+Determination	O
+of	O
+the	O
+Fe	B-chemical
+/	O
+EncFtnsH	B-protein
+protein	O
+ratio	O
+by	O
+ICP	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+.	O
+
+EncFtnsH	B-protein
+was	O
+purified	O
+as	O
+a	O
+SeMet	B-chemical
+derivative	O
+from	O
+E	B-species
+.	I-species
+coli	I-species
+B834	I-species
+(	I-species
+DE3	I-species
+)	I-species
+cells	O
+grown	O
+in	O
+SeMet	B-chemical
+medium	O
+with	O
+1	O
+mM	O
+Fe	B-chemical
+(	I-chemical
+NH4	I-chemical
+)	I-chemical
+2	I-chemical
+(	I-chemical
+SO4	I-chemical
+)	I-chemical
+2	I-chemical
+.	O
+
+Fractions	O
+from	O
+SEC	B-experimental_method
+were	O
+collected	O
+,	O
+acidified	O
+and	O
+analysed	O
+by	O
+ICP	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+.	O
+
+EncFtnsH	B-protein
+concentration	O
+was	O
+calculated	O
+based	O
+on	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+two	O
+SeMet	B-chemical
+per	O
+mature	B-protein_state
+monomer	B-oligomeric_state
+.	O
+
+These	O
+data	O
+were	O
+collected	O
+from	O
+EncFtnsH	B-protein
+fractions	O
+from	O
+a	O
+single	O
+gel	B-experimental_method
+-	I-experimental_method
+filtration	I-experimental_method
+run	O
+.	O
+
+Peak	O
+EncFtnsHretention	B-protein
+volume	O
+(	O
+ml	O
+)	O
+Element	O
+concentration	O
+(	O
+µM	O
+)	O
+Derived	O
+EncFtnsHconcentration	B-protein
+(	O
+µM	O
+)	O
+Derived	O
+Fe	B-chemical
+/	O
+EncFtnsH	B-protein
+monomer	B-oligomeric_state
+Ca	B-chemical
+Fe	B-chemical
+Zn	B-chemical
+Se	B-chemical
+Decamer	B-oligomeric_state
+66	O
+.	O
+5	O
+n	O
+.	O
+d	O
+.	O
+
+Estimates	O
+of	O
+EncFtnsH	B-protein
+molecular	B-evidence
+weight	I-evidence
+from	O
+SEC	B-experimental_method
+-	I-experimental_method
+MALLS	I-experimental_method
+analysis	O
+.	O
+
+EncFtnsH	B-protein
+was	O
+purified	O
+from	O
+E	B-species
+.	I-species
+coli	I-species
+BL21	I-species
+(	I-species
+DE3	I-species
+)	I-species
+grown	O
+in	O
+minimal	B-experimental_method
+medium	I-experimental_method
+(	O
+MM	B-experimental_method
+)	O
+by	O
+nickel	B-experimental_method
+affinity	I-experimental_method
+chromatography	I-experimental_method
+and	O
+size	B-experimental_method
+-	I-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+.	O
+
+Fractions	O
+from	O
+two	O
+peaks	B-evidence
+(	O
+decamer	B-oligomeric_state
+and	O
+monomer	B-oligomeric_state
+)	O
+were	O
+pooled	O
+separately	O
+(	O
+Figure	O
+1C	O
+)	O
+and	O
+analysed	O
+by	O
+SEC	B-experimental_method
+-	I-experimental_method
+MALLS	I-experimental_method
+using	O
+a	O
+Superdex	O
+200	O
+10	O
+/	O
+300	O
+GL	O
+column	O
+(	O
+GE	O
+Healthcare	O
+)	O
+and	O
+Viscotek	O
+SEC	B-experimental_method
+-	I-experimental_method
+MALLS	I-experimental_method
+instruments	O
+(	O
+Malvern	O
+Instruments	O
+)	O
+(	O
+Figure	O
+2C	O
+).	O
+
+The	O
+decamer	B-oligomeric_state
+and	O
+monomer	B-oligomeric_state
+peaks	B-evidence
+were	O
+both	O
+symmetric	O
+and	O
+monodisperse	O
+,	O
+allowing	O
+the	O
+estimation	O
+of	O
+the	O
+molecular	B-evidence
+weight	I-evidence
+of	O
+the	O
+species	O
+in	O
+these	O
+fractions	O
+.	O
+
+The	O
+proteins	O
+analyzed	O
+by	O
+SEC	B-experimental_method
+-	I-experimental_method
+MALLS	I-experimental_method
+came	O
+from	O
+single	O
+protein	O
+preparation	O
+.	O
+
+Molecular	B-evidence
+Weight	I-evidence
+(	O
+kDa	O
+)	O
+Decamer	B-oligomeric_state
+peak	O
+Monomer	B-oligomeric_state
+peak	O
+Theoretical	O
+133	O
+13	O
+EncFtnsH	B-protein
+-	O
+decamer	B-oligomeric_state
+fractions	O
+132	O
+15	O
+EncFtnsH	B-protein
+-	O
+monomer	B-oligomeric_state
+fractions	O
+126	O
+13	O
+
+We	O
+purified	O
+recombinant	O
+R	B-species
+.	I-species
+rubrum	I-species
+EncFtn	B-protein
+as	O
+both	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+sequence	O
+(	O
+140	B-residue_range
+amino	I-residue_range
+acids	I-residue_range
+)	O
+and	O
+a	O
+truncated	B-protein_state
+C	O
+-	O
+terminal	O
+hexahistidine	B-protein_state
+-	I-protein_state
+tagged	I-protein_state
+variant	O
+(	O
+amino	O
+acids	O
+1	B-residue_range
+–	I-residue_range
+96	I-residue_range
+plus	O
+the	O
+tag	O
+;	O
+herein	O
+EncFtnsH	B-protein
+).	O
+
+In	O
+both	O
+cases	O
+the	O
+elution	B-evidence
+profile	I-evidence
+from	O
+size	B-experimental_method
+-	I-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+(	O
+SEC	B-experimental_method
+)	O
+displayed	O
+two	O
+peaks	B-evidence
+(	O
+Figure	O
+2A	O
+).	O
+
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+analysis	O
+of	O
+fractions	O
+from	O
+these	O
+peaks	B-evidence
+showed	O
+that	O
+the	O
+high	O
+molecular	B-evidence
+weight	I-evidence
+peak	O
+was	O
+partially	O
+resistant	O
+to	O
+SDS	O
+and	O
+heat	O
+-	O
+induced	O
+denaturation	O
+;	O
+in	O
+contrast	O
+,	O
+the	O
+low	O
+molecular	B-evidence
+weight	I-evidence
+peak	O
+was	O
+consistent	O
+with	O
+monomeric	B-oligomeric_state
+mass	O
+of	O
+13	O
+kDa	O
+(	O
+Figure	O
+2B	O
+).	O
+
+MALDI	B-experimental_method
+peptide	I-experimental_method
+mass	I-experimental_method
+fingerprinting	I-experimental_method
+of	O
+these	O
+bands	O
+confirmed	O
+the	O
+identity	O
+of	O
+both	O
+as	O
+EncFtn	B-protein
+.	O
+
+Inductively	B-experimental_method
+coupled	I-experimental_method
+plasma	I-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+(	O
+ICP	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+)	O
+analysis	O
+of	O
+the	O
+SEC	B-experimental_method
+fractions	O
+showed	O
+100	O
+times	O
+more	O
+iron	B-chemical
+in	O
+the	O
+oligomeric	O
+fraction	O
+than	O
+the	O
+monomer	B-oligomeric_state
+(	O
+Figure	O
+2A	O
+,	O
+blue	O
+scatter	O
+points	O
+;	O
+Table	O
+1	O
+),	O
+suggesting	O
+that	O
+EncFtn	B-protein
+oligomerization	O
+is	O
+associated	O
+with	O
+iron	B-chemical
+binding	O
+.	O
+
+In	O
+order	O
+to	O
+determine	O
+the	O
+iron	B-chemical
+-	O
+loading	O
+stoichiometry	O
+in	O
+the	O
+EncFtn	B-protein
+complex	O
+,	O
+further	O
+ICP	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+experiments	O
+were	O
+performed	O
+using	O
+selenomethionine	B-chemical
+(	O
+SeMet	B-chemical
+)-	O
+labelled	O
+protein	O
+EncFtn	B-protein
+(	O
+Table	O
+1	O
+).	O
+
+In	O
+these	O
+experiments	O
+,	O
+we	O
+observed	O
+sub	O
+-	O
+stoichiometric	O
+metal	O
+binding	O
+,	O
+which	O
+is	O
+in	O
+contrast	O
+to	O
+the	O
+classical	B-protein_state
+ferritins	B-protein_type
+.	O
+
+Size	B-experimental_method
+-	I-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+with	O
+multi	B-experimental_method
+-	I-experimental_method
+angle	I-experimental_method
+laser	I-experimental_method
+light	I-experimental_method
+scattering	I-experimental_method
+(	O
+SEC	B-experimental_method
+-	I-experimental_method
+MALLS	I-experimental_method
+)	O
+analysis	O
+of	O
+samples	O
+taken	O
+from	O
+each	O
+peak	O
+gave	O
+calculated	O
+molecular	O
+weights	O
+consistent	O
+with	O
+a	O
+decamer	B-oligomeric_state
+for	O
+the	O
+high	O
+molecular	B-evidence
+weight	I-evidence
+peak	O
+and	O
+a	O
+monomer	B-oligomeric_state
+for	O
+the	O
+low	O
+molecular	B-evidence
+weight	I-evidence
+peak	O
+(	O
+Figure	O
+2C	O
+,	O
+Table	O
+2	O
+).	O
+
+Effect	O
+of	O
+metal	O
+ions	O
+on	O
+the	O
+oligomeric	O
+state	O
+of	O
+EncFtnsH	B-protein
+in	O
+solution	O
+.	O
+
+(	O
+A	O
+/	O
+B	O
+)	O
+EncFtnsH	B-protein
+-	O
+monomer	B-oligomeric_state
+was	O
+incubated	B-experimental_method
+with	O
+one	O
+mole	O
+equivalent	O
+of	O
+various	O
+metal	O
+salts	O
+for	O
+two	O
+hours	O
+prior	O
+to	O
+analytical	B-experimental_method
+gel	I-experimental_method
+-	I-experimental_method
+filtration	I-experimental_method
+using	O
+a	O
+Superdex	O
+200	O
+PC	O
+3	O
+.	O
+2	O
+/	O
+30	O
+column	O
+.	O
+
+Co2	B-chemical
++	I-chemical
+and	O
+Zn2	B-chemical
++	I-chemical
+induced	O
+the	O
+formation	O
+of	O
+the	O
+decameric	B-oligomeric_state
+form	O
+of	O
+EncFtnsH	B-protein
+;	O
+while	O
+Mn2	B-chemical
++,	I-chemical
+Mg2	B-chemical
++	I-chemical
+and	O
+Fe3	B-chemical
++	I-chemical
+did	O
+not	O
+significantly	O
+alter	O
+the	O
+oligomeric	O
+state	O
+of	O
+EncFtnsH	B-protein
+.	O
+
+PAGE	B-experimental_method
+analysis	O
+of	O
+the	O
+effect	O
+of	O
+metal	O
+ions	O
+on	O
+the	O
+oligomeric	O
+state	O
+of	O
+EncFtnsH	B-protein
+.	O
+
+50	O
+µM	O
+EncFtnsH	B-protein
+monomer	B-oligomeric_state
+or	O
+decamer	B-oligomeric_state
+samples	O
+were	O
+mixed	O
+with	O
+equal	O
+molar	O
+metal	O
+ions	O
+including	O
+Fe2	B-chemical
++,	I-chemical
+Co2	B-chemical
++,	I-chemical
+Zn2	B-chemical
++,	I-chemical
+Mn2	B-chemical
++,	I-chemical
+Ca2	B-chemical
++,	I-chemical
+Mg2	B-chemical
++	I-chemical
+and	O
+Fe3	B-chemical
++,	I-chemical
+which	O
+were	O
+analyzed	O
+by	O
+Native	B-experimental_method
+PAGE	I-experimental_method
+alongside	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+.	O
+
+(	O
+A	O
+)	O
+10	O
+%	O
+Native	B-experimental_method
+PAGE	I-experimental_method
+analysis	O
+of	O
+EncFtnsH	B-protein
+monomer	B-oligomeric_state
+fractions	O
+mixed	O
+with	O
+various	O
+metal	O
+solutions	O
+;	O
+(	O
+B	O
+)	O
+10	O
+%	O
+Native	B-experimental_method
+PAGE	I-experimental_method
+analysis	O
+of	O
+EncFtnsH	B-protein
+decamer	B-oligomeric_state
+fractions	O
+mixed	O
+with	O
+various	O
+metal	O
+solutions	O
+;	O
+(	O
+C	O
+)	O
+15	O
+%	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+analysis	O
+on	O
+the	O
+mixtures	O
+of	O
+EncFtnsH	B-protein
+monomer	B-oligomeric_state
+fractions	O
+and	O
+metal	O
+solutions	O
+;	O
+(	O
+D	O
+)	O
+15	O
+%	O
+SDS	B-experimental_method
+-	I-experimental_method
+PAGE	I-experimental_method
+analysis	O
+on	O
+the	O
+mixtures	O
+of	O
+EncFtnsH	B-protein
+decamer	B-oligomeric_state
+fractions	O
+and	O
+metal	O
+solutions	O
+.	O
+
+Effect	O
+of	O
+Fe2	B-chemical
++	I-chemical
+and	O
+protein	O
+concentration	O
+on	O
+the	O
+oligomeric	O
+state	O
+of	O
+EncFtnsH	B-protein
+in	O
+solution	O
+.	O
+
+(	O
+A	O
+)	O
+Recombinant	O
+EncFtnsH	B-protein
+was	O
+purified	O
+by	O
+Gel	B-experimental_method
+filtration	I-experimental_method
+Superdex	O
+200	O
+chromatography	O
+from	O
+E	B-species
+.	I-species
+coli	I-species
+BL21	I-species
+(	I-species
+DE3	I-species
+)	I-species
+grown	O
+in	O
+MM	B-experimental_method
+or	O
+in	O
+MM	B-experimental_method
+supplemented	O
+with	O
+1	O
+mM	O
+Fe	B-chemical
+(	I-chemical
+NH4	I-chemical
+)	I-chemical
+2	I-chemical
+(	I-chemical
+SO4	I-chemical
+)	I-chemical
+2	I-chemical
+(	O
+MM	B-experimental_method
++	O
+Fe2	B-chemical
++).	I-chemical
+
+A	O
+higher	O
+proportion	O
+of	O
+decamer	B-oligomeric_state
+(	O
+peak	O
+between	O
+65	O
+and	O
+75	O
+ml	O
+)	O
+is	O
+seen	O
+in	O
+the	O
+sample	O
+purified	O
+from	O
+MM	B-experimental_method
++	O
+Fe2	B-chemical
++	I-chemical
+compared	O
+to	O
+EncFtnsH	B-protein
+-	O
+MM	B-experimental_method
+,	O
+indicating	O
+that	O
+Fe2	B-chemical
++	I-chemical
+facilitates	O
+the	O
+multimerization	O
+of	O
+EncFtnsH	B-protein
+in	O
+vivo	O
+.	O
+(	O
+B	O
+)	O
+EncFtnsH	B-protein
+-	O
+monomer	B-oligomeric_state
+was	O
+incubated	O
+with	O
+one	O
+molar	O
+equivalent	O
+of	O
+Fe2	B-chemical
++	I-chemical
+salts	O
+for	O
+two	O
+hours	O
+prior	O
+to	O
+analytical	B-experimental_method
+gel	I-experimental_method
+-	I-experimental_method
+filtration	I-experimental_method
+using	O
+a	O
+Superdex	O
+200	O
+PC	O
+3	O
+.	O
+2	O
+/	O
+30	O
+column	O
+(	O
+GE	O
+Healthcare	O
+).	O
+
+Both	O
+Fe2	B-chemical
++	I-chemical
+salts	O
+tested	O
+induced	O
+the	O
+formation	O
+of	O
+decamer	B-oligomeric_state
+indicated	O
+by	O
+the	O
+peak	O
+between	O
+1	O
+.	O
+2	O
+and	O
+1	O
+.	O
+6	O
+ml	O
+.	O
+
+Monomeric	B-oligomeric_state
+and	O
+decameric	B-oligomeric_state
+samples	O
+of	O
+EncFtnsH	B-protein
+are	O
+shown	O
+as	O
+controls	O
+.	O
+
+Peaks	B-evidence
+around	O
+0	O
+.	O
+8	O
+ml	O
+were	O
+seen	O
+as	O
+protein	O
+aggregation	O
+.	O
+
+(	O
+C	O
+)	O
+Analytical	B-experimental_method
+gel	I-experimental_method
+filtration	I-experimental_method
+of	O
+EncFtn	B-protein
+monomer	B-oligomeric_state
+at	O
+different	O
+concentrations	O
+to	O
+illustrate	O
+the	O
+effect	O
+of	O
+protein	O
+concentration	O
+on	O
+multimerization	O
+.	O
+
+The	O
+major	O
+peak	O
+shows	O
+a	O
+shift	O
+towards	O
+a	O
+dimer	B-oligomeric_state
+species	O
+at	O
+high	O
+concentration	O
+of	O
+protein	O
+,	O
+but	O
+the	O
+ratio	O
+of	O
+this	O
+peak	O
+(	O
+1	O
+.	O
+5	O
+–	O
+1	O
+.	O
+8	O
+ml	O
+)	O
+to	O
+the	O
+decamer	B-oligomeric_state
+peak	O
+(	O
+1	O
+.	O
+2	O
+–	O
+1	O
+.	O
+5	O
+ml	O
+)	O
+does	O
+not	O
+change	O
+when	O
+compared	O
+to	O
+the	O
+low	O
+concentration	O
+sample	O
+.	O
+
+Gel	B-experimental_method
+-	I-experimental_method
+filtration	I-experimental_method
+peak	B-evidence
+area	I-evidence
+ratios	I-evidence
+for	O
+EncFtnsH	B-protein
+decamer	B-oligomeric_state
+and	O
+monomer	B-oligomeric_state
+on	O
+addition	O
+of	O
+different	O
+metal	O
+ions	O
+.	O
+
+EncFtnsH	B-protein
+was	O
+produced	O
+in	O
+E	B-species
+.	I-species
+coli	I-species
+BL21	I-species
+(	I-species
+DE3	I-species
+)	I-species
+cultured	O
+in	O
+MM	B-experimental_method
+and	O
+MM	B-experimental_method
+with	O
+1	O
+mM	O
+Fe	B-chemical
+(	I-chemical
+NH4	I-chemical
+)	I-chemical
+2	I-chemical
+(	I-chemical
+SO4	I-chemical
+)	I-chemical
+2	I-chemical
+(	O
+MM	B-experimental_method
++	O
+Fe2	B-chemical
++)	I-chemical
+and	O
+purified	O
+by	O
+gel	B-experimental_method
+-	I-experimental_method
+filtration	I-experimental_method
+chromatography	I-experimental_method
+using	O
+an	O
+Superdex	O
+200	O
+16	O
+/	O
+60	O
+column	O
+(	O
+GE	O
+Healthcare	O
+).	O
+
+Monomer	B-oligomeric_state
+fractions	O
+of	O
+EncFtnsH	B-protein
+purified	O
+from	O
+MM	B-experimental_method
+were	O
+pooled	O
+and	O
+run	O
+in	O
+subsequent	O
+analytical	B-experimental_method
+gel	I-experimental_method
+-	I-experimental_method
+filtration	I-experimental_method
+runs	O
+over	O
+the	O
+course	O
+of	O
+three	O
+days	O
+.	O
+
+Samples	O
+of	O
+EncFtnsH	B-protein
+monomer	B-oligomeric_state
+were	O
+incubated	O
+with	O
+one	O
+molar	O
+equivalent	O
+of	O
+metal	O
+ion	O
+salts	O
+at	O
+room	O
+temperature	O
+for	O
+two	O
+hours	O
+before	O
+analysis	O
+by	O
+analytical	B-experimental_method
+gel	I-experimental_method
+filtration	I-experimental_method
+chromatography	I-experimental_method
+(	O
+AGF	B-experimental_method
+)	O
+using	O
+a	O
+Superdex	O
+200	O
+10	O
+/	O
+300	O
+GL	O
+column	O
+.	O
+
+The	O
+area	O
+for	O
+resulting	O
+protein	O
+peaks	B-evidence
+were	O
+calculated	O
+using	O
+the	O
+Unicorn	O
+software	O
+(	O
+GE	O
+Healthcare	O
+);	O
+peak	B-evidence
+ratios	I-evidence
+were	O
+calculated	O
+to	O
+quantify	O
+the	O
+propensity	O
+of	O
+EncFtnsH	B-protein
+to	O
+multimerize	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+the	O
+different	O
+metal	O
+ions	O
+.	O
+
+The	O
+change	O
+in	O
+the	O
+ratios	O
+of	O
+monomer	B-oligomeric_state
+to	O
+decamer	B-oligomeric_state
+over	O
+the	O
+three	O
+days	O
+of	O
+experiments	O
+may	O
+be	O
+a	O
+consequence	O
+of	O
+experimental	O
+variability	O
+,	O
+or	O
+the	O
+propensity	O
+of	O
+this	O
+protein	O
+to	O
+equilibrate	O
+towards	O
+decamer	B-oligomeric_state
+over	O
+time	O
+.	O
+
+The	O
+increased	O
+decamer	B-oligomeric_state
+:	O
+monomer	B-oligomeric_state
+ratio	O
+seen	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+Fe2	B-chemical
++,	I-chemical
+Co2	B-chemical
++,	I-chemical
+and	O
+Zn2	B-chemical
++	I-chemical
+indicates	O
+that	O
+these	O
+metal	O
+ions	O
+facilitate	O
+multimerization	O
+of	O
+the	O
+EncFtnsH	B-protein
+protein	O
+,	O
+while	O
+the	O
+other	O
+metal	O
+ions	O
+tested	O
+do	O
+not	O
+appear	O
+to	O
+induce	O
+multimerization	O
+.	O
+
+The	O
+analytical	B-experimental_method
+gel	I-experimental_method
+filtration	I-experimental_method
+experiment	O
+was	O
+repeated	O
+twice	O
+using	O
+two	O
+independent	O
+preparations	O
+of	O
+protein	O
+,	O
+of	O
+which	O
+values	O
+calculated	O
+from	O
+one	O
+sample	O
+are	O
+presented	O
+here	O
+.	O
+
+Method	O
+Sample	O
+Monomer	B-oligomeric_state
+area	O
+Decamer	B-oligomeric_state
+area	O
+Decamer	B-oligomeric_state
+/	O
+Monomer	B-oligomeric_state
+Gel	B-experimental_method
+filtration	I-experimental_method
+Superdex	O
+200	O
+chromatography	O
+EncFtnsH	B-protein
+-	O
+MM	B-experimental_method
+64	O
+.	O
+3	O
+583	O
+.	O
+6	O
+0	O
+.	O
+1	O
+EncFtnsH	B-protein
+-	O
+MM	B-experimental_method
++	O
+Fe2	B-chemical
++	I-chemical
+1938	O
+.	O
+4	O
+426	O
+.	O
+4	O
+4	O
+.	O
+5	O
+Analytical	B-experimental_method
+Gel	I-experimental_method
+filtration	I-experimental_method
+Day1	O
+EncFtnsH	B-protein
+-	O
+decamer	B-oligomeric_state
+fractions	O
+20	O
+.	O
+2	O
+1	O
+.	O
+8	O
+11	O
+.	O
+2	O
+EncFtnsH	B-protein
+-	O
+monomer	B-oligomeric_state
+fractions	O
+2	O
+.	O
+9	O
+21	O
+.	O
+9	O
+0	O
+.	O
+1	O
+Fe	B-chemical
+(	I-chemical
+NH4	I-chemical
+)	I-chemical
+2	I-chemical
+(	I-chemical
+SO4	I-chemical
+)	I-chemical
+2	I-chemical
+/	O
+EncFtnsH	B-protein
+-	O
+monomer	B-oligomeric_state
+11	O
+.	O
+0	O
+13	O
+.	O
+0	O
+0	O
+.	O
+8	O
+FeSO4	B-chemical
+-	I-chemical
+HCl	I-chemical
+/	O
+EncFtnsH	B-protein
+-	O
+monomer	B-oligomeric_state
+11	O
+.	O
+3	O
+11	O
+.	O
+4	O
+1	O
+.	O
+0	O
+Analytical	B-experimental_method
+Gel	I-experimental_method
+filtration	I-experimental_method
+Day2	O
+EncFtnsH	B-protein
+-	O
+monomer	B-oligomeric_state
+fractions	O
+8	O
+.	O
+3	O
+22	O
+.	O
+8	O
+0	O
+.	O
+4	O
+CoCl2	B-chemical
+/	O
+EncFtnsH	B-protein
+-	O
+monomer	B-oligomeric_state
+17	O
+.	O
+7	O
+14	O
+.	O
+5	O
+1	O
+.	O
+2	O
+MnCl2	B-chemical
+/	O
+EncFtnsH	B-protein
+-	O
+monomer	B-oligomeric_state
+3	O
+.	O
+1	O
+30	O
+.	O
+5	O
+0	O
+.	O
+1	O
+ZnSO4	B-chemical
+/	O
+EncFtnsH	B-protein
+-	O
+monomer	B-oligomeric_state
+20	O
+.	O
+4	O
+9	O
+.	O
+0	O
+2	O
+.	O
+3	O
+FeCl3	B-chemical
+/	O
+EncFtnsH	B-protein
+-	O
+monomer	B-oligomeric_state
+3	O
+.	O
+9	O
+28	O
+.	O
+6	O
+0	O
+.	O
+1	O
+Analytical	B-experimental_method
+Gel	I-experimental_method
+filtration	I-experimental_method
+Day3	O
+EncFtnsH	B-protein
+-	O
+monomer	B-oligomeric_state
+fractions	O
+6	O
+.	O
+3	O
+23	O
+.	O
+4	O
+0	O
+.	O
+3	O
+MgSO4	B-chemical
+/	O
+EncFtnsH	B-protein
+-	O
+monomer	B-oligomeric_state
+5	O
+.	O
+8	O
+30	O
+.	O
+2	O
+0	O
+.	O
+2	O
+Ca	B-chemical
+acetate	I-chemical
+/	O
+EncFtnsH	B-protein
+-	O
+monomer	B-oligomeric_state
+5	O
+.	O
+6	O
+25	O
+.	O
+2	O
+0	O
+.	O
+2	O
+
+We	O
+purified	O
+EncFtnsH	B-protein
+from	O
+E	B-species
+.	I-species
+coli	I-species
+grown	O
+in	O
+MM	B-experimental_method
+with	O
+or	O
+without	O
+the	O
+addition	O
+of	O
+1	O
+mM	O
+Fe	B-chemical
+(	I-chemical
+NH4	I-chemical
+)	I-chemical
+2	I-chemical
+(	I-chemical
+SO4	I-chemical
+)	I-chemical
+2	I-chemical
+.	O
+
+The	O
+decamer	B-oligomeric_state
+to	O
+monomer	B-oligomeric_state
+ratio	O
+in	O
+the	O
+sample	O
+purified	O
+from	O
+cells	O
+grown	O
+in	O
+iron	B-chemical
+-	O
+supplemented	O
+media	O
+was	O
+4	O
+.	O
+5	O
+,	O
+while	O
+that	O
+from	O
+the	O
+iron	B-protein_state
+-	I-protein_state
+free	I-protein_state
+media	O
+was	O
+0	O
+.	O
+11	O
+,	O
+suggesting	O
+that	O
+iron	B-chemical
+induces	O
+the	O
+oligomerization	O
+of	O
+EncFtnsH	B-protein
+in	O
+vivo	O
+(	O
+Figure	O
+3A	O
+,	O
+Table	O
+3	O
+).	O
+
+To	O
+test	O
+the	O
+metal	O
+-	O
+dependent	O
+oligomerization	O
+of	O
+EncFtnsH	B-protein
+in	O
+vitro	O
+,	O
+we	O
+incubated	B-experimental_method
+the	O
+protein	O
+with	O
+various	O
+metal	O
+cations	O
+and	O
+subjected	O
+samples	O
+to	O
+analytical	B-experimental_method
+SEC	I-experimental_method
+and	O
+non	B-experimental_method
+-	I-experimental_method
+denaturing	I-experimental_method
+PAGE	I-experimental_method
+.	O
+
+Of	O
+the	O
+metals	O
+tested	O
+,	O
+only	O
+Fe2	B-chemical
++,	I-chemical
+Zn2	B-chemical
++	I-chemical
+and	O
+Co2	B-chemical
++	I-chemical
+induced	O
+the	O
+formation	O
+of	O
+significant	O
+amounts	O
+of	O
+the	O
+decamer	B-oligomeric_state
+(	O
+Figure	O
+3B	O
+,	O
+Figure	O
+3	O
+—	O
+figure	O
+supplement	O
+1	O
+/	O
+2	O
+).	O
+
+While	O
+Fe2	B-chemical
++	I-chemical
+induces	O
+the	O
+multimerization	O
+of	O
+EncFtnsH	B-protein
+,	O
+Fe3	B-chemical
++	I-chemical
+in	O
+the	O
+form	O
+of	O
+FeCl3	B-chemical
+does	O
+not	O
+have	O
+this	O
+effect	O
+on	O
+the	O
+protein	O
+,	O
+highlighting	O
+the	O
+apparent	O
+preference	O
+this	O
+protein	O
+has	O
+for	O
+the	O
+ferrous	B-chemical
+form	I-chemical
+of	I-chemical
+iron	I-chemical
+.	O
+
+To	O
+determine	O
+if	O
+the	O
+oligomerization	O
+of	O
+EncFtnsH	B-protein
+was	O
+concentration	O
+dependent	O
+we	O
+performed	O
+analytical	B-experimental_method
+SEC	I-experimental_method
+at	O
+90	O
+and	O
+700	O
+µM	O
+protein	O
+concentration	O
+(	O
+Figure	O
+3C	O
+).	O
+
+At	O
+the	O
+higher	O
+concentration	O
+,	O
+no	O
+increase	O
+in	O
+the	O
+decameric	B-oligomeric_state
+form	O
+of	O
+EncFtn	B-protein
+was	O
+observed	O
+;	O
+however	O
+,	O
+the	O
+shift	O
+in	O
+the	O
+major	O
+peak	O
+from	O
+the	O
+position	O
+of	O
+the	O
+monomer	B-oligomeric_state
+species	O
+indicated	O
+a	O
+tendency	O
+to	O
+dimerize	B-oligomeric_state
+at	O
+high	O
+concentration	O
+.	O
+
+Crystal	B-evidence
+structure	I-evidence
+of	O
+EncFtnsH	B-protein
+
+Electrostatic	O
+surface	O
+of	O
+EncFtnsH	B-protein
+.	O
+
+The	O
+solvent	O
+accessible	O
+surface	O
+of	O
+EncFtnsH	B-protein
+is	O
+shown	O
+,	O
+colored	O
+by	O
+electrostatic	O
+potential	O
+as	O
+calculated	O
+using	O
+the	O
+APBS	O
+plugin	O
+in	O
+PyMOL	O
+.	O
+
+Negatively	O
+charged	O
+regions	O
+are	O
+colored	O
+red	O
+and	O
+positive	O
+regions	O
+in	O
+blue	O
+,	O
+neutral	O
+regions	O
+in	O
+grey	O
+.	O
+(	O
+A	O
+)	O
+View	O
+of	O
+the	O
+surface	O
+of	O
+the	O
+EncFtnsH	B-protein
+decamer	B-oligomeric_state
+looking	O
+down	O
+the	O
+central	O
+axis	O
+.	O
+
+(	O
+B	O
+)	O
+Orthogonal	O
+view	O
+of	O
+(	O
+A	O
+).	O
+(	O
+C	O
+)	O
+Cutaway	O
+view	O
+of	O
+(	O
+B	O
+)	O
+showing	O
+the	O
+charge	O
+distribution	O
+within	O
+the	O
+central	B-site
+cavity	I-site
+.	O
+
+Crystal	B-evidence
+structure	I-evidence
+of	O
+EncFtnsH	B-protein
+.	O
+
+(	O
+A	O
+)	O
+Overall	O
+architecture	O
+of	O
+EncFtnsH	B-protein
+.	O
+Transparent	O
+solvent	O
+accessible	O
+surface	O
+view	O
+with	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+shown	O
+as	O
+tubes	O
+and	O
+bound	O
+metal	O
+ions	O
+as	O
+spheres	O
+.	O
+
+Alternating	O
+subunits	B-structure_element
+are	O
+colored	O
+blue	O
+and	O
+green	O
+for	O
+clarity	O
+.	O
+
+The	O
+doughnut	B-structure_element
+-	I-structure_element
+like	I-structure_element
+decamer	B-oligomeric_state
+is	O
+7	O
+nm	O
+in	O
+diameter	O
+and	O
+4	O
+.	O
+5	O
+nm	O
+thick	O
+.	O
+(	O
+B	O
+)	O
+Monomer	B-oligomeric_state
+of	O
+EncFtnsH	B-protein
+shown	O
+as	O
+a	O
+secondary	O
+structure	O
+cartoon	O
+.	O
+(	O
+C	O
+/	O
+D	O
+)	O
+Dimer	B-site
+interfaces	I-site
+formed	O
+in	O
+the	O
+decameric	B-oligomeric_state
+ring	B-structure_element
+of	O
+EncFtnsH	B-protein
+.	O
+Subunits	B-structure_element
+are	O
+shown	O
+as	O
+secondary	O
+structure	O
+cartoons	O
+and	O
+colored	O
+blue	O
+and	O
+green	O
+for	O
+clarity	O
+.	O
+
+Bound	O
+metal	O
+ions	O
+are	O
+shown	O
+as	O
+orange	O
+spheres	O
+for	O
+Fe3	B-chemical
++	I-chemical
+and	O
+grey	O
+and	O
+white	O
+spheres	O
+for	O
+Ca2	B-chemical
++.	I-chemical
+
+We	O
+determined	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+EncFtnsH	B-protein
+by	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+to	O
+2	O
+.	O
+0	O
+Å	O
+resolution	O
+(	O
+see	O
+Table	O
+1	O
+for	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+data	I-evidence
+collection	I-evidence
+and	I-evidence
+refinement	I-evidence
+statistics	I-evidence
+).	O
+
+The	O
+crystallographic	O
+asymmetric	O
+unit	O
+contained	O
+thirty	O
+monomers	B-oligomeric_state
+of	O
+EncFtn	B-protein
+with	O
+visible	O
+electron	B-evidence
+density	I-evidence
+for	O
+residues	O
+7	B-residue_range
+–	I-residue_range
+96	I-residue_range
+in	O
+each	O
+chain	O
+.	O
+
+The	O
+protein	O
+chains	O
+were	O
+arranged	O
+as	O
+three	O
+identical	O
+annular	B-structure_element
+decamers	B-oligomeric_state
+,	O
+each	O
+with	O
+D5	O
+symmetry	O
+.	O
+
+The	O
+decamer	B-oligomeric_state
+has	O
+a	O
+diameter	O
+of	O
+7	O
+nm	O
+and	O
+thickness	O
+of	O
+4	O
+nm	O
+(	O
+Figure	O
+4A	O
+).	O
+
+The	O
+monomer	B-oligomeric_state
+of	O
+EncFtn	B-protein
+has	O
+an	O
+N	O
+-	O
+terminal	O
+310	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+that	O
+precedes	O
+two	O
+4	O
+nm	O
+long	O
+antiparallel	B-structure_element
+α	I-structure_element
+-	I-structure_element
+helices	I-structure_element
+arranged	O
+with	O
+their	O
+long	O
+axes	O
+at	O
+25	O
+°	O
+to	O
+each	O
+other	O
+;	O
+these	O
+helices	B-structure_element
+are	O
+followed	O
+by	O
+a	O
+shorter	O
+1	O
+.	O
+4	O
+nm	O
+helix	B-structure_element
+projecting	O
+at	O
+70	O
+°	O
+from	O
+α2	B-structure_element
+(	O
+Figure	O
+4B	O
+).	O
+
+The	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+the	O
+crystallized	O
+construct	O
+extends	O
+from	O
+the	O
+outer	O
+circumference	O
+of	O
+the	O
+ring	B-structure_element
+,	O
+indicating	O
+that	O
+the	O
+encapsulin	B-site
+localization	I-site
+sequence	I-site
+in	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+protein	O
+is	O
+on	O
+the	O
+exterior	O
+of	O
+the	O
+ring	B-structure_element
+and	O
+is	O
+thus	O
+free	O
+to	O
+interact	O
+with	O
+its	O
+binding	B-site
+site	I-site
+on	O
+the	O
+encapsulin	B-protein
+shell	B-structure_element
+protein	O
+.	O
+
+The	O
+monomer	B-oligomeric_state
+of	O
+EncFtnsH	B-protein
+forms	O
+two	O
+distinct	O
+dimer	B-site
+interfaces	I-site
+within	O
+the	O
+decamer	B-oligomeric_state
+(	O
+Figure	O
+4	O
+C	O
+/	O
+D	O
+).	O
+
+The	O
+first	O
+dimer	B-oligomeric_state
+is	O
+formed	O
+from	O
+two	O
+monomers	B-oligomeric_state
+arranged	O
+antiparallel	O
+to	O
+each	O
+other	O
+,	O
+with	O
+α1	B-structure_element
+from	O
+each	O
+monomer	B-oligomeric_state
+interacting	O
+along	O
+their	O
+lengths	O
+and	O
+α3	B-structure_element
+interdigitating	O
+with	O
+α2	B-structure_element
+and	O
+α3	B-structure_element
+of	O
+the	O
+partner	O
+chain	O
+.	O
+
+This	O
+interface	B-site
+buries	O
+one	O
+third	O
+of	O
+the	O
+surface	O
+area	O
+from	O
+each	O
+partner	O
+and	O
+is	O
+stabilized	O
+by	O
+thirty	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+and	O
+fourteen	O
+salt	B-bond_interaction
+bridges	I-bond_interaction
+(	O
+Figure	O
+4C	O
+).	O
+
+The	O
+second	O
+dimer	B-site
+interface	I-site
+forms	O
+an	O
+antiparallel	B-structure_element
+four	I-structure_element
+-	I-structure_element
+helix	I-structure_element
+bundle	I-structure_element
+between	O
+helices	B-structure_element
+1	I-structure_element
+and	I-structure_element
+2	I-structure_element
+from	O
+each	O
+monomer	B-oligomeric_state
+(	O
+Figure	O
+4D	O
+).	O
+
+This	O
+interface	B-site
+is	O
+less	O
+extensive	O
+than	O
+the	O
+first	O
+and	O
+is	O
+stabilized	O
+by	O
+twenty	O
+-	O
+one	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+,	O
+six	O
+salt	B-bond_interaction
+bridges	I-bond_interaction
+,	O
+and	O
+a	O
+number	O
+of	O
+metal	O
+ions	O
+.	O
+
+The	O
+arrangement	O
+of	O
+ten	O
+monomers	B-oligomeric_state
+in	O
+alternating	O
+orientation	O
+forms	O
+the	O
+decamer	B-oligomeric_state
+of	O
+EncFtn	B-protein
+,	O
+which	O
+assembles	O
+as	O
+a	O
+pentamer	B-oligomeric_state
+of	O
+dimers	B-oligomeric_state
+(	O
+Figure	O
+4A	O
+).	O
+
+Each	O
+monomer	B-oligomeric_state
+lies	O
+at	O
+45	O
+°	O
+relative	O
+to	O
+the	O
+vertical	O
+central	O
+-	O
+axis	O
+of	O
+the	O
+ring	B-structure_element
+,	O
+with	O
+the	O
+N	O
+-	O
+termini	O
+of	O
+alternating	O
+subunits	B-structure_element
+capping	O
+the	O
+center	O
+of	O
+the	O
+ring	B-structure_element
+at	O
+each	O
+end	O
+,	O
+while	O
+the	O
+C	O
+-	O
+termini	O
+are	O
+arranged	O
+around	O
+the	O
+circumference	O
+.	O
+
+The	O
+central	B-site
+hole	I-site
+in	O
+the	O
+ring	B-structure_element
+is	O
+2	O
+.	O
+5	O
+nm	O
+at	O
+its	O
+widest	O
+in	O
+the	O
+center	O
+of	O
+the	O
+complex	O
+,	O
+and	O
+1	O
+.	O
+5	O
+nm	O
+at	O
+its	O
+narrowest	O
+point	O
+near	O
+the	O
+outer	O
+surface	O
+,	O
+although	O
+it	O
+should	O
+be	O
+noted	O
+that	O
+a	O
+number	O
+of	O
+residues	O
+at	O
+the	O
+N	O
+-	O
+terminus	O
+are	O
+not	O
+visible	O
+in	O
+the	O
+crystallographic	B-evidence
+electron	I-evidence
+density	I-evidence
+and	O
+these	O
+may	O
+occupy	O
+the	O
+central	B-site
+channel	I-site
+.	O
+
+The	O
+surface	O
+of	O
+the	O
+decamer	B-oligomeric_state
+has	O
+distinct	O
+negatively	B-site
+charged	I-site
+patches	I-site
+,	O
+both	O
+within	O
+the	O
+central	B-site
+hole	I-site
+and	O
+on	O
+the	O
+outer	O
+circumference	O
+,	O
+which	O
+form	O
+spokes	B-structure_element
+through	O
+the	O
+radius	O
+of	O
+the	O
+complex	O
+(	O
+Figure	O
+4	O
+—	O
+figure	O
+supplement	O
+1	O
+).	O
+
+EncFtn	B-protein
+ferroxidase	B-site
+center	I-site
+
+Putative	O
+ligand	B-site
+-	I-site
+binding	I-site
+site	I-site
+in	O
+EncFtnsH	B-protein
+.	O
+
+(	O
+A	O
+)	O
+Wall	O
+-	O
+eyed	O
+stereo	O
+view	O
+of	O
+the	O
+dimer	B-site
+interface	I-site
+of	O
+EncFtn	B-protein
+.	O
+
+Protein	O
+chains	O
+are	O
+shown	O
+as	O
+sticks	O
+,	O
+with	O
+2mFo	B-evidence
+-	I-evidence
+DFc	I-evidence
+electron	I-evidence
+density	I-evidence
+shown	O
+in	O
+blue	O
+mesh	O
+and	O
+contoured	O
+at	O
+1	O
+.	O
+5	O
+σ	O
+and	O
+mFo	B-evidence
+-	I-evidence
+DFc	I-evidence
+shown	O
+in	O
+green	O
+mesh	O
+and	O
+contoured	O
+at	O
+3	O
+σ	O
+.	O
+(	O
+B	O
+)	O
+Wall	O
+-	O
+eyed	O
+stereo	O
+view	O
+of	O
+putative	O
+metal	B-site
+binding	I-site
+site	I-site
+at	O
+the	O
+external	O
+surface	O
+of	O
+EncFtnsH	B-protein
+.	O
+Protein	O
+chains	O
+and	O
+electron	B-evidence
+density	I-evidence
+maps	I-evidence
+are	O
+shown	O
+as	O
+in	O
+(	O
+A	O
+).	O
+
+EncFtnsH	B-protein
+metal	B-site
+binding	I-site
+sites	I-site
+.	O
+
+(	O
+A	O
+)	O
+Wall	O
+-	O
+eyed	O
+stereo	O
+view	O
+of	O
+the	O
+metal	B-site
+-	I-site
+binding	I-site
+dimerization	I-site
+interface	I-site
+of	O
+EncFtnsH	B-protein
+.	O
+Protein	O
+residues	O
+are	O
+shown	O
+as	O
+sticks	O
+with	O
+blue	O
+and	O
+green	O
+carbons	O
+for	O
+the	O
+different	O
+subunits	B-structure_element
+,	O
+iron	B-chemical
+ions	O
+are	O
+shown	O
+as	O
+orange	O
+spheres	O
+and	O
+calcium	B-chemical
+as	O
+grey	O
+spheres	O
+,	O
+and	O
+the	O
+glycolic	B-chemical
+acid	I-chemical
+ligand	O
+is	O
+shown	O
+with	O
+yellow	O
+carbon	O
+atoms	O
+coordinated	O
+above	O
+the	O
+di	B-site
+-	I-site
+iron	I-site
+center	I-site
+.	O
+
+The	O
+2mFo	B-evidence
+-	I-evidence
+DFc	I-evidence
+electron	I-evidence
+density	I-evidence
+map	I-evidence
+is	O
+shown	O
+as	O
+a	O
+blue	O
+mesh	O
+contoured	O
+at	O
+1	O
+.	O
+5	O
+σ	O
+and	O
+the	O
+NCS	B-evidence
+-	I-evidence
+averaged	I-evidence
+anomalous	I-evidence
+difference	I-evidence
+map	I-evidence
+is	O
+shown	O
+as	O
+an	O
+orange	O
+mesh	O
+and	O
+contoured	O
+at	O
+10	O
+σ	O
+.	O
+(	O
+B	O
+)	O
+Iron	B-chemical
+coordination	B-bond_interaction
+within	O
+the	O
+FOC	B-site
+including	O
+residues	O
+Glu32	B-residue_name_number
+,	O
+Glu62	B-residue_name_number
+,	O
+His65	B-residue_name_number
+and	O
+Tyr39	B-residue_name_number
+from	O
+two	O
+chains	O
+.	O
+
+Protein	O
+and	O
+metal	O
+ions	O
+are	O
+shown	O
+as	O
+in	O
+A	O
+.	O
+Coordination	B-bond_interaction
+between	O
+the	O
+protein	O
+and	O
+iron	B-chemical
+ions	O
+is	O
+shown	O
+as	O
+yellow	O
+dashed	O
+lines	O
+with	O
+distances	O
+indicated	O
+.	O
+(	O
+C	O
+)	O
+Coordination	B-bond_interaction
+of	O
+calcium	B-chemical
+within	O
+the	O
+dimer	B-site
+interface	I-site
+by	O
+four	O
+glutamic	B-residue_name
+acid	I-residue_name
+residues	O
+(	O
+E31	B-residue_name_number
+and	O
+E34	B-residue_name_number
+from	O
+two	O
+chains	O
+).	O
+
+The	O
+calcium	B-chemical
+ion	O
+is	O
+shown	O
+as	O
+a	O
+grey	O
+sphere	O
+and	O
+water	B-chemical
+molecules	O
+involved	O
+in	O
+the	O
+coordination	B-bond_interaction
+of	O
+the	O
+calcium	B-chemical
+ion	O
+are	O
+shown	O
+as	O
+crosses	O
+.	O
+(	O
+D	O
+)	O
+Metal	B-site
+coordination	I-site
+site	I-site
+on	O
+the	O
+outer	O
+surface	O
+of	O
+EncFtnsH	B-protein
+.	O
+The	O
+two	O
+calcium	B-chemical
+ions	O
+are	O
+coordinated	B-bond_interaction
+by	I-bond_interaction
+residues	O
+His57	B-residue_name_number
+,	O
+Glu61	B-residue_name_number
+and	O
+Glu64	B-residue_name_number
+from	O
+the	O
+two	O
+chains	O
+of	O
+the	O
+FOC	B-site
+dimer	B-oligomeric_state
+,	O
+and	O
+are	O
+located	O
+at	O
+the	O
+outer	O
+surface	O
+of	O
+the	O
+complex	O
+,	O
+positioned	O
+10	O
+Å	O
+away	O
+from	O
+the	O
+FOC	B-site
+iron	B-chemical
+.	O
+
+The	O
+electron	B-evidence
+density	I-evidence
+maps	I-evidence
+of	O
+the	O
+initial	O
+EncFtnsH	B-protein
+model	O
+displayed	O
+significant	O
+positive	O
+peaks	O
+in	O
+the	O
+mFo	B-evidence
+-	I-evidence
+DFc	I-evidence
+map	I-evidence
+at	O
+the	O
+center	O
+of	O
+the	O
+4	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+bundle	I-structure_element
+dimer	B-oligomeric_state
+(	O
+Figure	O
+5	O
+—	O
+figure	O
+supplement	O
+1	O
+).	O
+
+Informed	O
+by	O
+the	O
+ICP	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+data	O
+indicating	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+iron	B-chemical
+in	O
+the	O
+protein	O
+we	O
+collected	O
+diffraction	B-evidence
+data	I-evidence
+at	O
+the	O
+experimentally	O
+determined	O
+iron	B-chemical
+absorption	O
+edge	O
+(	O
+1	O
+.	O
+74	O
+Å	O
+)	O
+and	O
+calculated	O
+an	O
+anomalous	B-evidence
+difference	I-evidence
+Fourier	I-evidence
+map	I-evidence
+using	O
+this	O
+data	O
+.	O
+
+Inspection	O
+of	O
+this	O
+map	B-evidence
+showed	O
+two	O
+10	O
+-	O
+sigma	O
+peaks	B-evidence
+between	O
+residues	O
+Glu32	B-residue_name_number
+,	O
+Glu62	B-residue_name_number
+and	O
+His65	B-residue_name_number
+of	O
+two	O
+adjacent	O
+chains	O
+,	O
+and	O
+a	O
+statistically	O
+smaller	O
+5	O
+-	O
+sigma	O
+peak	O
+between	O
+residues	O
+Glu31	B-residue_name_number
+and	O
+Glu34	B-residue_name_number
+of	O
+the	O
+two	O
+chains	O
+.	O
+
+Modeling	O
+metal	O
+ions	O
+into	O
+these	O
+peaks	O
+and	O
+refinement	B-experimental_method
+of	O
+the	O
+anomalous	B-evidence
+scattering	I-evidence
+parameters	I-evidence
+allowed	O
+us	O
+to	O
+identify	O
+these	O
+as	O
+two	O
+iron	B-chemical
+ions	O
+and	O
+a	O
+calcium	B-chemical
+ion	O
+respectively	O
+(	O
+Figure	O
+5A	O
+).	O
+
+An	O
+additional	O
+region	O
+of	O
+asymmetric	O
+electron	B-evidence
+density	I-evidence
+near	O
+the	O
+di	B-site
+-	I-site
+iron	I-site
+binding	I-site
+site	I-site
+in	O
+the	O
+mFo	B-evidence
+-	I-evidence
+DFc	I-evidence
+map	I-evidence
+was	O
+modeled	O
+as	O
+glycolic	B-chemical
+acid	I-chemical
+,	O
+presumably	O
+a	O
+breakdown	O
+product	O
+of	O
+the	O
+PEG	B-chemical
+3350	I-chemical
+used	O
+for	O
+crystallization	O
+.	O
+
+This	O
+di	B-site
+-	I-site
+iron	I-site
+center	I-site
+has	O
+an	O
+Fe	B-evidence
+-	I-evidence
+Fe	I-evidence
+distance	I-evidence
+of	O
+3	O
+.	O
+5	O
+Å	O
+,	O
+Fe	B-evidence
+-	I-evidence
+Glu	I-evidence
+-	I-evidence
+O	I-evidence
+distances	I-evidence
+between	O
+2	O
+.	O
+3	O
+and	O
+2	O
+.	O
+5	O
+Å	O
+,	O
+and	O
+Fe	B-evidence
+-	I-evidence
+His	I-evidence
+-	I-evidence
+N	I-evidence
+distances	I-evidence
+of	O
+2	O
+.	O
+5	O
+Å	O
+(	O
+Figure	O
+5B	O
+).	O
+
+This	O
+coordination	B-bond_interaction
+geometry	O
+is	O
+consistent	O
+with	O
+the	O
+di	B-site
+-	I-site
+nuclear	I-site
+ferroxidase	I-site
+center	I-site
+(	O
+FOC	B-site
+)	O
+found	O
+in	O
+ferritin	B-protein_type
+.	O
+
+It	O
+is	O
+interesting	O
+to	O
+note	O
+that	O
+although	O
+we	O
+did	O
+not	O
+add	O
+any	O
+additional	O
+iron	B-chemical
+to	O
+the	O
+crystallization	B-experimental_method
+trials	I-experimental_method
+,	O
+the	O
+FOC	B-site
+was	O
+fully	O
+occupied	O
+with	O
+iron	B-chemical
+in	O
+the	O
+final	O
+structure	B-evidence
+,	O
+implying	O
+that	O
+this	O
+site	O
+has	O
+a	O
+very	O
+high	O
+affinity	B-evidence
+for	O
+iron	B-chemical
+.	O
+
+The	O
+calcium	B-chemical
+ion	O
+coordinated	B-bond_interaction
+by	I-bond_interaction
+Glu31	B-residue_name_number
+and	O
+Glu34	B-residue_name_number
+adopts	O
+heptacoordinate	B-protein_state
+geometry	O
+,	O
+with	O
+coordination	B-bond_interaction
+distances	O
+of	O
+2	O
+.	O
+5	O
+Å	O
+between	O
+the	O
+metal	O
+ion	O
+and	O
+carboxylate	O
+oxygens	O
+of	O
+Glu31	B-residue_name_number
+and	O
+Glu34	B-residue_name_number
+(	O
+E31	B-site
+/	I-site
+34	I-site
+-	I-site
+site	I-site
+).	O
+
+A	O
+number	O
+of	O
+ordered	O
+solvent	O
+molecules	O
+are	O
+also	O
+coordinated	B-bond_interaction
+to	O
+this	O
+metal	O
+ion	O
+at	O
+a	O
+distance	O
+of	O
+2	O
+.	O
+5	O
+Å	O
+.	O
+This	O
+heptacoordinate	B-protein_state
+geometry	O
+is	O
+common	O
+in	O
+crystal	B-evidence
+structures	I-evidence
+with	O
+calcium	B-chemical
+ions	O
+(	O
+Figure	O
+5C	O
+).	O
+
+While	O
+ICP	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+indicated	O
+that	O
+there	O
+were	O
+negligible	O
+amounts	O
+of	O
+calcium	B-chemical
+in	O
+the	O
+purified	O
+protein	O
+,	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+140	O
+mM	O
+calcium	B-chemical
+acetate	I-chemical
+in	O
+the	O
+crystallization	O
+mother	O
+liquor	O
+favors	O
+the	O
+coordination	B-bond_interaction
+of	O
+calcium	B-chemical
+at	O
+this	O
+site	O
+.	O
+
+The	O
+fact	O
+that	O
+the	O
+protein	O
+does	O
+not	O
+multimerize	O
+in	O
+solution	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+Fe3	B-chemical
++	I-chemical
+may	O
+indicate	O
+that	O
+these	O
+metal	B-site
+binding	I-site
+sites	I-site
+have	O
+a	O
+lower	O
+affinity	O
+for	O
+the	O
+ferric	O
+form	O
+of	O
+iron	B-chemical
+,	O
+which	O
+is	O
+the	O
+product	O
+of	O
+the	O
+ferroxidase	B-protein_type
+reaction	O
+.	O
+
+A	O
+number	O
+of	O
+additional	O
+metal	O
+-	O
+ions	O
+were	O
+present	O
+at	O
+the	O
+outer	O
+circumference	O
+of	O
+at	O
+least	O
+one	O
+decamer	B-oligomeric_state
+in	O
+the	O
+asymmetric	O
+unit	O
+(	O
+Figure	O
+5D	O
+).	O
+
+These	O
+ions	O
+are	O
+coordinated	B-bond_interaction
+by	I-bond_interaction
+His57	B-residue_name_number
+,	O
+Glu61	B-residue_name_number
+and	O
+Glu64	B-residue_name_number
+from	O
+both	O
+chains	O
+in	O
+the	O
+FOC	B-site
+dimer	B-oligomeric_state
+and	O
+are	O
+4	O
+.	O
+5	O
+Å	O
+apart	O
+;	O
+Fe	B-evidence
+-	I-evidence
+Glu	I-evidence
+-	I-evidence
+O	I-evidence
+distances	O
+are	O
+between	O
+2	O
+.	O
+5	O
+and	O
+3	O
+.	O
+5	O
+Å	O
+and	O
+the	O
+Fe	B-evidence
+-	I-evidence
+His	I-evidence
+-	I-evidence
+N	I-evidence
+distances	I-evidence
+are	O
+4	O
+and	O
+4	O
+.	O
+5	O
+Å	O
+.	O
+
+Comparison	O
+of	O
+quaternary	O
+structure	O
+of	O
+EncFtnsH	B-protein
+and	O
+ferritin	B-protein_type
+.	O
+
+(	O
+A	O
+)	O
+Aligned	B-experimental_method
+FOC	B-site
+of	O
+EncFtnsH	B-protein
+and	O
+Pseudo	B-species
+-	I-species
+nitzschia	I-species
+multiseries	I-species
+ferritin	B-protein
+(	O
+PmFtn	B-protein
+).	O
+
+The	O
+metal	B-site
+binding	I-site
+site	I-site
+residues	O
+from	O
+two	O
+EncFtnsH	B-protein
+chains	O
+are	O
+shown	O
+in	O
+green	O
+and	O
+blue	O
+,	O
+while	O
+the	O
+PmFtn	B-protein
+is	O
+shown	O
+in	O
+orange	O
+.	O
+
+Fe2	B-chemical
++	I-chemical
+in	O
+the	O
+FOC	B-site
+is	O
+shown	O
+as	O
+orange	O
+spheres	O
+and	O
+Ca2	B-chemical
++	I-chemical
+in	O
+EncFtnsH	B-protein
+is	O
+shown	O
+as	O
+a	O
+grey	O
+sphere	O
+.	O
+
+The	O
+two	O
+-	O
+fold	O
+symmetry	O
+axis	O
+of	O
+the	O
+EncFtn	B-protein
+FOC	B-site
+is	O
+shown	O
+with	O
+a	O
+grey	O
+arrow	O
+(	O
+B	O
+)	O
+Cross	O
+-	O
+section	O
+surface	O
+view	O
+of	O
+quaternary	O
+structure	O
+of	O
+EncFtnsH	B-protein
+and	O
+PmFtn	B-protein
+as	O
+aligned	O
+in	O
+(	O
+A	O
+)	O
+(	O
+dashed	O
+black	O
+box	O
+).	O
+
+The	O
+central	B-site
+channel	I-site
+of	O
+EncFtnsH	B-protein
+is	O
+spatially	O
+equivalent	O
+to	O
+the	O
+outer	O
+surface	O
+of	O
+ferritin	B-protein_type
+and	O
+its	O
+outer	O
+surface	O
+corresponds	O
+to	O
+the	O
+mineralization	B-site
+surface	I-site
+within	O
+ferritin	B-protein_type
+.	O
+
+Comparison	B-experimental_method
+of	O
+the	O
+symmetric	O
+metal	B-site
+ion	I-site
+binding	I-site
+site	I-site
+of	O
+EncFtnsH	B-protein
+and	O
+the	O
+ferritin	B-protein_type
+FOC	B-site
+.	O
+
+(	O
+A	O
+)	O
+Structural	B-experimental_method
+alignment	I-experimental_method
+of	O
+the	O
+FOC	B-site
+residues	O
+in	O
+a	O
+dimer	B-oligomeric_state
+of	O
+EncFtnsH	B-protein
+(	O
+green	O
+/	O
+blue	O
+)	O
+with	O
+a	O
+monomer	B-oligomeric_state
+of	O
+Pseudo	B-species
+-	I-species
+nitzschia	I-species
+multiseries	I-species
+ferritin	B-protein
+(	O
+PmFtn	B-protein
+)	O
+(	O
+PDBID	O
+:	O
+4ITW	O
+)	O
+(	O
+orange	O
+).	O
+
+Iron	B-chemical
+ions	O
+are	O
+shown	O
+as	O
+orange	O
+spheres	O
+and	O
+a	O
+single	O
+calcium	B-chemical
+ion	O
+as	O
+a	O
+grey	O
+sphere	O
+.	O
+
+Residues	O
+within	O
+the	O
+FOC	B-site
+are	O
+conserved	B-protein_state
+between	O
+EncFtn	B-protein
+and	O
+ferritin	B-protein_type
+PmFtn	B-protein
+,	O
+with	O
+the	O
+exception	O
+of	O
+residues	O
+in	O
+the	O
+position	O
+equivalent	O
+to	O
+H65	B-residue_name_number
+’	O
+in	O
+the	O
+second	O
+subunit	B-oligomeric_state
+in	O
+the	O
+dimer	B-oligomeric_state
+(	O
+blue	O
+).	O
+
+The	O
+site	O
+in	O
+EncFtn	B-protein
+with	O
+bound	B-protein_state
+calcium	B-chemical
+is	O
+not	O
+present	O
+in	O
+other	O
+family	O
+members	O
+.	O
+
+(	O
+B	O
+)	O
+Secondary	O
+structure	O
+of	O
+aligned	B-experimental_method
+dimeric	B-oligomeric_state
+EncFtnsH	B-protein
+and	O
+monomeric	B-oligomeric_state
+ferritin	B-protein_type
+highlighting	O
+the	O
+conserved	B-protein_state
+four	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+bundle	I-structure_element
+.	O
+
+EncFtnsH	B-protein
+monomers	B-oligomeric_state
+are	O
+shown	O
+in	O
+green	O
+and	O
+blue	O
+and	O
+aligned	B-experimental_method
+PmFtn	B-protein
+monomer	B-oligomeric_state
+in	O
+orange	O
+as	O
+in	O
+A	O
+.	O
+(	O
+C	O
+)	O
+Cartoon	O
+of	O
+secondary	O
+structure	O
+elements	O
+in	O
+EncFtn	B-protein
+dimer	B-oligomeric_state
+and	O
+ferritin	B-protein_type
+.	O
+
+In	O
+the	O
+dimer	B-oligomeric_state
+of	O
+EncFtn	B-protein
+that	O
+forms	O
+the	O
+FOC	B-site
+,	O
+the	O
+C	O
+-	O
+terminus	O
+of	O
+the	O
+first	O
+monomer	B-oligomeric_state
+(	O
+green	O
+)	O
+and	O
+N	O
+-	O
+terminus	O
+of	O
+the	O
+second	O
+monomer	B-oligomeric_state
+(	O
+blue	O
+)	O
+correspond	O
+to	O
+the	O
+position	O
+of	O
+the	O
+long	B-structure_element
+linker	I-structure_element
+between	O
+α2	B-structure_element
+and	O
+α3	B-structure_element
+in	O
+ferritin	B-protein_type
+PmFtn	B-protein
+.	O
+
+Structural	B-experimental_method
+alignment	I-experimental_method
+of	O
+the	O
+di	B-site
+-	I-site
+iron	I-site
+binding	I-site
+site	I-site
+of	O
+EncFtnsH	B-protein
+to	O
+the	O
+FOC	B-site
+of	O
+Pseudo	B-species
+-	I-species
+nitzschia	I-species
+multiseries	I-species
+ferritin	B-protein_type
+(	O
+PmFtn	B-protein
+,	O
+PDB	O
+ID	O
+:	O
+4ITW	O
+)	O
+reveals	O
+a	O
+striking	O
+similarity	O
+between	O
+the	O
+metal	B-site
+binding	I-site
+sites	I-site
+of	O
+EncFtnsH	B-protein
+and	O
+the	O
+classical	B-protein_state
+ferritins	B-protein_type
+(	O
+Figure	O
+6A	O
+).	O
+
+The	O
+di	B-site
+-	I-site
+iron	I-site
+site	I-site
+of	O
+EncFtnsH	B-protein
+is	O
+by	O
+necessity	O
+symmetrical	O
+,	O
+as	O
+it	O
+is	O
+formed	O
+through	O
+a	O
+dimer	B-site
+interface	I-site
+,	O
+while	O
+the	O
+FOC	B-site
+of	O
+ferritin	B-protein_type
+does	O
+not	O
+have	O
+these	O
+constraints	O
+and	O
+varies	O
+in	O
+different	O
+species	O
+at	O
+a	O
+position	O
+equivalent	O
+to	O
+His65	B-residue_name_number
+of	O
+the	O
+second	O
+EncFtn	B-protein
+monomer	B-oligomeric_state
+in	O
+the	O
+FOC	B-site
+interface	I-site
+(	O
+His65	B-residue_name_number
+’)	O
+(	O
+Figure	O
+6A	O
+).	O
+
+Structural	B-experimental_method
+superimposition	I-experimental_method
+of	O
+the	O
+FOCs	B-site
+of	O
+ferritin	B-protein_type
+and	O
+EncFtn	B-protein
+brings	O
+the	O
+four	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+bundle	I-structure_element
+of	O
+the	O
+ferritin	B-protein_type
+fold	O
+into	O
+close	O
+alignment	O
+with	O
+the	O
+EncFtn	B-protein
+dimer	B-oligomeric_state
+,	O
+showing	O
+that	O
+the	O
+two	O
+families	O
+of	O
+proteins	O
+have	O
+essentially	O
+the	O
+same	O
+architecture	O
+around	O
+the	O
+di	B-site
+-	I-site
+iron	I-site
+center	I-site
+(	O
+Figure	O
+6B	O
+).	O
+
+The	O
+linker	B-structure_element
+connecting	O
+helices	B-structure_element
+2	I-structure_element
+and	I-structure_element
+3	I-structure_element
+of	O
+ferritin	B-protein_type
+is	O
+congruent	O
+with	O
+the	O
+start	O
+of	O
+the	O
+C	O
+-	O
+terminal	O
+helix	B-structure_element
+of	O
+one	O
+EncFtn	B-protein
+monomer	B-oligomeric_state
+and	O
+the	O
+N	O
+-	O
+terminal	O
+310	B-structure_element
+helix	I-structure_element
+of	O
+the	O
+second	O
+monomer	B-oligomeric_state
+(	O
+Figure	O
+6C	O
+).	O
+
+Mass	B-experimental_method
+spectrometry	I-experimental_method
+of	O
+the	O
+EncFtn	B-protein
+assembly	O
+
+Native	B-experimental_method
+IM	I-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+analysis	O
+of	O
+the	O
+apo	B-protein_state
+-	O
+EncFtnsH	B-protein
+monomer	B-oligomeric_state
+.	O
+
+(	O
+A	O
+)	O
+Mass	B-evidence
+spectrum	I-evidence
+of	O
+apo	B-protein_state
+-	O
+EncFtnsH	B-protein
+acquired	O
+from	O
+100	O
+mM	O
+ammonium	O
+acetate	O
+pH	O
+8	O
+.	O
+0	O
+under	O
+native	B-experimental_method
+MS	I-experimental_method
+conditions	O
+.	O
+
+The	O
+charge	B-evidence
+state	I-evidence
+distribution	O
+observed	O
+is	O
+bimodal	O
+,	O
+with	O
+peaks	B-evidence
+corresponding	O
+to	O
+the	O
+6	O
++	O
+to	O
+15	O
++	O
+charge	B-evidence
+states	I-evidence
+of	O
+apo	B-protein_state
+-	O
+monomer	B-oligomeric_state
+EncFtnsH	B-protein
+(	O
+neutral	O
+average	O
+mass	O
+13	O
+,	O
+194	O
+.	O
+3	O
+Da	O
+).	O
+(	O
+B	O
+)	O
+The	O
+arrival	B-evidence
+time	I-evidence
+distributions	I-evidence
+(	O
+ion	B-evidence
+mobility	I-evidence
+data	I-evidence
+)	O
+of	O
+all	O
+ions	O
+in	O
+the	O
+apo	B-protein_state
+-	O
+EncFtnsH	B-protein
+charge	B-evidence
+state	I-evidence
+distribution	O
+displayed	O
+as	O
+a	O
+greyscale	O
+heat	O
+map	O
+(	O
+linear	O
+intensity	O
+scale	O
+).	O
+(	O
+B	O
+)	O
+Right	O
+,	O
+the	O
+arrival	B-evidence
+time	I-evidence
+distribution	I-evidence
+of	O
+the	O
+6	O
++	O
+(	O
+orange	O
+)	O
+and	O
+7	O
++	O
+(	O
+green	O
+)	O
+charge	B-evidence
+state	I-evidence
+(	O
+dashed	O
+colored	O
+‐	O
+box	O
+)	O
+has	O
+been	O
+extracted	O
+and	O
+plotted	O
+;	O
+The	O
+arrival	B-evidence
+time	I-evidence
+distributions	I-evidence
+for	O
+these	O
+ion	O
+is	O
+shown	O
+(	O
+ms	O
+),	O
+along	O
+with	O
+the	O
+calibrated	O
+collision	B-evidence
+cross	I-evidence
+section	I-evidence
+,	O
+Ω	B-evidence
+(	O
+nm2	O
+).	O
+(	O
+C	O
+)	O
+The	O
+collision	B-evidence
+cross	I-evidence
+section	I-evidence
+of	O
+a	O
+single	O
+monomer	B-oligomeric_state
+unit	O
+from	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+Fe	B-protein_state
+-	I-protein_state
+loaded	I-protein_state
+EncFtnsH	B-protein
+decamer	B-oligomeric_state
+was	O
+calculated	O
+to	O
+be	O
+15	O
+.	O
+8	O
+nm2	O
+using	O
+IMPACT	O
+v	O
+.	O
+0	O
+.	O
+9	O
+.	O
+1	O
+.	O
+
+The	O
++	O
+8	O
+to	O
++	O
+15	O
+protein	O
+charge	B-evidence
+states	I-evidence
+have	O
+observed	O
+CCS	B-evidence
+between	O
+20	O
+–	O
+26	O
+nm2	O
+,	O
+which	O
+is	O
+significantly	O
+higher	O
+than	O
+the	O
+calculated	O
+CCS	B-evidence
+for	O
+an	O
+EncFtnsH	B-protein
+monomer	B-oligomeric_state
+taken	O
+from	O
+the	O
+decameric	B-oligomeric_state
+assembly	O
+crystal	B-evidence
+structure	I-evidence
+(	O
+15	O
+.	O
+8	O
+nm2	O
+).	O
+
+The	O
+mobility	B-evidence
+of	O
+the	O
++	O
+7	O
+charge	B-evidence
+state	I-evidence
+displays	O
+broad	O
+drift	B-evidence
+-	I-evidence
+time	I-evidence
+distribution	I-evidence
+with	O
+maxima	O
+consistent	O
+with	O
+CCS	B-evidence
+of	O
+15	O
+.	O
+9	O
+and	O
+17	O
+.	O
+9	O
+nm2	O
+.	O
+
+Finally	O
+,	O
+the	O
+6	O
++	O
+charge	B-evidence
+state	I-evidence
+of	O
+EncFtnsH	B-protein
+has	O
+mobility	B-evidence
+consistent	O
+with	O
+a	O
+CCS	B-evidence
+of	O
+12	O
+.	O
+3	O
+nm2	O
+,	O
+indicating	O
+a	O
+more	O
+compact	B-protein_state
+/	O
+collapsed	B-protein_state
+structure	O
+.	O
+
+It	O
+is	O
+clear	O
+from	O
+this	O
+data	O
+that	O
+apo	B-protein_state
+-	O
+EncFtnsH	B-protein
+exists	O
+in	O
+several	O
+gas	O
+phase	O
+conformations	O
+.	O
+
+The	O
+range	O
+of	O
+charge	B-evidence
+states	I-evidence
+occupied	O
+by	O
+the	O
+protein	O
+(	O
+6	O
++	O
+to	O
+15	O
++)	O
+and	O
+the	O
+range	O
+of	O
+CCS	B-evidence
+in	O
+which	O
+the	O
+protein	O
+is	O
+observed	O
+(	O
+12	O
+.	O
+3	O
+nm2	O
+–	O
+26	O
+nm2	O
+)	O
+are	O
+both	O
+large	O
+.	O
+
+In	O
+addition	O
+,	O
+many	O
+of	O
+the	O
+charge	B-evidence
+states	I-evidence
+observed	O
+have	O
+higher	O
+charge	O
+than	O
+the	O
+theoretical	O
+maximal	O
+charge	O
+on	O
+spherical	O
+globular	B-protein_state
+protein	O
+,	O
+as	O
+determined	O
+by	O
+the	O
+De	B-experimental_method
+La	I-experimental_method
+Mora	I-experimental_method
+relationship	I-experimental_method
+(	O
+ZR	B-evidence
+=	O
+0	O
+.	O
+0778m	O
+;	O
+for	O
+the	O
+EncFtnsH	B-protein
+monomer	B-oligomeric_state
+ZR	B-evidence
+=	O
+8	O
+.	O
+9	O
+)	O
+Fernandez	O
+.	O
+
+As	O
+described	O
+by	O
+Beveridge	O
+et	O
+al	O
+.,	O
+all	O
+these	O
+factors	O
+are	O
+indicative	O
+of	O
+a	O
+disordered	B-protein_state
+protein	O
+.	O
+
+Gas	O
+-	O
+phase	O
+disassembly	O
+of	O
+the	O
+holo	B-protein_state
+-	O
+EncFtnsH	B-protein
+decameric	B-oligomeric_state
+assembly	O
+.	O
+
+The	O
+entire	O
+charge	B-evidence
+state	I-evidence
+distribution	O
+of	O
+the	O
+Fe	B-protein_state
+-	I-protein_state
+loaded	I-protein_state
+holo	B-protein_state
+-	O
+EncFtnsH	B-protein
+assembly	O
+(	O
+green	O
+circles	O
+)	O
+was	O
+subject	O
+to	O
+collisional	B-experimental_method
+-	I-experimental_method
+induced	I-experimental_method
+dissociation	I-experimental_method
+(	O
+CID	B-experimental_method
+)	O
+by	O
+increasing	O
+the	O
+source	O
+cone	O
+voltage	O
+to	O
+200	O
+V	O
+and	O
+the	O
+trap	O
+voltage	O
+to	O
+50	O
+V	O
+.	O
+The	O
+resulting	O
+CID	B-experimental_method
+mass	B-evidence
+spectrum	I-evidence
+(	O
+A	O
+)	O
+revealed	O
+that	O
+dissociation	O
+of	O
+the	O
+holo	B-protein_state
+-	O
+EncFtnsH	B-protein
+decamer	B-oligomeric_state
+primarily	O
+occurred	O
+via	O
+ejection	O
+of	O
+a	O
+highly	O
+charged	O
+monomer	B-oligomeric_state
+(	O
+blue	O
+circles	O
+),	O
+leaving	O
+the	O
+‘	O
+stripped	B-protein_state
+’	O
+complex	O
+(	O
+a	O
+9mer	B-oligomeric_state
+;	O
+118	O
+.	O
+7	O
+kDa	O
+;	O
+yellow	O
+circles	O
+).	O
+
+The	O
+mass	O
+of	O
+the	O
+ejected	O
+-	O
+monomer	B-oligomeric_state
+is	O
+consistent	O
+with	O
+apo	B-protein_state
+-	O
+EncFtnsH	B-protein
+(	O
+13	O
+.	O
+2	O
+kDa	O
+),	O
+suggesting	O
+unfolding	O
+of	O
+the	O
+monomer	B-oligomeric_state
+(	O
+and	O
+loss	B-protein_state
+of	I-protein_state
+Fe	B-chemical
+)	O
+occurs	O
+during	O
+ejection	O
+from	O
+the	O
+complex	O
+.	O
+
+This	O
+observation	O
+of	O
+asymmetric	O
+charge	O
+partitioning	O
+of	O
+the	O
+sub	O
+-	O
+complexes	O
+with	O
+respect	O
+to	O
+the	O
+mass	O
+of	O
+the	O
+complex	O
+is	O
+consistent	O
+with	O
+the	O
+'	O
+typical	O
+'	O
+pathway	O
+of	O
+dissociation	O
+of	O
+protein	O
+assemblies	O
+by	O
+CID	B-experimental_method
+,	O
+as	O
+described	O
+by	O
+.	O
+
+In	O
+addition	O
+,	O
+a	O
+third	O
+,	O
+lower	O
+abundance	O
+,	O
+charge	B-evidence
+state	I-evidence
+distribution	O
+is	O
+observed	O
+which	O
+overlaps	O
+the	O
+EncFtn	B-protein
+ejected	O
+monomer	B-oligomeric_state
+charge	B-evidence
+state	I-evidence
+distribution	O
+;	O
+this	O
+region	O
+of	O
+the	O
+spectrum	O
+is	O
+highlighted	O
+in	O
+(	O
+B	O
+).	O
+
+This	O
+distribution	O
+is	O
+consistent	O
+with	O
+an	O
+ejected	O
+EncFtnsH	B-protein
+dimer	B-oligomeric_state
+(	O
+orange	O
+circles	O
+).	O
+
+Interestingly	O
+,	O
+closer	O
+analysis	O
+of	O
+the	O
+individual	O
+charge	B-evidence
+state	I-evidence
+of	O
+this	O
+dimeric	B-oligomeric_state
+CID	B-experimental_method
+product	O
+shows	O
+that	O
+this	O
+sub	O
+-	O
+complex	O
+exists	O
+in	O
+three	O
+forms	O
+–	O
+displaying	O
+mass	O
+consistent	O
+with	O
+an	O
+EncFtnsH	B-protein
+dimer	B-oligomeric_state
+binding	O
+0	O
+,	O
+1	O
+,	O
+and	O
+2	O
+Fe	B-chemical
+ions	O
+.	O
+
+This	O
+is	O
+highlighted	O
+in	O
+(	O
+C	O
+),	O
+where	O
+the	O
+15	O
++	O
+charge	B-evidence
+state	I-evidence
+of	O
+the	O
+EncFtnsH	B-protein
+dimer	B-oligomeric_state
+is	O
+shown	O
+;	O
+3	O
+peaks	B-evidence
+are	O
+observed	O
+with	O
+m	O
+/	O
+z	O
+1760	O
+.	O
+5	O
+,	O
+1763	O
+.	O
+8	O
+,	O
+and	O
+1767	O
+.	O
+0	O
+Th	O
+–	O
+the	O
+lowest	O
+peak	O
+corresponds	O
+to	O
+neutral	O
+masses	O
+of	O
+26392	O
+.	O
+5	O
+Da	O
+[	O
+predicted	O
+EncFtnsH	B-protein
+dimer	B-oligomeric_state
+,	O
+(	O
+C572H884N172O185S2	O
+)	O
+2	O
+;	O
+26388	O
+.	O
+6	O
+Da	O
+].	O
+
+The	O
+two	O
+further	O
+peaks	B-evidence
+have	O
+a	O
+delta	O
+-	O
+mass	O
+of	O
+~+	O
+50	O
+Da	O
+,	O
+consistent	O
+with	O
+Fe	B-chemical
+binding	O
+.	O
+
+We	O
+interpret	O
+these	O
+observations	O
+as	O
+partial	O
+‘	O
+atypical	O
+’	O
+CID	B-experimental_method
+fragmentation	O
+of	O
+the	O
+decameric	B-oligomeric_state
+complex	O
+–	O
+i	O
+.	O
+e	O
+.	O
+fragmentation	O
+of	O
+the	O
+initial	O
+complex	O
+with	O
+retention	O
+of	O
+subunit	O
+and	O
+ligand	O
+interactions	O
+.	O
+
+We	O
+postulate	O
+the	O
+high	O
+stability	O
+of	O
+this	O
+iron	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+dimer	B-oligomeric_state
+sub	O
+-	O
+complex	O
+is	O
+due	O
+to	O
+the	O
+metal	B-chemical
+coordination	B-bond_interaction
+at	O
+the	O
+dimer	B-site
+interface	I-site
+,	O
+increasing	O
+the	O
+strength	O
+of	O
+the	O
+dimer	B-site
+interface	I-site
+.	O
+
+Taken	O
+together	O
+,	O
+these	O
+observations	O
+support	O
+our	O
+findings	O
+that	O
+the	O
+topology	O
+of	O
+the	O
+decameric	B-oligomeric_state
+EncFtnsH	B-protein
+assembly	O
+is	O
+arranged	O
+as	O
+a	O
+pentamer	B-oligomeric_state
+of	O
+dimers	B-oligomeric_state
+,	O
+with	O
+two	O
+Fe	B-chemical
+ions	O
+at	O
+each	O
+dimer	B-site
+interface	I-site
+.	O
+
+Native	B-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+and	O
+ion	B-experimental_method
+mobility	I-experimental_method
+analysis	I-experimental_method
+of	O
+iron	B-chemical
+loading	O
+in	O
+EncFtnsH	B-protein
+.	O
+
+All	O
+spectra	B-evidence
+were	O
+acquired	O
+in	O
+100	O
+mM	O
+ammonium	O
+acetate	B-chemical
+,	O
+pH	O
+8	O
+.	O
+0	O
+with	O
+a	O
+protein	O
+concentration	O
+of	O
+5	O
+µM	O
+.	O
+(	O
+A	O
+)	O
+Native	B-experimental_method
+nanoelectrospray	I-experimental_method
+ionization	I-experimental_method
+(	O
+nESI	B-experimental_method
+)	O
+mass	B-experimental_method
+spectrometry	I-experimental_method
+of	O
+EncFtnsH	B-protein
+at	O
+varying	O
+iron	B-chemical
+concentrations	O
+.	O
+
+A1	O
+,	O
+nESI	B-experimental_method
+spectrum	B-evidence
+of	O
+iron	B-protein_state
+-	I-protein_state
+free	I-protein_state
+EncFtnsH	B-protein
+displays	O
+a	O
+charge	B-evidence
+state	I-evidence
+distribution	O
+consistent	O
+with	O
+EncFtnsH	B-protein
+monomer	B-oligomeric_state
+(	O
+blue	O
+circles	O
+,	O
+13	O
+,	O
+194	O
+Da	O
+).	O
+
+Addition	O
+of	O
+100	O
+µM	O
+(	O
+A2	O
+)	O
+and	O
+300	O
+µM	O
+(	O
+A3	O
+)	O
+Fe2	B-chemical
++	I-chemical
+results	O
+in	O
+the	O
+appearance	O
+of	O
+a	O
+second	O
+higher	O
+molecular	B-evidence
+weight	I-evidence
+charge	B-evidence
+state	I-evidence
+distribution	O
+consistent	O
+with	O
+a	O
+decameric	B-oligomeric_state
+assembly	O
+of	O
+EncFtnsH	B-protein
+(	O
+green	O
+circles	O
+,	O
+132	O
+.	O
+6	O
+kDa	O
+).	O
+
+(	O
+B	O
+)	O
+Ion	B-experimental_method
+mobility	I-experimental_method
+(	I-experimental_method
+IM	I-experimental_method
+)-	I-experimental_method
+MS	I-experimental_method
+of	O
+the	O
+iron	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+holo	B-protein_state
+-	O
+EncFtnsH	B-protein
+decamer	B-oligomeric_state
+.	O
+
+Top	O
+,	O
+Peaks	B-evidence
+corresponding	O
+to	O
+the	O
+22	O
++	O
+to	O
+26	O
++	O
+charge	B-evidence
+states	I-evidence
+of	O
+a	O
+homo	B-oligomeric_state
+-	I-oligomeric_state
+decameric	I-oligomeric_state
+assembly	O
+of	O
+EncFtnsH	B-protein
+are	O
+observed	O
+(	O
+132	O
+.	O
+6	O
+kDa	O
+).	O
+
+Top	O
+Insert	O
+,	O
+Analysis	O
+of	O
+the	O
+24	O
++	O
+charge	B-evidence
+state	I-evidence
+of	O
+the	O
+assembly	O
+at	O
+m	O
+/	O
+z	O
+5528	O
+.	O
+2	O
+Th	O
+.	O
+
+The	O
+theoretical	O
+average	O
+m	O
+/	O
+z	O
+of	O
+the	O
+24	O
++	O
+charge	B-evidence
+state	I-evidence
+with	O
+no	O
+additional	O
+metals	O
+bound	O
+is	O
+marked	O
+by	O
+a	O
+red	O
+line	O
+(	O
+5498	O
+.	O
+7	O
+Th	O
+);	O
+the	O
+observed	O
+m	O
+/	O
+z	O
+of	O
+the	O
+24	O
++	O
+charge	B-evidence
+state	I-evidence
+indicates	O
+that	O
+the	O
+EncFtnsH	B-protein
+assembly	O
+binds	O
+between	O
+10	O
+(	O
+green	O
+line	O
+,	O
+5521	O
+.	O
+1	O
+Th	O
+)	O
+and	O
+15	O
+Fe	B-chemical
+ions	O
+(	O
+blue	O
+line	O
+,	O
+5532	O
+.	O
+4	O
+Th	O
+)	O
+per	O
+decamer	B-oligomeric_state
+.	O
+
+Bottom	O
+,	O
+The	O
+arrival	B-evidence
+time	I-evidence
+distributions	I-evidence
+(	O
+ion	B-evidence
+mobility	I-evidence
+data	I-evidence
+)	O
+of	O
+all	O
+ions	O
+in	O
+the	O
+EncFtnsH	B-protein
+charge	B-evidence
+state	I-evidence
+distribution	O
+displayed	O
+as	O
+a	O
+greyscale	O
+heat	O
+map	O
+(	O
+linear	O
+intensity	O
+scale	O
+).	O
+
+Bottom	O
+right	O
+,	O
+The	O
+arrival	B-evidence
+time	I-evidence
+distribution	I-evidence
+of	O
+the	O
+24	O
++	O
+charge	B-evidence
+state	I-evidence
+(	O
+dashed	O
+blue	O
+box	O
+)	O
+has	O
+been	O
+extracted	O
+and	O
+plotted	O
+.	O
+
+The	O
+drift	B-evidence
+time	I-evidence
+for	O
+this	O
+ion	O
+is	O
+shown	O
+(	O
+ms	O
+),	O
+along	O
+with	O
+the	O
+calibrated	O
+collision	B-evidence
+cross	I-evidence
+section	I-evidence
+(	O
+CCS	B-evidence
+),	O
+Ω	B-evidence
+(	O
+nm2	O
+).	O
+
+In	O
+order	O
+to	O
+confirm	O
+the	O
+assignment	O
+of	O
+the	O
+oligomeric	O
+state	O
+of	O
+EncFtnsH	B-protein
+and	O
+investigate	O
+further	O
+the	O
+Fe2	B-chemical
++-	I-chemical
+dependent	O
+assembly	O
+,	O
+we	O
+used	O
+native	B-experimental_method
+nano	I-experimental_method
+-	I-experimental_method
+electrospray	I-experimental_method
+ionization	I-experimental_method
+(	O
+nESI	B-experimental_method
+)	O
+and	O
+ion	B-experimental_method
+-	I-experimental_method
+mobility	I-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+(	O
+IM	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+).	O
+
+As	O
+described	O
+above	O
+,	O
+by	O
+recombinant	B-experimental_method
+production	I-experimental_method
+of	O
+EncFtnsH	B-protein
+in	O
+minimal	O
+media	O
+we	O
+were	O
+able	O
+to	O
+limit	O
+the	O
+bioavailability	O
+of	O
+iron	B-chemical
+.	O
+
+Native	B-experimental_method
+MS	I-experimental_method
+analysis	O
+of	O
+EncFtnsH	B-protein
+produced	O
+in	O
+this	O
+way	O
+displayed	O
+a	O
+charge	B-evidence
+state	I-evidence
+distribution	O
+consistent	O
+with	O
+an	O
+EncFtnsH	B-protein
+monomer	B-oligomeric_state
+(	O
+blue	O
+circles	O
+,	O
+Figure	O
+7A1	O
+)	O
+with	O
+an	O
+average	O
+neutral	O
+mass	O
+of	O
+13	O
+,	O
+194	O
+Da	O
+,	O
+in	O
+agreement	O
+with	O
+the	O
+predicted	O
+mass	O
+of	O
+the	O
+EncFtnsH	B-protein
+protein	O
+(	O
+13	O
+,	O
+194	O
+.	O
+53	O
+Da	O
+).	O
+
+Titration	B-experimental_method
+with	O
+Fe2	B-chemical
++	I-chemical
+directly	O
+before	O
+native	B-experimental_method
+MS	I-experimental_method
+analysis	O
+resulted	O
+in	O
+the	O
+appearance	O
+of	O
+a	O
+new	O
+charge	B-evidence
+state	I-evidence
+distribution	O
+,	O
+consistent	O
+with	O
+an	O
+EncFtnsH	B-protein
+decameric	B-oligomeric_state
+assembly	O
+(+	O
+22	O
+to	O
++	O
+26	O
+;	O
+132	O
+.	O
+65	O
+kDa	O
+)	O
+(	O
+Figure	O
+7A2	O
+/	O
+3	O
+).	O
+
+After	O
+instrument	O
+optimization	O
+,	O
+the	O
+mass	O
+resolving	O
+power	O
+achieved	O
+was	O
+sufficient	O
+to	O
+assign	O
+iron	B-chemical
+-	O
+loading	O
+in	O
+the	O
+complex	O
+to	O
+between	O
+10	O
+and	O
+15	O
+Fe	B-chemical
+ions	O
+per	O
+decamer	B-oligomeric_state
+(	O
+Figure	O
+7B	O
+,	O
+inset	O
+top	O
+right	O
+),	O
+consistent	O
+with	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+10	O
+irons	B-chemical
+in	O
+the	O
+FOC	B-site
+and	O
+the	O
+coordination	B-bond_interaction
+of	O
+iron	B-chemical
+in	O
+the	O
+Glu31	B-site
+/	I-site
+34	I-site
+-	I-site
+site	I-site
+occupied	O
+by	O
+calcium	B-chemical
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+(	O
+Δmass	B-evidence
+observed	O
+~	O
+0	O
+.	O
+67	O
+kDa	O
+).	O
+
+MS	B-experimental_method
+analysis	O
+of	O
+EncFtnsH	B-protein
+after	O
+addition	O
+of	O
+further	O
+Fe2	B-chemical
++	I-chemical
+did	O
+not	O
+result	O
+in	O
+iron	B-chemical
+loading	O
+above	O
+this	O
+stoichiometry	O
+.	O
+
+Therefore	O
+,	O
+the	O
+extent	O
+of	O
+iron	B-chemical
+binding	O
+seen	O
+is	O
+limited	O
+to	O
+the	O
+FOC	B-site
+and	O
+Glu31	B-site
+/	I-site
+34	I-site
+secondary	I-site
+metal	I-site
+binding	I-site
+site	I-site
+.	O
+
+These	O
+data	O
+suggest	O
+that	O
+the	O
+decameric	B-oligomeric_state
+assembly	O
+of	O
+EncFtnsH	B-protein
+does	O
+not	O
+accrue	O
+iron	B-chemical
+in	O
+the	O
+same	O
+manner	O
+as	O
+classical	B-protein_state
+ferritin	B-protein_type
+,	O
+which	O
+is	O
+able	O
+to	O
+sequester	O
+around	O
+4500	O
+iron	B-chemical
+ions	O
+within	O
+its	O
+nanocage	B-complex_assembly
+.	O
+
+Ion	B-experimental_method
+mobility	I-experimental_method
+analysis	I-experimental_method
+of	O
+the	O
+EncFtnsH	B-protein
+decameric	B-oligomeric_state
+assembly	O
+,	O
+collected	O
+with	O
+minimal	O
+collisional	O
+activation	O
+,	O
+suggested	O
+that	O
+it	O
+consists	O
+of	O
+a	O
+single	O
+conformation	O
+with	O
+a	O
+collision	B-evidence
+cross	I-evidence
+section	I-evidence
+(	O
+CCS	B-evidence
+)	O
+of	O
+58	O
+.	O
+2	O
+nm2	O
+(	O
+Figure	O
+7B	O
+).	O
+
+This	O
+observation	O
+is	O
+in	O
+agreement	O
+with	O
+the	O
+calculated	O
+CCS	B-evidence
+of	O
+58	O
+.	O
+7	O
+nm2derived	O
+from	O
+our	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+EncFtnsH	B-protein
+decamer	B-oligomeric_state
+.	O
+
+By	O
+contrast	O
+,	O
+IM	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+measurements	O
+of	O
+the	O
+monomeric	B-oligomeric_state
+EncFtnsH	B-protein
+at	O
+pH	B-protein_state
+8	I-protein_state
+.	I-protein_state
+0	I-protein_state
+under	O
+the	O
+same	O
+instrumental	O
+conditions	O
+revealed	O
+that	O
+the	O
+metal	B-protein_state
+-	I-protein_state
+free	I-protein_state
+protein	B-protein
+monomer	B-oligomeric_state
+exists	O
+in	O
+a	O
+wide	O
+range	O
+of	O
+charge	B-evidence
+states	I-evidence
+(+	O
+6	O
+to	O
++	O
+16	O
+)	O
+and	O
+adopts	O
+many	O
+conformations	O
+in	O
+the	O
+gas	O
+phase	O
+with	O
+collision	O
+cross	O
+sections	O
+ranging	O
+from	O
+12	O
+nm2	O
+to	O
+26	O
+nm2	O
+(	O
+Figure	O
+7	O
+—	O
+figure	O
+supplement	O
+1	O
+).	O
+
+Thus	O
+,	O
+IM	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+studies	O
+highlight	O
+that	O
+higher	O
+order	O
+structure	O
+in	O
+EncFtnsH	B-protein
+is	O
+mediated	O
+/	O
+stabilized	O
+by	O
+metal	O
+binding	O
+,	O
+an	O
+observation	O
+that	O
+is	O
+in	O
+agreement	O
+with	O
+our	O
+solution	O
+studies	O
+.	O
+
+Taken	O
+together	O
+,	O
+these	O
+results	O
+suggest	O
+that	O
+di	O
+-	O
+iron	B-chemical
+binding	O
+,	O
+forming	O
+the	O
+FOC	B-site
+in	O
+EncFtnsH	B-protein
+,	O
+is	O
+required	O
+to	O
+stabilize	O
+the	O
+4	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+bundle	I-structure_element
+dimer	B-site
+interface	I-site
+,	O
+essentially	O
+reconstructing	O
+the	O
+classical	B-protein_state
+ferritin	B-protein_type
+-	O
+like	O
+fold	O
+;	O
+once	O
+stabilized	O
+,	O
+these	O
+dimers	B-oligomeric_state
+readily	O
+associate	O
+as	O
+pentamers	O
+,	O
+and	O
+the	O
+overall	O
+assembly	O
+adopts	O
+the	O
+decameric	B-oligomeric_state
+ring	O
+arrangement	O
+observed	O
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+.	O
+
+We	O
+subsequently	O
+performed	O
+gas	O
+phase	O
+disassembly	O
+of	O
+the	O
+decameric	B-oligomeric_state
+EncFtnsH	B-protein
+using	O
+collision	B-experimental_method
+-	I-experimental_method
+induced	I-experimental_method
+dissociation	I-experimental_method
+(	O
+CID	B-experimental_method
+)	O
+tandem	B-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+.	O
+
+Under	O
+the	O
+correct	O
+CID	B-experimental_method
+conditions	O
+,	O
+protein	O
+assemblies	O
+can	O
+dissociate	O
+with	O
+retention	O
+of	O
+subunit	O
+and	O
+ligand	O
+interactions	O
+,	O
+and	O
+thus	O
+provide	O
+structurally	O
+-	O
+informative	O
+evidence	O
+as	O
+to	O
+the	O
+topology	O
+of	O
+the	O
+original	O
+assembly	O
+;	O
+this	O
+has	O
+been	O
+termed	O
+‘	O
+atypical	O
+’	O
+dissociation	O
+.	O
+
+For	O
+EncFtnsH	B-protein
+,	O
+this	O
+atypical	O
+dissociation	O
+pathway	O
+was	O
+clearly	O
+evident	O
+;	O
+CID	B-experimental_method
+of	O
+the	O
+EncFtnsH	B-protein
+decamer	B-oligomeric_state
+resulted	O
+in	O
+the	O
+appearance	O
+of	O
+a	O
+dimeric	B-oligomeric_state
+EncFtnsH	B-protein
+subcomplex	O
+containing	O
+0	O
+,	O
+1	O
+,	O
+or	O
+2	O
+iron	B-chemical
+ions	O
+(	O
+Figure	O
+7	O
+—	O
+figure	O
+supplement	O
+2	O
+).	O
+
+In	O
+light	O
+of	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+,	O
+this	O
+observation	O
+can	O
+be	O
+rationalized	O
+as	O
+dissociation	O
+of	O
+the	O
+EncFtnsH	B-protein
+decamer	B-oligomeric_state
+by	O
+disruption	O
+of	O
+the	O
+non	B-site
+-	I-site
+FOC	I-site
+interface	I-site
+with	O
+at	O
+least	O
+partial	O
+retention	O
+of	O
+the	O
+FOC	B-site
+interface	I-site
+and	O
+the	O
+FOC	B-site
+-	O
+Fe	B-chemical
+.	O
+
+Thus	O
+,	O
+this	O
+observation	O
+supports	O
+our	O
+crystallographic	O
+assignment	O
+of	O
+the	O
+overall	O
+topology	O
+of	O
+the	O
+EncFtnsH	B-protein
+assembly	O
+as	O
+a	O
+pentameric	B-oligomeric_state
+assembly	O
+of	O
+dimers	B-oligomeric_state
+with	O
+two	O
+iron	B-chemical
+ions	O
+located	O
+at	O
+the	O
+FOC	B-site
+dimer	I-site
+interface	I-site
+.	O
+
+In	O
+addition	O
+,	O
+this	O
+analysis	O
+provides	O
+evidence	O
+that	O
+the	O
+overall	O
+architecture	O
+of	O
+the	O
+complex	O
+is	O
+consistent	O
+in	O
+the	O
+crystal	B-evidence
+,	O
+solution	O
+and	O
+gas	O
+phases	O
+.	O
+
+Ferroxidase	B-protein_type
+activity	O
+
+TEM	B-experimental_method
+visualization	O
+of	O
+iron	B-protein_state
+-	I-protein_state
+loaded	I-protein_state
+bacterial	B-taxonomy_domain
+nanocompartments	B-complex_assembly
+and	O
+ferritin	B-protein_type
+.	O
+
+Decameric	B-oligomeric_state
+EncFtnsH	B-protein
+,	O
+encapsulin	B-protein
+,	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+and	O
+apoferritin	B-protein_state
+,	O
+at	O
+8	O
+.	O
+5	O
+µM	O
+,	O
+were	O
+mixed	O
+with	O
+147	O
+µM	O
+,	O
+1	O
+mM	O
+,	O
+1	O
+mM	O
+and	O
+215	O
+µM	O
+acidic	O
+Fe	B-chemical
+(	I-chemical
+NH4	I-chemical
+)	I-chemical
+2	I-chemical
+(	I-chemical
+SO4	I-chemical
+)	I-chemical
+2	I-chemical
+,	O
+respectively	O
+.	O
+
+Protein	O
+mixtures	O
+were	O
+incubated	O
+at	O
+room	O
+temperature	O
+for	O
+1	O
+hr	O
+prior	O
+to	O
+TEM	B-experimental_method
+analysis	O
+with	O
+or	O
+without	O
+uranyl	B-chemical
+acetate	I-chemical
+stain	O
+.	O
+
+(	O
+A	O
+–	O
+D	O
+)	O
+Unstained	O
+EncFtnsH	B-protein
+,	O
+encapsulin	B-protein
+,	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+,	O
+apoferritin	B-protein_state
+loaded	B-protein_state
+with	I-protein_state
+Fe2	B-chemical
++,	I-chemical
+respectively	O
+,	O
+with	O
+35	O
+,	O
+000	O
+x	O
+magnification	O
+and	O
+scale	O
+bars	O
+indicate	O
+100	O
+nm	O
+.	O
+(	O
+E	O
+)	O
+Protein	O
+-	O
+free	O
+sample	O
+as	O
+a	O
+control	O
+.	O
+(	O
+F	O
+–	O
+I	O
+)	O
+Stained	B-experimental_method
+EncFtnsH	B-protein
+,	O
+encapsulin	B-protein
+,	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+,	O
+apoferritin	B-protein_state
+loaded	B-protein_state
+with	I-protein_state
+Fe2	B-chemical
++,	I-chemical
+respectively	O
+,	O
+with	O
+140	O
+,	O
+000	O
+x	O
+magnification	O
+and	O
+scale	O
+bars	O
+indicate	O
+25	O
+nm	O
+.	O
+
+Spectroscopic	O
+evidence	O
+for	O
+the	O
+ferroxidase	B-protein_type
+activity	O
+and	O
+comparison	O
+of	O
+iron	B-chemical
+loading	O
+capacity	O
+of	O
+apoferritin	B-protein_state
+,	O
+EncFtnsH	B-protein
+,	O
+encapsulin	B-protein
+,	O
+and	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+.	O
+
+(	O
+A	O
+)	O
+Apoferritin	B-protein_state
+(	O
+10	O
+μM	O
+monomer	B-oligomeric_state
+concentration	O
+)	O
+and	O
+EncFtnsH	B-protein
+decamer	B-oligomeric_state
+fractions	O
+(	O
+20	O
+μM	O
+monomer	B-oligomeric_state
+concentration	O
+,	O
+10	O
+μM	O
+FOC	B-site
+concentration	O
+)	O
+were	O
+incubated	O
+with	O
+20	O
+and	O
+100	O
+μM	O
+iron	B-chemical
+(	O
+2	O
+and	O
+10	O
+times	O
+molar	O
+equivalent	O
+Fe2	B-chemical
++	I-chemical
+per	O
+FOC	B-site
+)	O
+and	O
+progress	B-evidence
+curves	I-evidence
+of	O
+the	O
+oxidation	O
+of	O
+Fe2	B-chemical
++	I-chemical
+to	O
+Fe3	B-chemical
++	I-chemical
+at	O
+315	O
+nm	O
+were	O
+recorded	O
+in	O
+a	O
+spectrophotometer	O
+.	O
+
+The	O
+background	O
+oxidation	O
+of	O
+iron	B-chemical
+at	O
+20	O
+and	O
+100	O
+μM	O
+in	O
+enzyme	O
+-	O
+free	O
+controls	O
+are	O
+shown	O
+for	O
+reference	O
+.	O
+(	O
+B	O
+)	O
+Encapsulin	B-protein
+and	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+complexes	O
+at	O
+10	O
+μM	O
+asymmetric	O
+unit	O
+concentration	O
+were	O
+incubated	B-experimental_method
+with	O
+Fe2	B-chemical
++	I-chemical
+at	O
+20	O
+and	O
+100	O
+μM	O
+and	O
+progress	B-evidence
+curves	I-evidence
+for	O
+iron	B-chemical
+oxidation	O
+at	O
+A315	O
+were	O
+measured	O
+in	O
+a	O
+UV	B-experimental_method
+/	I-experimental_method
+visible	I-experimental_method
+spectrophotometer	I-experimental_method
+.	O
+
+Enzyme	O
+free	O
+controls	O
+for	O
+background	O
+oxidation	O
+of	O
+Fe2	B-chemical
++	I-chemical
+are	O
+shown	O
+for	O
+reference	O
+.	O
+(	O
+C	O
+)	O
+Histogram	O
+of	O
+the	O
+iron	B-chemical
+loading	O
+capacity	O
+per	O
+biological	O
+assembly	O
+of	O
+EncFtnsH	B-protein
+,	O
+encapsulin	B-protein
+,	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+and	O
+apoferritin	B-protein_state
+.	O
+
+The	O
+results	O
+shown	O
+are	O
+for	O
+three	O
+technical	O
+replicates	O
+and	O
+represent	O
+the	O
+optimal	O
+iron	B-chemical
+loading	O
+by	O
+the	O
+complexes	O
+after	O
+three	O
+hours	O
+when	O
+incubated	O
+with	O
+Fe2	B-chemical
++.	I-chemical
+
+In	O
+light	O
+of	O
+the	O
+identification	O
+of	O
+an	O
+iron	B-protein_state
+-	I-protein_state
+loaded	I-protein_state
+FOC	B-site
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+EncFtn	B-protein
+and	O
+our	O
+native	B-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+data	O
+,	O
+we	O
+performed	O
+ferroxidase	B-experimental_method
+and	I-experimental_method
+peroxidase	I-experimental_method
+assays	I-experimental_method
+to	O
+demonstrate	O
+the	O
+catalytic	O
+activity	O
+of	O
+this	O
+protein	O
+.	O
+
+In	O
+addition	O
+,	O
+we	O
+also	O
+assayed	O
+equine	B-taxonomy_domain
+apoferritin	B-protein_state
+,	O
+an	O
+example	O
+of	O
+a	O
+classical	B-protein_state
+ferritin	B-protein_type
+enzyme	O
+,	O
+as	O
+a	O
+positive	O
+control	O
+.	O
+
+Unlike	O
+the	O
+Dps	B-protein_type
+family	I-protein_type
+of	O
+ferritin	B-protein_type
+-	I-protein_type
+like	I-protein_type
+proteins	I-protein_type
+,	O
+EncFtn	B-protein
+showed	O
+no	O
+peroxidase	O
+activity	O
+when	O
+assayed	O
+with	O
+the	O
+substrate	O
+ortho	B-chemical
+-	I-chemical
+phenylenediamine	I-chemical
+.	O
+
+The	O
+ferroxidase	B-protein_type
+activity	O
+of	O
+EncFtnsH	B-protein
+was	O
+measured	O
+by	O
+recording	O
+the	O
+progress	B-evidence
+curve	I-evidence
+of	O
+Fe2	B-chemical
++	I-chemical
+oxidation	O
+to	O
+Fe3	B-chemical
++	I-chemical
+at	O
+315	O
+nm	O
+after	O
+addition	O
+of	O
+20	O
+and	O
+100	O
+µM	O
+Fe2	B-chemical
++	I-chemical
+(	O
+2	O
+and	O
+10	O
+times	O
+molar	O
+ratio	O
+Fe2	B-chemical
++/	I-chemical
+FOC	B-site
+).	O
+
+In	O
+both	O
+experiments	O
+the	O
+rate	O
+of	O
+oxidation	O
+was	O
+faster	O
+than	O
+background	O
+oxidation	O
+of	O
+Fe2	B-chemical
++	I-chemical
+by	O
+molecular	O
+oxygen	B-chemical
+,	O
+and	O
+was	O
+highest	O
+for	O
+100	O
+µM	O
+Fe2	B-chemical
++	I-chemical
+(	O
+Figure	O
+8A	O
+).	O
+
+These	O
+data	O
+show	O
+that	O
+recombinant	O
+EncFtnsH	B-protein
+acts	O
+as	O
+an	O
+active	B-protein_state
+ferroxidase	B-protein_type
+enzyme	O
+.	O
+
+When	O
+compared	O
+to	O
+apoferritin	B-protein_state
+,	O
+EncFtnsH	B-protein
+oxidized	O
+Fe2	B-chemical
++	I-chemical
+at	O
+a	O
+slower	O
+rate	O
+and	O
+the	O
+reaction	O
+did	O
+not	O
+run	O
+to	O
+completion	O
+over	O
+the	O
+1800	O
+s	O
+of	O
+the	O
+experiment	O
+.	O
+
+Addition	O
+of	O
+higher	O
+quantities	O
+of	O
+iron	B-chemical
+resulted	O
+in	O
+the	O
+formation	O
+of	O
+a	O
+yellow	O
+/	O
+red	O
+precipitate	O
+at	O
+the	O
+end	O
+of	O
+the	O
+reaction	O
+.	O
+
+We	O
+also	O
+performed	O
+these	O
+assays	O
+on	O
+purified	O
+recombinant	O
+encapsulin	B-protein
+;	O
+which	O
+,	O
+when	O
+assayed	O
+alone	O
+,	O
+did	O
+not	O
+display	O
+ferroxidase	B-protein_type
+activity	O
+above	O
+background	O
+Fe2	B-chemical
++	I-chemical
+oxidation	O
+(	O
+Figure	O
+8B	O
+).	O
+
+In	O
+contrast	O
+,	O
+complexes	O
+of	O
+the	O
+full	B-protein_state
+EncFtn	B-protein
+encapsulin	B-protein
+nanocompartment	B-complex_assembly
+(	O
+i	O
+.	O
+e	O
+.	O
+the	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+protein	O
+complex	O
+)	O
+displayed	O
+ferroxidase	B-protein_type
+activity	O
+comparable	O
+to	O
+apoferritin	B-protein_state
+without	O
+the	O
+formation	O
+of	O
+precipitates	O
+(	O
+Figure	O
+8B	O
+).	O
+
+We	O
+attributed	O
+the	O
+precipitates	O
+observed	O
+in	O
+the	O
+EncFtnsH	B-protein
+ferroxidase	B-experimental_method
+assay	I-experimental_method
+to	O
+the	O
+production	O
+of	O
+insoluble	O
+Fe3	B-chemical
++	I-chemical
+complexes	O
+,	O
+which	O
+led	O
+us	O
+to	O
+propose	O
+that	O
+EncFtn	B-protein
+does	O
+not	O
+directly	O
+store	O
+Fe3	B-chemical
++	I-chemical
+in	O
+a	O
+mineral	O
+form	O
+.	O
+
+This	O
+observation	O
+agrees	O
+with	O
+native	B-experimental_method
+MS	I-experimental_method
+results	O
+,	O
+which	O
+indicates	O
+a	O
+maximum	O
+iron	B-chemical
+loading	O
+of	O
+10	O
+–	O
+15	O
+iron	B-chemical
+ions	O
+per	O
+decameric	B-oligomeric_state
+EncFtn	B-protein
+;	O
+and	O
+the	O
+structure	B-evidence
+,	O
+which	O
+does	O
+not	O
+possess	O
+the	O
+enclosed	O
+iron	B-site
+-	I-site
+storage	I-site
+cavity	I-site
+characteristic	O
+of	O
+classical	B-protein_state
+ferritins	B-protein_type
+and	O
+Dps	B-protein_type
+family	I-protein_type
+proteins	I-protein_type
+that	O
+can	O
+directly	O
+accrue	O
+mineralized	O
+Fe3	B-chemical
++	I-chemical
+within	O
+their	O
+nanocompartment	B-complex_assembly
+structures	B-evidence
+.	O
+
+To	O
+analyze	O
+the	O
+products	O
+of	O
+these	O
+reactions	O
+and	O
+determine	O
+whether	O
+the	O
+EncFtn	B-protein
+and	O
+encapsulin	B-protein
+were	O
+able	O
+to	O
+store	O
+iron	B-chemical
+in	O
+a	O
+mineral	O
+form	O
+,	O
+we	O
+performed	O
+TEM	B-experimental_method
+on	O
+the	O
+reaction	O
+mixtures	O
+from	O
+the	O
+ferroxidase	B-experimental_method
+assay	I-experimental_method
+.	O
+
+The	O
+EncFtnsH	B-protein
+reaction	O
+mixture	O
+showed	O
+the	O
+formation	O
+of	O
+large	O
+,	O
+irregular	O
+electron	O
+-	O
+dense	O
+precipitates	O
+(	O
+Figure	O
+8	O
+—	O
+figure	O
+supplement	O
+1A	O
+).	O
+
+A	O
+similar	O
+distribution	O
+of	O
+particles	O
+was	O
+observed	O
+after	O
+addition	O
+of	O
+Fe2	B-chemical
++	I-chemical
+to	O
+the	O
+encapsulin	B-protein
+protein	O
+(	O
+Figure	O
+8	O
+—	O
+figure	O
+supplement	O
+1B	O
+).	O
+
+In	O
+contrast	O
+,	O
+addition	O
+of	O
+Fe2	B-chemical
++	I-chemical
+to	O
+the	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+nanocompartment	B-complex_assembly
+resulted	O
+in	O
+small	O
+,	O
+highly	O
+regular	O
+,	O
+electron	O
+dense	O
+particles	O
+of	O
+approximately	O
+5	O
+nm	O
+in	O
+diameter	O
+(	O
+Figure	O
+8	O
+—	O
+figure	O
+supplement	O
+1C	O
+);	O
+we	O
+interpret	O
+these	O
+observations	O
+as	O
+controlled	O
+mineralization	O
+of	O
+iron	B-chemical
+within	O
+the	O
+nanocompartment	B-complex_assembly
+.	O
+
+Addition	O
+of	O
+Fe2	B-chemical
++	I-chemical
+to	O
+apoferritin	B-protein_state
+resulted	O
+in	O
+a	O
+mixture	O
+of	O
+large	O
+particles	O
+and	O
+small	O
+(~	O
+2	O
+nm	O
+)	O
+particles	O
+consistent	O
+with	O
+partial	O
+mineralization	O
+by	O
+the	O
+ferritin	B-protein_type
+and	O
+some	O
+background	O
+oxidation	O
+of	O
+the	O
+iron	B-chemical
+(	O
+Figure	O
+8	O
+—	O
+figure	O
+supplement	O
+1D	O
+).	O
+
+Negative	B-experimental_method
+stain	I-experimental_method
+TEM	I-experimental_method
+of	O
+these	O
+samples	O
+revealed	O
+that	O
+upon	O
+addition	O
+of	O
+iron	B-chemical
+,	O
+the	O
+EncFtnsH	B-protein
+protein	O
+showed	O
+significant	O
+aggregation	O
+(	O
+Figure	O
+8	O
+—	O
+figure	O
+supplement	O
+1F	O
+);	O
+while	O
+the	O
+encapsulin	B-protein
+,	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+system	O
+,	O
+and	O
+apoferritin	B-protein_state
+are	O
+present	O
+as	O
+distinct	O
+nanocompartments	B-complex_assembly
+without	O
+significant	O
+protein	O
+aggregation	O
+(	O
+Figure	O
+8	O
+—	O
+figure	O
+supplement	O
+1G	O
+–	O
+I	O
+).	O
+
+Iron	B-chemical
+storage	O
+in	O
+encapsulin	B-protein
+nanocompartments	B-complex_assembly
+
+The	O
+results	O
+of	O
+the	O
+ferroxidase	B-experimental_method
+assay	I-experimental_method
+and	O
+micrographs	B-evidence
+of	O
+the	O
+reaction	O
+products	O
+suggest	O
+that	O
+the	O
+oxidation	O
+and	O
+mineralization	O
+function	O
+of	O
+the	O
+classical	B-protein_state
+ferritins	B-protein_type
+are	O
+split	O
+between	O
+the	O
+EncFtn	B-protein
+and	O
+encapsulin	B-protein
+proteins	O
+,	O
+with	O
+the	O
+EncFtn	B-protein
+acting	O
+as	O
+a	O
+ferroxidase	B-protein_type
+and	O
+the	O
+encapsulin	B-protein
+shell	B-structure_element
+providing	O
+an	O
+environment	O
+and	O
+template	O
+for	O
+iron	B-chemical
+mineralization	O
+and	O
+storage	O
+.	O
+
+To	O
+investigate	O
+this	O
+further	O
+,	O
+we	O
+added	O
+Fe2	B-chemical
++	I-chemical
+at	O
+various	O
+concentrations	O
+to	O
+samples	O
+of	O
+apo	B-protein_state
+-	O
+ferritin	B-protein_type
+,	O
+EncFtn	B-protein
+,	O
+isolated	O
+encapsulin	B-protein
+,	O
+and	O
+the	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+protein	O
+complex	O
+,	O
+and	O
+subjected	O
+these	O
+samples	O
+to	O
+a	O
+ferrozine	B-experimental_method
+assay	I-experimental_method
+to	O
+quantify	O
+the	O
+amount	O
+of	O
+iron	B-chemical
+associated	O
+with	O
+the	O
+proteins	O
+after	O
+three	O
+hours	O
+of	O
+incubation	O
+.	O
+
+The	O
+maximum	O
+iron	B-chemical
+loading	O
+capacity	O
+of	O
+these	O
+systems	O
+was	O
+calculated	O
+as	O
+the	O
+quantity	O
+of	O
+iron	B-chemical
+per	O
+biological	O
+assembly	O
+(	O
+Figure	O
+8C	O
+).	O
+
+In	O
+this	O
+assay	O
+,	O
+the	O
+EncFtnsH	B-protein
+decamer	B-oligomeric_state
+binds	O
+a	O
+maximum	O
+of	O
+around	O
+48	O
+iron	B-chemical
+ions	O
+before	O
+excess	O
+iron	B-chemical
+induces	O
+protein	O
+precipitation	O
+.	O
+
+The	O
+encapsulin	B-protein
+shell	B-structure_element
+protein	O
+can	O
+sequester	O
+about	O
+2200	O
+iron	B-chemical
+ions	O
+before	O
+significant	O
+protein	O
+loss	O
+occurs	O
+,	O
+and	O
+the	O
+reconstituted	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+nanocompartment	B-complex_assembly
+sequestered	O
+about	O
+4150	O
+iron	B-chemical
+ions	O
+.	O
+
+This	O
+latter	O
+result	O
+is	O
+significantly	O
+more	O
+than	O
+the	O
+apoferritin	B-protein_state
+used	O
+in	O
+our	O
+assay	O
+,	O
+which	O
+sequesters	O
+approximately	O
+570	O
+iron	B-chemical
+ions	O
+in	O
+this	O
+assay	O
+(	O
+Figure	O
+8C	O
+,	O
+Table	O
+5	O
+).	O
+
+Consideration	O
+of	O
+the	O
+functional	O
+oligomeric	O
+states	O
+of	O
+these	O
+proteins	O
+,	O
+where	O
+EncFtn	B-protein
+is	O
+a	O
+decamer	B-oligomeric_state
+and	O
+encapsulin	B-protein
+forms	O
+an	O
+icosahedral	B-protein_state
+cage	B-complex_assembly
+,	O
+and	O
+estimation	O
+of	O
+the	O
+iron	B-chemical
+loading	O
+capacity	O
+of	O
+these	O
+complexes	O
+gives	O
+insight	O
+into	O
+the	O
+role	O
+of	O
+the	O
+two	O
+proteins	O
+in	O
+iron	B-chemical
+storage	O
+and	O
+mineralization	O
+.	O
+
+EncFtn	B-protein
+decamers	B-oligomeric_state
+bind	O
+up	O
+to	O
+48	O
+iron	B-chemical
+ions	O
+(	O
+Figure	O
+8C	O
+),	O
+which	O
+is	O
+significantly	O
+higher	O
+than	O
+the	O
+stoichiometry	O
+of	O
+fifteen	O
+metal	O
+ions	O
+visible	O
+in	O
+the	O
+FOC	B-site
+and	O
+E31	B-site
+/	I-site
+34	I-site
+-	I-site
+site	I-site
+of	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+EncFtnsH	B-protein
+decamer	B-oligomeric_state
+and	O
+our	O
+MS	B-experimental_method
+analysis	O
+.	O
+
+The	O
+discrepancy	O
+between	O
+these	O
+solution	B-experimental_method
+measurements	I-experimental_method
+and	O
+our	O
+MS	B-experimental_method
+analysis	O
+may	O
+indicate	O
+that	O
+there	O
+are	O
+additional	O
+metal	B-site
+-	I-site
+binding	I-site
+sites	I-site
+on	O
+the	O
+interior	O
+channel	B-site
+and	O
+exterior	O
+faces	O
+of	O
+the	O
+protein	O
+;	O
+this	O
+is	O
+consistent	O
+with	O
+our	O
+identification	O
+of	O
+a	O
+number	O
+of	O
+weak	O
+metal	B-site
+-	I-site
+binding	I-site
+sites	I-site
+at	O
+the	O
+surface	O
+of	O
+the	O
+protein	O
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+(	O
+Figure	O
+5D	O
+).	O
+
+These	O
+observations	O
+are	O
+consistent	O
+with	O
+hydrated	O
+Fe2	B-chemical
++	I-chemical
+ions	O
+being	O
+channeled	O
+to	O
+the	O
+active	B-site
+site	I-site
+from	O
+the	O
+E31	B-site
+/	I-site
+34	I-site
+-	I-site
+site	I-site
+and	O
+the	O
+subsequent	O
+exit	O
+of	O
+Fe3	B-chemical
++	I-chemical
+products	O
+on	O
+the	O
+outer	O
+surface	O
+,	O
+as	O
+is	O
+seen	O
+in	O
+other	O
+ferritin	B-protein_type
+family	O
+proteins	O
+.	O
+
+While	O
+the	O
+isolated	O
+encapsulin	B-protein
+shell	B-structure_element
+does	O
+not	O
+display	O
+any	O
+ferroxidase	B-protein_type
+activity	O
+,	O
+it	O
+binds	O
+around	O
+2200	O
+iron	B-chemical
+ions	O
+in	O
+our	O
+assay	O
+(	O
+Table	O
+5	O
+).	O
+
+This	O
+implies	O
+that	O
+the	O
+shell	B-structure_element
+can	O
+bind	O
+a	O
+significant	O
+amount	O
+of	O
+iron	B-chemical
+on	O
+its	O
+outer	O
+and	O
+inner	O
+surfaces	O
+.	O
+
+While	O
+the	O
+maximum	O
+reported	O
+loading	O
+capacity	O
+of	O
+classical	B-protein_state
+ferritins	B-protein_type
+is	O
+approximately	O
+4500	O
+iron	B-chemical
+ions	O
+,	O
+in	O
+our	O
+assay	O
+system	O
+we	O
+were	O
+only	O
+able	O
+to	O
+load	O
+apoferritin	B-protein_state
+with	O
+around	O
+570	O
+iron	B-chemical
+ions	O
+.	O
+
+However	O
+,	O
+the	O
+recombinant	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+nanocompartment	B-complex_assembly
+was	O
+able	O
+to	O
+bind	O
+over	O
+4100	O
+iron	B-chemical
+ions	O
+in	O
+the	O
+same	O
+time	O
+period	O
+,	O
+over	O
+seven	O
+times	O
+the	O
+amount	O
+seen	O
+for	O
+the	O
+apoferritin	B-protein_state
+.	O
+
+We	O
+note	O
+we	O
+do	O
+not	O
+reach	O
+the	O
+experimental	O
+maximum	O
+iron	B-chemical
+loading	O
+for	O
+apoferritin	B-protein_state
+and	O
+therefore	O
+the	O
+total	O
+iron	B-chemical
+-	O
+loading	O
+capacity	O
+of	O
+our	O
+system	O
+may	O
+be	O
+significantly	O
+higher	O
+than	O
+in	O
+this	O
+experimental	O
+system	O
+.	O
+
+Taken	O
+together	O
+,	O
+our	O
+data	O
+show	O
+that	O
+EncFtn	B-protein
+can	O
+catalytically	O
+oxidize	O
+Fe2	B-chemical
++	I-chemical
+to	O
+Fe3	B-chemical
++;	I-chemical
+however	O
+,	O
+iron	B-chemical
+binding	O
+in	O
+EncFtn	B-protein
+is	O
+limited	O
+to	O
+the	O
+FOC	B-site
+and	O
+several	O
+surface	O
+metal	B-site
+binding	I-site
+sites	I-site
+.	O
+
+In	O
+contrast	O
+,	O
+the	O
+encapsulin	B-protein
+protein	O
+displays	O
+no	O
+catalytic	O
+activity	O
+,	O
+but	O
+has	O
+the	O
+ability	O
+to	O
+bind	O
+a	O
+considerable	O
+amount	O
+of	O
+iron	B-chemical
+.	O
+
+Finally	O
+,	O
+the	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+nanocompartment	B-complex_assembly
+complex	O
+retains	O
+the	O
+catalytic	O
+activity	O
+of	O
+EncFtn	B-protein
+,	O
+and	O
+sequesters	O
+iron	B-chemical
+within	O
+the	O
+encapsulin	B-protein
+shell	B-structure_element
+at	O
+a	O
+higher	O
+level	O
+than	O
+the	O
+isolated	O
+components	O
+of	O
+the	O
+system	O
+,	O
+and	O
+at	O
+a	O
+significantly	O
+higher	O
+level	O
+than	O
+the	O
+classical	B-protein_state
+ferritins	B-protein_type
+.	O
+
+Furthermore	O
+,	O
+our	O
+recombinant	O
+nanocompartments	B-complex_assembly
+may	O
+not	O
+have	O
+the	O
+physiological	O
+subunit	O
+stoichiometry	O
+,	O
+and	O
+the	O
+iron	B-chemical
+-	O
+loading	O
+capacity	O
+of	O
+native	B-protein_state
+nanocompartments	B-complex_assembly
+is	O
+potentially	O
+much	O
+higher	O
+than	O
+the	O
+level	O
+we	O
+have	O
+observed	O
+.	O
+
+Mutagenesis	B-experimental_method
+of	O
+the	O
+EncFtnsHferroxidase	B-protein
+center	B-site
+
+Purification	O
+of	O
+recombinant	O
+R	B-species
+.	I-species
+rubrum	I-species
+EncFtnsH	B-protein
+FOC	B-site
+mutants	B-protein_state
+.	O
+
+Single	O
+mutants	B-protein_state
+E32A	B-mutant
+,	O
+E62A	B-mutant
+,	O
+and	O
+H65A	B-mutant
+of	O
+EncFtnsH	B-protein
+produced	O
+from	O
+E	B-species
+.	I-species
+coli	I-species
+BL21	I-species
+(	I-species
+DE3	I-species
+)	I-species
+cells	O
+grown	O
+in	O
+MM	B-experimental_method
+and	O
+MM	B-experimental_method
+supplemented	O
+with	O
+iron	B-chemical
+were	O
+subjected	O
+to	O
+Superdex	O
+200	O
+size	B-experimental_method
+-	I-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+.	O
+
+(	O
+A	O
+)	O
+Gel	B-evidence
+-	I-evidence
+filtration	I-evidence
+chromatogram	I-evidence
+of	O
+the	O
+E32A	B-mutant
+mutant	B-protein_state
+form	O
+of	O
+EncFtnsH	B-protein
+resulted	O
+in	O
+an	O
+elution	B-evidence
+profile	I-evidence
+with	O
+a	O
+majority	O
+of	O
+the	O
+protein	O
+eluting	O
+as	O
+the	O
+decameric	B-oligomeric_state
+form	O
+of	O
+the	O
+protein	O
+and	O
+a	O
+small	O
+proportion	O
+of	O
+monomer	B-oligomeric_state
+.	O
+(	O
+B	O
+)	O
+Gel	B-experimental_method
+-	I-experimental_method
+filtration	I-experimental_method
+chromatograhy	I-experimental_method
+of	O
+the	O
+E62A	B-mutant
+mutant	B-protein_state
+form	O
+of	O
+EncFtnsH	B-protein
+resulted	O
+in	O
+an	O
+elution	B-evidence
+profile	I-evidence
+with	O
+a	O
+single	O
+major	O
+decameric	B-oligomeric_state
+peak	O
+.	O
+(	O
+C	O
+)	O
+Gel	B-experimental_method
+-	I-experimental_method
+filtration	I-experimental_method
+chromatography	I-experimental_method
+of	O
+the	O
+H65A	B-mutant
+mutant	B-protein_state
+form	O
+of	O
+EncFtnsH	B-protein
+resulted	O
+in	O
+a	O
+single	O
+peak	O
+corresponding	O
+to	O
+the	O
+protein	O
+monomer	B-oligomeric_state
+.	O
+
+To	O
+investigate	O
+the	O
+structural	O
+and	O
+biochemical	O
+role	O
+played	O
+by	O
+the	O
+metal	B-site
+binding	I-site
+residues	I-site
+in	O
+the	O
+di	B-site
+-	I-site
+iron	I-site
+FOC	I-site
+of	O
+EncFtnsH	B-protein
+we	O
+produced	O
+alanine	B-experimental_method
+mutations	I-experimental_method
+in	O
+each	O
+of	O
+these	O
+residues	O
+:	O
+Glu32	B-residue_name_number
+,	O
+Glu62	B-residue_name_number
+,	O
+and	O
+His65	B-residue_name_number
+.	O
+
+These	O
+EncFtnsH	B-protein
+mutants	B-protein_state
+were	O
+produced	O
+in	O
+E	B-species
+.	I-species
+coli	I-species
+cells	O
+grown	O
+in	O
+MM	B-experimental_method
+,	O
+both	O
+in	O
+the	O
+absence	B-protein_state
+and	O
+presence	B-protein_state
+of	I-protein_state
+additional	O
+iron	B-chemical
+.	O
+
+The	O
+E32A	B-mutant
+and	O
+E62A	B-mutant
+mutants	B-protein_state
+eluted	O
+from	O
+SEC	B-experimental_method
+at	O
+a	O
+volume	O
+consistent	O
+with	O
+the	O
+decameric	B-oligomeric_state
+form	O
+of	O
+EncFtnsH	B-protein
+,	O
+with	O
+a	O
+small	O
+proportion	O
+of	O
+monomer	B-oligomeric_state
+;	O
+the	O
+H65A	B-mutant
+mutant	B-protein_state
+eluted	O
+at	O
+a	O
+volume	O
+consistent	O
+with	O
+the	O
+monomeric	B-oligomeric_state
+form	O
+of	O
+EncFtnsH	B-protein
+(	O
+Figure	O
+9	O
+).	O
+
+For	O
+all	O
+of	O
+the	O
+mutants	B-protein_state
+studied	O
+,	O
+no	O
+change	O
+in	O
+oligomerization	O
+state	O
+was	O
+apparent	O
+upon	O
+addition	O
+of	O
+Fe2	B-chemical
++	I-chemical
+in	O
+vitro	O
+.	O
+
+Native	B-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+of	O
+EncFtnsH	B-protein
+mutants	B-protein_state
+.	O
+
+All	O
+spectra	B-evidence
+were	O
+acquired	O
+in	O
+100	O
+mM	O
+ammonium	O
+acetate	B-chemical
+,	O
+pH	O
+8	O
+.	O
+0	O
+with	O
+a	O
+protein	O
+concentration	O
+of	O
+5	O
+µM	O
+.	O
+(	O
+A	O
+)	O
+Wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+EncFtnsH	B-protein
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+iron	B-chemical
+displays	O
+a	O
+charge	B-evidence
+state	I-evidence
+distribution	I-evidence
+consistent	O
+with	O
+a	O
+monomer	B-oligomeric_state
+(	O
+see	O
+also	O
+Figure	O
+8	O
+).	O
+(	O
+B	O
+)	O
+E32A	B-mutant
+EncFtnsH	B-protein
+displays	O
+a	O
+charge	B-evidence
+states	I-evidence
+consistent	O
+with	O
+a	O
+decamer	B-oligomeric_state
+(	O
+green	O
+circles	O
+);	O
+a	O
+minor	O
+species	O
+,	O
+consistent	O
+with	O
+the	O
+monomer	B-oligomeric_state
+of	O
+E32A	B-mutant
+mutant	B-protein_state
+is	O
+also	O
+observed	O
+(	O
+blue	O
+circles	O
+).	O
+
+(	O
+C	O
+)	O
+E62A	B-mutant
+EncFtnsH	B-protein
+displays	O
+charge	B-evidence
+states	I-evidence
+consistent	O
+with	O
+a	O
+decamer	B-oligomeric_state
+(	O
+green	O
+circles	O
+).	O
+(	O
+D	O
+)	O
+H65A	B-mutant
+EncFtnsH	B-protein
+displays	O
+charge	B-evidence
+states	I-evidence
+consistent	O
+with	O
+both	O
+monomer	B-oligomeric_state
+(	O
+blue	O
+circles	O
+)	O
+and	O
+dimer	B-oligomeric_state
+(	O
+purple	O
+circles	O
+).	O
+
+In	O
+addition	O
+to	O
+SEC	B-experimental_method
+studies	O
+,	O
+native	B-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+of	O
+the	O
+apo	B-protein_state
+-	O
+EncFtnsH	B-protein
+mutants	B-protein_state
+was	O
+performed	O
+and	O
+compared	O
+with	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+apo	B-protein_state
+-	O
+EncFtnsH	B-protein
+protein	O
+(	O
+Figure	O
+10	O
+).	O
+
+As	O
+described	O
+above	O
+,	O
+the	O
+apo	B-protein_state
+-	O
+EncFtnsH	B-protein
+has	O
+a	O
+charge	B-evidence
+state	I-evidence
+distribution	O
+consistent	O
+with	O
+an	O
+unstructured	B-protein_state
+monomer	B-oligomeric_state
+,	O
+and	O
+decamer	B-oligomeric_state
+formation	O
+is	O
+only	O
+initiated	O
+upon	O
+addition	O
+of	O
+ferrous	O
+iron	B-chemical
+.	O
+
+Both	O
+the	O
+E32A	B-mutant
+mutant	B-protein_state
+and	O
+E62A	B-mutant
+mutant	B-protein_state
+displayed	O
+charge	B-evidence
+state	I-evidence
+distributions	O
+consistent	O
+with	O
+decamers	B-oligomeric_state
+,	O
+even	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+Fe2	B-chemical
++.	I-chemical
+
+This	O
+gas	O
+-	O
+phase	O
+observation	O
+is	O
+consistent	O
+with	O
+SEC	B-experimental_method
+measurements	O
+,	O
+which	O
+indicate	O
+both	O
+of	O
+these	O
+variants	O
+were	O
+also	O
+decamers	B-oligomeric_state
+in	O
+solution	O
+.	O
+
+Thus	O
+it	O
+seems	O
+that	O
+these	O
+mutations	O
+allow	O
+the	O
+decamer	B-oligomeric_state
+to	O
+form	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+iron	B-chemical
+in	O
+the	O
+FOC	B-site
+.	O
+
+In	O
+contrast	O
+to	O
+the	O
+glutamic	B-residue_name
+acid	I-residue_name
+mutants	B-protein_state
+,	O
+MS	B-experimental_method
+analysis	O
+of	O
+the	O
+H65A	B-mutant
+mutant	B-protein_state
+is	O
+similar	O
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+apo	B-protein_state
+-	O
+EncFtnsH	B-protein
+and	O
+is	O
+present	O
+as	O
+a	O
+monomer	B-oligomeric_state
+;	O
+interestingly	O
+a	O
+minor	O
+population	O
+of	O
+dimeric	B-oligomeric_state
+H65A	B-mutant
+was	O
+also	O
+observed	O
+.	O
+
+We	O
+propose	O
+that	O
+the	O
+observed	O
+differences	O
+in	O
+the	O
+oligomerization	O
+state	O
+of	O
+the	O
+E32A	B-mutant
+and	O
+E62A	B-mutant
+mutants	B-protein_state
+compared	O
+to	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+are	O
+due	O
+to	O
+the	O
+changes	O
+in	O
+the	O
+electrostatic	O
+environment	O
+within	O
+the	O
+FOC	B-site
+.	O
+
+At	O
+neutral	B-protein_state
+pH	I-protein_state
+the	O
+glutamic	B-residue_name
+acid	I-residue_name
+residues	O
+are	O
+negatively	O
+charged	O
+,	O
+while	O
+the	O
+histidine	B-residue_name
+residues	O
+are	O
+predominantly	O
+in	O
+their	O
+uncharged	O
+state	O
+.	O
+
+In	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+(	O
+WT	B-protein_state
+)	O
+EncFtnsH	B-protein
+this	O
+leads	O
+to	O
+electrostatic	O
+repulsion	O
+between	O
+subunits	B-structure_element
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+iron	B-chemical
+.	O
+
+Coordination	B-bond_interaction
+of	O
+Fe2	B-chemical
++	I-chemical
+in	O
+this	O
+site	O
+stabilizes	O
+the	O
+dimer	B-oligomeric_state
+and	O
+reconstitutes	O
+the	O
+active	B-protein_state
+FOC	B-site
+.	O
+
+The	O
+geometric	O
+arrangement	O
+of	O
+Glu32	B-residue_name_number
+and	O
+Glu62	B-residue_name_number
+in	O
+the	O
+FOC	B-site
+explains	O
+their	O
+behavior	O
+in	O
+solution	O
+and	O
+the	O
+gas	O
+phase	O
+,	O
+where	O
+they	O
+both	O
+favor	O
+the	O
+formation	O
+of	O
+decamers	B-oligomeric_state
+due	O
+to	O
+the	O
+loss	O
+of	O
+a	O
+repulsive	O
+negative	O
+charge	O
+.	O
+
+The	O
+FOC	B-site
+in	O
+the	O
+H65A	B-mutant
+mutant	B-protein_state
+is	O
+destabilized	O
+through	O
+the	O
+loss	B-protein_state
+of	I-protein_state
+this	O
+metal	B-site
+coordinating	I-site
+residue	I-site
+and	O
+potential	O
+positive	O
+charge	O
+carrier	O
+,	O
+thus	O
+favoring	O
+the	O
+monomer	B-oligomeric_state
+in	O
+solution	O
+and	O
+the	O
+gas	O
+phase	O
+.	O
+
+Data	B-evidence
+collection	I-evidence
+and	I-evidence
+refinement	I-evidence
+statistics	I-evidence
+.	O
+
+WT	B-protein_state
+E32A	B-mutant
+E62A	B-mutant
+H65A	B-mutant
+Data	O
+collection	O
+Wavelength	O
+(	O
+Å	O
+)	O
+1	O
+.	O
+74	O
+1	O
+.	O
+73	O
+1	O
+.	O
+73	O
+1	O
+.	O
+74	O
+Resolution	O
+range	O
+(	O
+Å	O
+)	O
+49	O
+.	O
+63	O
+-	O
+2	O
+.	O
+06	O
+(	O
+2	O
+.	O
+10	O
+-	O
+2	O
+.	O
+06	O
+)	O
+48	O
+.	O
+84	O
+-	O
+2	O
+.	O
+59	O
+(	O
+2	O
+.	O
+683	O
+-	O
+2	O
+.	O
+59	O
+)	O
+48	O
+.	O
+87	O
+-	O
+2	O
+.	O
+21	O
+(	O
+2	O
+.	O
+29	O
+-	O
+2	O
+.	O
+21	O
+)	O
+48	O
+.	O
+86	O
+-	O
+2	O
+.	O
+97	O
+(	O
+3	O
+.	O
+08	O
+-	O
+2	O
+.	O
+97	O
+)	O
+Space	O
+group	O
+P	O
+1	O
+21	O
+1	O
+P	O
+1	O
+21	O
+1	O
+P	O
+1	O
+21	O
+1	O
+P	O
+1	O
+21	O
+1	O
+Unit	O
+cell	O
+(	O
+Å	O
+)	O
+a	O
+b	O
+c	O
+β	O
+(°)	O
+98	O
+.	O
+18	O
+120	O
+.	O
+53	O
+140	O
+.	O
+30	O
+95	O
+.	O
+36	O
+97	O
+.	O
+78	O
+120	O
+.	O
+28	O
+140	O
+.	O
+53	O
+95	O
+.	O
+41	O
+98	O
+.	O
+09	O
+120	O
+.	O
+23	O
+140	O
+.	O
+36	O
+95	O
+.	O
+50	O
+98	O
+.	O
+03	O
+120	O
+.	O
+29	O
+140	O
+.	O
+43	O
+95	O
+.	O
+39	O
+Total	O
+reflections	O
+1	O
+,	O
+264	O
+,	O
+922	O
+(	O
+41	O
+,	O
+360	O
+)	O
+405	O
+,	O
+488	O
+(	O
+36	O
+,	O
+186	O
+)	O
+1	O
+,	O
+069	O
+,	O
+345	O
+(	O
+95	O
+,	O
+716	O
+)	O
+323	O
+,	O
+853	O
+(	O
+32	O
+,	O
+120	O
+)	O
+Unique	O
+reflections	O
+197	O
+,	O
+873	O
+(	O
+8	O
+,	O
+766	O
+)	O
+100	O
+,	O
+067	O
+(	O
+9	O
+,	O
+735	O
+)	O
+162	O
+,	O
+379	O
+(	O
+15	O
+,	O
+817	O
+)	O
+66	O
+,	O
+658	O
+(	O
+6	O
+,	O
+553	O
+)	O
+Multiplicity	O
+6	O
+.	O
+4	O
+(	O
+4	O
+.	O
+7	O
+)	O
+4	O
+.	O
+1	O
+(	O
+3	O
+.	O
+7	O
+)	O
+6	O
+.	O
+6	O
+(	O
+6	O
+.	O
+1	O
+)	O
+4	O
+.	O
+9	O
+(	O
+4	O
+.	O
+9	O
+)	O
+Anomalous	O
+multiplicity	O
+3	O
+.	O
+2	O
+(	O
+2	O
+.	O
+6	O
+)	O
+N	O
+/	O
+A	O
+N	O
+/	O
+A	O
+N	O
+/	O
+A	O
+Completeness	O
+(%)	O
+99	O
+.	O
+2	O
+(	O
+88	O
+.	O
+6	O
+)	O
+99	O
+.	O
+0	O
+(	O
+97	O
+.	O
+0	O
+)	O
+100	O
+(	O
+97	O
+.	O
+0	O
+)	O
+100	O
+(	O
+99	O
+.	O
+0	O
+)	O
+Anomalous	O
+completeness	O
+(%)	O
+96	O
+.	O
+7	O
+(	O
+77	O
+.	O
+2	O
+)	O
+N	O
+/	O
+A	O
+N	O
+/	O
+A	O
+N	O
+/	O
+A	O
+Mean	O
+I	O
+/	O
+sigma	O
+(	O
+I	O
+)	O
+10	O
+.	O
+6	O
+(	O
+1	O
+.	O
+60	O
+)	O
+8	O
+.	O
+46	O
+(	O
+1	O
+.	O
+79	O
+)	O
+13	O
+.	O
+74	O
+(	O
+1	O
+.	O
+80	O
+)	O
+8	O
+.	O
+09	O
+(	O
+1	O
+.	O
+74	O
+)	O
+Wilson	O
+B	O
+-	O
+factor	O
+26	O
+.	O
+98	O
+40	O
+.	O
+10	O
+33	O
+.	O
+97	O
+52	O
+.	O
+20	O
+Rmerge	O
+0	O
+.	O
+123	O
+(	O
+0	O
+.	O
+790	O
+)	O
+0	O
+.	O
+171	O
+(	O
+0	O
+.	O
+792	O
+)	O
+0	O
+.	O
+0979	O
+(	O
+1	O
+.	O
+009	O
+)	O
+0	O
+.	O
+177	O
+(	O
+0	O
+.	O
+863	O
+)	O
+Rmeas	O
+0	O
+.	O
+147	O
+(	O
+0	O
+.	O
+973	O
+)	O
+0	O
+.	O
+196	O
+(	O
+0	O
+.	O
+923	O
+)	O
+0	O
+.	O
+1064	O
+(	O
+1	O
+.	O
+107	O
+)	O
+0	O
+.	O
+199	O
+(	O
+0	O
+.	O
+966	O
+)	O
+CC1	O
+/	O
+2	O
+0	O
+.	O
+995	O
+(	O
+0	O
+.	O
+469	O
+)	O
+0	O
+.	O
+985	O
+(	O
+0	O
+.	O
+557	O
+)	O
+0	O
+.	O
+998	O
+(	O
+0	O
+.	O
+642	O
+)	O
+0	O
+.	O
+989	O
+(	O
+0	O
+.	O
+627	O
+)	O
+CC	O
+*	O
+0	O
+.	O
+999	O
+(	O
+0	O
+.	O
+846	O
+)	O
+0	O
+.	O
+996	O
+(	O
+0	O
+.	O
+846	O
+)	O
+0	O
+.	O
+999	O
+(	O
+0	O
+.	O
+884	O
+)	O
+0	O
+.	O
+997	O
+(	O
+0	O
+.	O
+878	O
+)	O
+Image	O
+DOI	O
+10	O
+.	O
+7488	O
+/	O
+ds	O
+/	O
+1342	O
+10	O
+.	O
+7488	O
+/	O
+ds	O
+/	O
+1419	O
+10	O
+.	O
+7488	O
+/	O
+ds	O
+/	O
+1420	O
+10	O
+.	O
+7488	O
+/	O
+ds	O
+/	O
+1421	O
+Refinement	O
+Rwork	O
+0	O
+.	O
+171	O
+(	O
+0	O
+.	O
+318	O
+)	O
+0	O
+.	O
+183	O
+(	O
+0	O
+.	O
+288	O
+)	O
+0	O
+.	O
+165	O
+(	O
+0	O
+.	O
+299	O
+)	O
+0	O
+.	O
+186	O
+(	O
+0	O
+.	O
+273	O
+)	O
+Rfree	O
+0	O
+.	O
+206	O
+(	O
+0	O
+.	O
+345	O
+)	O
+0	O
+.	O
+225	O
+(	O
+0351	O
+)	O
+0	O
+.	O
+216	O
+(	O
+0	O
+.	O
+364	O
+)	O
+0	O
+.	O
+237	O
+(	O
+0	O
+.	O
+325	O
+)	O
+Number	O
+of	O
+non	O
+-	O
+hydrogen	O
+atoms	O
+23	O
+,	O
+222	O
+22	O
+,	O
+366	O
+22	O
+,	O
+691	O
+22	O
+,	O
+145	O
+macromolecules	O
+22	O
+,	O
+276	O
+22	O
+,	O
+019	O
+21	O
+,	O
+965	O
+22	O
+,	O
+066	O
+ligands	O
+138	O
+8	O
+24	O
+74	O
+water	B-chemical
+808	O
+339	O
+702	O
+5	O
+Protein	O
+residues	O
+2	O
+,	O
+703	O
+2	O
+,	O
+686	O
+2	O
+,	O
+675	O
+2	O
+,	O
+700	O
+RMS	O
+(	O
+bonds	O
+)	O
+(	O
+Å	O
+)	O
+0	O
+.	O
+012	O
+0	O
+.	O
+005	O
+0	O
+.	O
+011	O
+0	O
+.	O
+002	O
+RMS	O
+(	O
+angles	O
+)	O
+(°)	O
+1	O
+.	O
+26	O
+0	O
+.	O
+58	O
+1	O
+.	O
+02	O
+0	O
+.	O
+40	O
+Ramachandran	O
+favored	O
+(%)	O
+100	O
+99	O
+100	O
+99	O
+Ramachandran	O
+allowed	O
+(%)	O
+0	O
+1	O
+0	O
+1	O
+Ramachandran	O
+outliers	O
+(%)	O
+0	O
+0	O
+0	O
+0	O
+Clash	O
+score	O
+1	O
+.	O
+42	O
+1	O
+.	O
+42	O
+1	O
+.	O
+79	O
+0	O
+.	O
+97	O
+Average	O
+B	O
+-	O
+factor	O
+(	O
+Å2	O
+)	O
+33	O
+.	O
+90	O
+42	O
+.	O
+31	O
+41	O
+.	O
+34	O
+47	O
+.	O
+68	O
+macromolecules	O
+33	O
+.	O
+80	O
+42	O
+.	O
+35	O
+41	O
+.	O
+31	O
+47	O
+.	O
+60	O
+ligands	O
+40	O
+.	O
+40	O
+72	O
+.	O
+80	O
+65	O
+.	O
+55	O
+72	O
+.	O
+34	O
+solvent	O
+36	O
+.	O
+20	O
+38	O
+.	O
+95	O
+41	O
+.	O
+46	O
+33	O
+.	O
+85	O
+PDB	O
+ID	O
+5DA5	O
+5L89	O
+5L8B	O
+5L8G	O
+
+Iron	B-chemical
+loading	O
+capacity	O
+of	O
+EncFtn	B-protein
+,	O
+encapsulin	B-protein
+and	O
+ferritin	B-protein_type
+.	O
+
+Protein	O
+samples	O
+(	O
+at	O
+8	O
+.	O
+5	O
+µM	O
+)	O
+including	O
+decameric	B-oligomeric_state
+EncFtnsH	B-protein
+,	O
+encapsulin	B-protein
+,	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+and	O
+apoferritin	B-protein_state
+were	O
+mixed	O
+with	O
+Fe	B-chemical
+(	I-chemical
+NH4	I-chemical
+)	I-chemical
+2	I-chemical
+(	I-chemical
+SO4	I-chemical
+)	I-chemical
+(	O
+in	O
+0	O
+.	O
+1	O
+%	O
+(	O
+v	O
+/	O
+v	O
+)	O
+HCl	B-chemical
+)	O
+of	O
+different	O
+concentrations	O
+in	O
+50	O
+mM	O
+Tris	O
+-	O
+HCl	O
+(	O
+pH	O
+8	O
+.	O
+0	O
+),	O
+150	O
+mM	O
+NaCl	B-chemical
+buffer	O
+at	O
+room	O
+temperature	O
+for	O
+3	O
+hrs	O
+in	O
+the	O
+air	O
+.	O
+
+Protein	O
+-	O
+Fe	B-chemical
+mixtures	O
+were	O
+centrifuged	O
+at	O
+13	O
+,	O
+000	O
+x	O
+g	O
+to	O
+remove	O
+precipitated	O
+material	O
+and	O
+desalted	O
+prior	O
+to	O
+the	O
+Fe	B-chemical
+and	O
+protein	O
+content	O
+analysis	O
+by	O
+ferrozine	B-experimental_method
+assay	I-experimental_method
+and	O
+BCA	B-experimental_method
+microplate	I-experimental_method
+assay	I-experimental_method
+,	O
+respectively	O
+.	O
+
+Fe	B-chemical
+to	O
+protein	O
+ratio	O
+was	O
+calculated	O
+to	O
+indicate	O
+the	O
+Fe	B-chemical
+binding	O
+capacity	O
+of	O
+the	O
+protein	O
+.	O
+
+Protein	O
+stability	O
+was	O
+compromised	O
+at	O
+high	O
+iron	B-chemical
+concentrations	O
+;	O
+therefore	O
+,	O
+the	O
+highest	O
+iron	B-chemical
+loading	O
+with	O
+the	O
+least	O
+protein	O
+precipitation	O
+was	O
+used	O
+to	O
+derive	O
+the	O
+maximum	O
+iron	B-chemical
+loading	O
+capacity	O
+per	O
+biological	O
+assembly	O
+(	O
+underlined	O
+and	O
+highlighted	O
+in	O
+bold	O
+).	O
+
+The	O
+biological	O
+unit	O
+assemblies	O
+are	O
+a	O
+decamer	B-oligomeric_state
+for	O
+EncFtnsH	B-protein
+,	O
+a	O
+60mer	B-oligomeric_state
+for	O
+encapsulin	B-protein
+,	O
+a	O
+60mer	B-oligomeric_state
+of	O
+encapsulin	B-protein
+loaded	B-protein_state
+with	I-protein_state
+12	O
+copies	O
+of	O
+decameric	B-oligomeric_state
+EncFtn	B-protein
+in	O
+the	O
+complex	O
+,	O
+and	O
+24mer	B-oligomeric_state
+for	O
+horse	B-taxonomy_domain
+spleen	O
+apoferritin	B-protein_state
+.	O
+
+Errors	O
+are	O
+quoted	O
+as	O
+the	O
+standard	O
+deviation	O
+of	O
+three	O
+technical	O
+repeats	O
+in	O
+both	O
+the	O
+ferrozine	B-experimental_method
+and	I-experimental_method
+BCA	I-experimental_method
+microplate	I-experimental_method
+assays	I-experimental_method
+.	O
+
+The	O
+proteins	O
+used	O
+in	O
+Fe	B-chemical
+loading	O
+experiment	O
+came	O
+from	O
+a	O
+single	O
+preparation	O
+.	O
+
+Protein	O
+sample	O
+Fe	B-chemical
+(	I-chemical
+NH4	I-chemical
+)	I-chemical
+2	I-chemical
+(	I-chemical
+SO4	I-chemical
+)	I-chemical
+2	I-chemical
+loading	O
+(	O
+µM	O
+)	O
+Fe	B-chemical
+detected	O
+by	O
+ferrozine	B-experimental_method
+assay	I-experimental_method
+(	O
+µM	O
+)	O
+Protein	O
+detected	O
+by	O
+BCA	B-experimental_method
+microplate	I-experimental_method
+assay	I-experimental_method
+(	O
+µM	O
+)	O
+Fe	B-chemical
+/	O
+monomeric	O
+protein	O
+Maximum	O
+Fe	B-chemical
+loading	O
+per	O
+biological	O
+assembly	O
+unit	O
+8	O
+.	O
+46	O
+µM	O
+EncFtnsH	B-protein
+-	O
+10mer	B-oligomeric_state
+0	O
+4	O
+.	O
+73	O
+±	O
+2	O
+.	O
+32	O
+5	O
+.	O
+26	O
+±	O
+0	O
+.	O
+64	O
+0	O
+.	O
+90	O
+±	O
+0	O
+.	O
+44	O
+39	O
+.	O
+9	O
+9	O
+.	O
+93	O
+±	O
+1	O
+.	O
+20	O
+5	O
+.	O
+36	O
+±	O
+0	O
+.	O
+69	O
+1	O
+.	O
+85	O
+±	O
+0	O
+.	O
+22	O
+84	O
+17	O
+.	O
+99	O
+±	O
+2	O
+.	O
+01	O
+4	O
+.	O
+96	O
+±	O
+0	O
+.	O
+04	O
+3	O
+.	O
+63	O
+±	O
+0	O
+.	O
+41	O
+147	O
+21	O
+.	O
+09	O
+±	O
+1	O
+.	O
+94	O
+4	O
+.	O
+44	O
+±	O
+0	O
+.	O
+21	O
+4	O
+.	O
+75	O
+±	O
+0	O
+.	O
+44	O
+48	O
+±	O
+4	O
+224	O
+28	O
+.	O
+68	O
+±	O
+0	O
+.	O
+30	O
+3	O
+.	O
+73	O
+±	O
+0	O
+.	O
+53	O
+7	O
+.	O
+68	O
+±	O
+0	O
+.	O
+08	O
+301	O
+11	O
+.	O
+27	O
+±	O
+1	O
+.	O
+10	O
+2	O
+.	O
+50	O
+±	O
+0	O
+.	O
+05	O
+4	O
+.	O
+51	O
+±	O
+0	O
+.	O
+44	O
+8	O
+.	O
+50	O
+µM	O
+Encapsulin	B-protein
+0	O
+-	O
+1	O
+.	O
+02	O
+±	O
+0	O
+.	O
+54	O
+8	O
+.	O
+63	O
+±	O
+0	O
+.	O
+17	O
+-	O
+0	O
+.	O
+12	O
+±	O
+0	O
+.	O
+06	O
+224	O
+62	O
+.	O
+24	O
+±	O
+2	O
+.	O
+49	O
+10	O
+.	O
+01	O
+±	O
+0	O
+.	O
+58	O
+6	O
+.	O
+22	O
+±	O
+0	O
+.	O
+35	O
+301	O
+67	O
+.	O
+94	O
+±	O
+3	O
+.	O
+15	O
+8	O
+.	O
+69	O
+±	O
+0	O
+.	O
+42	O
+7	O
+.	O
+81	O
+±	O
+0	O
+.	O
+36	O
+450	O
+107	O
+.	O
+96	O
+±	O
+8	O
+.	O
+88	O
+8	O
+.	O
+50	O
+±	O
+0	O
+.	O
+69	O
+12	O
+.	O
+71	O
+±	O
+1	O
+.	O
+05	O
+700	O
+97	O
+.	O
+51	O
+±	O
+3	O
+.	O
+19	O
+7	O
+.	O
+26	O
+±	O
+0	O
+.	O
+20	O
+13	O
+.	O
+44	O
+±	O
+0	O
+.	O
+44	O
+1000	O
+308	O
+.	O
+63	O
+±	O
+2	O
+.	O
+06	O
+8	O
+.	O
+42	O
+±	O
+0	O
+.	O
+34	O
+36	O
+.	O
+66	O
+±	O
+0	O
+.	O
+24	O
+2199	O
+±	O
+15	O
+1500	O
+57	O
+.	O
+09	O
+±	O
+0	O
+.	O
+90	O
+1	O
+.	O
+44	O
+±	O
+0	O
+.	O
+21	O
+39	O
+.	O
+77	O
+±	O
+0	O
+.	O
+62	O
+2000	O
+9	O
+.	O
+2	O
+±	O
+1	O
+.	O
+16	O
+0	O
+.	O
+21	O
+±	O
+0	O
+.	O
+14	O
+44	O
+.	O
+73	O
+±	O
+5	O
+.	O
+63	O
+8	O
+.	O
+70	O
+µM	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+0	O
+3	O
+.	O
+31	O
+±	O
+1	O
+.	O
+57	O
+6	O
+.	O
+85	O
+±	O
+0	O
+.	O
+07	O
+0	O
+.	O
+48	O
+±	O
+0	O
+.	O
+23	O
+224	O
+116	O
+.	O
+27	O
+±	O
+3	O
+.	O
+74	O
+7	O
+.	O
+63	O
+±	O
+0	O
+.	O
+12	O
+15	O
+.	O
+25	O
+±	O
+0	O
+.	O
+49	O
+301	O
+132	O
+.	O
+86	O
+±	O
+4	O
+.	O
+03	O
+6	O
+.	O
+66	O
+±	O
+0	O
+.	O
+31	O
+19	O
+.	O
+96	O
+±	O
+0	O
+.	O
+61	O
+450	O
+220	O
+.	O
+57	O
+±	O
+27	O
+.	O
+33	O
+6	O
+.	O
+12	O
+±	O
+1	O
+.	O
+07	O
+36	O
+.	O
+06	O
+±	O
+4	O
+.	O
+47	O
+700	O
+344	O
+.	O
+03	O
+±	O
+40	O
+.	O
+38	O
+6	O
+.	O
+94	O
+±	O
+0	O
+.	O
+17	O
+49	O
+.	O
+58	O
+±	O
+5	O
+.	O
+82	O
+1000	O
+496	O
+.	O
+00	O
+±	O
+38	O
+.	O
+48	O
+7	O
+.	O
+19	O
+±	O
+0	O
+.	O
+08	O
+68	O
+.	O
+94	O
+±	O
+5	O
+.	O
+35	O
+4137	O
+±	O
+321	O
+1500	O
+569	O
+.	O
+98	O
+±	O
+73	O
+.	O
+63	O
+5	O
+.	O
+73	O
+±	O
+0	O
+.	O
+03	O
+99	O
+.	O
+44	O
+±	O
+12	O
+.	O
+84	O
+2000	O
+584	O
+.	O
+30	O
+±	O
+28	O
+.	O
+33	O
+4	O
+.	O
+88	O
+±	O
+0	O
+.	O
+22	O
+119	O
+.	O
+62	O
+±	O
+5	O
+.	O
+80	O
+8	O
+.	O
+50	O
+µM	O
+Apoferritin	B-protein_state
+0	O
+3	O
+.	O
+95	O
+±	O
+2	O
+.	O
+26	O
+9	O
+.	O
+37	O
+±	O
+0	O
+.	O
+24	O
+0	O
+.	O
+42	O
+±	O
+0	O
+.	O
+25	O
+42	O
+.	O
+5	O
+10	O
+.	O
+27	O
+±	O
+1	O
+.	O
+12	O
+8	O
+.	O
+27	O
+±	O
+0	O
+.	O
+30	O
+1	O
+.	O
+24	O
+±	O
+0	O
+.	O
+18	O
+212	O
+.	O
+5	O
+44	O
+.	O
+48	O
+±	O
+2	O
+.	O
+76	O
+7	O
+.	O
+85	O
+±	O
+0	O
+.	O
+77	O
+5	O
+.	O
+67	O
+±	O
+0	O
+.	O
+83	O
+637	O
+.	O
+5	O
+160	O
+.	O
+93	O
+±	O
+4	O
+.	O
+27	O
+6	O
+.	O
+76	O
+±	O
+0	O
+.	O
+81	O
+23	O
+.	O
+79	O
+±	O
+3	O
+.	O
+12	O
+571	O
+±	O
+75	O
+1275	O
+114	O
+.	O
+92	O
+±	O
+3	O
+.	O
+17	O
+3	O
+.	O
+84	O
+±	O
+0	O
+.	O
+30	O
+29	O
+.	O
+91	O
+±	O
+2	O
+.	O
+95	O
+1700	O
+91	O
+.	O
+40	O
+±	O
+3	O
+.	O
+37	O
+3	O
+.	O
+14	O
+±	O
+0	O
+.	O
+35	O
+29	O
+.	O
+13	O
+±	O
+3	O
+.	O
+86	O
+
+To	O
+understand	O
+the	O
+impact	O
+of	O
+the	O
+mutants	B-protein_state
+on	O
+the	O
+organization	O
+and	O
+metal	O
+binding	O
+of	O
+the	O
+FOC	B-site
+,	O
+we	O
+determined	O
+the	O
+X	B-evidence
+-	I-evidence
+ray	I-evidence
+crystal	I-evidence
+structures	I-evidence
+of	O
+each	O
+of	O
+the	O
+EncFtnsH	B-protein
+mutants	B-protein_state
+(	O
+See	O
+Table	O
+4	O
+for	O
+data	O
+collection	O
+and	O
+refinement	O
+statistics	O
+).	O
+
+The	O
+crystal	O
+packing	O
+of	O
+all	O
+of	O
+the	O
+mutants	B-protein_state
+in	O
+this	O
+study	O
+is	O
+essentially	O
+isomorphous	O
+to	O
+the	O
+EncFtnsH	B-protein
+structure	B-evidence
+.	O
+
+All	O
+of	O
+the	O
+mutants	B-protein_state
+display	O
+the	O
+same	O
+decameric	B-oligomeric_state
+arrangement	O
+in	O
+the	O
+crystals	B-evidence
+as	O
+the	O
+EncFtnsH	B-protein
+structure	B-evidence
+,	O
+and	O
+the	O
+monomers	B-oligomeric_state
+superimpose	B-experimental_method
+with	O
+an	O
+average	O
+RMSDCα	B-evidence
+of	O
+less	O
+than	O
+0	O
+.	O
+2	O
+Å	O
+.	O
+
+FOC	B-site
+dimer	B-site
+interface	I-site
+of	O
+EncFtnsH	B-mutant
+-	I-mutant
+E32A	I-mutant
+mutant	B-protein_state
+.	O
+
+(	O
+A	O
+)	O
+Wall	O
+-	O
+eyed	O
+stereo	O
+view	O
+of	O
+the	O
+metal	B-site
+-	I-site
+binding	I-site
+dimerization	I-site
+interface	I-site
+of	O
+EncFtnsH	B-mutant
+-	I-mutant
+E32A	I-mutant
+.	O
+
+Protein	O
+residues	O
+are	O
+shown	O
+as	O
+sticks	O
+with	O
+blue	O
+and	O
+green	O
+carbons	O
+for	O
+the	O
+different	O
+subunits	B-structure_element
+.	O
+
+The	O
+2mFo	B-evidence
+-	I-evidence
+DFc	I-evidence
+electron	I-evidence
+density	I-evidence
+map	I-evidence
+is	O
+shown	O
+as	O
+a	O
+blue	O
+mesh	O
+contoured	O
+at	O
+1	O
+.	O
+5	O
+σ	O
+.	O
+
+(	O
+B	O
+)	O
+Views	O
+of	O
+the	O
+FOC	B-site
+of	O
+the	O
+EncFtnsH	B-mutant
+-	I-mutant
+E32Amutant	I-mutant
+.	O
+
+FOC	B-site
+dimer	I-site
+interface	I-site
+of	O
+EncFtnsH	B-mutant
+-	I-mutant
+E62A	I-mutant
+mutant	B-protein_state
+.	O
+
+(	O
+A	O
+)	O
+Wall	O
+-	O
+eyed	O
+stereo	O
+view	O
+of	O
+the	O
+metal	B-site
+-	I-site
+binding	I-site
+dimerization	I-site
+interface	I-site
+of	O
+EncFtnsH	B-mutant
+-	I-mutant
+E62A	I-mutant
+.	O
+
+The	O
+single	O
+coordinated	O
+calcium	B-chemical
+ion	O
+is	O
+shown	O
+as	O
+a	O
+grey	O
+sphere	O
+.	O
+(	O
+B	O
+)	O
+Views	O
+of	O
+the	O
+FOC	B-site
+of	O
+the	O
+EncFtnsH	B-mutant
+-	I-mutant
+E62A	I-mutant
+mutant	B-protein_state
+.	O
+
+FOC	B-site
+dimer	I-site
+interface	I-site
+of	O
+EncFtnsH	B-mutant
+-	I-mutant
+H65A	I-mutant
+mutant	B-protein_state
+.	O
+
+(	O
+A	O
+)	O
+Wall	O
+-	O
+eyed	O
+stereo	O
+view	O
+of	O
+the	O
+metal	B-site
+-	I-site
+binding	I-site
+dimerization	I-site
+interface	I-site
+of	O
+EncFtnsH	B-mutant
+-	I-mutant
+H65A	I-mutant
+.	O
+
+The	O
+coordinated	O
+calcium	B-chemical
+ions	O
+are	O
+shown	O
+as	O
+a	O
+grey	O
+spheres	O
+with	O
+coordination	B-bond_interaction
+distances	O
+in	O
+the	O
+FOC	B-site
+highlighted	O
+with	O
+yellow	O
+dashed	O
+lines	O
+.	O
+
+(	O
+B	O
+)	O
+Views	O
+of	O
+the	O
+FOC	B-site
+of	O
+the	O
+EncFtnsH	B-mutant
+-	I-mutant
+H65A	I-mutant
+mutant	B-protein_state
+.	O
+
+Comparison	O
+of	O
+the	O
+EncFtnsH	B-protein
+FOC	B-site
+mutants	B-protein_state
+vs	O
+wild	B-protein_state
+type	I-protein_state
+.	O
+
+The	O
+structures	B-evidence
+of	O
+the	O
+three	O
+EncFtnsH	B-protein
+mutants	B-protein_state
+were	O
+all	O
+determined	O
+by	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallography	I-experimental_method
+.	O
+
+The	O
+E32A	B-mutant
+,	O
+E62A	B-mutant
+and	O
+H65A	B-mutant
+mutants	B-protein_state
+were	O
+crystallized	B-experimental_method
+in	O
+identical	O
+conditions	O
+to	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+.	O
+
+EncFtnsH	B-protein
+structure	B-evidence
+and	O
+were	O
+essentially	O
+isomorphous	O
+in	O
+terms	O
+of	O
+their	O
+unit	O
+cell	O
+dimensions	O
+.	O
+
+The	O
+FOC	B-site
+residues	O
+of	O
+the	O
+mutants	B-protein_state
+and	O
+native	B-protein_state
+EncFtnsH	B-protein
+structures	B-evidence
+are	O
+shown	O
+as	O
+sticks	O
+with	O
+coordinated	B-bond_interaction
+Fe2	B-chemical
++	I-chemical
+as	O
+orange	O
+and	O
+Ca2	B-chemical
++	I-chemical
+as	O
+grey	O
+spheres	O
+and	O
+are	O
+colored	O
+as	O
+follows	O
+:	O
+wild	B-protein_state
+type	I-protein_state
+,	O
+grey	O
+;	O
+E32A	B-mutant
+,	O
+pink	O
+;	O
+E62A	B-mutant
+,	O
+green	O
+;	O
+H65A	B-mutant
+,	O
+blue	O
+.	O
+
+Of	O
+the	O
+mutants	B-protein_state
+,	O
+only	O
+H65A	B-mutant
+has	O
+any	O
+coordinated	B-bond_interaction
+metal	O
+ions	O
+,	O
+which	O
+appear	O
+to	O
+be	O
+calcium	B-chemical
+ions	O
+from	O
+the	O
+crystallization	O
+condition	O
+.	O
+
+The	O
+overall	O
+organization	O
+of	O
+FOC	B-site
+residues	O
+is	O
+retained	O
+in	O
+the	O
+mutants	B-protein_state
+,	O
+with	O
+almost	O
+no	O
+backbone	O
+movements	O
+.	O
+
+Significant	O
+differences	O
+center	O
+around	O
+Tyr39	B-residue_name_number
+,	O
+which	O
+moves	O
+to	O
+coordinate	B-bond_interaction
+the	O
+bound	B-protein_state
+calcium	B-chemical
+ions	O
+in	O
+the	O
+H65A	B-mutant
+mutant	B-protein_state
+;	O
+and	O
+Glu32	B-residue_name_number
+,	O
+which	O
+moves	O
+away	O
+from	O
+the	O
+metal	O
+ions	O
+in	O
+this	O
+structure	B-evidence
+.	O
+
+Close	O
+inspection	O
+of	O
+the	O
+region	O
+of	O
+the	O
+protein	O
+around	O
+the	O
+FOC	B-site
+in	O
+each	O
+of	O
+the	O
+mutants	B-protein_state
+highlights	O
+their	O
+effect	O
+on	O
+metal	O
+binding	O
+(	O
+Figure	O
+11	O
+and	O
+Figure	O
+11	O
+—	O
+figure	O
+supplement	O
+1	O
+–	O
+3	O
+).	O
+
+In	O
+the	O
+E32A	B-mutant
+mutant	B-protein_state
+the	O
+position	O
+of	O
+the	O
+side	O
+chains	O
+of	O
+the	O
+remaining	O
+iron	B-site
+coordinating	I-site
+residues	I-site
+in	O
+the	O
+FOC	B-site
+is	O
+essentially	O
+unchanged	O
+,	O
+but	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+the	O
+axial	O
+-	O
+metal	O
+coordinating	B-bond_interaction
+ligand	O
+provided	O
+by	O
+the	O
+Glu32	B-residue_name_number
+side	O
+chain	O
+abrogates	B-protein_state
+metal	I-protein_state
+binding	I-protein_state
+in	O
+this	O
+site	O
+.	O
+
+The	O
+Glu31	B-site
+/	I-site
+34	I-site
+-	I-site
+site	I-site
+also	O
+lacks	B-protein_state
+metal	B-chemical
+,	O
+with	O
+the	O
+side	O
+chain	O
+of	O
+Glu31	B-residue_name_number
+rotated	O
+by	O
+180	O
+°	O
+at	O
+the	O
+Cβ	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+metal	B-chemical
+(	O
+Figure	O
+11	O
+—	O
+figure	O
+supplement	O
+1	O
+).	O
+
+The	O
+E62A	B-mutant
+mutant	B-protein_state
+has	O
+a	O
+similar	O
+effect	O
+on	O
+the	O
+FOC	B-site
+to	O
+the	O
+E32A	B-mutant
+mutant	B-protein_state
+,	O
+however	O
+the	O
+entry	B-site
+site	I-site
+still	O
+has	O
+a	O
+calcium	B-chemical
+ion	O
+coordinated	B-bond_interaction
+between	O
+residues	O
+Glu31	B-residue_name_number
+and	O
+Glu34	B-residue_name_number
+(	O
+Figure	O
+11	O
+—	O
+figure	O
+supplement	O
+2	O
+).	O
+
+The	O
+H65A	B-mutant
+mutant	B-protein_state
+diverges	O
+significantly	O
+from	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+in	O
+the	O
+position	O
+of	O
+the	O
+residues	O
+Glu32	B-residue_name_number
+and	O
+Tyr39	B-residue_name_number
+in	O
+the	O
+FOC	B-site
+.	O
+
+E32	B-residue_name_number
+appears	O
+in	O
+either	O
+the	O
+original	O
+orientation	O
+as	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+and	O
+coordinates	B-bond_interaction
+Ca2	B-chemical
++	I-chemical
+in	O
+this	O
+position	O
+,	O
+or	O
+it	O
+is	O
+flipped	O
+by	O
+180	O
+°	O
+at	O
+the	O
+Cβ	O
+,	O
+moving	O
+away	O
+from	O
+the	O
+coordinated	B-bond_interaction
+calcium	B-chemical
+ion	O
+in	O
+the	O
+FOC	B-site
+.	O
+
+Tyr39	B-residue_name_number
+moves	O
+closer	O
+to	O
+Ca2	B-chemical
++	I-chemical
+compared	O
+to	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+coordinates	B-bond_interaction
+the	O
+calcium	B-chemical
+ion	O
+(	O
+Figure	O
+11	O
+—	O
+figure	O
+supplement	O
+3	O
+).	O
+
+A	O
+single	O
+calcium	B-chemical
+ion	O
+is	O
+present	O
+in	O
+the	O
+entry	B-site
+site	I-site
+of	O
+this	O
+mutant	B-protein_state
+;	O
+however	O
+,	O
+Glu31	B-residue_name_number
+of	O
+one	O
+chain	O
+is	O
+rotated	O
+away	O
+from	O
+the	O
+metal	O
+ion	O
+and	O
+is	O
+not	O
+involved	O
+in	O
+coordination	B-bond_interaction
+.	O
+
+Taken	O
+together	O
+the	O
+results	O
+of	O
+our	O
+data	O
+show	O
+that	O
+these	O
+changes	O
+to	O
+the	O
+FOC	B-site
+of	O
+EncFtn	B-protein
+still	O
+permit	O
+the	O
+formation	O
+of	O
+the	O
+decameric	B-oligomeric_state
+form	O
+of	O
+the	O
+protein	O
+.	O
+
+While	O
+the	O
+proteins	O
+all	O
+appear	O
+decameric	B-oligomeric_state
+in	O
+crystals	B-evidence
+,	O
+their	O
+solution	O
+and	O
+gas	O
+-	O
+phase	O
+behavior	O
+differs	O
+considerably	O
+and	O
+the	O
+mutants	B-protein_state
+no	O
+longer	O
+show	O
+metal	O
+-	O
+dependent	O
+oligomerization	O
+.	O
+
+These	O
+results	O
+highlight	O
+the	O
+importance	O
+of	O
+metal	B-chemical
+coordination	B-bond_interaction
+in	O
+the	O
+FOC	B-site
+for	O
+the	O
+stability	O
+and	O
+assembly	O
+of	O
+the	O
+EncFtn	B-protein
+protein	O
+.	O
+
+Progress	B-evidence
+curves	I-evidence
+recording	O
+ferroxidase	B-protein_type
+activity	O
+of	O
+EncFtnsH	B-protein
+mutants	B-protein_state
+.	O
+
+20	O
+µM	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+EncFtnsH	B-protein
+,	O
+E32A	B-mutant
+,	O
+E62A	B-mutant
+and	O
+H65A	B-mutant
+mutants	B-protein_state
+were	O
+mixed	O
+with	O
+20	O
+µM	O
+or	O
+100	O
+µM	O
+acidic	O
+Fe	B-chemical
+(	I-chemical
+NH4	I-chemical
+)	I-chemical
+2	I-chemical
+(	I-chemical
+SO4	I-chemical
+)	I-chemical
+2	I-chemical
+,	O
+respectively	O
+.	O
+
+Absorbance	O
+at	O
+315	O
+nm	O
+was	O
+recorded	O
+for	O
+1800	O
+s	O
+at	O
+25	O
+°	O
+C	O
+as	O
+an	O
+indication	O
+of	O
+Fe3	B-chemical
++	I-chemical
+formation	O
+.	O
+
+Protein	O
+free	O
+samples	O
+(	O
+dashed	O
+and	O
+dotted	O
+lines	O
+)	O
+were	O
+measured	O
+for	O
+Fe2	B-chemical
++	I-chemical
+background	O
+oxidation	O
+as	O
+controls	O
+.	O
+
+Relative	O
+ferroxidase	B-protein_type
+activity	O
+of	O
+EncFtnsH	B-protein
+mutants	B-protein_state
+.	O
+
+EncFtnsH	B-protein
+,	O
+and	O
+the	O
+mutant	B-protein_state
+forms	O
+E32A	B-mutant
+,	O
+E62A	B-mutant
+and	O
+H65A	B-mutant
+,	O
+each	O
+at	O
+20	O
+µM	O
+,	O
+were	O
+mixed	O
+with	O
+100	O
+µM	O
+acidic	O
+Fe	B-chemical
+(	I-chemical
+NH4	I-chemical
+)	I-chemical
+2	I-chemical
+(	I-chemical
+SO4	I-chemical
+)	I-chemical
+2	I-chemical
+.	O
+
+Ferroxidase	B-protein_type
+activity	O
+of	O
+the	O
+mutant	B-protein_state
+forms	O
+is	O
+determined	O
+by	O
+measuring	B-experimental_method
+the	I-experimental_method
+absorbance	I-experimental_method
+at	I-experimental_method
+315	I-experimental_method
+nm	I-experimental_method
+for	O
+1800	O
+s	O
+at	O
+25	O
+°	O
+C	O
+as	O
+an	O
+indication	O
+of	O
+Fe3	B-chemical
++	I-chemical
+formation	O
+.	O
+
+The	O
+relative	O
+ferroxidase	B-protein_type
+activity	O
+of	O
+mutants	B-protein_state
+is	O
+plotted	O
+as	O
+a	O
+proportion	O
+of	O
+the	O
+activity	O
+of	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+protein	O
+using	O
+the	O
+endpoint	O
+measurement	B-experimental_method
+of	I-experimental_method
+A315	I-experimental_method
+.	O
+
+The	O
+FOC	B-site
+mutants	B-protein_state
+showed	O
+reduced	O
+ferroxidase	B-protein_type
+activity	O
+to	O
+varied	O
+extents	O
+,	O
+among	O
+which	O
+E62A	B-mutant
+significantly	O
+abrogated	O
+the	O
+ferroxidase	B-protein_type
+activity	O
+.	O
+
+To	O
+address	O
+the	O
+question	O
+of	O
+how	O
+mutagenesis	B-experimental_method
+of	O
+the	O
+iron	B-site
+coordinating	I-site
+residues	I-site
+affects	O
+the	O
+enzymatic	O
+activity	O
+of	O
+the	O
+EncFtnsH	B-protein
+protein	O
+we	O
+recorded	O
+progress	B-evidence
+curves	I-evidence
+for	O
+the	O
+oxidation	O
+of	O
+Fe2	B-chemical
++	I-chemical
+to	O
+Fe3	B-chemical
++	I-chemical
+by	O
+the	O
+different	O
+mutants	B-protein_state
+as	O
+before	O
+.	O
+
+Mutagenesis	B-experimental_method
+of	O
+E32A	B-mutant
+and	O
+H65A	B-mutant
+reduces	O
+the	O
+activity	O
+of	O
+EncFtnsH	B-protein
+by	O
+about	O
+40	O
+%-	O
+55	O
+%;	O
+the	O
+E62A	B-mutant
+mutant	B-protein_state
+completely	O
+abrogates	O
+activity	O
+,	O
+presumably	O
+through	O
+the	O
+loss	B-protein_state
+of	I-protein_state
+the	O
+bridging	O
+coordination	B-bond_interaction
+for	O
+the	O
+formation	O
+of	O
+the	O
+di	B-site
+-	I-site
+nuclear	I-site
+iron	I-site
+center	I-site
+of	O
+the	O
+FOC	B-site
+(	O
+Figure	O
+12	O
+).	O
+
+Collectively	O
+,	O
+the	O
+effect	O
+of	O
+mutating	B-experimental_method
+these	O
+residues	O
+in	O
+the	O
+FOC	B-site
+confirms	O
+the	O
+importance	O
+of	O
+the	O
+iron	B-site
+coordinating	I-site
+residues	I-site
+for	O
+the	O
+ferroxidase	B-protein_type
+activity	O
+of	O
+the	O
+EncFtnsH	B-protein
+protein	O
+.	O
+
+Phylogenetic	B-evidence
+tree	I-evidence
+of	O
+ferritin	B-protein_type
+family	O
+proteins	O
+.	O
+
+The	O
+tree	O
+was	O
+built	O
+using	O
+the	O
+Neighbor	B-experimental_method
+-	I-experimental_method
+Joining	I-experimental_method
+method	I-experimental_method
+based	O
+on	O
+step	B-experimental_method
+-	I-experimental_method
+wise	I-experimental_method
+amino	I-experimental_method
+acid	I-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+of	O
+the	O
+four	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+bundle	I-structure_element
+portions	O
+of	O
+ferritin	B-protein_type
+family	O
+proteins	O
+(	O
+Supplementary	O
+file	O
+1	O
+).	O
+
+The	O
+evolutionary	B-evidence
+distances	I-evidence
+were	O
+computed	O
+using	O
+the	O
+p	B-experimental_method
+-	I-experimental_method
+distance	I-experimental_method
+method	I-experimental_method
+and	O
+are	O
+in	O
+the	O
+units	O
+of	O
+the	O
+number	O
+of	O
+amino	O
+acid	O
+differences	O
+per	O
+site	O
+.	O
+
+Our	O
+study	O
+reports	O
+on	O
+a	O
+new	O
+class	O
+of	O
+ferritin	B-protein_type
+-	O
+like	O
+proteins	O
+(	O
+EncFtn	B-protein
+),	O
+which	O
+are	O
+associated	O
+with	O
+bacterial	B-taxonomy_domain
+encapsulin	B-protein
+nanocompartments	B-complex_assembly
+(	O
+Enc	B-protein
+).	O
+
+By	O
+studying	O
+the	O
+EncFtn	B-protein
+from	O
+R	B-species
+.	I-species
+rubrum	I-species
+we	O
+demonstrate	O
+that	O
+iron	B-chemical
+binding	O
+results	O
+in	O
+assembly	O
+of	O
+EncFtn	B-protein
+decamers	B-oligomeric_state
+,	O
+which	O
+display	O
+a	O
+unique	O
+annular	O
+architecture	O
+.	O
+
+Despite	O
+a	O
+radically	O
+different	O
+quaternary	O
+structure	O
+to	O
+the	O
+classical	B-protein_state
+ferritins	B-protein_type
+,	O
+the	O
+four	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+bundle	I-structure_element
+scaffold	I-structure_element
+and	O
+FOC	B-site
+of	O
+EncFtnsH	B-protein
+are	O
+strikingly	O
+similar	O
+to	O
+ferritin	B-protein_type
+(	O
+Figure	O
+6A	O
+).	O
+
+A	O
+sequence	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+phylogenetic	I-experimental_method
+tree	I-experimental_method
+for	O
+proteins	O
+in	O
+the	O
+ferritin	B-protein_type
+family	O
+was	O
+constructed	O
+;	O
+in	O
+addition	O
+to	O
+the	O
+classical	B-protein_state
+ferritins	B-protein_type
+,	O
+bacterioferritins	B-protein_type
+and	O
+Dps	B-protein_type
+proteins	O
+,	O
+our	O
+analysis	O
+included	O
+the	O
+encapsulin	B-protein_type
+-	I-protein_type
+associated	I-protein_type
+ferritin	I-protein_type
+-	I-protein_type
+like	I-protein_type
+proteins	I-protein_type
+(	O
+EncFtns	B-protein_type
+)	O
+and	O
+a	O
+group	O
+related	O
+to	O
+these	O
+,	O
+but	O
+lacking	O
+the	O
+encapsulin	B-protein
+sequence	O
+(	O
+Non	B-protein_type
+-	I-protein_type
+EncFtn	I-protein_type
+).	O
+
+The	O
+analysis	O
+revealed	O
+that	O
+the	O
+EncFtn	B-protein
+and	O
+Non	B-protein_type
+-	I-protein_type
+EncFtn	I-protein_type
+proteins	O
+form	O
+groups	O
+distinct	O
+from	O
+the	O
+other	O
+clearly	O
+delineated	O
+groups	O
+of	O
+ferritins	B-protein_type
+,	O
+and	O
+represent	O
+outliers	O
+in	O
+the	O
+tree	O
+(	O
+Figure	O
+13	O
+).	O
+
+While	O
+it	O
+is	O
+difficult	O
+to	O
+infer	O
+ancestral	O
+lineages	O
+in	O
+protein	O
+families	O
+,	O
+the	O
+similarity	O
+seen	O
+in	O
+the	O
+active	B-site
+site	I-site
+scaffold	I-site
+of	O
+these	O
+proteins	O
+highlights	O
+a	O
+shared	O
+evolutionary	O
+relationship	O
+between	O
+EncFtn	B-protein_type
+proteins	O
+and	O
+other	O
+members	O
+of	O
+the	O
+ferritin	B-protein_type
+superfamily	O
+that	O
+has	O
+been	O
+noted	O
+in	O
+previous	O
+studies	O
+(;	O
+).	O
+
+From	O
+this	O
+analysis	O
+,	O
+we	O
+propose	O
+that	O
+the	O
+four	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+fold	I-structure_element
+of	O
+the	O
+classical	B-protein_state
+ferritins	B-protein_type
+may	O
+have	O
+arisen	O
+through	O
+gene	O
+duplication	O
+of	O
+an	O
+ancestor	O
+of	O
+EncFtn	B-protein
+.	O
+
+This	O
+gene	O
+duplication	O
+would	O
+result	O
+in	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+one	O
+EncFtn	B-protein
+monomer	B-oligomeric_state
+being	O
+linked	O
+to	O
+the	O
+N	O
+-	O
+terminus	O
+of	O
+another	O
+and	O
+thus	O
+stabilizing	O
+the	O
+four	B-structure_element
+-	I-structure_element
+helix	I-structure_element
+bundle	I-structure_element
+fold	I-structure_element
+within	O
+a	O
+single	O
+polypeptide	O
+chain	O
+(	O
+Figure	O
+6B	O
+).	O
+
+Linking	O
+the	O
+protein	O
+together	O
+in	O
+this	O
+way	O
+relaxes	O
+the	O
+requirement	O
+for	O
+the	O
+maintenance	O
+of	O
+a	O
+symmetrical	O
+FOC	B-site
+and	O
+thus	O
+provides	O
+a	O
+path	O
+to	O
+the	O
+diversity	O
+in	O
+active	B-site
+-	I-site
+site	I-site
+residues	I-site
+seen	O
+across	O
+the	O
+ferritin	B-protein_type
+family	O
+(	O
+Figure	O
+6A	O
+,	O
+residues	O
+Glu95	B-residue_name_number
+,	O
+Gln128	B-residue_name_number
+and	O
+Glu131	B-residue_name_number
+in	O
+PmFtn	B-protein
+,	O
+Supplementary	O
+file	O
+1	O
+).	O
+
+Relationship	O
+between	O
+ferritin	B-protein_type
+structure	B-evidence
+and	O
+activity	O
+
+The	O
+quaternary	O
+arrangement	O
+of	O
+classical	B-protein_state
+ferritins	B-protein_type
+into	O
+an	O
+octahedral	B-protein_state
+nanocage	B-complex_assembly
+and	O
+Dps	B-protein
+into	O
+a	O
+dodecamer	B-oligomeric_state
+is	O
+absolutely	O
+required	O
+for	O
+their	O
+function	O
+as	O
+iron	B-chemical
+storage	O
+compartments	O
+.	O
+
+The	O
+oxidation	O
+and	O
+mineralization	O
+of	O
+iron	B-chemical
+must	O
+be	O
+spatially	O
+separated	O
+from	O
+the	O
+host	O
+cytosol	O
+to	O
+prevent	O
+the	O
+formation	O
+of	O
+damaging	O
+hydroxyl	O
+radicals	O
+in	O
+the	O
+Fenton	O
+and	O
+Haber	O
+-	O
+Weiss	O
+reactions	O
+.	O
+
+This	O
+is	O
+achieved	O
+in	O
+all	O
+ferritins	B-protein_type
+by	O
+confining	O
+the	O
+oxidation	O
+of	O
+iron	B-chemical
+to	O
+the	O
+interior	O
+of	O
+the	O
+protein	O
+complex	O
+,	O
+thus	O
+achieving	O
+sequestration	O
+of	O
+the	O
+Fe3	B-chemical
++	I-chemical
+mineralization	O
+product	O
+.	O
+
+A	O
+structural	B-experimental_method
+alignment	I-experimental_method
+of	O
+the	O
+FOC	B-site
+of	O
+EncFtn	B-protein
+with	O
+the	O
+classical	B-protein_state
+ferritin	B-protein_type
+PmFtn	B-protein
+shows	O
+that	O
+the	O
+central	B-structure_element
+ring	I-structure_element
+of	O
+EncFtn	B-protein
+corresponds	O
+to	O
+the	O
+external	O
+surface	O
+of	O
+ferritin	B-protein_type
+,	O
+while	O
+the	O
+outer	O
+circumference	O
+of	O
+EncFtn	B-protein
+is	O
+congruent	O
+with	O
+the	O
+inner	O
+mineralization	B-site
+surface	I-site
+of	O
+ferritin	B-protein_type
+(	O
+Figure	O
+6	O
+—	O
+figure	O
+supplement	O
+1A	O
+).	O
+
+This	O
+overlay	B-experimental_method
+highlights	O
+the	O
+fact	O
+that	O
+the	O
+ferroxidase	B-site
+center	I-site
+of	O
+EncFtn	B-protein
+faces	O
+in	O
+the	O
+opposite	O
+direction	O
+relative	O
+to	O
+the	O
+classical	B-protein_state
+ferritins	B-protein_type
+and	O
+is	O
+essentially	O
+inside	O
+out	O
+regarding	O
+iron	B-chemical
+storage	O
+space	O
+(	O
+Figure	O
+6	O
+—	O
+figure	O
+supplement	O
+1B	O
+,	O
+boxed	O
+region	O
+).	O
+
+Analysis	O
+of	O
+each	O
+of	O
+the	O
+single	O
+mutations	B-experimental_method
+(	O
+E32A	B-mutant
+,	O
+E62A	B-mutant
+and	O
+H65A	B-mutant
+)	O
+made	O
+in	O
+the	O
+FOC	B-site
+highlights	O
+the	O
+importance	O
+of	O
+the	O
+iron	B-site
+-	I-site
+coordinating	I-site
+residues	I-site
+in	O
+the	O
+catalytic	O
+activity	O
+of	O
+EncFtn	B-protein
+.	O
+
+Furthermore	O
+,	O
+the	O
+position	O
+of	O
+the	O
+calcium	B-chemical
+ion	O
+coordinated	B-bond_interaction
+by	I-bond_interaction
+Glu31	B-residue_name_number
+and	O
+Glu34	B-residue_name_number
+seen	O
+in	O
+the	O
+EncFtnsH	B-protein
+structure	B-evidence
+suggests	O
+an	O
+entry	B-site
+site	I-site
+to	O
+channel	O
+metal	O
+ions	O
+into	O
+the	O
+FOC	B-site
+;	O
+we	O
+propose	O
+that	O
+this	O
+site	O
+binds	O
+hydrated	O
+iron	B-chemical
+ions	O
+in	O
+vivo	O
+and	O
+acts	O
+as	O
+a	O
+selectivity	O
+filter	O
+and	O
+gate	O
+for	O
+the	O
+FOC	B-site
+.	O
+
+The	O
+constellation	O
+of	O
+charged	O
+residues	O
+on	O
+the	O
+outer	O
+circumference	O
+of	O
+EncFtn	B-protein
+(	O
+His57	B-residue_name_number
+,	O
+Glu61	B-residue_name_number
+and	O
+Glu64	B-residue_name_number
+)	O
+could	O
+function	O
+in	O
+the	O
+same	O
+way	O
+as	O
+the	O
+residues	O
+lining	O
+the	O
+mineralization	B-site
+surface	I-site
+within	O
+the	O
+classical	B-protein_state
+ferritin	B-protein_type
+nanocage	B-complex_assembly
+,	O
+and	O
+given	O
+their	O
+proximity	O
+to	O
+the	O
+FOC	B-site
+these	O
+sites	O
+may	O
+be	O
+the	O
+exit	B-site
+portal	I-site
+and	O
+mineralization	B-site
+site	I-site
+.	O
+
+The	O
+absolute	O
+requirement	O
+for	O
+the	O
+spatial	O
+separation	O
+of	O
+oxidation	O
+and	O
+mineralization	O
+in	O
+ferritins	B-protein_type
+suggests	O
+that	O
+the	O
+EncFtn	B-protein_type
+family	O
+proteins	O
+are	O
+not	O
+capable	O
+of	O
+storing	O
+iron	B-chemical
+minerals	O
+due	O
+to	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+an	O
+enclosed	O
+compartment	O
+in	O
+their	O
+structure	O
+(	O
+Figure	O
+6	O
+—	O
+figure	O
+supplement	O
+1B	O
+).	O
+
+Our	O
+biochemical	B-experimental_method
+characterization	I-experimental_method
+of	O
+EncFtn	B-protein
+supports	O
+this	O
+hypothesis	O
+,	O
+indicating	O
+that	O
+while	O
+this	O
+protein	O
+is	O
+capable	O
+of	O
+oxidizing	O
+iron	B-chemical
+,	O
+it	O
+does	O
+not	O
+accrue	O
+mineralized	O
+iron	B-chemical
+in	O
+an	O
+analogous	O
+manner	O
+to	O
+classical	B-protein_state
+ferritins	B-protein_type
+.	O
+
+While	O
+EncFtn	B-protein
+does	O
+not	O
+store	O
+iron	B-chemical
+itself	O
+,	O
+its	O
+association	O
+with	O
+the	O
+encapsulin	B-protein
+nanocage	B-complex_assembly
+suggests	O
+that	O
+mineralization	O
+occurs	O
+within	O
+the	O
+cavity	B-site
+of	O
+the	O
+encapsulin	B-protein
+shell	B-structure_element
+.	O
+
+Our	O
+ferroxidase	B-experimental_method
+assay	I-experimental_method
+data	O
+on	O
+the	O
+recombinant	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+nanocompartments	B-complex_assembly
+,	O
+which	O
+accrue	O
+over	O
+4100	O
+iron	B-chemical
+ions	O
+per	O
+complex	O
+and	O
+form	O
+regular	O
+nanoparticles	B-complex_assembly
+,	O
+are	O
+consistent	O
+with	O
+the	O
+encapsulin	B-protein
+protein	O
+acting	O
+as	O
+the	O
+store	O
+for	O
+iron	B-chemical
+oxidized	O
+by	O
+the	O
+EncFtn	B-protein
+enzyme	O
+.	O
+
+TEM	B-experimental_method
+analysis	O
+of	O
+the	O
+reaction	O
+products	O
+shows	O
+the	O
+production	O
+of	O
+homogeneous	O
+iron	B-chemical
+nanoparticles	O
+only	O
+in	O
+the	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+nanocompartment	B-complex_assembly
+(	O
+Figure	O
+8	O
+—	O
+figure	O
+supplement	O
+1	O
+).	O
+
+Model	O
+of	O
+iron	B-chemical
+oxidation	O
+in	O
+encapsulin	B-protein
+nanocompartments	B-complex_assembly
+.	O
+
+(	O
+A	O
+)	O
+Model	O
+of	O
+EncFtnsH	B-protein
+docking	B-experimental_method
+to	O
+the	O
+encapsulin	B-protein
+shell	B-structure_element
+.	O
+
+A	O
+single	O
+pentamer	B-oligomeric_state
+of	O
+the	O
+icosahedral	B-protein_state
+T	B-species
+.	I-species
+maritima	I-species
+encapsulin	B-protein
+structure	B-evidence
+(	O
+PDBID	O
+:	O
+3DKT	O
+)	O
+is	O
+shown	O
+as	O
+a	O
+blue	O
+surface	O
+with	O
+the	O
+encapsulin	B-protein
+localization	B-structure_element
+sequence	I-structure_element
+of	O
+EncFtn	B-protein
+shown	O
+as	O
+a	O
+purple	O
+surface	O
+.	O
+
+The	O
+C	O
+-	O
+terminal	O
+regions	O
+of	O
+the	O
+EncFtn	B-protein
+subunits	B-structure_element
+correspond	O
+to	O
+the	O
+position	O
+of	O
+the	O
+localization	B-structure_element
+sequences	I-structure_element
+seen	O
+in	O
+3DKT	O
+.	O
+
+Alignment	B-experimental_method
+of	O
+EncFtnsH	B-protein
+with	O
+3DKT	O
+positions	O
+the	O
+central	B-site
+channel	I-site
+directly	O
+above	O
+the	O
+pore	B-site
+in	O
+the	O
+3DKT	O
+pentamer	B-oligomeric_state
+axis	O
+(	O
+shown	O
+as	O
+a	O
+grey	O
+pentagon	O
+).	O
+(	O
+B	O
+)	O
+Surface	O
+view	O
+of	O
+EncFtn	B-protein
+within	O
+the	O
+encapsulin	B-protein
+nanocompartment	B-complex_assembly
+(	O
+grey	O
+and	O
+blue	O
+respectively	O
+).	O
+
+The	O
+lumen	O
+of	O
+the	O
+encapsulin	B-protein
+nanocompartment	B-complex_assembly
+is	O
+considerably	O
+larger	O
+than	O
+the	O
+interior	O
+of	O
+ferritin	B-protein_type
+(	O
+shown	O
+in	O
+orange	O
+behind	O
+the	O
+encapsulin	B-protein
+for	O
+reference	O
+)	O
+and	O
+thus	O
+allows	O
+the	O
+storage	O
+of	O
+significantly	O
+more	O
+iron	B-chemical
+.	O
+
+The	O
+proposed	O
+pathway	O
+for	O
+iron	B-chemical
+movement	O
+through	O
+the	O
+encapsulin	B-protein
+shell	B-structure_element
+and	O
+EncFtn	B-protein
+FOC	B-site
+is	O
+shown	O
+with	O
+arrows	O
+.	O
+(	O
+C	O
+)	O
+Model	O
+ofiron	O
+oxidation	O
+within	O
+an	O
+encapsulin	B-protein
+nanocompartment	B-complex_assembly
+.	O
+
+As	O
+EncFtn	B-protein
+is	O
+unable	O
+to	O
+mineralize	O
+iron	B-chemical
+on	O
+its	O
+surface	O
+directly	O
+,	O
+Fe2	B-chemical
++	I-chemical
+must	O
+pass	O
+through	O
+the	O
+encapsulin	B-protein
+shell	B-structure_element
+to	O
+access	O
+the	O
+first	O
+metal	B-site
+binding	I-site
+site	I-site
+within	O
+the	O
+central	B-site
+channel	I-site
+of	O
+EncFtnsH	B-protein
+(	O
+entry	B-site
+site	I-site
+)	O
+prior	O
+to	O
+oxidation	O
+within	O
+the	O
+FOC	B-site
+and	O
+release	O
+as	O
+Fe3	B-chemical
++	I-chemical
+to	O
+the	O
+outer	O
+surface	O
+of	O
+the	O
+protein	O
+where	O
+it	O
+can	O
+be	O
+mineralized	O
+within	O
+the	O
+lumen	O
+of	O
+the	O
+encapsulin	B-protein
+cage	O
+.	O
+
+Docking	B-experimental_method
+the	O
+decamer	B-oligomeric_state
+structure	B-evidence
+of	O
+EncFtnsH	B-protein
+into	O
+the	O
+pentamer	B-oligomeric_state
+of	O
+the	O
+T	B-species
+.	I-species
+maritima	I-species
+encapsulin	B-protein
+Tmari_0786	B-gene
+(	O
+PDB	O
+ID	O
+:	O
+3DKT	O
+)	O
+shows	O
+that	O
+the	O
+position	O
+of	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+extensions	I-structure_element
+of	O
+our	O
+EncFtnsH	B-protein
+structure	B-evidence
+are	O
+consistent	O
+with	O
+the	O
+localization	B-structure_element
+sequences	I-structure_element
+seen	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+encapsulin	B-protein
+protein	O
+(	O
+Figure	O
+14A	O
+).	O
+
+Thus	O
+,	O
+it	O
+appears	O
+that	O
+the	O
+EncFtn	B-protein
+decamer	B-oligomeric_state
+is	O
+the	O
+physiological	O
+state	O
+of	O
+this	O
+protein	O
+.	O
+
+This	O
+arrangement	O
+positions	O
+the	O
+central	B-structure_element
+ring	I-structure_element
+of	O
+EncFtn	B-protein
+directly	O
+above	O
+the	O
+pore	B-site
+at	O
+the	O
+five	O
+-	O
+fold	O
+symmetry	O
+axis	O
+of	O
+the	O
+encapsulin	B-protein
+shell	B-structure_element
+and	O
+highlights	O
+a	O
+potential	O
+route	O
+for	O
+the	O
+entry	O
+of	O
+iron	B-chemical
+into	O
+the	O
+encapsulin	B-protein
+and	O
+towards	O
+the	O
+active	B-site
+site	I-site
+of	O
+EncFtn	B-protein
+.	O
+
+A	O
+comparison	O
+of	O
+the	O
+encapsulin	B-protein
+nanocompartment	B-complex_assembly
+and	O
+the	O
+ferritin	B-protein_type
+nanocage	B-complex_assembly
+highlights	O
+the	O
+size	O
+differential	O
+between	O
+the	O
+two	O
+complexes	O
+(	O
+Figure	O
+14B	O
+)	O
+that	O
+allows	O
+the	O
+encapsulin	B-protein
+to	O
+store	O
+significantly	O
+more	O
+iron	B-chemical
+.	O
+
+The	O
+presence	B-protein_state
+of	I-protein_state
+five	O
+FOCs	B-site
+per	O
+EncFtnsH	B-protein
+decamer	B-oligomeric_state
+and	O
+the	O
+fact	O
+that	O
+the	O
+icosahedral	B-protein_state
+encapsulin	B-protein
+nanocage	B-complex_assembly
+can	O
+hold	O
+up	O
+to	O
+twelve	O
+of	O
+decameric	B-oligomeric_state
+EncFtn	B-protein
+between	O
+each	O
+of	O
+the	O
+internal	O
+five	O
+-	O
+fold	O
+vertices	O
+means	O
+that	O
+they	O
+can	O
+achieve	O
+a	O
+high	O
+rate	O
+of	O
+iron	B-chemical
+mineralization	O
+across	O
+the	O
+entire	O
+nanocompartment	B-complex_assembly
+.	O
+
+This	O
+arrangement	O
+of	O
+multiple	O
+reaction	O
+centers	O
+in	O
+a	O
+single	O
+protein	O
+assembly	O
+is	O
+reminiscent	O
+of	O
+classical	B-protein_state
+ferritins	B-protein_type
+,	O
+which	O
+has	O
+24	O
+FOCs	B-site
+distributed	O
+around	O
+the	O
+nanocage	B-complex_assembly
+.	O
+
+Our	O
+structural	B-evidence
+data	I-evidence
+,	O
+coupled	O
+with	O
+biochemical	B-experimental_method
+and	I-experimental_method
+ICP	I-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+analysis	O
+,	O
+suggest	O
+a	O
+model	O
+for	O
+the	O
+activity	O
+of	O
+the	O
+encapsulin	B-protein
+iron	B-complex_assembly
+-	I-complex_assembly
+megastore	I-complex_assembly
+(	O
+Figure	O
+14C	O
+).	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+T	B-species
+.	I-species
+maritima	I-species
+encapsulin	B-protein
+shell	B-structure_element
+protein	O
+has	O
+a	O
+negatively	B-site
+charged	I-site
+pore	I-site
+positioned	O
+to	O
+allow	O
+the	O
+passage	O
+of	O
+Fe2	B-chemical
++	I-chemical
+into	O
+the	O
+encapsulin	B-protein
+and	O
+directs	O
+the	O
+metal	O
+towards	O
+the	O
+central	O
+,	O
+negatively	B-site
+charged	I-site
+hole	I-site
+of	O
+the	O
+EncFtn	B-protein
+ring	B-structure_element
+(	O
+Figure	O
+4	O
+—	O
+figure	O
+supplement	O
+1	O
+).	O
+
+The	O
+five	O
+metal	B-site
+-	I-site
+binding	I-site
+sites	I-site
+on	O
+the	O
+interior	O
+of	O
+the	O
+ring	B-structure_element
+(	O
+Glu31	B-site
+/	I-site
+34	I-site
+-	I-site
+sites	I-site
+)	O
+may	O
+select	O
+for	O
+the	O
+Fe2	B-chemical
++	I-chemical
+ion	O
+and	O
+direct	O
+it	O
+towards	O
+their	O
+cognate	O
+FOCs	B-site
+.	O
+
+We	O
+propose	O
+that	O
+the	O
+oxidation	O
+of	O
+Fe2	B-chemical
++	I-chemical
+to	O
+Fe3	B-chemical
++	I-chemical
+occurs	O
+within	O
+the	O
+FOC	B-site
+according	O
+to	O
+the	O
+model	O
+postulated	O
+by	O
+in	O
+which	O
+the	O
+FOC	B-site
+acts	O
+as	O
+a	O
+substrate	B-site
+site	I-site
+through	O
+which	O
+iron	B-chemical
+passes	O
+and	O
+is	O
+released	O
+on	O
+to	O
+weakly	B-site
+coordinating	I-site
+sites	I-site
+at	O
+the	O
+outer	O
+circumference	O
+of	O
+the	O
+protein	O
+(	O
+His57	B-residue_name_number
+,	O
+Glu61	B-residue_name_number
+and	O
+Glu64	B-residue_name_number
+),	O
+where	O
+it	O
+is	O
+able	O
+to	O
+form	O
+ferrihydrite	B-chemical
+minerals	O
+which	O
+can	O
+be	O
+safely	O
+deposited	O
+within	O
+the	O
+lumen	O
+of	O
+the	O
+encapsulin	B-protein
+nanocompartment	B-complex_assembly
+(	O
+Figure	O
+14	O
+).	O
+
+Here	O
+we	O
+describe	O
+for	O
+the	O
+first	O
+time	O
+the	O
+structure	B-evidence
+and	O
+biochemistry	O
+of	O
+a	O
+new	O
+class	O
+of	O
+encapsulin	B-protein_type
+-	I-protein_type
+associated	I-protein_type
+ferritin	I-protein_type
+-	I-protein_type
+like	I-protein_type
+protein	I-protein_type
+and	O
+demonstrate	O
+that	O
+it	O
+has	O
+an	O
+absolute	O
+requirement	O
+for	O
+compartmentalization	O
+within	O
+an	O
+encapsulin	B-protein
+nanocage	B-complex_assembly
+to	O
+act	O
+as	O
+an	O
+iron	B-chemical
+store	O
+.	O
+
+Further	O
+work	O
+on	O
+the	O
+EncFtn	B-complex_assembly
+-	I-complex_assembly
+Enc	I-complex_assembly
+nanocompartment	B-complex_assembly
+will	O
+establish	O
+the	O
+structural	O
+basis	O
+for	O
+the	O
+movement	O
+of	O
+iron	B-chemical
+through	O
+the	O
+encapsulin	B-protein
+shell	B-structure_element
+,	O
+the	O
+mechanism	O
+of	O
+iron	B-chemical
+oxidation	O
+by	O
+the	O
+EncFtn	B-protein
+FOC	B-site
+and	O
+its	O
+subsequent	O
+storage	O
+in	O
+the	O
+lumen	O
+of	O
+the	O
+encapsulin	B-protein
+nanocompartment	B-complex_assembly
+.	O
+
diff --git a/annotation_IOB/PMC5014086.tsv b/annotation_IOB/PMC5014086.tsv
new file mode 100644
index 0000000000000000000000000000000000000000..0d8253e391169f206677a5de0ef40802501b94c5
--- /dev/null
+++ b/annotation_IOB/PMC5014086.tsv
@@ -0,0 +1,4243 @@
+Structure	B-evidence
+of	O
+the	O
+Dual	O
+-	O
+Mode	O
+Wnt	B-protein_type
+Regulator	O
+Kremen1	B-protein
+and	O
+Insight	O
+into	O
+Ternary	O
+Complex	O
+Formation	O
+with	O
+LRP6	B-protein
+and	O
+Dickkopf	B-protein_type
+
+Kremen	B-protein_type
+1	I-protein_type
+and	I-protein_type
+2	I-protein_type
+have	O
+been	O
+identified	O
+as	O
+co	B-protein_type
+-	I-protein_type
+receptors	I-protein_type
+for	O
+Dickkopf	B-protein_type
+(	O
+Dkk	B-protein_type
+)	O
+proteins	O
+,	O
+hallmark	O
+secreted	O
+antagonists	O
+of	O
+canonical	O
+Wnt	B-protein_type
+signaling	O
+.	O
+
+We	O
+present	O
+here	O
+three	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+ectodomain	B-structure_element
+of	O
+human	B-species
+Kremen1	B-protein
+(	O
+KRM1ECD	B-protein
+)	O
+at	O
+resolutions	O
+between	O
+1	O
+.	O
+9	O
+and	O
+3	O
+.	O
+2	O
+Å	O
+.	O
+KRM1ECD	B-protein
+emerges	O
+as	O
+a	O
+rigid	O
+molecule	O
+with	O
+tight	O
+interactions	O
+stabilizing	O
+a	O
+triangular	B-protein_state
+arrangement	I-protein_state
+of	O
+its	O
+Kringle	B-structure_element
+,	O
+WSC	B-structure_element
+,	O
+and	O
+CUB	B-structure_element
+structural	O
+domains	O
+.	O
+
+The	O
+structures	B-evidence
+reveal	O
+an	O
+unpredicted	O
+homology	O
+of	O
+the	O
+WSC	B-structure_element
+domain	O
+to	O
+hepatocyte	B-protein_type
+growth	I-protein_type
+factor	I-protein_type
+.	O
+
+We	O
+further	O
+report	O
+the	O
+general	O
+architecture	O
+of	O
+the	O
+ternary	O
+complex	O
+formed	O
+by	O
+the	O
+Wnt	B-protein_type
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+Lrp5	B-protein_type
+/	I-protein_type
+6	I-protein_type
+,	O
+Dkk	B-protein_type
+,	O
+and	O
+Krm	B-protein_type
+,	O
+determined	O
+from	O
+a	O
+low	O
+-	O
+resolution	O
+complex	O
+crystal	B-evidence
+structure	I-evidence
+between	O
+β	B-structure_element
+-	I-structure_element
+propeller	I-structure_element
+/	I-structure_element
+EGF	I-structure_element
+repeats	I-structure_element
+(	I-structure_element
+PE	I-structure_element
+)	I-structure_element
+3	I-structure_element
+and	I-structure_element
+4	I-structure_element
+of	O
+the	O
+Wnt	B-protein_type
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+LRP6	B-protein
+(	O
+LRP6PE3PE4	B-protein
+),	O
+the	O
+cysteine	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+domain	I-structure_element
+2	I-structure_element
+(	O
+CRD2	B-structure_element
+)	O
+of	O
+DKK1	B-protein
+,	O
+and	O
+KRM1ECD	B-protein
+.	O
+
+DKK1CRD2	B-protein
+is	O
+sandwiched	O
+between	O
+LRP6PE3	B-protein
+and	O
+KRM1Kringle	B-protein
+-	B-structure_element
+WSC	I-structure_element
+.	O
+
+Modeling	B-experimental_method
+studies	O
+supported	O
+by	O
+surface	B-experimental_method
+plasmon	I-experimental_method
+resonance	I-experimental_method
+suggest	O
+a	O
+direct	O
+interaction	B-site
+site	I-site
+between	O
+Krm1CUB	B-protein
+and	O
+Lrp6PE2	B-protein
+.	O
+
+The	O
+structure	B-evidence
+of	O
+the	O
+KREMEN	B-protein
+1	I-protein
+ectodomain	B-structure_element
+is	O
+solved	B-experimental_method
+from	O
+three	O
+crystal	B-evidence
+forms	I-evidence
+
+Kringle	B-structure_element
+,	O
+WSC	B-structure_element
+,	O
+and	O
+CUB	B-structure_element
+subdomains	O
+interact	O
+tightly	O
+to	O
+form	O
+a	O
+single	O
+structural	O
+unit	O
+
+The	O
+interface	B-site
+to	O
+DKKs	B-protein_type
+is	O
+formed	O
+from	O
+the	O
+Kringle	B-structure_element
+and	O
+WSC	B-structure_element
+domains	O
+
+The	O
+CUB	B-structure_element
+domain	O
+is	O
+found	O
+to	O
+interact	O
+directly	O
+with	O
+LRP6PE1PE2	B-protein
+
+Zebisch	O
+et	O
+al	O
+.	O
+describe	O
+the	O
+ectodomain	B-structure_element
+structure	B-evidence
+of	O
+KREMEN	B-protein
+1	I-protein
+,	O
+a	O
+receptor	B-protein_type
+for	O
+Wnt	B-protein_type
+antagonists	O
+of	O
+the	O
+DKK	B-protein_type
+family	O
+.	O
+
+Apo	B-protein_state
+structures	B-evidence
+and	O
+a	O
+complex	B-protein_state
+with	I-protein_state
+functional	B-protein_state
+fragments	I-protein_state
+of	O
+DKK1	B-protein
+and	O
+LRP6	B-protein
+shed	O
+light	O
+on	O
+the	O
+function	O
+of	O
+this	O
+dual	O
+-	O
+mode	O
+regulator	O
+of	O
+Wnt	B-protein_type
+signaling	O
+.	O
+
+Signaling	O
+by	O
+Wnt	B-protein_type
+morphogens	O
+is	O
+renowned	O
+for	O
+its	O
+fundamental	O
+roles	O
+in	O
+embryonic	O
+development	O
+,	O
+tissue	O
+homeostasis	O
+,	O
+and	O
+stem	O
+cell	O
+maintenance	O
+.	O
+
+Due	O
+to	O
+these	O
+functions	O
+,	O
+generation	O
+,	O
+delivery	O
+,	O
+and	O
+interpretation	O
+of	O
+Wnt	B-protein_type
+signals	O
+are	O
+all	O
+heavily	O
+regulated	O
+in	O
+the	O
+animal	O
+body	O
+.	O
+
+Vertebrate	B-taxonomy_domain
+Dickkopf	B-protein_type
+proteins	O
+(	O
+Dkk1	B-protein_type
+,	O
+2	B-protein_type
+,	O
+and	O
+4	B-protein_type
+)	O
+are	O
+one	O
+of	O
+many	O
+secreted	O
+antagonists	O
+of	O
+Wnt	B-protein_type
+and	O
+function	O
+by	O
+blocking	O
+access	O
+to	O
+the	O
+Wnt	B-protein_type
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+LRP5	B-protein
+/	I-protein
+6	I-protein
+.	O
+
+Kremen	B-protein_type
+proteins	O
+(	O
+Krm1	B-protein_type
+and	O
+Krm2	B-protein_type
+)	O
+have	O
+been	O
+identified	O
+as	O
+additional	O
+high	O
+-	O
+affinity	O
+transmembrane	B-protein_type
+receptors	I-protein_type
+for	O
+Dkk	B-protein_type
+.	O
+
+Krm	B-protein_type
+and	O
+Dkk	B-protein_type
+synergize	O
+in	O
+Wnt	B-protein_type
+inhibition	O
+during	O
+Xenopus	B-taxonomy_domain
+embryogenesis	O
+to	O
+regulate	O
+anterior	O
+-	O
+posterior	O
+patterning	O
+.	O
+
+Mechanistically	O
+it	O
+is	O
+thought	O
+that	O
+,	O
+in	O
+the	O
+presence	B-protein_state
+of	I-protein_state
+Dkk	B-protein_type
+,	O
+Krm	B-protein_type
+forms	O
+a	O
+ternary	O
+complex	B-protein_state
+with	I-protein_state
+Lrp6	B-protein_type
+,	O
+which	O
+is	O
+then	O
+rapidly	O
+endocytosed	O
+.	O
+
+This	O
+amplifies	O
+the	O
+intrinsic	O
+Wnt	B-protein_type
+antagonistic	O
+activity	O
+of	O
+Dkk	B-protein_type
+by	O
+efficiently	O
+depleting	O
+the	O
+cell	O
+surface	O
+of	O
+the	O
+Wnt	B-protein_type
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+.	O
+
+In	O
+accordance	O
+with	O
+this	O
+,	O
+Krm1	B-protein_type
+−/−	O
+and	O
+Krm2	B-protein_type
+−/−	O
+double	B-experimental_method
+knockout	I-experimental_method
+mice	B-taxonomy_domain
+show	O
+a	O
+high	O
+bone	O
+mass	O
+phenotype	O
+typical	O
+of	O
+increased	O
+Wnt	B-protein_type
+signaling	O
+,	O
+as	O
+well	O
+as	O
+growth	O
+of	O
+ectopic	O
+forelimb	O
+digits	O
+.	O
+
+Growth	O
+of	O
+ectopic	O
+digits	O
+is	O
+further	O
+enhanced	O
+upon	O
+additional	O
+loss	O
+of	O
+dkk	B-protein_type
+expression	O
+.	O
+
+The	O
+Wnt	B-protein_type
+antagonistic	O
+activity	O
+of	O
+Krm1	B-protein_type
+is	O
+also	O
+linked	O
+to	O
+its	O
+importance	O
+for	O
+correct	O
+thymus	O
+epithelium	O
+formation	O
+in	O
+mice	B-taxonomy_domain
+.	O
+
+The	O
+importance	O
+of	O
+intact	B-protein_state
+KRM1	B-protein
+for	O
+normal	O
+human	B-species
+development	O
+and	O
+health	O
+is	O
+highlighted	O
+by	O
+the	O
+recent	O
+finding	O
+that	O
+a	O
+homozygous	O
+mutation	O
+in	O
+the	O
+ectodomain	B-structure_element
+of	O
+KRM1	B-protein
+leads	O
+to	O
+severe	O
+ectodermal	O
+dysplasia	O
+including	O
+oligodontia	O
+.	O
+
+Interestingly	O
+,	O
+the	O
+Wnt	B-protein_type
+antagonistic	O
+activity	O
+of	O
+Krm	B-protein_type
+is	O
+context	O
+dependent	O
+,	O
+and	O
+Krm	B-protein_type
+proteins	O
+are	O
+actually	O
+dual	O
+-	O
+mode	O
+Wnt	B-protein_type
+regulators	O
+.	O
+
+In	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+Dkk	B-protein_type
+,	O
+Krm1	B-protein_type
+and	O
+2	B-protein_type
+change	O
+their	O
+function	O
+from	O
+inhibition	O
+to	O
+enhancement	O
+of	O
+Lrp6	B-protein_type
+-	O
+mediated	O
+signaling	O
+.	O
+
+By	O
+direct	O
+binding	O
+to	O
+Lrp6	B-protein_type
+via	O
+the	O
+ectodomains	B-structure_element
+,	O
+Krm	B-protein_type
+proteins	O
+promote	O
+Lrp6	B-protein_type
+cell	O
+-	O
+surface	O
+localization	O
+and	O
+hence	O
+increase	O
+receptor	O
+availability	O
+.	O
+
+Further	O
+increasing	O
+the	O
+complexity	O
+of	O
+Krm	B-protein_type
+functionality	O
+,	O
+it	O
+was	O
+recently	O
+found	O
+that	O
+Krm1	B-protein_type
+(	O
+but	O
+not	O
+Krm2	B-protein_type
+)	O
+can	O
+also	O
+act	O
+independently	O
+of	O
+LRP5	B-protein
+/	I-protein
+6	I-protein
+and	O
+Wnt	B-protein_type
+as	O
+a	O
+dependence	O
+receptor	O
+,	O
+triggering	O
+apoptosis	O
+unless	O
+bound	B-protein_state
+to	I-protein_state
+Dkk	B-protein_type
+.	O
+
+Structurally	O
+,	O
+Krm1	B-protein_type
+and	O
+2	B-protein_type
+are	O
+type	B-protein_type
+I	I-protein_type
+transmembrane	I-protein_type
+proteins	I-protein_type
+with	O
+a	O
+40	O
+kDa	O
+ectodomain	B-structure_element
+and	O
+a	O
+flexible	B-protein_state
+cytoplasmic	B-structure_element
+tail	I-structure_element
+consisting	O
+of	O
+60	B-residue_range
+–	O
+75	B-residue_range
+residues	O
+.	O
+
+The	O
+ectodomain	B-structure_element
+consists	O
+of	O
+three	O
+similarly	O
+sized	O
+structural	O
+domains	O
+of	O
+around	O
+10	O
+kDa	O
+each	O
+:	O
+the	O
+N	O
+-	O
+terminal	O
+Kringle	B-structure_element
+domain	O
+(	O
+KR	B-structure_element
+)	O
+is	O
+followed	O
+by	O
+a	O
+WSC	B-structure_element
+domain	O
+of	O
+unknown	O
+fold	O
+.	O
+
+The	O
+third	O
+structural	O
+domain	O
+is	O
+a	O
+CUB	B-structure_element
+domain	O
+.	O
+
+An	O
+approximately	B-residue_range
+70	I-residue_range
+-	I-residue_range
+residue	I-residue_range
+linker	B-structure_element
+connects	O
+the	O
+CUB	B-structure_element
+domain	O
+to	O
+the	O
+transmembrane	B-structure_element
+span	I-structure_element
+.	O
+
+An	O
+intact	B-protein_state
+KR	B-structure_element
+-	I-structure_element
+WSC	I-structure_element
+-	I-structure_element
+CUB	I-structure_element
+domain	O
+triplet	O
+and	O
+membrane	O
+attachment	O
+is	O
+required	O
+for	O
+Wnt	B-protein_type
+antagonism	O
+.	O
+
+The	O
+transmembrane	B-structure_element
+span	I-structure_element
+and	O
+cytoplasmic	B-structure_element
+tail	I-structure_element
+can	O
+be	O
+replaced	O
+with	O
+a	O
+GPI	B-structure_element
+linker	B-structure_element
+without	O
+impact	O
+on	O
+Wnt	B-protein_type
+antagonism	O
+.	O
+
+The	O
+structures	B-evidence
+presented	O
+here	O
+reveal	O
+the	O
+unknown	O
+fold	O
+of	O
+the	O
+WSC	B-structure_element
+domain	O
+and	O
+the	O
+tight	O
+interactions	O
+of	O
+all	O
+three	O
+domains	O
+.	O
+
+We	O
+further	O
+succeeded	O
+in	O
+determination	O
+of	O
+a	O
+low	O
+-	O
+resolution	O
+LRP6PE3PE4	B-complex_assembly
+-	I-complex_assembly
+DKK1CRD2	I-complex_assembly
+-	I-complex_assembly
+KRM1ECD	I-complex_assembly
+complex	O
+,	O
+defining	O
+the	O
+architecture	O
+of	O
+the	O
+Wnt	B-protein_type
+inhibitory	B-complex_assembly
+complex	I-complex_assembly
+that	O
+leads	O
+to	O
+Lrp6	B-protein
+cell	O
+-	O
+surface	O
+depletion	O
+.	O
+
+The	O
+recombinant	O
+production	O
+of	O
+the	O
+extracellular	B-structure_element
+domain	I-structure_element
+of	O
+Krm	B-protein_type
+for	O
+structural	B-experimental_method
+studies	I-experimental_method
+proved	O
+challenging	O
+(	O
+see	O
+Experimental	O
+Procedures	O
+).	O
+
+We	O
+succeeded	O
+in	O
+purifying	O
+KRM1ECD	B-protein
+complexes	B-protein_state
+with	I-protein_state
+DKK1fl	B-protein
+,	O
+DKK1Linker	B-protein
+-	B-structure_element
+CRD2	I-structure_element
+,	O
+and	O
+DKK1CRD2	B-protein
+that	O
+were	O
+monodisperse	O
+and	O
+stable	O
+in	O
+gel	B-experimental_method
+filtration	I-experimental_method
+,	O
+hence	O
+indicating	O
+at	O
+least	O
+micromolar	O
+affinity	O
+(	O
+data	O
+not	O
+shown	O
+).	O
+
+Several	O
+crystal	B-evidence
+forms	I-evidence
+were	O
+obtained	O
+from	O
+these	O
+complexes	O
+,	O
+however	O
+,	O
+crystals	B-evidence
+always	O
+contained	O
+only	O
+KRM1	B-protein
+protein	O
+.	O
+
+We	O
+solved	B-experimental_method
+the	O
+structure	B-evidence
+of	O
+KRM1ECD	B-protein
+in	O
+three	O
+crystal	O
+forms	O
+at	O
+1	O
+.	O
+9	O
+,	O
+2	O
+.	O
+8	O
+,	O
+and	O
+3	O
+.	O
+2	O
+Å	O
+resolution	O
+(	O
+Table	O
+1	O
+).	O
+
+The	O
+high	O
+-	O
+resolution	O
+structure	B-evidence
+is	O
+a	O
+near	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+model	O
+(	O
+Figure	O
+1	O
+).	O
+
+The	O
+small	B-protein_state
+,	O
+flexible	B-protein_state
+,	O
+and	O
+charged	B-protein_state
+98AEHED102	B-structure_element
+loop	I-structure_element
+could	O
+only	O
+be	O
+modeled	O
+in	O
+a	O
+slightly	O
+lower	O
+resolution	O
+structure	B-evidence
+and	O
+in	O
+crystal	O
+form	O
+III	O
+.	O
+
+The	O
+KR	B-structure_element
+,	O
+WSC	B-structure_element
+,	O
+and	O
+CUB	B-structure_element
+are	O
+arranged	O
+in	O
+a	O
+roughly	O
+triangular	O
+fashion	O
+with	O
+tight	O
+interactions	O
+between	O
+all	O
+three	O
+domains	O
+.	O
+
+The	O
+KR	B-structure_element
+domain	O
+,	O
+which	O
+bears	O
+two	O
+of	O
+the	O
+four	O
+glycosylation	B-site
+sites	I-site
+,	O
+contains	O
+the	O
+canonical	O
+three	O
+disulfide	B-ptm
+bridges	I-ptm
+(	O
+C32	B-residue_name_number
+-	O
+C114	B-residue_name_number
+,	O
+C55	B-residue_name_number
+-	O
+C95	B-residue_name_number
+,	O
+C84	B-residue_name_number
+-	O
+C109	B-residue_name_number
+)	O
+and	O
+,	O
+like	O
+other	O
+Kringle	B-structure_element
+domains	O
+,	O
+is	O
+low	O
+in	O
+secondary	O
+structure	O
+elements	O
+.	O
+
+The	O
+structurally	O
+most	O
+similar	O
+Kringle	B-structure_element
+domain	O
+is	O
+that	O
+of	O
+human	B-species
+plasminogen	B-protein
+(	O
+PDB	O
+:	O
+1PKR	O
+)	O
+with	O
+an	O
+root	B-evidence
+-	I-evidence
+mean	I-evidence
+-	I-evidence
+square	I-evidence
+deviation	I-evidence
+(	O
+RMSD	B-evidence
+)	O
+of	O
+1	O
+.	O
+7	O
+Å	O
+for	O
+73	O
+aligned	O
+Cα	O
+(	O
+Figure	O
+1B	O
+).	O
+
+The	O
+KRM1	B-protein
+structure	B-evidence
+reveals	O
+the	O
+fold	O
+of	O
+the	O
+WSC	B-structure_element
+domain	O
+for	O
+the	O
+first	O
+time	O
+.	O
+
+The	O
+structure	B-evidence
+is	O
+best	O
+described	O
+as	O
+a	O
+sandwich	B-structure_element
+of	O
+a	O
+β1	B-structure_element
+-	I-structure_element
+β5	I-structure_element
+-	I-structure_element
+β3	I-structure_element
+-	I-structure_element
+β4	I-structure_element
+-	I-structure_element
+β2	I-structure_element
+antiparallel	I-structure_element
+β	I-structure_element
+sheet	I-structure_element
+and	O
+a	O
+single	O
+α	B-structure_element
+helix	I-structure_element
+.	O
+
+The	O
+structure	B-evidence
+is	O
+also	O
+rich	O
+in	O
+loops	B-structure_element
+and	O
+is	O
+stabilized	O
+by	O
+four	O
+disulfide	B-ptm
+bridges	I-ptm
+(	O
+C122	B-residue_name_number
+-	O
+C186	B-residue_name_number
+,	O
+C147	B-residue_name_number
+-	O
+C167	B-residue_name_number
+,	O
+C151	B-residue_name_number
+-	O
+C169	B-residue_name_number
+,	O
+C190	B-residue_name_number
+-	O
+C198	B-residue_name_number
+).	O
+
+Using	O
+the	O
+PDBeFold	B-experimental_method
+server	I-experimental_method
+,	O
+we	O
+detected	O
+a	O
+surprising	O
+yet	O
+significant	O
+homology	O
+to	O
+PAN	B-structure_element
+module	I-structure_element
+domains	I-structure_element
+.	O
+
+The	O
+closest	O
+structural	O
+relative	O
+is	O
+hepatocyte	B-protein_type
+growth	I-protein_type
+factor	I-protein_type
+(	O
+HGF	B-protein_type
+,	O
+PDB	O
+:	O
+1GP9	O
+),	O
+which	O
+superposes	B-experimental_method
+with	O
+an	O
+RMSD	B-evidence
+of	O
+2	O
+.	O
+3	O
+Å	O
+for	O
+58	O
+aligned	O
+Cα	O
+(	O
+Figure	O
+1B	O
+).	O
+
+The	O
+CUB	B-structure_element
+domain	O
+bears	O
+two	O
+glycosylation	B-site
+sites	I-site
+.	O
+
+Although	O
+present	O
+,	O
+the	O
+quality	O
+of	O
+the	O
+electron	B-evidence
+density	I-evidence
+around	O
+N217	B-residue_name_number
+did	O
+not	O
+allow	O
+modeling	O
+of	O
+the	O
+sugar	O
+moiety	O
+.	O
+
+In	O
+crystal	B-evidence
+form	I-evidence
+I	I-evidence
+,	O
+a	O
+calcium	B-chemical
+ion	O
+is	O
+present	O
+at	O
+the	O
+canonical	O
+position	O
+coordinated	B-bond_interaction
+by	I-bond_interaction
+the	O
+carboxylates	O
+of	O
+D263	B-residue_name_number
+,	O
+D266	B-residue_name_number
+(	O
+bidentate	O
+),	O
+and	O
+D306	B-residue_name_number
+,	O
+as	O
+well	O
+as	O
+the	O
+carbonyl	O
+of	O
+N309	B-residue_name_number
+and	O
+a	O
+water	B-chemical
+molecule	O
+.	O
+
+The	O
+coordination	B-site
+sphere	I-site
+deviates	O
+significantly	O
+from	O
+perfectly	O
+octahedral	O
+(	O
+not	O
+shown	O
+).	O
+
+This	O
+might	O
+result	O
+in	O
+the	O
+site	O
+having	O
+a	O
+low	O
+affinity	O
+and	O
+may	O
+explain	O
+why	O
+calcium	B-chemical
+is	O
+not	O
+present	O
+in	O
+the	O
+two	O
+low	O
+-	O
+resolution	O
+crystal	B-evidence
+forms	I-evidence
+.	O
+
+Loss	B-protein_state
+of	I-protein_state
+calcium	B-chemical
+has	O
+led	O
+to	O
+loop	B-structure_element
+rearrangements	O
+and	O
+partial	O
+disorder	O
+in	O
+these	O
+crystal	B-evidence
+forms	I-evidence
+.	O
+
+The	O
+closest	O
+structural	O
+relative	O
+is	O
+the	O
+CUB_C	B-structure_element
+domain	O
+of	O
+Tsg	B-protein
+-	I-protein
+6	I-protein
+(	O
+PDB	O
+:	O
+2WNO	O
+),	O
+which	O
+superposes	B-experimental_method
+with	O
+KRMCUB	B-protein
+with	O
+an	O
+RMSD	B-evidence
+of	O
+1	O
+.	O
+6	O
+Å	O
+for	O
+104	O
+Cα	O
+(	O
+Figure	O
+1B	O
+).	O
+
+A	O
+superposition	B-experimental_method
+of	O
+the	O
+three	O
+KRM1	B-protein
+structures	B-evidence
+reveals	O
+no	O
+major	O
+structural	O
+differences	O
+(	O
+Figure	O
+1C	O
+)	O
+as	O
+anticipated	O
+from	O
+the	O
+plethora	O
+of	O
+interactions	O
+between	O
+the	O
+three	O
+domains	O
+.	O
+
+Minor	O
+differences	O
+are	O
+caused	O
+by	O
+the	O
+collapse	O
+of	O
+the	O
+Ca2	B-site
++	I-site
+binding	I-site
+site	I-site
+in	O
+crystal	B-evidence
+forms	I-evidence
+II	I-evidence
+and	I-evidence
+III	I-evidence
+and	O
+loop	B-structure_element
+flexibility	O
+in	O
+the	O
+KR	B-structure_element
+domain	O
+.	O
+
+The	O
+F207S	B-mutant
+mutation	O
+recently	O
+found	O
+to	O
+cause	O
+ectodermal	O
+dysplasia	O
+in	O
+Palestinian	O
+families	O
+maps	O
+to	O
+the	O
+hydrophobic	B-site
+core	I-site
+of	O
+the	O
+protein	O
+at	O
+the	O
+interface	B-site
+of	O
+the	O
+three	O
+subdomains	O
+(	O
+Figure	O
+1A	O
+).	O
+
+Such	O
+a	O
+mutation	O
+is	O
+bound	B-protein_state
+to	I-protein_state
+severely	O
+destabilize	O
+the	O
+protein	O
+structure	O
+of	O
+KRM1	B-protein
+,	O
+leading	O
+to	O
+disturbance	O
+of	O
+its	O
+Wnt	B-protein_type
+antagonistic	O
+,	O
+Wnt	B-protein_type
+stimulatory	O
+,	O
+and	O
+Wnt	B-protein_type
+independent	O
+activity	O
+.	O
+
+Co	B-experimental_method
+-	I-experimental_method
+crystallization	I-experimental_method
+of	O
+LRP6PE3PE4	B-protein
+with	O
+DKK1CRD2	B-protein
+,	O
+and	O
+LRP6PE1	B-protein
+with	O
+an	O
+N	O
+-	O
+terminal	O
+peptide	O
+of	O
+DKK1	B-protein
+has	O
+provided	O
+valuable	O
+structural	O
+insight	O
+into	O
+direct	O
+Wnt	B-protein_type
+inhibition	O
+by	O
+Dkk	B-protein_type
+ligands	O
+.	O
+
+One	O
+face	O
+of	O
+the	O
+rather	O
+flat	B-protein_state
+DKK1CRD2	B-protein
+fragment	O
+binds	B-protein_state
+to	I-protein_state
+the	O
+third	B-structure_element
+β	I-structure_element
+propeller	I-structure_element
+of	O
+LRP6	B-protein
+.	O
+
+Mutational	B-experimental_method
+analyses	I-experimental_method
+further	O
+implied	O
+that	O
+the	O
+LRP6PE3	B-protein
+-	O
+averted	O
+face	O
+of	O
+DKK1CRD2	B-protein
+bears	O
+the	O
+Krm	B-site
+binding	I-site
+site	I-site
+,	O
+hence	O
+suggesting	O
+how	O
+Dkk	B-protein_type
+can	O
+recruit	O
+both	O
+receptors	B-protein_type
+into	O
+a	O
+ternary	O
+complex	O
+.	O
+
+To	O
+obtain	O
+direct	O
+insight	O
+into	O
+ternary	O
+complex	O
+formation	O
+by	O
+Lrp5	B-protein_type
+/	I-protein_type
+6	I-protein_type
+,	O
+Dkk	B-protein_type
+,	O
+and	O
+Krm	B-protein_type
+,	O
+we	O
+subjected	O
+an	O
+LRP6PE3PE4	B-complex_assembly
+-	I-complex_assembly
+DKK1fl	I-complex_assembly
+-	I-complex_assembly
+KRM1ECD	I-complex_assembly
+complex	O
+to	O
+crystallization	B-experimental_method
+trials	I-experimental_method
+.	O
+
+Diffraction	B-evidence
+data	I-evidence
+collected	O
+from	O
+the	O
+resulting	O
+crystals	B-evidence
+were	O
+highly	O
+anisotropic	O
+with	O
+diffraction	O
+extending	O
+in	O
+the	O
+best	O
+directions	O
+to	O
+3	O
+.	O
+5	O
+Å	O
+and	O
+3	O
+.	O
+7	O
+Å	O
+but	O
+only	O
+to	O
+6	O
+.	O
+4	O
+Å	O
+in	O
+the	O
+third	O
+direction	O
+.	O
+
+Despite	O
+the	O
+lack	O
+of	O
+high	O
+-	O
+resolution	O
+diffraction	B-evidence
+,	O
+the	O
+general	O
+architecture	O
+of	O
+the	O
+ternary	O
+complex	O
+is	O
+revealed	O
+(	O
+Figure	O
+2A	O
+).	O
+
+DKK1CRD2	B-protein
+binds	B-protein_state
+to	I-protein_state
+the	O
+top	O
+face	O
+of	O
+the	O
+LRP6	B-protein
+PE3	B-structure_element
+β	B-structure_element
+propeller	I-structure_element
+as	O
+described	O
+earlier	O
+for	O
+the	O
+binary	O
+complex	O
+.	O
+
+KRM1ECD	B-protein
+does	O
+indeed	O
+bind	B-protein_state
+on	I-protein_state
+the	O
+opposite	O
+side	O
+of	O
+DKK1CRD2	B-protein
+with	O
+only	O
+its	O
+KR	B-structure_element
+and	O
+WSC	B-structure_element
+domains	O
+engaged	O
+in	O
+binding	O
+(	O
+Figure	O
+2A	O
+).	O
+
+Although	O
+present	O
+in	O
+the	O
+complex	O
+subjected	O
+to	O
+crystallization	B-experimental_method
+,	O
+we	O
+observe	O
+no	O
+density	B-evidence
+that	O
+could	O
+correspond	O
+to	O
+CRD1	B-structure_element
+or	O
+the	O
+domain	B-structure_element
+linker	I-structure_element
+(	O
+L	B-structure_element
+).	O
+
+We	O
+confirm	O
+that	O
+the	O
+CRD2	B-structure_element
+of	O
+DKK1	B-protein
+is	O
+required	O
+and	O
+sufficient	O
+for	O
+binding	O
+to	O
+KRM1	B-protein
+:	O
+In	O
+surface	B-experimental_method
+plasmon	I-experimental_method
+resonance	I-experimental_method
+(	O
+SPR	B-experimental_method
+),	O
+we	O
+measured	O
+low	O
+micromolar	O
+affinity	B-evidence
+between	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+DKK1	B-protein
+and	O
+immobilized	O
+KRM1ECD	B-protein
+(	O
+Figure	O
+2B	O
+).	O
+
+A	O
+SUMO	B-experimental_method
+fusion	I-experimental_method
+of	O
+DKK1L	B-structure_element
+-	I-structure_element
+CRD2	I-structure_element
+displayed	O
+a	O
+similar	O
+(	O
+slightly	O
+higher	O
+)	O
+affinity	B-evidence
+.	O
+
+In	O
+contrast	O
+,	O
+a	O
+SUMO	B-experimental_method
+fusion	I-experimental_method
+of	O
+DKK1CRD1	B-structure_element
+-	I-structure_element
+L	I-structure_element
+did	O
+not	O
+display	O
+binding	O
+for	O
+concentrations	O
+tested	O
+up	O
+to	O
+325	O
+μM	O
+(	O
+Figure	O
+2B	O
+).	O
+
+Overall	O
+,	O
+the	O
+DKK1	B-site
+-	I-site
+KRM1	I-site
+interface	I-site
+is	O
+characterized	O
+by	O
+a	O
+large	O
+number	O
+of	O
+polar	B-bond_interaction
+interactions	I-bond_interaction
+but	O
+only	O
+few	O
+hydrophobic	B-bond_interaction
+contacts	I-bond_interaction
+(	O
+Figure	O
+2C	O
+).	O
+
+The	O
+crystal	B-evidence
+structure	I-evidence
+gives	O
+an	O
+explanation	O
+for	O
+DKK1	B-protein
+loss	O
+-	O
+of	O
+-	O
+binding	O
+mutations	O
+identified	O
+previously	O
+:	O
+R191	B-residue_name_number
+of	O
+DKK1	B-protein
+forms	O
+a	O
+double	O
+salt	B-bond_interaction
+bridge	I-bond_interaction
+to	O
+D125	B-residue_name_number
+and	O
+E162	B-residue_name_number
+of	O
+KRM1	B-protein
+(	O
+Figure	O
+2C	O
+).	O
+
+A	O
+charge	B-experimental_method
+reversal	I-experimental_method
+as	O
+in	O
+the	O
+mouse	B-taxonomy_domain
+Dkk1	B-protein
+(	O
+mDkk1	B-protein
+)	O
+R197E	B-mutant
+variant	O
+would	O
+severely	O
+disrupt	O
+the	O
+binding	O
+.	O
+
+Similarly	O
+,	O
+the	O
+K226	B-residue_name_number
+side	O
+chain	O
+of	O
+DKK1	B-protein
+,	O
+which	O
+points	O
+to	O
+a	O
+small	O
+hydrophobic	B-site
+pocket	I-site
+on	O
+the	O
+surface	O
+of	O
+KRM1	B-protein
+formed	O
+by	O
+Y108	B-residue_name_number
+,	O
+W94	B-residue_name_number
+,	O
+and	O
+W106	B-residue_name_number
+,	O
+forms	O
+salt	B-bond_interaction
+bridges	I-bond_interaction
+with	O
+the	O
+side	O
+chains	O
+of	O
+KRM1	B-protein
+D88	B-residue_name_number
+and	O
+D90	B-residue_name_number
+.	O
+
+Again	O
+,	O
+a	O
+charge	B-experimental_method
+reversal	I-experimental_method
+as	O
+shown	O
+before	O
+for	O
+mDkk1	B-protein
+K232E	B-mutant
+would	O
+be	O
+incompatible	O
+with	O
+binding	O
+.	O
+
+The	O
+side	O
+chain	O
+of	O
+DKK1	B-protein
+S192	B-residue_name_number
+was	O
+also	O
+predicted	O
+to	O
+be	O
+involved	O
+in	O
+Krm	B-protein_type
+binding	O
+.	O
+
+Indeed	O
+,	O
+we	O
+found	O
+(	O
+Figure	O
+2C	O
+)	O
+that	O
+the	O
+side	O
+chain	O
+of	O
+D201	B-residue_name_number
+of	O
+KRM1	B-protein
+forms	O
+two	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+to	O
+the	O
+side	O
+-	O
+chain	O
+hydroxyl	O
+and	O
+the	O
+backbone	O
+amide	O
+of	O
+S192	B-residue_name_number
+(	O
+mouse	B-taxonomy_domain
+,	O
+S198	B-residue_name_number
+).	O
+
+Additional	O
+polar	B-bond_interaction
+interactions	I-bond_interaction
+are	O
+formed	O
+between	O
+the	O
+N140	B-residue_name_number
+,	O
+S163	B-residue_name_number
+,	O
+and	O
+Y165	B-residue_name_number
+side	O
+chains	O
+of	O
+KRM1	B-protein
+and	O
+DKK1	B-protein
+backbone	O
+carbonyls	O
+of	O
+W206	B-residue_name_number
+,	O
+L190	B-residue_name_number
+,	O
+and	O
+C189	B-residue_name_number
+,	O
+respectively	O
+.	O
+
+The	O
+carbonyl	O
+of	O
+DKK1	B-protein
+R224	B-residue_name_number
+is	O
+hydrogen	B-bond_interaction
+bonded	I-bond_interaction
+to	O
+Y105	B-residue_name_number
+and	O
+W106	B-residue_name_number
+of	O
+KRM1	B-protein
+.	O
+
+We	O
+suspect	O
+that	O
+the	O
+Dkk	B-protein_type
+charge	B-experimental_method
+reversal	I-experimental_method
+mutations	I-experimental_method
+performed	O
+in	O
+the	O
+murine	B-taxonomy_domain
+background	O
+and	O
+shown	O
+to	O
+diminish	O
+Krm	B-protein_type
+binding	O
+K211E	B-mutant
+and	O
+R203E	B-mutant
+(	O
+mouse	B-taxonomy_domain
+K217E	B-mutant
+and	O
+R209E	B-mutant
+)	O
+do	O
+so	O
+likely	O
+indirectly	O
+by	O
+disruption	O
+of	O
+the	O
+Dkk	B-protein_type
+fold	O
+.	O
+
+We	O
+further	O
+validated	O
+the	O
+DKK1	B-site
+binding	I-site
+site	I-site
+on	O
+KRM1	B-protein
+by	O
+introducing	B-experimental_method
+glycosylation	B-site
+sites	I-site
+at	O
+the	O
+KR	B-structure_element
+(	O
+90DVS92	B-mutant
+→	I-mutant
+NVS	I-mutant
+)	O
+and	O
+WSC	B-structure_element
+(	O
+189VCF191	B-mutant
+→	I-mutant
+NCS	I-mutant
+)	O
+domains	O
+pointing	O
+toward	O
+DKK	B-protein
+(	O
+Figures	O
+2A	O
+and	O
+2D	O
+).	O
+
+Introduction	O
+of	O
+N	B-ptm
+-	I-ptm
+linked	I-ptm
+glycans	I-ptm
+in	O
+protein	B-site
+-	I-site
+protein	I-site
+-	I-site
+binding	I-site
+sites	I-site
+is	O
+an	O
+established	O
+way	O
+of	O
+disrupting	O
+protein	B-site
+-	I-site
+binding	I-site
+interfaces	I-site
+.	O
+
+Both	O
+ectodomain	B-structure_element
+mutants	B-protein_state
+were	O
+secreted	O
+comparably	O
+with	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+,	O
+indicating	O
+correct	O
+folding	O
+,	O
+but	O
+failed	O
+to	O
+achieve	O
+any	O
+detectable	O
+binding	O
+in	O
+SPR	B-experimental_method
+using	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+DKK1	B-protein
+as	O
+analyte	O
+.	O
+
+In	O
+contrast	O
+,	O
+a	O
+mutant	B-protein_state
+carrying	O
+an	O
+additional	O
+N	B-ptm
+-	I-ptm
+glycan	I-ptm
+outside	O
+the	O
+interface	B-site
+at	O
+the	O
+CUB	B-structure_element
+domain	O
+(	O
+309NQA311	B-mutant
+→	I-mutant
+NQS	I-mutant
+),	O
+was	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+-	O
+like	O
+in	O
+DKK1	B-protein
+binding	O
+(	O
+Figure	O
+2D	O
+).	O
+
+Identification	O
+of	O
+a	O
+Direct	O
+LRP6	B-site
+-	I-site
+KRM1	I-site
+Binding	I-site
+Site	I-site
+
+The	O
+LRP6PE3PE4	B-complex_assembly
+-	I-complex_assembly
+DKK1CRD2	I-complex_assembly
+-	I-complex_assembly
+KRM1ECD	I-complex_assembly
+complex	O
+structure	B-evidence
+reveals	O
+no	O
+direct	O
+interactions	O
+between	O
+KRM1	B-protein
+and	O
+LRP6	B-protein
+.	O
+
+We	O
+constructed	O
+in	O
+silico	O
+a	O
+ternary	O
+complex	B-protein_state
+with	I-protein_state
+a	O
+close	O
+to	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+LRP6	B-protein
+ectodomain	B-structure_element
+(	O
+PE1PE2PE3PE4	B-structure_element
+horse	B-structure_element
+shoe	I-structure_element
+)	O
+similar	O
+to	O
+but	O
+without	O
+refinement	O
+against	O
+electron	B-experimental_method
+microscopy	I-experimental_method
+(	O
+EM	B-experimental_method
+)	O
+or	O
+small	B-experimental_method
+-	I-experimental_method
+angle	I-experimental_method
+X	I-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+scattering	I-experimental_method
+data	O
+.	O
+
+An	O
+auxiliary	O
+PE3PE4	B-structure_element
+fragment	O
+was	O
+superimposed	B-experimental_method
+via	O
+PE4	B-structure_element
+onto	O
+PE3	B-structure_element
+of	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+,	O
+and	O
+the	O
+LRP6PE1PE2	B-protein
+structure	B-evidence
+was	O
+superimposed	B-experimental_method
+via	O
+PE2	B-structure_element
+onto	O
+PE3	B-structure_element
+of	O
+this	O
+auxiliary	O
+fragment	O
+(	O
+Figure	O
+3A	O
+).	O
+
+For	O
+this	O
+crude	O
+approximation	O
+of	O
+a	O
+true	O
+ternary	O
+complex	O
+,	O
+we	O
+noted	O
+very	O
+close	O
+proximity	O
+between	O
+the	O
+Ca2	B-site
++-	I-site
+binding	I-site
+region	I-site
+of	O
+KRM1	B-protein
+and	O
+the	O
+top	O
+face	O
+of	O
+the	O
+PE2	B-structure_element
+β	B-structure_element
+propeller	I-structure_element
+of	O
+LRP6	B-protein
+.	O
+
+The	O
+solvent	B-protein_state
+-	I-protein_state
+exposed	I-protein_state
+residues	O
+R307	B-residue_name_number
+,	O
+I308	B-residue_name_number
+,	O
+and	O
+N309	B-residue_name_number
+of	O
+the	O
+central	O
+Ca2	B-structure_element
++-	I-structure_element
+binding	I-structure_element
+β	I-structure_element
+connection	I-structure_element
+loop	I-structure_element
+of	O
+KRM1	B-protein
+would	O
+be	O
+almost	O
+ideally	O
+positioned	O
+for	O
+binding	O
+to	O
+this	O
+face	O
+,	O
+which	O
+is	O
+commonly	O
+used	O
+as	O
+a	O
+binding	B-site
+site	I-site
+on	O
+β	B-structure_element
+propellers	I-structure_element
+.	O
+
+Peptides	O
+containing	O
+arginine	B-residue_name
+/	O
+lysine	B-residue_name
+,	O
+isoleucine	B-residue_name
+,	O
+and	O
+asparagine	B-residue_name
+(	O
+consensus	O
+sequence	O
+N	B-structure_element
+-	I-structure_element
+X	I-structure_element
+-	I-structure_element
+I	I-structure_element
+-(	I-structure_element
+G	I-structure_element
+)-	I-structure_element
+R	I-structure_element
+/	I-structure_element
+K	I-structure_element
+)	O
+are	O
+also	O
+employed	O
+by	O
+DKK1	B-protein
+and	O
+SOST	B-protein
+to	O
+bind	O
+to	O
+LRP6	B-protein
+(	O
+albeit	O
+to	O
+propeller	B-structure_element
+1	I-structure_element
+;	O
+Figure	O
+3B	O
+).	O
+
+To	O
+support	O
+the	O
+hypothesis	O
+that	O
+KRM1CUB	B-protein
+binds	B-protein_state
+to	I-protein_state
+LRP6PE2	B-protein
+,	O
+we	O
+used	O
+SPR	B-experimental_method
+and	O
+compared	O
+binding	O
+of	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+the	O
+GlycoCUB	B-protein_state
+mutant	I-protein_state
+of	O
+KRM1ECD	B-protein
+(	O
+bearing	O
+an	O
+N	B-site
+-	I-site
+glycosylation	I-site
+site	I-site
+at	O
+N309	B-residue_name_number
+)	O
+with	O
+a	O
+purified	O
+LRP6PE1PE2	B-protein
+fragment	O
+.	O
+
+Indeed	O
+,	O
+we	O
+found	O
+that	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+Dkk	B-protein_type
+,	O
+KRM1ECD	B-protein
+bound	B-protein_state
+with	O
+considerable	O
+affinity	O
+to	B-protein_state
+LRP6PE1PE2	B-protein
+(	O
+Figure	O
+3C	O
+).	O
+
+In	O
+contrast	O
+,	O
+no	O
+saturable	O
+binding	O
+was	O
+observed	O
+between	O
+KRM1	B-protein
+and	O
+LRP6PE3PE4	B-protein
+.	O
+
+Introduction	B-experimental_method
+of	I-experimental_method
+an	O
+N	B-site
+-	I-site
+glycosylation	I-site
+site	I-site
+at	O
+N309	B-residue_name_number
+in	O
+KRM1ECD	B-protein
+abolished	O
+LRP6PE1PE2	B-protein
+binding	O
+(	O
+Figure	O
+3C	O
+),	O
+while	O
+binding	O
+to	O
+DKK1	B-protein
+was	O
+unaffected	O
+(	O
+Figure	O
+2D	O
+).	O
+
+We	O
+conclude	O
+that	O
+the	O
+predicted	O
+binding	B-site
+site	I-site
+between	O
+KRM1CUB	B-protein
+and	O
+LRP6PE2	B-protein
+is	O
+a	O
+strong	O
+candidate	O
+for	O
+mediating	O
+the	O
+direct	O
+Lrp6	B-complex_assembly
+-	I-complex_assembly
+Krm	I-complex_assembly
+interaction	O
+,	O
+which	O
+is	O
+thought	O
+to	O
+increase	O
+Wnt	B-protein_type
+responsiveness	O
+by	O
+stabilizing	O
+Lrp6	B-protein
+at	O
+the	O
+cell	O
+surface	O
+.	O
+
+Further	O
+experiments	O
+are	O
+required	O
+to	O
+pinpoint	O
+the	O
+exact	O
+binding	B-site
+site	I-site
+.	O
+
+Although	O
+LRP6PE1	B-protein
+appears	O
+somewhat	O
+out	O
+of	O
+reach	O
+in	O
+the	O
+modeled	O
+ternary	O
+complex	O
+,	O
+it	O
+cannot	O
+be	O
+excluded	O
+as	O
+the	O
+Krm	B-site
+binding	I-site
+site	I-site
+in	O
+the	O
+ternary	O
+complex	O
+and	O
+LRP6	B-complex_assembly
+-	I-complex_assembly
+Krm	I-complex_assembly
+binary	O
+complex	O
+.	O
+
+The	O
+presence	B-protein_state
+of	I-protein_state
+DKK	B-protein
+may	O
+govern	O
+which	O
+propeller	B-structure_element
+(	O
+PE1	B-structure_element
+versus	O
+PE2	B-structure_element
+)	O
+of	O
+LRP6	B-protein
+is	O
+available	O
+for	O
+Krm	B-protein_type
+binding	O
+.	O
+
+Apparent	O
+binding	O
+across	O
+the	O
+proposed	O
+KRM1CUB	B-site
+-	I-site
+LRP6PE2	I-site
+interface	I-site
+is	O
+expected	O
+to	O
+be	O
+higher	O
+once	O
+Krm	B-protein_type
+is	O
+also	O
+cross	O
+-	O
+linked	O
+to	O
+LRP6PE3	B-protein
+via	O
+DKK1CRD2	B-protein
+(	O
+Figure	O
+3D	O
+).	O
+
+Low	O
+-	O
+resolution	O
+negative	B-experimental_method
+-	I-experimental_method
+stain	I-experimental_method
+EM	I-experimental_method
+and	O
+small	B-experimental_method
+-	I-experimental_method
+angle	I-experimental_method
+X	I-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+scattering	I-experimental_method
+studies	O
+of	O
+LRP6PE1PE2PE3PE4	B-protein
+,	O
+in	B-protein_state
+isolation	I-protein_state
+and	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+Dkk1	B-protein_type
+,	O
+plus	O
+negative	B-experimental_method
+-	I-experimental_method
+stain	I-experimental_method
+EM	I-experimental_method
+of	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+LRP6	B-protein
+ectodomain	B-structure_element
+,	O
+have	O
+indicated	O
+curved	B-protein_state
+,	O
+platform	B-protein_state
+-	I-protein_state
+like	I-protein_state
+conformations	O
+but	O
+also	O
+potential	O
+flexibility	O
+between	O
+PE2	B-structure_element
+and	O
+PE3	B-structure_element
+.	O
+
+It	O
+is	O
+therefore	O
+possible	O
+that	O
+the	O
+interplay	O
+of	O
+Krm	B-protein_type
+and	O
+Dkk	B-protein_type
+binding	O
+can	O
+promote	O
+changes	O
+in	O
+LRP6	B-protein
+ectodomain	B-structure_element
+conformation	O
+with	O
+functional	O
+consequences	O
+;	O
+however	O
+,	O
+such	O
+ideas	O
+await	O
+investigation	O
+.	O
+
+Taken	O
+together	O
+,	O
+the	O
+structural	B-experimental_method
+and	I-experimental_method
+biophysical	I-experimental_method
+studies	I-experimental_method
+we	O
+report	O
+here	O
+extend	O
+our	O
+mechanistic	O
+understanding	O
+of	O
+Wnt	B-protein_type
+signal	O
+regulation	O
+.	O
+
+We	O
+describe	O
+the	O
+ectodomain	B-structure_element
+structure	B-evidence
+of	O
+the	O
+dual	O
+Wnt	B-protein_type
+regulator	O
+Krm1	B-protein_type
+,	O
+providing	O
+an	O
+explanation	O
+for	O
+the	O
+detrimental	O
+effect	O
+on	O
+health	O
+and	O
+development	O
+of	O
+a	O
+homozygous	O
+KRM1	B-protein
+mutation	O
+.	O
+
+We	O
+also	O
+reveal	O
+the	O
+interaction	O
+mode	O
+of	O
+Krm	B-complex_assembly
+-	I-complex_assembly
+Dkk	I-complex_assembly
+and	O
+the	O
+architecture	O
+of	O
+the	O
+ternary	O
+complex	O
+formed	O
+by	O
+Lrp5	B-protein_type
+/	I-protein_type
+6	I-protein_type
+,	O
+Dkk	B-protein_type
+,	O
+and	O
+Krm	B-protein_type
+.	O
+
+Furthermore	O
+,	O
+the	O
+ternary	O
+crystal	B-evidence
+structure	I-evidence
+has	O
+guided	O
+in	B-experimental_method
+silico	I-experimental_method
+and	I-experimental_method
+biophysical	I-experimental_method
+analyses	I-experimental_method
+to	O
+suggest	O
+a	O
+direct	O
+LRP6	B-site
+-	I-site
+KRM1	I-site
+interaction	I-site
+site	I-site
+.	O
+
+Our	O
+findings	O
+provide	O
+a	O
+solid	O
+foundation	O
+for	O
+additional	O
+studies	O
+to	O
+probe	O
+how	O
+ternary	O
+complex	O
+formation	O
+triggers	O
+internalization	O
+,	O
+whereas	O
+Krm	B-protein_type
+binding	O
+in	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+Dkk	B-protein_type
+stabilizes	O
+the	O
+Wnt	B-protein_type
+co	B-protein_type
+-	I-protein_type
+receptor	I-protein_type
+at	O
+the	O
+cell	O
+surface	O
+.	O
+
+Structure	B-evidence
+of	O
+Unliganded	B-protein_state
+KRM1ECD	B-protein
+
+(	O
+A	O
+)	O
+The	O
+KRM1ECD	B-protein
+fold	O
+(	O
+crystal	B-evidence
+form	I-evidence
+I	I-evidence
+)	O
+colored	O
+blue	O
+to	O
+red	O
+from	O
+the	O
+N	O
+to	O
+C	O
+terminus	O
+.	O
+
+Cysteines	B-residue_name
+as	O
+ball	O
+and	O
+sticks	O
+,	O
+glycosylation	B-site
+sites	I-site
+as	O
+sticks	O
+.	O
+
+The	O
+bound	O
+calcium	B-chemical
+is	O
+shown	O
+as	O
+a	O
+gray	O
+sphere	O
+.	O
+
+The	O
+site	O
+of	O
+the	O
+F207S	B-mutant
+mutation	O
+associated	O
+with	O
+ectodermal	O
+dysplasia	O
+in	O
+humans	B-species
+is	O
+shown	O
+as	O
+mesh	O
+.	O
+
+(	O
+B	O
+)	O
+Superposition	B-experimental_method
+of	O
+the	O
+three	O
+KRM1ECD	B-protein
+subdomains	O
+(	O
+solid	O
+)	O
+with	O
+their	O
+next	O
+structurally	O
+characterized	O
+homologs	O
+(	O
+half	O
+transparent	O
+).	O
+
+(	O
+C	O
+)	O
+Superposition	B-experimental_method
+of	O
+KRM1ECD	B-protein
+from	O
+the	O
+three	O
+crystal	B-evidence
+forms	I-evidence
+.	O
+
+Alignment	B-evidence
+scores	I-evidence
+for	O
+each	O
+pairing	O
+are	O
+indicated	O
+on	O
+the	O
+dashed	O
+triangle	O
+.	O
+
+(	O
+A	O
+)	O
+The	O
+structure	B-evidence
+of	O
+the	O
+ternary	O
+LRP6PE3PE4	B-complex_assembly
+-	I-complex_assembly
+DKK1CRD2	I-complex_assembly
+-	I-complex_assembly
+KRM1ECD	I-complex_assembly
+complex	O
+.	O
+
+DKK1	B-protein
+(	O
+orange	O
+)	O
+is	O
+sandwiched	O
+between	O
+the	O
+PE3	B-structure_element
+module	O
+of	O
+LRP6	B-protein
+(	O
+blue	O
+)	O
+and	O
+the	O
+KR	B-structure_element
+-	I-structure_element
+WSC	I-structure_element
+domain	O
+pair	O
+of	O
+KRM1	B-protein
+(	O
+green	O
+).	O
+
+Colored	O
+symbols	O
+indicate	O
+introduced	O
+N	B-site
+-	I-site
+glycan	I-site
+attachment	I-site
+sites	I-site
+(	O
+see	O
+D	O
+).	O
+
+(	O
+B	O
+)	O
+SPR	B-experimental_method
+data	O
+comparing	O
+binding	O
+of	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+DKK1	B-protein
+and	O
+SUMO	B-experimental_method
+fusions	I-experimental_method
+of	O
+DKK1	B-protein
+truncations	O
+for	O
+binding	O
+to	O
+immobilized	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+KRM1ECD	B-protein
+.	O
+
+(	O
+C	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+DKK1CRD2	B-site
+-	I-site
+KRM1ECD	I-site
+interface	I-site
+.	O
+
+Residues	O
+involved	O
+in	O
+interface	B-site
+formation	O
+are	O
+shown	O
+as	O
+sticks	O
+;	O
+those	O
+mentioned	O
+in	O
+the	O
+text	O
+are	O
+labeled	O
+.	O
+
+Salt	B-bond_interaction
+bridges	I-bond_interaction
+are	O
+in	O
+pink	O
+and	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+in	O
+black	O
+.	O
+
+(	O
+D	O
+)	O
+SPR	B-experimental_method
+binding	B-evidence
+data	I-evidence
+comparing	O
+DKK1	B-protein
+analyte	O
+binding	O
+with	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+KRM1ECD	B-protein
+and	O
+three	O
+variants	O
+bearing	O
+engineered	B-protein_state
+glycosylation	B-site
+sites	I-site
+on	O
+the	O
+KR	B-structure_element
+and	O
+WSC	B-structure_element
+domains	O
+(	O
+green	O
+and	O
+blue	O
+pointing	O
+to	O
+DKK1	B-protein
+)	O
+and	O
+on	O
+the	O
+CUB	B-structure_element
+domain	O
+(	O
+orange	O
+).	O
+
+LRP6	B-complex_assembly
+-	I-complex_assembly
+KRM1	I-complex_assembly
+Direct	O
+Interaction	O
+and	O
+Summary	O
+
+(	O
+A	O
+)	O
+In	O
+a	O
+construction	O
+of	O
+a	O
+ternary	O
+complex	B-protein_state
+with	I-protein_state
+all	O
+four	O
+β	B-structure_element
+propellers	I-structure_element
+of	O
+LRP6	B-protein
+intact	B-protein_state
+,	O
+the	O
+CUB	B-structure_element
+domain	O
+points	O
+via	O
+its	O
+Ca2	B-site
++-	I-site
+binding	I-site
+region	I-site
+toward	O
+the	O
+top	O
+face	O
+of	O
+the	O
+second	B-structure_element
+β	I-structure_element
+propeller	I-structure_element
+.	O
+
+(	O
+B	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+potential	O
+interaction	B-site
+site	I-site
+.	O
+
+In	O
+addition	O
+,	O
+LRP6PE2	B-protein
+has	O
+been	O
+superimposed	B-experimental_method
+with	O
+DKK1	B-protein
+(	O
+yellow	O
+)	O
+and	O
+SOST	B-protein
+(	O
+pink	O
+)	O
+peptide	O
+complexes	O
+of	O
+LRP6PE1	B-protein
+.	O
+
+(	O
+C	O
+)	O
+SPR	B-experimental_method
+measurements	I-experimental_method
+comparing	O
+LRP6PE1PE2	B-protein
+binding	O
+with	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+KRM1ECD	B-protein
+and	O
+the	O
+GlycoCUB	B-protein_state
+mutant	I-protein_state
+bearing	O
+an	O
+N	B-ptm
+-	I-ptm
+glycan	I-ptm
+at	O
+N309	B-residue_name_number
+.	O
+
+(	O
+D	O
+)	O
+Schematic	O
+representation	O
+of	O
+structural	O
+and	O
+biophysical	O
+findings	O
+and	O
+their	O
+implications	O
+for	O
+Wnt	B-protein_type
+-	O
+dependent	O
+(	O
+left	O
+,	O
+middle	O
+)	O
+and	O
+independent	O
+(	O
+right	O
+)	O
+signaling	O
+.	O
+
+Conformational	O
+differences	O
+in	O
+the	O
+depictions	O
+of	O
+LRP6	B-protein
+are	O
+included	O
+purely	O
+for	O
+ease	O
+of	O
+representation	O
+.	O
+
+Diffraction	B-evidence
+and	I-evidence
+Refinement	I-evidence
+Statistics	I-evidence
+
+KRM1ECD	B-protein
+KRM1ECD	B-protein
+KRM1ECD	B-protein
+KRM1ECD	B-protein
+LRP6PE3PE4	B-complex_assembly
+-	I-complex_assembly
+DKKCRD2	I-complex_assembly
+-	I-complex_assembly
+KRM1ECD	I-complex_assembly
+Crystal	O
+form	O
+I	O
+I	O
+II	O
+III	O
+I	O
+X	O
+-	O
+ray	O
+source	O
+Diamond	O
+i04	O
+Diamond	O
+i03	O
+Diamond	O
+i03	O
+Diamond	O
+i04	O
+Diamond	O
+i04	O
+Wavelength	O
+(	O
+Å	O
+)	O
+0	O
+.	O
+9793	O
+0	O
+.	O
+9700	O
+0	O
+.	O
+9700	O
+0	O
+.	O
+9795	O
+0	O
+.	O
+9795	O
+Space	O
+group	O
+P3121	O
+P3121	O
+P43	O
+P41212	O
+C2221	O
+Unit	O
+cell	O
+a	O
+/	O
+α	O
+(	O
+Å	O
+/°)	O
+50	O
+.	O
+9	O
+/	O
+90	O
+50	O
+.	O
+5	O
+/	O
+90	O
+65	O
+.	O
+8	O
+/	O
+90	O
+67	O
+.	O
+8	O
+/	O
+90	O
+86	O
+.	O
+9	O
+/	O
+90	O
+b	O
+/	O
+β	O
+(	O
+Å	O
+/°)	O
+50	O
+.	O
+9	O
+/	O
+90	O
+50	O
+.	O
+5	O
+/	O
+90	O
+65	O
+.	O
+8	O
+/	O
+90	O
+67	O
+.	O
+8	O
+/	O
+90	O
+100	O
+.	O
+1	O
+/	O
+90	O
+c	O
+/	O
+γ	O
+(	O
+Å	O
+/°)	O
+188	O
+.	O
+4	O
+/	O
+120	O
+187	O
+.	O
+4	O
+/	O
+120	O
+75	O
+.	O
+0	O
+/	O
+90	O
+198	O
+.	O
+2	O
+/	O
+90	O
+270	O
+.	O
+7	O
+/	O
+90	O
+Wilson	O
+B	O
+factor	O
+(	O
+Å2	O
+)	O
+31	O
+41	O
+76	O
+77	O
+NA	O
+Resolution	O
+range	O
+(	O
+Å	O
+)	O
+47	O
+.	O
+10	O
+–	O
+1	O
+.	O
+90	O
+(	O
+1	O
+.	O
+95	O
+–	O
+1	O
+.	O
+90	O
+)	O
+62	O
+.	O
+47	O
+–	O
+2	O
+.	O
+10	O
+(	O
+2	O
+.	O
+16	O
+–	O
+2	O
+.	O
+10	O
+)	O
+75	O
+.	O
+00	O
+–	O
+2	O
+.	O
+80	O
+(	O
+2	O
+.	O
+99	O
+–	O
+2	O
+.	O
+80	O
+)	O
+67	O
+.	O
+80	O
+–	O
+3	O
+.	O
+20	O
+(	O
+3	O
+.	O
+42	O
+–	O
+3	O
+.	O
+20	O
+)	O
+67	O
+.	O
+68	O
+–	O
+3	O
+.	O
+50	O
+(	O
+7	O
+.	O
+16	O
+–	O
+6	O
+.	O
+40	O
+,	O
+3	O
+.	O
+92	O
+–	O
+3	O
+.	O
+50	O
+)	O
+Unique	O
+reflections	O
+23	O
+,	O
+300	O
+(	O
+1	O
+,	O
+524	O
+)	O
+17	O
+,	O
+089	O
+(	O
+1	O
+,	O
+428	O
+)	O
+7	O
+,	O
+964	O
+(	O
+1	O
+,	O
+448	O
+)	O
+8	O
+,	O
+171	O
+(	O
+1	O
+,	O
+343	O
+)	O
+8	O
+,	O
+070	O
+(	O
+723	O
+,	O
+645	O
+)	O
+Average	O
+multiplicity	O
+9	O
+.	O
+1	O
+(	O
+9	O
+.	O
+2	O
+)	O
+5	O
+.	O
+2	O
+(	O
+5	O
+.	O
+3	O
+)	O
+3	O
+.	O
+7	O
+(	O
+3	O
+.	O
+7	O
+)	O
+22	O
+.	O
+7	O
+(	O
+12	O
+.	O
+6	O
+)	O
+3	O
+.	O
+8	O
+(	O
+3	O
+.	O
+5	O
+,	O
+4	O
+.	O
+4	O
+)	O
+Completeness	O
+(%)	O
+99	O
+.	O
+8	O
+(	O
+98	O
+.	O
+5	O
+)	O
+100	O
+(	O
+100	O
+)	O
+99	O
+.	O
+8	O
+(	O
+100	O
+)	O
+98	O
+.	O
+8	O
+(	O
+93	O
+.	O
+4	O
+)	O
+51	O
+.	O
+6	O
+(	O
+98	O
+.	O
+5	O
+,	O
+14	O
+.	O
+1	O
+)	O
+<	O
+I	O
+/	O
+σI	O
+>	O
+11	O
+.	O
+4	O
+(	O
+1	O
+.	O
+7	O
+)	O
+12	O
+.	O
+0	O
+(	O
+1	O
+.	O
+7	O
+)	O
+14	O
+.	O
+9	O
+(	O
+1	O
+.	O
+5	O
+)	O
+13	O
+.	O
+1	O
+(	O
+1	O
+.	O
+9	O
+)	O
+4	O
+.	O
+6	O
+(	O
+4	O
+.	O
+1	O
+,	O
+2	O
+.	O
+2	O
+)	O
+Rmerge	O
+(%)	O
+14	O
+.	O
+8	O
+(	O
+158	O
+.	O
+3	O
+)	O
+9	O
+.	O
+3	O
+(	O
+98	O
+.	O
+0	O
+)	O
+6	O
+.	O
+2	O
+(	O
+98	O
+.	O
+9	O
+)	O
+29	O
+.	O
+8	O
+(	O
+142	O
+.	O
+2	O
+)	O
+44	O
+.	O
+9	O
+(	O
+40	O
+.	O
+5	O
+,	O
+114	O
+.	O
+2	O
+)	O
+Rpim	O
+(%)	O
+15	O
+.	O
+7	O
+(	O
+55	O
+.	O
+3	O
+)	O
+10	O
+.	O
+3	O
+(	O
+109	O
+.	O
+0	O
+)	O
+3	O
+.	O
+7	O
+(	O
+53	O
+.	O
+8	O
+)	O
+6	O
+.	O
+3	O
+(	O
+40	O
+.	O
+0	O
+)	O
+24	O
+.	O
+7	O
+(	O
+23	O
+.	O
+9	O
+,	O
+59	O
+.	O
+9	O
+)	O
+Refinement	O
+Rwork	O
+(%)	O
+17	O
+.	O
+9	O
+18	O
+.	O
+4	O
+21	O
+.	O
+6	O
+20	O
+.	O
+2	O
+32	O
+.	O
+1	O
+Rfree	O
+(%)	O
+22	O
+.	O
+7	O
+23	O
+.	O
+2	O
+30	O
+.	O
+7	O
+27	O
+.	O
+1	O
+35	O
+.	O
+5	O
+No	O
+.	O
+of	O
+Non	O
+-	O
+Hydrogen	O
+Atoms	O
+Protein	O
+2	O
+,	O
+260	O
+2	O
+,	O
+301	O
+2	O
+,	O
+102	O
+2	O
+,	O
+305	O
+7	O
+,	O
+730	O
+N	O
+-	O
+glycans	O
+42	O
+42	O
+28	O
+28	O
+0	O
+Water	B-chemical
+79	O
+54	O
+0	O
+2	O
+0	O
+Ligands	O
+6	O
+6	O
+2	O
+5	O
+0	O
+Average	O
+B	O
+factor	O
+(	O
+Å2	O
+)	O
+Protein	O
+63	O
+65	O
+108	O
+84	O
+–	O
+N	O
+-	O
+glycans	O
+35	O
+46	O
+102	O
+18	O
+–	O
+Water	B-chemical
+68	O
+85	O
+–	O
+75	O
+–	O
+Ligands	O
+36	O
+47	O
+91	O
+75	O
+66	O
+RMSD	B-evidence
+from	O
+Ideality	O
+Bond	O
+lengths	O
+(	O
+Å	O
+)	O
+0	O
+.	O
+020	O
+0	O
+.	O
+016	O
+0	O
+.	O
+019	O
+0	O
+.	O
+016	O
+0	O
+.	O
+004	O
+Bond	O
+angles	O
+(°)	O
+2	O
+.	O
+050	O
+1	O
+.	O
+748	O
+1	O
+.	O
+952	O
+1	O
+.	O
+796	O
+0	O
+.	O
+770	O
+Ramachandran	O
+Plot	O
+Favored	O
+(%)	O
+96	O
+.	O
+8	O
+95	O
+.	O
+5	O
+96	O
+.	O
+9	O
+94	O
+.	O
+9	O
+92	O
+.	O
+3	O
+Allowed	O
+(%)	O
+99	O
+.	O
+7	O
+100	O
+.	O
+0	O
+100	O
+.	O
+0	O
+99	O
+.	O
+7	O
+99	O
+.	O
+8	O
+Number	O
+of	O
+outliers	O
+1	O
+0	O
+0	O
+1	O
+2	O
+PDB	O
+code	O
+5FWS	O
+5FWT	O
+5FWU	O
+5FWV	O
+5FWW	O
+
+An	O
+additional	O
+shell	O
+given	O
+for	O
+the	O
+ternary	O
+complex	O
+corresponds	O
+to	O
+the	O
+last	O
+shell	O
+with	O
+near	O
+-	O
+complete	O
+diffraction	B-evidence
+data	I-evidence
+.	O
+
diff --git a/annotation_IOB/PMC5063996.tsv b/annotation_IOB/PMC5063996.tsv
new file mode 100644
index 0000000000000000000000000000000000000000..8956ff6cdcec3ee2c838c0edf80c162cb0597320
--- /dev/null
+++ b/annotation_IOB/PMC5063996.tsv
@@ -0,0 +1,7314 @@
+The	O
+Mechanism	O
+by	O
+Which	O
+Arabinoxylanases	B-protein_type
+Can	O
+Recognize	O
+Highly	B-protein_state
+Decorated	I-protein_state
+Xylans	B-chemical
+*	O
+
+The	O
+enzymatic	O
+degradation	O
+of	O
+plant	B-taxonomy_domain
+cell	O
+walls	O
+is	O
+an	O
+important	O
+biological	O
+process	O
+of	O
+increasing	O
+environmental	O
+and	O
+industrial	O
+significance	O
+.	O
+
+Xylan	B-chemical
+,	O
+a	O
+major	O
+component	O
+of	O
+the	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+,	O
+consists	O
+of	O
+a	O
+backbone	O
+of	O
+β	B-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+xylose	I-chemical
+(	O
+Xylp	B-chemical
+)	O
+units	O
+that	O
+are	O
+often	O
+decorated	O
+with	O
+arabinofuranose	B-chemical
+(	O
+Araf	B-chemical
+)	O
+side	O
+chains	O
+.	O
+
+A	O
+large	O
+penta	B-protein_type
+-	I-protein_type
+modular	I-protein_type
+enzyme	I-protein_type
+,	O
+CtXyl5A	B-protein
+,	O
+was	O
+shown	O
+previously	O
+to	O
+specifically	O
+target	O
+arabinoxylans	B-chemical
+.	O
+
+Here	O
+we	O
+report	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+arabinoxylanase	B-protein_type
+and	O
+the	O
+enzyme	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+ligands	B-chemical
+.	O
+
+The	O
+data	O
+showed	O
+that	O
+four	O
+of	O
+the	O
+protein	O
+modules	O
+adopt	O
+a	O
+rigid	O
+structure	O
+,	O
+which	O
+stabilizes	O
+the	O
+catalytic	B-structure_element
+domain	I-structure_element
+.	O
+
+The	O
+C	O
+-	O
+terminal	O
+non	B-structure_element
+-	I-structure_element
+catalytic	I-structure_element
+carbohydrate	I-structure_element
+binding	I-structure_element
+module	I-structure_element
+could	O
+not	O
+be	O
+observed	O
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+,	O
+suggesting	O
+positional	O
+flexibility	O
+.	O
+
+The	O
+structure	B-evidence
+of	O
+the	O
+enzyme	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+Xylp	B-chemical
+-	I-chemical
+β	I-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+Xylp	I-chemical
+-	I-chemical
+β	I-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+Xylp	I-chemical
+-[	I-chemical
+α	I-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+Araf	I-chemical
+]-	I-chemical
+β	I-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+Xylp	I-chemical
+showed	O
+that	O
+the	O
+Araf	B-chemical
+decoration	O
+linked	O
+O3	O
+to	O
+the	O
+xylose	B-chemical
+in	O
+the	O
+active	B-site
+site	I-site
+is	O
+located	O
+in	O
+the	O
+pocket	B-site
+(−	O
+2	B-site
+*	I-site
+subsite	I-site
+)	O
+that	O
+abuts	O
+onto	O
+the	O
+catalytic	B-site
+center	I-site
+.	O
+
+The	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+can	O
+also	O
+bind	O
+to	O
+Xylp	B-chemical
+and	O
+Arap	B-chemical
+,	O
+explaining	O
+why	O
+the	O
+enzyme	O
+can	O
+utilize	O
+xylose	B-chemical
+and	O
+arabinose	B-chemical
+as	O
+specificity	O
+determinants	O
+.	O
+
+Alanine	B-experimental_method
+substitution	I-experimental_method
+of	O
+Glu68	B-residue_name_number
+,	O
+Tyr92	B-residue_name_number
+,	O
+or	O
+Asn139	B-residue_name_number
+,	O
+which	O
+interact	O
+with	O
+arabinose	B-chemical
+and	O
+xylose	B-chemical
+side	O
+chains	O
+at	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+,	O
+abrogates	O
+catalytic	O
+activity	O
+.	O
+
+Distal	O
+to	O
+the	O
+active	B-site
+site	I-site
+,	O
+the	O
+xylan	B-chemical
+backbone	O
+makes	O
+limited	O
+apolar	O
+contacts	O
+with	O
+the	O
+enzyme	O
+,	O
+and	O
+the	O
+hydroxyls	O
+are	O
+solvent	B-protein_state
+-	I-protein_state
+exposed	I-protein_state
+.	O
+
+This	O
+explains	O
+why	O
+CtXyl5A	B-protein
+is	O
+capable	O
+of	O
+hydrolyzing	O
+xylans	B-chemical
+that	O
+are	O
+extensively	O
+decorated	O
+and	O
+that	O
+are	O
+recalcitrant	O
+to	O
+classic	O
+endo	B-protein_type
+-	I-protein_type
+xylanase	I-protein_type
+attack	O
+.	O
+
+The	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+is	O
+an	O
+important	O
+biological	O
+substrate	O
+.	O
+
+This	O
+complex	O
+composite	O
+structure	O
+is	O
+depolymerized	O
+by	O
+microorganisms	B-taxonomy_domain
+that	O
+occupy	O
+important	O
+highly	O
+competitive	O
+ecological	O
+niches	O
+,	O
+whereas	O
+the	O
+process	O
+makes	O
+an	O
+important	O
+contribution	O
+to	O
+the	O
+carbon	O
+cycle	O
+.	O
+
+Given	O
+that	O
+the	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+is	O
+the	O
+most	O
+abundant	O
+source	O
+of	O
+renewable	O
+organic	O
+carbon	O
+on	O
+the	O
+planet	O
+,	O
+this	O
+macromolecular	O
+substrate	O
+has	O
+substantial	O
+industrial	O
+potential	O
+.	O
+
+An	O
+example	O
+of	O
+the	O
+chemical	O
+complexity	O
+of	O
+the	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+is	O
+provided	O
+by	O
+xylan	B-chemical
+,	O
+which	O
+is	O
+the	O
+major	O
+hemicellulosic	O
+component	O
+.	O
+
+This	O
+polysaccharide	B-chemical
+comprises	O
+a	O
+backbone	O
+of	O
+β	B-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+d	I-chemical
+-	I-chemical
+xylose	I-chemical
+residues	O
+in	O
+their	O
+pyranose	B-chemical
+configuration	O
+(	O
+Xylp	B-chemical
+)	O
+that	O
+are	O
+decorated	O
+at	O
+O2	O
+with	O
+4	B-chemical
+-	I-chemical
+O	I-chemical
+-	I-chemical
+methyl	I-chemical
+-	I-chemical
+d	I-chemical
+-	I-chemical
+glucuronic	I-chemical
+acid	I-chemical
+(	O
+GlcA	B-chemical
+)	O
+and	O
+at	O
+O2	O
+and	O
+/	O
+or	O
+O3	O
+with	O
+α	B-chemical
+-	I-chemical
+l	I-chemical
+-	I-chemical
+arabinofuranose	I-chemical
+(	O
+Araf	B-chemical
+)	O
+residues	O
+,	O
+whereas	O
+the	O
+polysaccharide	B-chemical
+can	O
+also	O
+be	O
+extensively	O
+acetylated	O
+.	O
+
+In	O
+addition	O
+,	O
+the	O
+Araf	B-chemical
+side	O
+chain	O
+decorations	O
+can	O
+also	O
+be	O
+esterified	O
+to	O
+ferulic	B-chemical
+acid	I-chemical
+that	O
+,	O
+in	O
+some	O
+species	O
+,	O
+provide	O
+a	O
+chemical	O
+link	O
+between	O
+hemicellulose	B-chemical
+and	O
+lignin	B-chemical
+.	O
+
+The	O
+precise	O
+structure	O
+of	O
+xylans	B-chemical
+varies	O
+between	O
+plant	B-taxonomy_domain
+species	O
+,	O
+in	O
+particular	O
+in	O
+different	O
+tissues	O
+and	O
+during	O
+cellular	O
+differentiation	O
+.	O
+
+In	O
+specialized	O
+plant	B-taxonomy_domain
+tissues	O
+,	O
+such	O
+as	O
+the	O
+outer	O
+layer	O
+of	O
+cereal	B-taxonomy_domain
+grains	O
+,	O
+xylans	B-chemical
+are	O
+extremely	O
+complex	O
+,	O
+and	O
+side	O
+chains	O
+may	O
+comprise	O
+a	O
+range	O
+of	O
+other	O
+sugars	B-chemical
+including	O
+l	B-chemical
+-	I-chemical
+and	I-chemical
+d	I-chemical
+-	I-chemical
+galactose	I-chemical
+and	O
+β	B-chemical
+-	I-chemical
+and	I-chemical
+α	I-chemical
+-	I-chemical
+Xylp	I-chemical
+units	O
+.	O
+
+Indeed	O
+,	O
+in	O
+these	O
+cereal	B-taxonomy_domain
+brans	O
+,	O
+xylans	B-chemical
+have	O
+very	O
+few	O
+backbone	O
+Xylp	B-chemical
+units	O
+that	O
+are	O
+undecorated	O
+,	O
+and	O
+the	O
+side	O
+chains	O
+can	O
+contain	O
+up	O
+to	O
+six	O
+sugars	B-chemical
+.	O
+
+Reflecting	O
+the	O
+chemical	O
+and	O
+physical	O
+complexity	O
+of	O
+the	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+,	O
+microorganisms	B-taxonomy_domain
+that	O
+utilize	O
+these	O
+composite	O
+structures	O
+express	O
+a	O
+large	O
+number	O
+of	O
+polysaccharide	B-protein_type
+-	I-protein_type
+degrading	I-protein_type
+enzymes	I-protein_type
+,	O
+primarily	O
+glycoside	B-protein_type
+hydrolases	I-protein_type
+,	O
+but	O
+also	O
+polysaccharide	B-protein_type
+lyases	I-protein_type
+,	O
+carbohydrate	B-protein_type
+esterases	I-protein_type
+,	O
+and	O
+lytic	B-protein_type
+polysaccharide	I-protein_type
+monooxygenases	I-protein_type
+.	O
+
+These	O
+carbohydrate	B-protein_type
+active	I-protein_type
+enzymes	I-protein_type
+are	O
+grouped	O
+into	O
+sequence	O
+-	O
+based	O
+families	O
+in	O
+the	O
+CAZy	O
+database	O
+.	O
+
+With	O
+respect	O
+to	O
+xylan	B-chemical
+degradation	O
+,	O
+the	O
+backbone	O
+of	O
+simple	O
+xylans	B-chemical
+is	O
+hydrolyzed	O
+by	O
+endo	B-protein_type
+-	I-protein_type
+acting	I-protein_type
+xylanases	I-protein_type
+,	O
+the	O
+majority	O
+of	O
+which	O
+are	O
+located	O
+in	O
+glycoside	B-protein_type
+hydrolase	I-protein_type
+(	O
+GH	B-protein_type
+)	O
+5	B-protein_type
+families	O
+GH10	B-protein_type
+and	O
+GH11	B-protein_type
+,	O
+although	O
+they	O
+are	O
+also	O
+present	O
+in	O
+GH8	B-protein_type
+.	O
+
+The	O
+extensive	O
+decoration	O
+of	O
+the	O
+xylan	B-chemical
+backbone	O
+generally	O
+restricts	O
+the	O
+capacity	O
+of	O
+these	O
+enzymes	O
+to	O
+attack	O
+the	O
+polysaccharide	B-chemical
+prior	O
+to	O
+removal	O
+of	O
+the	O
+side	O
+chains	O
+by	O
+a	O
+range	O
+of	O
+α	B-protein_type
+-	I-protein_type
+glucuronidases	I-protein_type
+,	O
+α	B-protein_type
+-	I-protein_type
+arabinofuranosidases	I-protein_type
+,	O
+and	O
+esterases	B-protein_type
+.	O
+
+Two	O
+xylanases	B-protein_type
+,	O
+however	O
+,	O
+utilize	O
+the	O
+side	O
+chains	O
+as	O
+essential	O
+specificity	O
+determinants	O
+and	O
+thus	O
+target	O
+decorated	O
+forms	O
+of	O
+the	O
+hemicellulose	B-chemical
+.	O
+
+The	O
+GH30	B-protein_type
+glucuronoxylanases	B-protein_type
+require	O
+the	O
+Xylp	B-chemical
+bound	B-protein_state
+at	I-protein_state
+the	O
+−	B-site
+2	I-site
+to	O
+contain	O
+a	O
+GlcA	B-chemical
+side	O
+chain	O
+(	O
+the	O
+scissile	O
+bond	O
+targeted	O
+by	O
+glycoside	B-protein_type
+hydrolases	I-protein_type
+is	O
+between	O
+subsites	B-site
+−	I-site
+1	I-site
+and	I-site
++	I-site
+1	I-site
+,	O
+and	O
+subsites	B-site
+that	O
+extend	O
+toward	O
+the	O
+non	O
+-	O
+reducing	O
+and	O
+reducing	O
+ends	O
+of	O
+the	O
+substrate	O
+are	O
+assigned	O
+increasing	O
+negative	O
+and	O
+positive	O
+numbers	O
+,	O
+respectively	O
+).	O
+
+The	O
+GH5	B-protein_type
+arabinoxylanase	B-protein_type
+(	O
+CtXyl5A	B-protein
+)	O
+derived	O
+from	O
+Clostridium	B-species
+thermocellum	I-species
+displays	O
+an	O
+absolute	O
+requirement	O
+for	O
+xylans	B-chemical
+that	O
+contain	O
+Araf	B-chemical
+side	O
+chains	O
+.	O
+
+In	O
+this	O
+enzyme	O
+,	O
+the	O
+key	O
+specificity	O
+determinant	O
+is	O
+the	O
+Araf	B-chemical
+appended	O
+to	O
+O3	O
+of	O
+the	O
+Xylp	B-chemical
+bound	B-protein_state
+in	I-protein_state
+the	O
+active	B-site
+site	I-site
+(−	O
+1	B-site
+subsite	I-site
+).	O
+
+The	O
+reaction	O
+products	O
+generated	O
+from	O
+arabinoxylans	B-chemical
+,	O
+however	O
+,	O
+suggest	O
+that	O
+Araf	B-chemical
+can	O
+be	O
+accommodated	O
+at	O
+subsites	B-site
+distal	O
+to	O
+the	O
+active	B-site
+site	I-site
+.	O
+
+CtXyl5A	B-protein
+is	O
+a	O
+multimodular	O
+enzyme	O
+containing	O
+,	O
+in	O
+addition	O
+to	O
+the	O
+GH5	B-protein_type
+catalytic	B-structure_element
+module	I-structure_element
+(	O
+CtGH5	B-structure_element
+);	O
+three	O
+non	B-structure_element
+-	I-structure_element
+catalytic	I-structure_element
+carbohydrate	I-structure_element
+binding	I-structure_element
+modules	I-structure_element
+(	O
+CBMs	B-structure_element
+)	O
+belonging	O
+to	O
+families	O
+6	B-protein_type
+(	O
+CtCBM6	B-structure_element
+),	O
+13	B-protein_type
+(	O
+CtCBM13	B-structure_element
+),	O
+and	O
+62	B-protein_type
+(	O
+CtCBM62	B-structure_element
+);	O
+fibronectin	B-protein_type
+type	I-protein_type
+3	I-protein_type
+(	O
+Fn3	B-structure_element
+)	O
+domain	O
+;	O
+and	O
+a	O
+C	O
+-	O
+terminal	O
+dockerin	B-structure_element
+domain	O
+Fig	O
+.	O
+1	O
+.	O
+
+Previous	O
+studies	O
+of	O
+Fn3	B-structure_element
+domains	O
+have	O
+indicated	O
+that	O
+they	O
+might	O
+function	O
+as	O
+ligand	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+modules	I-structure_element
+,	O
+as	O
+a	O
+compact	O
+form	O
+of	O
+peptide	O
+linkers	O
+or	O
+spacers	O
+between	O
+other	O
+domains	O
+,	O
+as	O
+cellulose	B-structure_element
+-	I-structure_element
+disrupting	I-structure_element
+modules	I-structure_element
+,	O
+or	O
+as	O
+proteins	O
+that	O
+help	O
+large	O
+enzyme	O
+complexes	O
+remain	O
+soluble	O
+.	O
+
+The	O
+dockerin	B-structure_element
+domain	O
+recruits	O
+the	O
+enzyme	O
+into	O
+the	O
+cellulosome	B-complex_assembly
+,	O
+a	O
+multienzyme	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+degrading	O
+complex	O
+presented	O
+on	O
+the	O
+surface	O
+of	O
+C	B-species
+.	I-species
+thermocellum	I-species
+.	O
+
+CtCBM6	B-structure_element
+stabilizes	O
+CtGH5	B-structure_element
+,	O
+and	O
+CtCBM62	B-structure_element
+binds	O
+to	O
+d	B-chemical
+-	I-chemical
+galactopyranose	I-chemical
+and	O
+l	B-chemical
+-	I-chemical
+arabinopyranose	I-chemical
+.	O
+
+The	O
+function	O
+of	O
+the	O
+CtCBM13	B-structure_element
+and	O
+Fn3	B-structure_element
+modules	O
+remains	O
+unclear	O
+.	O
+
+This	O
+report	O
+exploits	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+mature	B-protein_state
+CtXyl5A	B-protein
+lacking	B-protein_state
+its	O
+C	O
+-	O
+terminal	O
+dockerin	B-structure_element
+domain	O
+(	O
+CtXyl5A	B-mutant
+-	I-mutant
+Doc	I-mutant
+),	O
+and	O
+the	O
+enzyme	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+ligands	B-chemical
+,	O
+to	O
+explore	O
+the	O
+mechanism	O
+of	O
+substrate	O
+specificity	O
+.	O
+
+The	O
+data	O
+show	O
+that	O
+the	O
+plasticity	O
+in	O
+substrate	O
+recognition	O
+enables	O
+the	O
+enzyme	O
+to	O
+hydrolyze	O
+highly	O
+complex	O
+xylans	B-chemical
+that	O
+are	O
+not	O
+accessible	O
+to	O
+classical	O
+GH10	B-protein_type
+and	O
+GH11	B-protein_type
+endo	B-protein_type
+-	I-protein_type
+xylanases	I-protein_type
+.	O
+
+Molecular	O
+architecture	O
+of	O
+GH5_34	B-protein_type
+enzymes	O
+.	O
+
+Modules	O
+prefaced	O
+by	O
+GH	B-structure_element
+,	O
+CBM	B-structure_element
+,	O
+or	O
+CE	B-structure_element
+are	O
+modules	O
+in	O
+the	O
+indicated	O
+glycoside	B-protein_type
+hydrolase	I-protein_type
+,	O
+carbohydrate	B-structure_element
+binding	I-structure_element
+module	I-structure_element
+,	O
+or	O
+carbohydrate	B-protein_type
+esterase	I-protein_type
+families	O
+,	O
+respectively	O
+.	O
+
+Laminin_3_G	B-structure_element
+domain	O
+belongs	O
+to	O
+the	O
+concanavalin	B-protein_type
+A	I-protein_type
+lectin	I-protein_type
+superfamily	I-protein_type
+,	O
+and	O
+FN3	B-structure_element
+denotes	O
+a	O
+fibronectin	B-structure_element
+type	I-structure_element
+3	I-structure_element
+domain	I-structure_element
+.	O
+
+Segments	O
+labeled	O
+D	O
+are	O
+dockerin	B-structure_element
+domains	O
+.	O
+
+Substrate	O
+Specificity	O
+of	O
+CtXyl5A	B-protein
+
+Previous	O
+studies	O
+showed	O
+that	O
+CtXyl5A	B-protein
+is	O
+an	O
+arabinoxylan	B-protein_type
+-	I-protein_type
+specific	I-protein_type
+xylanase	I-protein_type
+that	O
+generates	O
+xylooligosaccharides	B-chemical
+with	O
+an	O
+arabinose	B-chemical
+linked	O
+O3	O
+to	O
+the	O
+reducing	O
+end	O
+xylose	B-chemical
+.	O
+
+The	O
+enzyme	O
+is	O
+active	O
+against	O
+both	O
+wheat	B-taxonomy_domain
+and	O
+rye	B-taxonomy_domain
+arabinoxylans	B-chemical
+(	O
+abbreviated	O
+as	O
+WAX	B-chemical
+and	O
+RAX	B-chemical
+,	O
+respectively	O
+).	O
+
+It	O
+was	O
+proposed	O
+that	O
+arabinose	B-chemical
+decorations	O
+make	O
+productive	O
+interactions	O
+with	O
+a	O
+pocket	B-site
+(−	O
+2	B-site
+*)	I-site
+that	O
+is	O
+abutted	O
+onto	O
+the	O
+active	B-site
+site	I-site
+or	O
+−	B-site
+1	I-site
+subsite	I-site
+.	O
+
+Arabinose	B-chemical
+side	O
+chains	O
+of	O
+the	O
+other	O
+backbone	O
+xylose	B-chemical
+units	O
+in	O
+the	O
+oligosaccharides	B-chemical
+generated	O
+by	O
+CtXyl5A	B-protein
+were	O
+essentially	O
+random	O
+.	O
+
+These	O
+data	O
+suggest	O
+that	O
+O3	O
+,	O
+and	O
+possibly	O
+O2	O
+,	O
+on	O
+the	O
+xylose	B-chemical
+residues	O
+at	O
+subsites	B-site
+distal	O
+to	O
+the	O
+active	B-site
+site	I-site
+and	O
+−	B-site
+2	I-site
+*	I-site
+pocket	I-site
+are	O
+solvent	B-protein_state
+-	I-protein_state
+exposed	I-protein_state
+,	O
+implying	O
+that	O
+the	O
+enzyme	O
+can	O
+access	O
+highly	O
+decorated	O
+xylans	B-chemical
+.	O
+
+To	O
+test	O
+this	O
+hypothesis	O
+,	O
+the	O
+activity	O
+of	O
+CtXyl5A	B-protein
+against	O
+xylans	B-chemical
+from	O
+cereal	B-taxonomy_domain
+brans	O
+was	O
+assessed	O
+.	O
+
+CtXyl5a	B-protein
+was	O
+incubated	B-experimental_method
+with	O
+a	O
+range	O
+of	O
+xylans	B-chemical
+for	O
+16	O
+h	O
+at	O
+60	O
+°	O
+C	O
+,	O
+and	O
+the	O
+limit	O
+products	O
+were	O
+visualized	O
+by	O
+TLC	B-experimental_method
+.	O
+
+These	O
+xylans	B-chemical
+are	O
+highly	O
+decorated	O
+not	O
+only	O
+with	O
+Araf	B-chemical
+and	O
+GlcA	B-chemical
+units	O
+but	O
+also	O
+with	O
+l	B-chemical
+-	I-chemical
+Gal	I-chemical
+,	O
+d	B-chemical
+-	I-chemical
+Gal	I-chemical
+,	O
+and	O
+d	B-chemical
+-	I-chemical
+Xyl	I-chemical
+.	O
+
+Indeed	O
+,	O
+very	O
+few	O
+xylose	B-chemical
+units	O
+in	O
+the	O
+backbone	O
+of	O
+bran	O
+xylans	B-chemical
+lack	O
+side	O
+chains	O
+.	O
+
+The	O
+data	O
+presented	O
+in	O
+Table	O
+1	O
+showed	O
+that	O
+CtXyl5A	B-protein
+was	O
+active	O
+against	O
+corn	B-taxonomy_domain
+bran	O
+xylan	B-chemical
+(	O
+CX	B-chemical
+).	O
+
+In	O
+contrast	O
+typical	O
+endo	B-protein_type
+-	I-protein_type
+xylanases	I-protein_type
+from	O
+GH10	B-protein_type
+and	O
+GH11	B-protein_type
+were	O
+unable	O
+to	O
+attack	O
+CX	B-chemical
+,	O
+reflecting	O
+the	O
+lack	B-protein_state
+of	I-protein_state
+undecorated	O
+xylose	B-chemical
+units	O
+in	O
+the	O
+backbone	O
+(	O
+the	O
+active	B-site
+site	I-site
+of	O
+these	O
+enzymes	O
+can	O
+only	O
+bind	B-protein_state
+to	I-protein_state
+non	O
+-	O
+substituted	O
+xylose	B-chemical
+residues	O
+).	O
+
+The	O
+limit	O
+products	O
+generated	O
+by	O
+CtXyl5A	B-protein
+from	O
+CX	B-chemical
+consisted	O
+of	O
+an	O
+extensive	O
+range	O
+of	O
+oligosaccharides	B-chemical
+.	O
+
+These	O
+data	O
+support	O
+the	O
+view	O
+that	O
+in	O
+subsites	B-site
+out	O
+with	O
+the	O
+active	B-site
+site	I-site
+the	O
+O2	O
+and	O
+O3	O
+groups	O
+of	O
+the	O
+bound	O
+xylose	B-chemical
+units	O
+are	O
+solvent	B-protein_state
+-	I-protein_state
+exposed	I-protein_state
+and	O
+will	O
+thus	O
+tolerate	O
+decoration	O
+.	O
+
+Kinetics	B-evidence
+of	O
+GH5_34	B-protein_type
+arabinoxylanases	B-protein_type
+
+Enzyme	O
+Variant	O
+kcat	B-evidence
+/	O
+Km	B-evidence
+WAX	B-chemical
+RAX	B-chemical
+CX	B-chemical
+min	O
+−	O
+1mg	O
+−	O
+1ml	O
+CtXyl5A	B-protein
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+-	I-structure_element
+CBM13	I-structure_element
+-	I-structure_element
+Fn3	I-structure_element
+-	I-structure_element
+CBM62	I-structure_element
+800	O
+ND	O
+460	O
+CtXyl5A	B-protein
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+-	I-structure_element
+CBM13	I-structure_element
+-	I-structure_element
+Fn3	I-structure_element
+1	O
+,	O
+232	O
+ND	O
+659	O
+CtXyl5A	B-protein
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+-	I-structure_element
+CBM13	I-structure_element
+1	O
+,	O
+307	O
+ND	O
+620	O
+CtXyl5A	B-protein
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+488	O
+ND	O
+102	O
+CtXyl5A	B-protein
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+:	O
+E68A	B-mutant
+NA	O
+NA	O
+NA	O
+CtXyl5A	B-protein
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+:	O
+Y92A	B-mutant
+NA	O
+NA	O
+NA	O
+CtXyl5A	B-protein
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+:	O
+N135A	B-mutant
+260	O
+ND	O
+ND	O
+CtXyl5A	B-protein
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+:	O
+N139A	B-mutant
+NA	O
+NA	O
+NA	O
+AcGH5	B-protein
+Wild	B-protein_state
+type	I-protein_state
+628	O
+1	O
+,	O
+641	O
+289	O
+GpGH5	B-protein
+Wild	B-protein_state
+type	I-protein_state
+2	O
+,	O
+600	O
+9	O
+,	O
+986	O
+314	O
+VbGH5	B-protein
+Wild	B-protein_state
+type	I-protein_state
+ND	O
+ND	O
+ND	O
+VbGH5	B-protein
+D45A	B-mutant
+102	O
+203	O
+23	O
+
+To	O
+explore	O
+whether	O
+substrate	O
+bound	B-protein_state
+only	I-protein_state
+at	I-protein_state
+−	B-site
+2	I-site
+*	I-site
+and	O
+−	B-site
+1	I-site
+in	O
+the	O
+negative	B-site
+subsites	I-site
+was	O
+hydrolyzed	O
+by	O
+CtXyl5A	B-protein
+,	O
+the	O
+limit	O
+products	O
+of	O
+CX	B-chemical
+digested	O
+by	O
+the	O
+arabinoxylanase	B-protein_type
+were	O
+subjected	O
+to	O
+size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+using	O
+a	O
+Bio	O
+-	O
+Gel	O
+P	O
+-	O
+2	O
+,	O
+and	O
+the	O
+smallest	O
+oligosaccharides	B-chemical
+(	O
+largest	O
+elution	O
+volume	O
+)	O
+were	O
+chosen	O
+for	O
+further	O
+study	O
+.	O
+
+HPAEC	B-experimental_method
+analysis	O
+of	O
+the	O
+smallest	O
+oligosaccharide	B-chemical
+fraction	O
+(	O
+pool	O
+4	O
+)	O
+contained	O
+two	O
+species	O
+with	O
+retention	O
+times	O
+of	O
+14	O
+.	O
+0	O
+min	O
+(	O
+oligosaccharide	B-chemical
+1	O
+)	O
+and	O
+20	O
+.	O
+8	O
+min	O
+(	O
+oligosaccharide	B-chemical
+2	O
+)	O
+(	O
+Fig	O
+.	O
+2	O
+).	O
+
+Positive	B-experimental_method
+mode	I-experimental_method
+electrospray	I-experimental_method
+mass	I-experimental_method
+spectrometry	I-experimental_method
+showed	O
+that	O
+pool	O
+4	O
+contained	O
+exclusively	O
+molecular	O
+ions	O
+with	O
+a	O
+m	O
+/	O
+z	O
+=	O
+305	O
+[	O
+M	O
++	O
+Na	O
+]+,	O
+which	O
+corresponds	O
+to	O
+a	O
+pentose	B-chemical
+-	O
+pentose	B-chemical
+disaccharide	B-chemical
+(	O
+molecular	O
+mass	O
+=	O
+282	O
+Da	O
+)	O
+as	O
+a	O
+sodium	O
+ion	O
+adduct	O
+,	O
+whereas	O
+a	O
+dimer	O
+of	O
+the	O
+disaccharide	B-chemical
+with	O
+a	O
+sodium	O
+adduct	O
+(	O
+m	O
+/	O
+z	O
+=	O
+587	O
+[	O
+2M	O
++	O
+Na	O
+]+)	O
+was	O
+also	O
+evident	O
+.	O
+
+The	O
+monosaccharide	O
+composition	O
+of	O
+pool	O
+4	O
+determined	O
+by	O
+TFA	B-experimental_method
+hydrolysis	I-experimental_method
+contained	O
+xylose	B-chemical
+and	O
+arabinose	B-chemical
+in	O
+a	O
+3	O
+:	O
+1	O
+ratio	O
+.	O
+
+This	O
+suggests	O
+that	O
+the	O
+two	O
+oligosaccharides	B-chemical
+consist	O
+of	O
+two	O
+disaccharides	B-chemical
+:	O
+one	O
+consisting	O
+of	O
+two	O
+xylose	B-chemical
+residues	O
+and	O
+the	O
+other	O
+consisting	O
+of	O
+an	O
+arabinose	B-chemical
+linked	O
+to	O
+a	O
+xylose	B-chemical
+.	O
+
+Treatment	O
+of	O
+pool	O
+4	O
+with	O
+the	O
+nonspecific	B-protein_type
+arabinofuranosidase	I-protein_type
+,	O
+CjAbf51A	B-protein
+,	O
+resulted	O
+in	O
+the	O
+loss	O
+of	O
+oligosaccharide	B-chemical
+2	O
+and	O
+the	O
+production	O
+of	O
+both	O
+xylose	B-chemical
+and	O
+arabinose	B-chemical
+,	O
+indicative	O
+of	O
+a	O
+disaccharide	B-chemical
+of	O
+xylose	B-chemical
+and	O
+arabinose	B-chemical
+.	O
+
+Incubation	O
+of	O
+pool	O
+4	O
+with	O
+a	O
+β	B-protein_type
+-	I-protein_type
+1	I-protein_type
+,	I-protein_type
+3	I-protein_type
+-	I-protein_type
+xylosidase	I-protein_type
+(	O
+XynB	B-protein
+)	O
+converted	O
+oligosaccharide	B-chemical
+1	O
+into	O
+xylose	B-chemical
+,	O
+demonstrating	O
+that	O
+this	O
+molecule	O
+is	O
+the	O
+disaccharide	B-chemical
+β	B-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+xylobiose	I-chemical
+.	O
+
+This	O
+view	O
+is	O
+supported	O
+by	O
+the	O
+inability	O
+of	O
+a	O
+β	B-protein_type
+-	I-protein_type
+1	I-protein_type
+,	I-protein_type
+4	I-protein_type
+-	I-protein_type
+specific	I-protein_type
+xylosidase	I-protein_type
+to	O
+hydrolyze	O
+oligosaccharide	B-chemical
+1	O
+or	O
+oligosaccharide	B-chemical
+2	O
+(	O
+data	O
+not	O
+shown	O
+).	O
+
+The	O
+crucial	O
+importance	O
+of	O
+occupancy	O
+of	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+pocket	I-site
+for	O
+catalytic	O
+competence	O
+is	O
+illustrated	O
+by	O
+the	O
+inability	O
+of	O
+the	O
+enzyme	O
+to	O
+hydrolyze	O
+linear	O
+β	B-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+xylooligosaccharides	I-chemical
+.	O
+
+The	O
+generation	O
+of	O
+Araf	B-chemical
+-	I-chemical
+Xylp	I-chemical
+and	O
+Xyl	B-chemical
+-	I-chemical
+β	I-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+Xyl	I-chemical
+as	O
+reaction	O
+products	O
+demonstrates	O
+that	O
+occupancy	O
+of	O
+the	O
+−	B-site
+2	I-site
+subsite	I-site
+is	O
+not	O
+essential	O
+for	O
+catalytic	O
+activity	O
+,	O
+which	O
+is	O
+in	O
+contrast	O
+to	O
+all	O
+endo	B-protein_type
+-	I-protein_type
+acting	I-protein_type
+xylanases	I-protein_type
+where	O
+this	O
+subsite	B-site
+plays	O
+a	O
+critical	O
+role	O
+in	O
+enzyme	O
+activity	O
+.	O
+
+Indeed	O
+,	O
+the	O
+data	O
+demonstrate	O
+that	O
+−	B-site
+2	I-site
+*	I-site
+plays	O
+a	O
+more	O
+important	O
+role	O
+in	O
+productive	O
+substrate	O
+binding	O
+than	O
+the	O
+−	B-site
+2	I-site
+subsite	I-site
+.	O
+
+Unfortunately	O
+,	O
+the	O
+inability	O
+to	O
+generate	O
+highly	O
+purified	O
+(	B-chemical
+Xyl	I-chemical
+-	I-chemical
+β	I-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+)	I-chemical
+n	I-chemical
+-[	I-chemical
+β	I-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+Xyl	I-chemical
+/	I-chemical
+Ara	I-chemical
+]-	I-chemical
+Xyl	I-chemical
+oligosaccharides	B-chemical
+from	O
+arabinoxylans	B-chemical
+prevented	O
+the	O
+precise	O
+binding	O
+energies	O
+at	O
+the	O
+negative	O
+subsites	O
+to	O
+be	O
+determined	O
+.	O
+
+Identification	O
+of	O
+the	O
+disaccharide	B-chemical
+reaction	O
+products	O
+generated	O
+from	O
+CX	B-chemical
+.	O
+
+The	O
+smallest	O
+reaction	O
+products	O
+were	O
+purified	O
+by	O
+size	B-experimental_method
+exclusion	I-experimental_method
+chromatography	I-experimental_method
+and	O
+analyzed	O
+by	O
+HPAEC	B-experimental_method
+(	O
+A	O
+)	O
+and	O
+positive	O
+mode	O
+ESI	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+(	O
+B	O
+),	O
+respectively	O
+.	O
+
+The	O
+samples	O
+were	O
+treated	O
+with	O
+a	O
+nonspecific	B-protein_type
+arabinofuranosidase	I-protein_type
+(	O
+CjAbf51A	B-protein
+)	O
+and	O
+a	O
+GH3	B-protein_type
+xylosidase	I-protein_type
+(	O
+XynB	B-protein
+)	O
+that	O
+targeted	O
+β	O
+-	O
+1	O
+,	O
+3	O
+-	O
+xylosidic	O
+bonds	O
+.	O
+
+X	O
+,	O
+xylose	B-chemical
+;	O
+A	O
+,	O
+arabinose	B-chemical
+.	O
+
+The	O
+m	O
+/	O
+z	O
+=	O
+305	O
+species	O
+denotes	O
+a	O
+pentose	B-chemical
+disaccharide	B-chemical
+as	O
+a	O
+sodium	O
+adduct	O
+[	O
+M	O
++	O
+Na	O
+]+,	O
+whereas	O
+the	O
+m	O
+/	O
+z	O
+=	O
+587	O
+signal	O
+corresponds	O
+to	O
+an	O
+ESI	B-experimental_method
+-	I-experimental_method
+MS	I-experimental_method
+dimer	O
+of	O
+the	O
+pentose	B-chemical
+disaccharide	B-chemical
+also	O
+as	O
+a	O
+sodium	O
+adduct	O
+[	O
+2M	O
++	O
+Na	O
+]+.	O
+
+Crystal	B-evidence
+Structure	I-evidence
+of	O
+the	O
+Catalytic	B-structure_element
+Module	I-structure_element
+of	O
+CtXyl5A	B-protein
+in	B-protein_state
+Complex	I-protein_state
+with	I-protein_state
+Ligands	B-chemical
+
+To	O
+understand	O
+the	O
+structural	O
+basis	O
+for	O
+the	O
+biochemical	O
+properties	O
+of	O
+CtXyl5A	B-protein
+,	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+enzyme	O
+with	O
+ligands	O
+that	O
+occupy	O
+the	O
+substrate	B-site
+binding	I-site
+cleft	I-site
+and	O
+the	O
+critical	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+were	O
+sought	O
+.	O
+
+The	O
+data	O
+presented	O
+in	O
+Fig	O
+.	O
+3A	O
+show	O
+the	O
+structure	B-evidence
+of	O
+the	O
+CtXyl5A	B-protein
+derivative	O
+CtGH5	B-structure_element
+-	I-structure_element
+CtCBM6	I-structure_element
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+arabinose	B-chemical
+bound	B-protein_state
+in	I-protein_state
+the	O
+−	B-site
+2	I-site
+*	I-site
+pocket	I-site
+.	O
+
+Interestingly	O
+,	O
+the	O
+bound	B-protein_state
+arabinose	B-chemical
+was	O
+in	O
+the	O
+pyranose	B-chemical
+conformation	O
+rather	O
+than	O
+in	O
+its	O
+furanose	B-chemical
+form	O
+found	O
+in	O
+arabinoxylans	B-chemical
+.	O
+
+O1	O
+was	O
+facing	O
+toward	O
+the	O
+active	B-site
+site	I-site
+−	B-site
+1	I-site
+subsite	I-site
+,	O
+indicative	O
+of	O
+the	O
+bound	B-protein_state
+arabinose	B-chemical
+being	O
+in	O
+the	O
+right	O
+orientation	O
+to	O
+be	O
+linked	O
+to	O
+the	O
+xylan	B-chemical
+backbone	O
+via	O
+an	O
+α	O
+-	O
+1	O
+,	O
+3	O
+linkage	O
+.	O
+
+As	O
+discussed	O
+on	O
+below	O
+,	O
+the	O
+axial	O
+O4	O
+of	O
+the	O
+Arap	B-chemical
+did	O
+not	O
+interact	O
+with	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+,	O
+suggesting	O
+that	O
+the	O
+pocket	B-site
+might	O
+be	O
+capable	O
+of	O
+binding	O
+a	O
+xylose	B-chemical
+molecule	O
+.	O
+
+Indeed	O
+,	O
+soaking	B-experimental_method
+apo	B-protein_state
+crystals	B-evidence
+with	O
+xylose	B-chemical
+showed	O
+that	O
+the	O
+pentose	B-chemical
+sugar	B-chemical
+also	O
+bound	B-protein_state
+in	I-protein_state
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+in	O
+its	O
+pyranose	B-chemical
+conformation	O
+(	O
+Fig	O
+.	O
+3B	O
+).	O
+
+These	O
+crystal	B-evidence
+structures	I-evidence
+support	O
+the	O
+biochemical	O
+data	O
+presented	O
+above	O
+showing	O
+that	O
+the	O
+enzyme	O
+generated	O
+β	B-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+3	I-chemical
+-	I-chemical
+xylobiose	I-chemical
+from	O
+CX	B-chemical
+,	O
+which	O
+would	O
+require	O
+the	O
+disaccharide	B-chemical
+to	O
+bind	O
+at	O
+the	O
+−	B-site
+1	I-site
+and	I-site
+−	I-site
+2	I-site
+*	I-site
+subsites	I-site
+.	O
+
+A	O
+third	O
+product	O
+complex	O
+was	O
+generated	O
+by	O
+co	B-experimental_method
+-	I-experimental_method
+crystallizing	I-experimental_method
+the	O
+nucleophile	B-protein_state
+inactive	I-protein_state
+mutant	B-protein_state
+CtGH5E279S	B-mutant
+-	O
+CtCBM6	B-structure_element
+with	O
+a	O
+WAX	B-chemical
+-	O
+derived	O
+oligosaccharide	B-chemical
+(	O
+Fig	O
+.	O
+3C	O
+).	O
+
+The	O
+data	O
+revealed	O
+a	O
+pentasaccharide	B-chemical
+bound	B-protein_state
+to	I-protein_state
+the	O
+enzyme	O
+,	O
+comprising	O
+β	B-chemical
+-	I-chemical
+1	I-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+xylotetraose	I-chemical
+with	O
+an	O
+Araf	B-chemical
+linked	O
+α	O
+-	O
+1	O
+,	O
+3	O
+to	O
+the	O
+reducing	O
+end	O
+xylose	B-chemical
+.	O
+
+The	O
+xylotetraose	B-chemical
+was	O
+positioned	O
+in	O
+subsites	B-site
+−	I-site
+1	I-site
+to	I-site
+−	I-site
+4	I-site
+and	O
+the	O
+Araf	B-chemical
+in	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+pocket	I-site
+.	O
+
+Analysis	O
+of	O
+the	O
+three	O
+structures	B-evidence
+showed	O
+that	O
+O1	O
+,	O
+O2	O
+,	O
+O3	O
+,	O
+and	O
+the	O
+endocyclic	O
+oxygen	O
+occupied	O
+identical	O
+positions	O
+in	O
+the	O
+Arap	B-chemical
+,	O
+Araf	B-chemical
+,	O
+and	O
+Xylp	B-chemical
+ligands	O
+bound	B-protein_state
+in	I-protein_state
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+and	O
+thus	O
+made	O
+identical	O
+interactions	O
+with	O
+the	O
+pocket	B-site
+.	O
+
+O1	O
+makes	O
+a	O
+polar	B-bond_interaction
+contact	I-bond_interaction
+with	O
+Nδ2	O
+of	O
+Asn139	B-residue_name_number
+,	O
+O2	O
+is	O
+within	O
+hydrogen	B-bond_interaction
+bonding	I-bond_interaction
+distance	O
+with	O
+Oδ1	O
+of	O
+Asn139	B-residue_name_number
+and	O
+the	O
+backbone	O
+N	O
+of	O
+Asn135	B-residue_name_number
+,	O
+and	O
+O3	O
+interacts	O
+with	O
+the	O
+N	O
+of	O
+Gly136	B-residue_name_number
+and	O
+Oϵ2	O
+of	O
+Glu68	B-residue_name_number
+.	O
+
+Although	O
+O4	O
+of	O
+Arap	B-chemical
+does	O
+not	O
+make	O
+a	O
+direct	O
+interaction	O
+with	O
+the	O
+enzyme	O
+,	O
+O4	O
+and	O
+O5	O
+of	O
+Xylp	B-chemical
+and	O
+Araf	B-chemical
+,	O
+respectively	O
+,	O
+form	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+Oϵ1	O
+of	O
+Glu68	B-residue_name_number
+.	O
+
+Finally	O
+Tyr92	B-residue_name_number
+makes	O
+apolar	O
+parallel	B-bond_interaction
+interactions	I-bond_interaction
+with	O
+the	O
+pyranose	B-chemical
+or	O
+furanose	B-chemical
+rings	O
+of	O
+the	O
+three	O
+sugars	O
+.	O
+
+Representation	O
+of	O
+the	O
+residues	O
+involved	O
+in	O
+the	O
+ligands	O
+recognition	O
+at	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+.	O
+
+Interacting	O
+residues	O
+are	O
+represented	O
+as	O
+stick	O
+in	O
+blue	O
+,	O
+and	O
+the	O
+catalytic	B-site
+residues	I-site
+and	O
+the	O
+mutated	B-experimental_method
+glutamate	B-residue_name
+(	O
+into	O
+a	O
+serine	B-residue_name
+)	O
+are	O
+in	O
+magenta	O
+.	O
+
+A	O
+,	O
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+an	O
+arabinopyranose	B-chemical
+.	O
+
+B	O
+,	O
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+a	O
+xylopyranose	B-chemical
+.	O
+
+C	O
+,	O
+CtGH5E279S	B-mutant
+-	O
+CBM6	B-structure_element
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+a	O
+pentasaccharide	B-chemical
+(	O
+β1	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+xylotetraose	I-chemical
+with	O
+an	O
+l	B-chemical
+-	I-chemical
+Araf	I-chemical
+linked	O
+α1	O
+,	O
+3	O
+to	O
+the	O
+reducing	O
+end	O
+xylose	B-chemical
+).	O
+
+The	O
+xylan	B-chemical
+backbone	O
+is	O
+shown	O
+transparently	O
+for	O
+more	O
+clarity	O
+.	O
+
+Densities	B-evidence
+shown	O
+in	O
+blue	O
+are	O
+RefMac	O
+maximum	B-evidence
+-	I-evidence
+likelihood	I-evidence
+σA	I-evidence
+-	I-evidence
+weighted	I-evidence
+2Fo	I-evidence
+−	I-evidence
+Fc	I-evidence
+at	I-evidence
+1	I-evidence
+.	I-evidence
+5	I-evidence
+σ	I-evidence
+.	O
+
+The	O
+importance	O
+of	O
+the	O
+interactions	O
+between	O
+the	O
+ligands	O
+and	O
+the	O
+side	O
+chains	O
+of	O
+the	O
+residues	O
+in	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+pocket	I-site
+were	O
+evaluated	O
+by	O
+alanine	B-experimental_method
+substitution	I-experimental_method
+of	O
+these	O
+amino	O
+acids	O
+.	O
+
+The	O
+mutants	B-protein_state
+E68A	B-mutant
+,	O
+Y92A	B-mutant
+,	O
+and	O
+N139A	B-mutant
+were	O
+all	O
+inactive	B-protein_state
+(	O
+Table	O
+1	O
+),	O
+demonstrating	O
+the	O
+importance	O
+of	O
+the	O
+interactions	O
+of	O
+these	O
+residues	O
+with	O
+the	O
+substrate	O
+and	O
+reinforcing	O
+the	O
+critical	O
+role	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+plays	O
+in	O
+the	O
+activity	O
+of	O
+the	O
+enzyme	O
+.	O
+
+N135A	B-mutant
+retained	O
+wild	B-protein_state
+type	I-protein_state
+activity	O
+because	O
+the	O
+O2	O
+of	O
+the	O
+sugars	O
+interacts	O
+with	O
+the	O
+backbone	O
+N	O
+of	O
+Asn135	B-residue_name_number
+and	O
+not	O
+with	O
+the	O
+side	O
+chain	O
+.	O
+
+Because	O
+the	O
+hydroxyls	O
+of	O
+Xylp	B-chemical
+or	O
+Araf	B-chemical
+in	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+pocket	I-site
+are	O
+not	O
+solvent	B-protein_state
+-	I-protein_state
+exposed	I-protein_state
+,	O
+the	O
+active	B-site
+site	I-site
+of	O
+the	O
+arabinoxylanase	B-protein_type
+can	O
+only	O
+bind	O
+to	O
+xylose	B-chemical
+residues	O
+that	O
+contain	O
+a	O
+single	O
+xylose	B-chemical
+or	O
+arabinose	B-chemical
+O3	O
+decoration	O
+.	O
+
+This	O
+may	O
+explain	O
+why	O
+the	O
+kcat	B-evidence
+/	O
+Km	B-evidence
+for	O
+CtXyl5A	B-protein
+against	O
+WAX	B-chemical
+was	O
+2	O
+-	O
+fold	O
+higher	O
+than	O
+against	O
+CX	B-chemical
+(	O
+Table	O
+1	O
+).	O
+
+WAX	B-chemical
+is	O
+likely	O
+to	O
+have	O
+a	O
+higher	O
+concentration	O
+of	O
+single	O
+Araf	B-chemical
+decorations	O
+compared	O
+with	O
+CX	B-chemical
+and	O
+thus	O
+contain	O
+more	O
+substrate	O
+available	O
+to	O
+the	O
+arabinoxylanase	B-protein_type
+.	O
+
+In	O
+the	O
+active	B-site
+site	I-site
+of	O
+CtXyl5A	B-protein
+the	O
+α	B-chemical
+-	I-chemical
+d	I-chemical
+-	I-chemical
+Xylp	I-chemical
+,	O
+which	O
+is	O
+in	O
+its	O
+relaxed	O
+4C1	O
+conformation	O
+,	O
+makes	O
+the	O
+following	O
+interactions	O
+with	O
+the	O
+enzyme	O
+(	O
+Fig	O
+.	O
+4	O
+,	O
+A	O
+–	O
+C	O
+):	O
+O1	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+the	O
+Nδ1	O
+of	O
+His253	B-residue_name_number
+and	O
+Oϵ2	O
+of	O
+Glu171	B-residue_name_number
+(	O
+catalytic	O
+acid	O
+-	O
+base	O
+)	O
+and	O
+makes	O
+a	O
+possible	O
+weak	O
+polar	B-bond_interaction
+contact	I-bond_interaction
+with	O
+the	O
+OH	O
+of	O
+Tyr255	B-residue_name_number
+and	O
+Oγ	O
+of	O
+Ser279	B-residue_name_number
+(	O
+mutation	O
+of	O
+the	O
+catalytic	O
+nucleophile	O
+);	O
+O2	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+Nδ2	O
+of	O
+Asn170	B-residue_name_number
+and	O
+OH	O
+of	O
+Tyr92	B-residue_name_number
+.	O
+
+O3	O
+(	O
+O1	O
+of	O
+the	O
+Araf	B-chemical
+at	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+)	O
+makes	O
+a	O
+polar	B-bond_interaction
+contact	I-bond_interaction
+with	O
+Nδ2	O
+of	O
+Asn139	B-residue_name_number
+;	O
+the	O
+endocyclic	O
+oxygen	O
+hydrogens	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+the	O
+OH	O
+of	O
+Tyr255	B-residue_name_number
+.	O
+
+The	O
+Xylp	B-chemical
+in	O
+the	O
+active	B-site
+site	I-site
+makes	O
+strong	O
+parallel	B-bond_interaction
+apolar	I-bond_interaction
+interactions	I-bond_interaction
+with	O
+Phe310	B-residue_name_number
+.	O
+
+Substrate	O
+recognition	O
+in	O
+the	O
+active	B-site
+site	I-site
+is	O
+conserved	B-protein_state
+between	O
+CtXyl5A	B-protein
+and	O
+the	O
+closest	O
+GH5	B-protein_type
+structural	O
+homolog	O
+,	O
+the	O
+endoglucanase	B-protein_type
+BaCel5A	B-protein
+(	O
+PDB	O
+code	O
+1qi2	O
+)	O
+as	O
+noted	O
+previously	O
+.	O
+
+Comparison	O
+of	O
+the	O
+ligand	O
+recognition	O
+at	O
+the	O
+distal	O
+negative	B-site
+subsites	I-site
+between	O
+CtGH5E279S	B-mutant
+-	O
+CBM6	B-structure_element
+,	O
+the	O
+cellulase	B-protein_type
+BaCel5A	B-protein
+,	O
+and	O
+the	O
+xylanase	B-protein_type
+GH10	B-protein_type
+.	O
+
+A	O
+–	O
+C	O
+show	O
+CtGH5E279S	B-mutant
+-	O
+CBM6	O
+is	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+a	O
+pentasaccharide	B-chemical
+(	O
+β1	B-chemical
+,	I-chemical
+4	I-chemical
+-	I-chemical
+xylotetraose	I-chemical
+with	O
+an	O
+l	B-chemical
+-	I-chemical
+Araf	I-chemical
+linked	O
+α1	O
+,	O
+3	O
+to	O
+the	O
+reducing	O
+end	O
+xylose	B-chemical
+).	O
+
+A	O
+,	O
+Poseview	O
+representation	O
+highlighting	O
+the	O
+hydrogen	B-bond_interaction
+bonding	I-bond_interaction
+and	O
+the	O
+hydrophobic	B-bond_interaction
+interactions	I-bond_interaction
+that	O
+occur	O
+in	O
+the	O
+negative	B-site
+subsites	I-site
+.	O
+
+C	O
+,	O
+density	B-evidence
+of	O
+the	O
+ligand	O
+shown	O
+in	O
+blue	O
+is	O
+RefMac	O
+maximum	B-evidence
+-	I-evidence
+likelihood	I-evidence
+σA	I-evidence
+-	I-evidence
+weighted	I-evidence
+2Fo	I-evidence
+−	I-evidence
+Fc	I-evidence
+at	I-evidence
+1	I-evidence
+.	I-evidence
+5	I-evidence
+σ	I-evidence
+.	O
+
+D	O
+and	O
+E	O
+display	O
+BaCel5A	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+deoxy	B-chemical
+-	I-chemical
+2	I-chemical
+-	I-chemical
+fluoro	I-chemical
+-	I-chemical
+β	I-chemical
+-	I-chemical
+d	I-chemical
+-	I-chemical
+cellotrioside	I-chemical
+(	O
+PDB	O
+code	O
+1qi2	O
+),	O
+and	O
+F	O
+and	O
+G	O
+show	O
+CmXyn10B	B-protein
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+a	O
+xylotriose	B-chemical
+(	O
+PDB	O
+code	O
+1uqy	O
+).	O
+
+B	O
+,	O
+D	O
+,	O
+and	O
+F	O
+are	O
+surface	O
+representations	O
+(	O
+CtGH5E279S	B-mutant
+-	O
+CBM6	O
+in	O
+gray	O
+,	O
+BaCel5A	B-protein
+in	O
+cyan	O
+,	O
+and	O
+the	O
+xylanase	B-protein_type
+GH10	B-protein_type
+in	O
+light	O
+brown	O
+).	O
+
+The	O
+black	O
+dashes	O
+represent	O
+the	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+.	O
+
+The	O
+capacity	O
+of	O
+CtXyl5A	B-protein
+to	O
+act	O
+on	O
+the	O
+highly	O
+decorated	O
+xylan	B-chemical
+CX	B-chemical
+indicates	O
+that	O
+O3	O
+and	O
+possibly	O
+O2	O
+of	O
+the	O
+backbone	O
+Xylp	B-chemical
+units	O
+are	O
+solvent	B-protein_state
+-	I-protein_state
+exposed	I-protein_state
+.	O
+
+This	O
+is	O
+consistent	O
+with	O
+the	O
+interaction	O
+of	O
+the	O
+xylotetraose	B-chemical
+backbone	O
+with	O
+the	O
+enzyme	O
+distal	O
+to	O
+the	O
+active	B-site
+site	I-site
+.	O
+
+A	O
+surface	O
+representation	O
+of	O
+the	O
+enzyme	O
+(	O
+Fig	O
+.	O
+4B	O
+)	O
+shows	O
+that	O
+O3	O
+and	O
+O2	O
+of	O
+xylose	B-chemical
+units	O
+at	O
+subsites	B-site
+−	I-site
+2	I-site
+to	I-site
+−	I-site
+4	I-site
+are	O
+solvent	B-protein_state
+-	I-protein_state
+exposed	I-protein_state
+and	O
+are	O
+thus	O
+available	O
+for	O
+decoration	O
+.	O
+
+Indeed	O
+,	O
+these	O
+pyranose	B-chemical
+sugars	B-chemical
+make	O
+very	O
+weak	O
+apolar	B-bond_interaction
+interactions	I-bond_interaction
+with	O
+the	O
+arabinoxylanase	B-protein_type
+.	O
+
+At	O
+−	B-site
+2	I-site
+,	O
+Xylp	B-chemical
+makes	O
+planar	B-bond_interaction
+apolar	I-bond_interaction
+interactions	I-bond_interaction
+with	O
+the	O
+Araf	B-chemical
+bound	B-protein_state
+to	I-protein_state
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+(	O
+Fig	O
+.	O
+4C	O
+).	O
+
+Xylp	B-chemical
+at	O
+subsites	B-site
+−	I-site
+2	I-site
+and	I-site
+−	I-site
+3	I-site
+,	O
+respectively	O
+,	O
+make	O
+weak	O
+hydrophobic	B-bond_interaction
+contact	I-bond_interaction
+with	O
+Val318	B-residue_name_number
+,	O
+the	O
+−	B-site
+3	I-site
+Xylp	B-chemical
+makes	O
+planar	B-bond_interaction
+apolar	I-bond_interaction
+interactions	I-bond_interaction
+with	O
+Ala137	B-residue_name_number
+,	O
+whereas	O
+the	O
+xylose	B-chemical
+at	O
+−	B-site
+4	I-site
+forms	O
+parallel	B-bond_interaction
+apolar	I-bond_interaction
+contacts	I-bond_interaction
+with	O
+Trp69	B-residue_name_number
+.	O
+
+Comparison	O
+of	O
+the	O
+distal	O
+negative	B-site
+subsites	I-site
+of	O
+CtXyl5A	B-protein
+with	O
+BaCel5A	B-protein
+and	O
+a	O
+typical	O
+GH10	B-protein_type
+xylanase	B-protein_type
+(	O
+CmXyn10B	B-protein
+,	O
+PDB	O
+code	O
+1uqy	O
+)	O
+highlights	O
+the	O
+paucity	O
+of	O
+interactions	O
+between	O
+the	O
+arabinoxylanase	B-protein_type
+and	O
+its	O
+substrate	O
+out	O
+with	O
+the	O
+active	B-site
+site	I-site
+(	O
+Fig	O
+.	O
+4	O
+).	O
+
+Thus	O
+,	O
+the	O
+cellulase	B-protein_type
+contains	O
+three	O
+negative	B-site
+subsites	I-site
+and	O
+the	O
+sugars	B-chemical
+bound	B-protein_state
+in	I-protein_state
+the	O
+−	B-site
+2	I-site
+and	I-site
+−	I-site
+3	I-site
+subsites	I-site
+make	O
+a	O
+total	O
+of	O
+9	O
+polar	B-bond_interaction
+interactions	I-bond_interaction
+with	O
+the	O
+enzyme	O
+(	O
+Fig	O
+.	O
+4	O
+,	O
+D	O
+and	O
+E	O
+).	O
+
+The	O
+GH10	B-protein_type
+xylanase	B-protein_type
+also	O
+contains	O
+a	O
+−	B-site
+2	I-site
+subsite	I-site
+that	O
+,	O
+similar	O
+to	O
+the	O
+cellulase	B-protein_type
+,	O
+makes	O
+numerous	O
+interactions	O
+with	O
+the	O
+substrate	O
+(	O
+Fig	O
+.	O
+4	O
+,	O
+F	O
+and	O
+G	O
+).	O
+
+The	O
+Influence	O
+of	O
+the	O
+Modular	O
+Architecture	O
+of	O
+CtXyl5A	B-protein
+on	O
+Catalytic	O
+Activity	O
+
+CtXyl5A	B-protein
+,	O
+in	O
+addition	O
+to	O
+its	O
+catalytic	B-structure_element
+module	I-structure_element
+,	O
+contains	O
+three	O
+CBMs	B-structure_element
+(	O
+CtCBM6	B-structure_element
+,	O
+CtCBM13	B-structure_element
+,	O
+and	O
+CtCBM62	B-structure_element
+)	O
+and	O
+a	O
+fibronectin	B-structure_element
+domain	I-structure_element
+(	O
+CtFn3	B-structure_element
+).	O
+
+A	O
+previous	O
+study	O
+showed	O
+that	O
+although	O
+the	O
+CBM6	B-structure_element
+bound	B-protein_state
+in	I-protein_state
+an	O
+exo	B-protein_state
+-	I-protein_state
+mode	I-protein_state
+to	O
+xylo	B-chemical
+-	I-chemical
+and	I-chemical
+cellulooligosaccharides	I-chemical
+,	O
+the	O
+primary	O
+role	O
+of	O
+this	O
+module	O
+was	O
+to	O
+stabilize	O
+the	O
+structure	O
+of	O
+the	O
+GH5	B-protein_type
+catalytic	B-structure_element
+module	I-structure_element
+.	O
+
+To	O
+explore	O
+the	O
+contribution	O
+of	O
+the	O
+other	O
+non	B-structure_element
+-	I-structure_element
+catalytic	I-structure_element
+modules	I-structure_element
+to	O
+CtXyl5A	B-protein
+function	O
+,	O
+the	O
+activity	O
+of	O
+a	O
+series	O
+of	O
+truncated	B-protein_state
+derivatives	O
+of	O
+the	O
+arabinoxylanase	B-protein_type
+were	O
+assessed	O
+.	O
+
+The	O
+data	O
+in	O
+Table	O
+1	O
+show	O
+that	O
+removal	B-experimental_method
+of	I-experimental_method
+CtCBM62	B-structure_element
+caused	O
+a	O
+modest	O
+increase	O
+in	O
+activity	O
+against	O
+both	O
+WAX	B-chemical
+and	O
+CX	B-chemical
+,	O
+whereas	O
+deletion	B-experimental_method
+of	I-experimental_method
+the	O
+Fn3	B-structure_element
+domain	O
+had	O
+no	O
+further	O
+impact	O
+on	O
+catalytic	O
+performance	O
+.	O
+
+Truncation	B-experimental_method
+of	O
+CtCBM13	B-structure_element
+,	O
+however	O
+,	O
+caused	O
+a	O
+4	O
+–	O
+5	O
+-	O
+fold	O
+reduction	O
+in	O
+activity	O
+against	O
+both	O
+substrates	O
+.	O
+
+Members	O
+of	O
+CBM13	B-structure_element
+have	O
+been	O
+shown	O
+to	O
+bind	O
+to	O
+xylans	B-chemical
+,	O
+mannose	B-chemical
+,	O
+and	O
+galactose	B-chemical
+residues	O
+in	O
+complex	B-chemical
+glycans	I-chemical
+,	O
+hinting	O
+that	O
+the	O
+function	O
+of	O
+CtCBM13	B-structure_element
+is	O
+to	O
+increase	O
+the	O
+proximity	O
+of	O
+substrate	O
+to	O
+the	O
+catalytic	B-structure_element
+module	I-structure_element
+of	O
+CtXyl5A	B-protein
+.	O
+
+Binding	B-experimental_method
+studies	I-experimental_method
+,	O
+however	O
+,	O
+showed	O
+that	O
+CtCBM13	B-structure_element
+displayed	O
+no	O
+affinity	O
+for	O
+a	O
+range	O
+of	O
+relevant	O
+glycans	B-chemical
+including	O
+WAX	B-chemical
+,	O
+CX	B-chemical
+,	O
+xylose	B-chemical
+,	O
+mannose	B-chemical
+,	O
+galactose	B-chemical
+,	O
+and	O
+birchwood	B-chemical
+xylan	I-chemical
+(	O
+BX	B-chemical
+)	O
+(	O
+data	O
+not	O
+shown	O
+).	O
+
+It	O
+would	O
+appear	O
+,	O
+therefore	O
+,	O
+that	O
+CtCBM13	B-structure_element
+makes	O
+a	O
+structural	O
+contribution	O
+to	O
+the	O
+function	O
+of	O
+CtXyl5A	B-protein
+.	O
+
+Crystal	B-evidence
+Structure	I-evidence
+of	O
+CtXyl5A	B-mutant
+-	I-mutant
+D	I-mutant
+
+To	O
+explore	O
+further	O
+the	O
+role	O
+of	O
+the	O
+non	B-structure_element
+-	I-structure_element
+catalytic	I-structure_element
+modules	I-structure_element
+in	O
+CtXyl5A	B-protein
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+CtXyl5A	B-protein
+extending	O
+from	O
+CtGH5	B-structure_element
+to	O
+CtCBM62	B-structure_element
+was	O
+sought	O
+.	O
+
+To	O
+obtain	O
+a	O
+construct	O
+that	O
+could	O
+potentially	O
+be	O
+crystallized	B-experimental_method
+,	O
+the	O
+protein	O
+was	O
+generated	O
+without	B-protein_state
+the	O
+C	O
+-	O
+terminal	O
+dockerin	B-structure_element
+domain	O
+because	O
+it	O
+is	O
+known	O
+to	O
+be	O
+unstable	O
+and	O
+prone	O
+to	O
+cleavage	O
+.	O
+
+Using	O
+this	O
+construct	O
+(	O
+CtXyl5A	B-mutant
+-	I-mutant
+D	I-mutant
+)	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+arabinoxylanase	B-protein_type
+was	O
+determined	O
+by	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+to	O
+a	O
+resolution	O
+of	O
+2	O
+.	O
+64	O
+Å	O
+with	O
+Rwork	B-evidence
+and	O
+Rfree	B-evidence
+at	O
+23	O
+.	O
+7	O
+%	O
+and	O
+27	O
+.	O
+8	O
+%,	O
+respectively	O
+.	O
+
+The	O
+structure	B-evidence
+comprises	O
+a	O
+continuous	O
+polypeptide	O
+extending	O
+from	O
+Ala36	B-residue_range
+to	I-residue_range
+Trp742	I-residue_range
+displaying	O
+four	O
+modules	O
+GH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+-	I-structure_element
+CBM13	I-structure_element
+-	I-structure_element
+Fn3	I-structure_element
+.	O
+
+Although	O
+there	O
+was	O
+some	O
+electron	B-evidence
+density	I-evidence
+for	O
+CtCBM62	B-structure_element
+,	O
+it	O
+was	O
+not	O
+sufficient	O
+to	O
+confidently	O
+build	O
+the	O
+module	O
+(	O
+Fig	O
+.	O
+5	O
+).	O
+
+Further	O
+investigation	O
+of	O
+the	O
+crystal	B-evidence
+packing	I-evidence
+revealed	O
+a	O
+large	O
+solvent	B-site
+channel	I-site
+adjacent	O
+to	O
+the	O
+area	O
+the	O
+CBM62	B-structure_element
+occupies	O
+.	O
+
+We	O
+postulate	O
+that	O
+the	O
+reason	O
+for	O
+the	O
+poor	O
+electron	B-evidence
+density	I-evidence
+is	O
+due	O
+to	O
+the	O
+CtCBM62	B-structure_element
+being	O
+mobile	B-protein_state
+compared	O
+with	O
+the	O
+rest	O
+of	O
+the	O
+protein	O
+.	O
+
+The	O
+structures	B-evidence
+of	O
+CtGH5	B-structure_element
+and	O
+CtCBM6	B-structure_element
+have	O
+been	O
+described	O
+previously	O
+.	O
+
+Surface	O
+representation	O
+of	O
+the	O
+tetra	O
+-	O
+modular	O
+arabinoxylanase	B-protein_type
+and	O
+zoom	O
+view	O
+on	O
+the	O
+CtGH5	B-structure_element
+loop	B-structure_element
+.	O
+
+The	O
+blue	O
+module	O
+is	O
+the	O
+CtGH5	B-structure_element
+catalytic	B-structure_element
+domain	I-structure_element
+,	O
+the	O
+green	O
+module	O
+corresponds	O
+to	O
+the	O
+CtCBM6	B-structure_element
+,	O
+the	O
+yellow	O
+module	O
+is	O
+the	O
+CtCBM13	B-structure_element
+,	O
+and	O
+the	O
+salmon	O
+module	O
+is	O
+the	O
+fibronectin	B-structure_element
+domain	I-structure_element
+.	O
+
+The	O
+CtGH5	B-structure_element
+loop	B-structure_element
+is	O
+stabilized	O
+between	O
+the	O
+CtCBM6	B-structure_element
+and	O
+the	O
+CtCBM13	B-structure_element
+modules	O
+.	O
+
+CtCBM13	B-structure_element
+extends	O
+from	O
+Gly567	B-residue_range
+to	I-residue_range
+Pro648	I-residue_range
+.	O
+
+Typical	O
+of	O
+CBM13	B-protein_type
+proteins	O
+CtCBM13	B-structure_element
+displays	O
+a	O
+β	B-structure_element
+-	I-structure_element
+trefoil	I-structure_element
+fold	I-structure_element
+comprising	O
+the	O
+canonical	O
+pseudo	O
+3	O
+-	O
+fold	O
+symmetry	O
+with	O
+a	O
+3	B-structure_element
+-	I-structure_element
+fold	I-structure_element
+repeating	I-structure_element
+unit	I-structure_element
+of	O
+40	B-residue_range
+–	I-residue_range
+50	I-residue_range
+amino	I-residue_range
+acid	I-residue_range
+residues	O
+characteristic	O
+of	O
+the	O
+Ricin	B-protein_type
+superfamily	I-protein_type
+.	O
+
+Each	O
+repeat	B-structure_element
+contains	O
+two	O
+pairs	O
+of	O
+antiparallel	B-structure_element
+β	I-structure_element
+-	I-structure_element
+strands	I-structure_element
+.	O
+
+A	O
+Dali	B-experimental_method
+search	I-experimental_method
+revealed	O
+structural	O
+homologs	O
+from	O
+the	O
+CBM13	B-protein_type
+family	O
+with	O
+an	O
+root	B-evidence
+mean	I-evidence
+square	I-evidence
+deviation	I-evidence
+less	O
+than	O
+2	O
+.	O
+0	O
+Å	O
+and	O
+sequence	O
+identities	O
+of	O
+less	O
+than	O
+20	O
+%	O
+that	O
+include	O
+the	O
+functionally	O
+relevant	O
+homologs	O
+C	B-species
+.	I-species
+thermocellum	I-species
+exo	B-protein_type
+-	I-protein_type
+β	I-protein_type
+-	I-protein_type
+1	I-protein_type
+,	I-protein_type
+3	I-protein_type
+-	I-protein_type
+galactanase	I-protein_type
+(	O
+PDB	O
+code	O
+3vsz	O
+),	O
+Streptomyces	B-species
+avermitilis	I-species
+β	B-protein_type
+-	I-protein_type
+l	I-protein_type
+-	I-protein_type
+arabinopyranosidase	I-protein_type
+(	O
+PDB	O
+code	O
+3a21	O
+),	O
+Streptomyces	B-species
+lividans	I-species
+xylanase	B-protein
+10A	I-protein
+(	O
+PDB	O
+code	O
+,	O
+1mc9	O
+),	O
+and	O
+Streptomyces	B-species
+olivaceoviridis	I-species
+E	I-species
+-	I-species
+86	I-species
+xylanase	B-protein
+10A	I-protein
+(	O
+PDB	O
+code	O
+1v6v	O
+).	O
+
+The	O
+Fn3	B-structure_element
+module	O
+displays	O
+a	O
+typical	O
+β	B-structure_element
+-	I-structure_element
+sandwich	I-structure_element
+fold	I-structure_element
+with	O
+the	O
+two	O
+sheets	B-structure_element
+comprising	O
+,	O
+primarily	O
+,	O
+three	O
+antiparallel	B-structure_element
+strands	I-structure_element
+in	O
+the	O
+order	O
+β1	B-structure_element
+-	I-structure_element
+β2	I-structure_element
+-	I-structure_element
+β5	I-structure_element
+in	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+1	I-structure_element
+and	O
+β4	B-structure_element
+-	I-structure_element
+β3	I-structure_element
+-	I-structure_element
+β6	I-structure_element
+in	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+2	I-structure_element
+.	O
+
+Although	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+2	I-structure_element
+presents	O
+a	O
+cleft	B-site
+-	O
+like	O
+topology	O
+,	O
+typical	O
+of	O
+endo	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+CBMs	I-protein_type
+,	O
+the	O
+surface	O
+lacks	O
+aromatic	O
+residues	O
+that	O
+play	O
+a	O
+key	O
+role	O
+in	O
+ligand	O
+recognition	O
+,	O
+and	O
+in	O
+the	O
+context	O
+of	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+enzyme	B-protein
+,	O
+the	O
+cleft	B-site
+abuts	O
+into	O
+CtCBM13	B-structure_element
+and	O
+thus	O
+would	O
+not	O
+be	O
+able	O
+to	O
+accommodate	O
+an	O
+extended	O
+polysaccharide	B-chemical
+chain	O
+(	O
+see	O
+below	O
+).	O
+
+In	O
+the	O
+structure	B-evidence
+of	O
+CtXyl5A	B-mutant
+-	I-mutant
+D	I-mutant
+,	O
+the	O
+four	O
+modules	B-structure_element
+form	O
+a	O
+three	O
+-	O
+leaf	O
+clover	O
+-	O
+like	O
+structure	O
+(	O
+Fig	O
+.	O
+5	O
+).	O
+
+Between	O
+the	O
+interfaces	B-site
+of	O
+CtGH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+-	I-structure_element
+CBM13	I-structure_element
+there	O
+are	O
+a	O
+number	O
+of	O
+interactions	O
+that	O
+maintain	O
+the	O
+modules	O
+in	O
+a	O
+fixed	O
+position	O
+relative	O
+to	O
+each	O
+other	O
+.	O
+
+The	O
+interaction	O
+of	O
+CtGH5	B-structure_element
+and	O
+CtCBM6	B-structure_element
+,	O
+which	O
+buries	O
+a	O
+substantial	O
+apolar	B-site
+solvent	I-site
+-	I-site
+exposed	I-site
+surface	I-site
+of	O
+the	O
+two	O
+modules	O
+,	O
+has	O
+been	O
+described	O
+previously	O
+.	O
+
+The	O
+polar	B-bond_interaction
+interactions	I-bond_interaction
+between	O
+these	O
+two	O
+modules	O
+comprise	O
+14	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+and	O
+5	O
+salt	B-bond_interaction
+bridges	I-bond_interaction
+.	O
+
+The	O
+apolar	B-bond_interaction
+and	I-bond_interaction
+polar	I-bond_interaction
+interactions	I-bond_interaction
+between	O
+these	O
+two	O
+modules	O
+likely	O
+explaining	O
+why	O
+they	O
+do	O
+not	O
+fold	O
+independently	O
+compared	O
+with	O
+other	O
+glycoside	B-protein_type
+hydrolases	I-protein_type
+that	O
+contain	O
+CBMs	B-structure_element
+.	O
+
+CtCBM13	B-structure_element
+acts	O
+as	O
+the	O
+central	B-structure_element
+domain	I-structure_element
+,	O
+which	O
+interacts	B-protein_state
+with	I-protein_state
+CtGH5	B-structure_element
+,	O
+CtCBM6	B-structure_element
+,	O
+and	O
+CtFn3	B-structure_element
+via	O
+2	O
+,	O
+5	O
+,	O
+and	O
+4	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+,	O
+respectively	O
+,	O
+burying	O
+a	O
+surface	O
+area	O
+of	O
+∼	O
+450	O
+,	O
+350	O
+,	O
+and	O
+500	O
+Å2	O
+,	O
+respectively	O
+,	O
+to	O
+form	O
+a	O
+compact	B-protein_state
+heterotetramer	B-oligomeric_state
+.	O
+
+With	O
+respect	O
+to	O
+the	O
+CtCBM6	B-site
+-	I-site
+CBM13	I-site
+interface	I-site
+,	O
+the	O
+linker	B-structure_element
+(	O
+SPISTGTIP	B-structure_element
+)	O
+between	O
+the	O
+two	O
+modules	B-structure_element
+,	O
+extending	O
+from	O
+Ser514	B-residue_name_number
+to	O
+Pro522	B-residue_name_number
+,	O
+adopts	O
+a	O
+fixed	B-protein_state
+conformation	I-protein_state
+.	O
+
+Such	O
+sequences	O
+are	O
+normally	O
+extremely	O
+flexible	O
+;	O
+however	O
+,	O
+the	O
+two	O
+Ile	B-residue_name
+residues	O
+make	O
+extensive	O
+apolar	B-bond_interaction
+contacts	I-bond_interaction
+within	O
+the	O
+linker	B-structure_element
+and	O
+with	O
+the	O
+two	O
+CBMs	B-structure_element
+,	O
+leading	O
+to	O
+conformational	O
+stabilization	O
+.	O
+
+The	O
+interactions	O
+between	O
+CtGH5	B-structure_element
+and	O
+the	O
+two	O
+CBMs	B-structure_element
+,	O
+which	O
+are	O
+mediated	O
+by	O
+the	O
+tip	O
+of	O
+the	O
+loop	B-structure_element
+between	O
+β	B-structure_element
+-	I-structure_element
+7	I-structure_element
+and	O
+α	B-structure_element
+-	I-structure_element
+7	I-structure_element
+(	O
+loop	B-structure_element
+7	I-structure_element
+)	O
+of	O
+CtGH5	B-structure_element
+,	O
+not	O
+only	O
+stabilize	O
+the	O
+trimodular	B-structure_element
+clover	I-structure_element
+-	O
+like	O
+structure	O
+but	O
+also	O
+make	O
+a	O
+contribution	O
+to	O
+catalytic	O
+function	O
+.	O
+
+Central	O
+to	O
+the	O
+interactions	O
+between	O
+the	O
+three	O
+modules	B-structure_element
+is	O
+Trp285	B-residue_name_number
+,	O
+which	O
+is	O
+intercalated	B-bond_interaction
+between	I-bond_interaction
+the	O
+two	O
+CBMs	B-structure_element
+.	O
+
+The	O
+Nϵ	O
+of	O
+this	O
+aromatic	O
+residue	O
+makes	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+the	O
+backbone	O
+carbonyl	O
+of	O
+Val615	B-residue_name_number
+and	O
+Gly616	B-residue_name_number
+in	O
+CtCBM13	B-structure_element
+,	O
+and	O
+the	O
+indole	O
+ring	O
+makes	O
+several	O
+apolar	B-bond_interaction
+contacts	I-bond_interaction
+with	O
+CtCBM6	B-structure_element
+(	O
+Pro440	B-residue_name_number
+,	O
+Phe489	B-residue_name_number
+,	O
+Gly491	B-residue_name_number
+,	O
+and	O
+Ala492	B-residue_name_number
+)	O
+(	O
+Fig	O
+.	O
+5	O
+).	O
+
+Indeed	O
+,	O
+loop	B-structure_element
+7	I-structure_element
+is	O
+completely	B-protein_state
+disordered	I-protein_state
+in	O
+the	O
+truncated	B-protein_state
+derivative	O
+of	O
+CtXyl5A	B-protein
+comprising	O
+CtGH5	B-structure_element
+and	O
+CtCBM6	B-structure_element
+,	O
+demonstrating	O
+that	O
+the	O
+interactions	O
+with	O
+CtCBM13	B-structure_element
+stabilize	O
+the	O
+conformation	O
+of	O
+this	O
+loop	B-structure_element
+.	O
+
+Although	O
+the	O
+tip	O
+of	O
+loop	B-structure_element
+7	I-structure_element
+does	O
+not	O
+directly	O
+contribute	O
+to	O
+the	O
+topology	O
+of	O
+the	O
+active	B-site
+site	I-site
+,	O
+it	O
+is	O
+only	O
+∼	O
+12	O
+Å	O
+from	O
+the	O
+catalytic	O
+nucleophile	O
+Glu279	B-residue_name_number
+.	O
+
+Thus	O
+,	O
+any	O
+perturbation	O
+of	O
+the	O
+loop	B-structure_element
+(	O
+through	O
+the	O
+removal	B-experimental_method
+of	O
+CtCBM13	B-structure_element
+)	O
+is	O
+likely	O
+to	O
+influence	O
+the	O
+electrostatic	O
+and	O
+apolar	O
+environment	O
+of	O
+the	O
+catalytic	O
+apparatus	O
+,	O
+which	O
+could	O
+explain	O
+the	O
+reduction	O
+in	O
+activity	O
+associated	O
+with	O
+the	O
+deletion	B-experimental_method
+of	O
+CtCBM13	B-structure_element
+.	O
+
+Similar	O
+to	O
+the	O
+interactions	O
+between	O
+CtCBM6	B-structure_element
+and	O
+CtCBM13	B-structure_element
+,	O
+there	O
+are	O
+extensive	O
+hydrophobic	B-bond_interaction
+interactions	I-bond_interaction
+between	O
+CtCBM13	B-structure_element
+and	O
+CtFn3	B-structure_element
+,	O
+resulting	O
+in	O
+very	O
+little	O
+flexibility	O
+between	O
+these	O
+modules	B-structure_element
+.	O
+
+As	O
+stated	O
+above	O
+,	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+CtCBM62	B-structure_element
+in	O
+the	O
+structure	B-evidence
+suggests	O
+that	O
+the	O
+module	B-structure_element
+can	O
+adopt	O
+multiple	O
+positions	O
+with	O
+respect	O
+to	O
+the	O
+rest	O
+of	O
+the	O
+protein	O
+.	O
+
+The	O
+CtCBM62	B-structure_element
+,	O
+by	O
+binding	B-protein_state
+to	I-protein_state
+its	O
+ligands	O
+(	O
+d	B-chemical
+-	I-chemical
+Galp	I-chemical
+and	O
+l	B-chemical
+-	I-chemical
+Arap	I-chemical
+)	O
+in	O
+plant	B-taxonomy_domain
+cell	O
+walls	O
+,	O
+may	O
+be	O
+able	O
+to	O
+recruit	O
+the	O
+enzyme	O
+onto	O
+its	O
+target	O
+substrate	O
+.	O
+
+Xylans	B-chemical
+are	O
+not	O
+generally	O
+thought	O
+to	O
+contain	O
+such	O
+sugars	B-chemical
+.	O
+
+d	B-chemical
+-	I-chemical
+Galp	I-chemical
+,	O
+however	O
+,	O
+has	O
+been	O
+detected	O
+in	O
+xylans	B-chemical
+in	O
+the	O
+outer	O
+layer	O
+of	O
+cereal	B-taxonomy_domain
+grains	O
+and	O
+in	O
+eucalyptus	B-taxonomy_domain
+trees	I-taxonomy_domain
+,	O
+which	O
+are	O
+substrates	O
+used	O
+by	O
+CtXyl5A	B-protein
+.	O
+
+Thus	O
+,	O
+CtCBM62	B-structure_element
+may	O
+direct	O
+the	O
+enzyme	O
+to	O
+particularly	O
+complex	O
+xylans	B-chemical
+containing	O
+d	B-chemical
+-	I-chemical
+Galp	I-chemical
+at	O
+the	O
+non	O
+-	O
+reducing	O
+termini	O
+of	O
+the	O
+side	O
+chains	O
+,	O
+consistent	O
+with	O
+the	O
+open	B-protein_state
+substrate	B-site
+binding	I-site
+cleft	I-site
+of	O
+the	O
+arabinoxylanase	B-protein_type
+that	O
+is	O
+optimized	O
+to	O
+bind	O
+highly	O
+decorated	O
+forms	O
+of	O
+the	O
+hemicellulose	B-chemical
+.	O
+
+In	O
+general	O
+CBMs	B-structure_element
+have	O
+little	O
+influence	O
+on	O
+enzyme	O
+activity	O
+against	O
+soluble	O
+substrates	O
+but	O
+have	O
+a	O
+significant	O
+impact	O
+on	O
+glycans	B-chemical
+within	O
+plant	B-taxonomy_domain
+cell	O
+walls	O
+.	O
+
+Thus	O
+,	O
+the	O
+role	O
+of	O
+CBM62	B-structure_element
+will	O
+likely	O
+only	O
+be	O
+evident	O
+against	O
+insoluble	O
+composite	O
+substrates	O
+.	O
+
+Exploring	O
+GH5	B-protein_type
+Subfamily	I-protein_type
+34	I-protein_type
+
+CtXyl5A	B-protein
+is	O
+a	O
+member	O
+of	O
+a	O
+seven	O
+-	O
+protein	O
+subfamily	O
+of	O
+GH5	B-protein_type
+,	O
+GH5_34	B-protein_type
+.	O
+
+Four	O
+of	O
+these	O
+proteins	O
+are	O
+distinct	O
+,	O
+whereas	O
+the	O
+other	O
+three	O
+members	O
+are	O
+essentially	O
+identical	O
+(	O
+derived	O
+from	O
+different	O
+strains	O
+of	O
+C	B-species
+.	I-species
+thermocellum	I-species
+).	O
+
+To	O
+investigate	O
+further	O
+the	O
+substrate	O
+specificity	O
+within	O
+this	O
+subfamily	O
+,	O
+recombinant	O
+forms	O
+of	O
+three	O
+members	O
+of	O
+GH5_34	B-protein_type
+that	O
+were	O
+distinct	O
+from	O
+CtXyl5A	B-protein
+were	O
+generated	O
+.	O
+
+AcGH5	B-protein
+has	O
+a	O
+similar	O
+molecular	O
+architecture	O
+to	O
+CtXyl5A	B-protein
+with	O
+the	O
+exception	O
+of	O
+an	O
+additional	O
+carbohydrate	B-structure_element
+esterase	I-structure_element
+family	I-structure_element
+6	I-structure_element
+module	I-structure_element
+at	O
+the	O
+C	O
+terminus	O
+(	O
+Fig	O
+.	O
+1	O
+).	O
+
+The	O
+GH5_34	B-protein_type
+from	O
+Verrucomicrobiae	B-taxonomy_domain
+bacterium	B-taxonomy_domain
+,	O
+VbGH5	B-protein
+,	O
+contains	O
+the	O
+GH5	B-structure_element
+-	I-structure_element
+CBM6	I-structure_element
+-	I-structure_element
+CBM13	I-structure_element
+core	O
+structure	O
+,	O
+but	O
+the	O
+C	O
+-	O
+terminal	O
+Fn3	B-structure_element
+-	I-structure_element
+CBM62	I-structure_element
+-	I-structure_element
+dockerin	I-structure_element
+modules	O
+,	O
+present	O
+in	O
+CtXyl5A	B-protein
+,	O
+are	O
+replaced	O
+with	O
+a	O
+Laminin_3_G	B-structure_element
+domain	I-structure_element
+,	O
+which	O
+,	O
+by	O
+analogy	O
+to	O
+homologous	O
+domains	O
+in	O
+other	O
+proteins	O
+that	O
+have	O
+affinity	O
+for	O
+carbohydrates	B-chemical
+,	O
+may	O
+display	O
+a	O
+glycan	B-chemical
+binding	O
+function	O
+.	O
+
+The	O
+Verrucomicobiae	B-taxonomy_domain
+enzyme	O
+also	O
+has	O
+an	O
+N	O
+-	O
+terminal	O
+GH43	B-protein_type
+subfamily	I-protein_type
+10	I-protein_type
+(	O
+GH43_10	B-protein_type
+)	O
+catalytic	B-structure_element
+module	I-structure_element
+.	O
+
+The	O
+fungal	B-taxonomy_domain
+GH5_34	B-protein_type
+,	O
+GpGH5	B-protein
+,	O
+unlike	O
+the	O
+two	O
+bacterial	B-taxonomy_domain
+homologs	O
+,	O
+comprises	O
+a	O
+single	O
+GH5	B-protein_type
+catalytic	B-structure_element
+module	I-structure_element
+lacking	O
+all	O
+of	O
+the	O
+other	O
+accessory	O
+modules	O
+(	O
+Fig	O
+.	O
+1	O
+).	O
+
+GpGh5	B-protein
+is	O
+particularly	O
+interesting	O
+as	O
+Gonapodya	B-species
+prolifera	I-species
+is	O
+the	O
+only	O
+fungus	B-taxonomy_domain
+of	O
+the	O
+several	O
+hundred	O
+fungal	B-taxonomy_domain
+genomes	O
+that	O
+encodes	O
+a	O
+GH5_34	B-protein_type
+enzyme	O
+.	O
+
+In	O
+fact	O
+there	O
+are	O
+four	O
+potential	O
+GH5_34	B-protein_type
+sequences	O
+in	O
+the	O
+G	B-species
+.	I-species
+prolifera	I-species
+genome	O
+,	O
+all	O
+of	O
+which	O
+show	O
+high	O
+sequence	O
+homology	O
+to	O
+Clostridium	B-taxonomy_domain
+GH5_34	B-protein_type
+sequences	O
+.	O
+
+G	B-species
+.	I-species
+prolifera	I-species
+and	O
+Clostridium	B-taxonomy_domain
+occupy	O
+similar	O
+environments	O
+,	O
+suggesting	O
+that	O
+the	O
+GpGH5_34	B-protein
+gene	O
+was	O
+acquired	O
+from	O
+a	O
+Clostridium	B-taxonomy_domain
+species	O
+,	O
+which	O
+was	O
+followed	O
+by	O
+duplication	O
+of	O
+the	O
+gene	O
+in	O
+the	O
+fungal	B-taxonomy_domain
+genome	O
+.	O
+
+The	O
+sequence	O
+identity	O
+of	O
+the	O
+GH5_34	B-protein_type
+catalytic	B-structure_element
+modules	I-structure_element
+with	O
+CtXyl5A	B-protein
+ranged	O
+from	O
+55	O
+to	O
+80	O
+%	O
+(	O
+supplemental	O
+Fig	O
+.	O
+S1	O
+).	O
+
+All	O
+the	O
+GH5_34	B-protein_type
+enzymes	O
+were	O
+active	O
+on	O
+the	O
+arabinoxylans	B-chemical
+RAX	B-chemical
+,	O
+WAX	B-chemical
+,	O
+and	O
+CX	B-chemical
+but	O
+displayed	O
+no	O
+activity	O
+on	O
+BX	B-chemical
+(	O
+Table	O
+1	O
+and	O
+Fig	O
+.	O
+6	O
+)	O
+and	O
+are	O
+thus	O
+defined	O
+as	O
+arabinoxylanases	B-protein_type
+.	O
+
+The	O
+limit	O
+products	O
+generated	O
+by	O
+CtXyl5A	B-protein
+,	O
+AcGH5	B-protein
+,	O
+and	O
+GpGH5	B-protein
+comprised	O
+a	O
+range	O
+of	O
+oligosaccharides	B-chemical
+with	O
+some	O
+high	O
+molecular	O
+weight	O
+material	O
+.	O
+
+The	O
+oligosaccharides	B-chemical
+with	O
+low	O
+degrees	O
+of	O
+polymerization	O
+were	O
+absent	O
+in	O
+the	O
+VbGH5	B-protein
+reaction	O
+products	O
+.	O
+
+However	O
+,	O
+the	O
+enzyme	O
+generated	O
+a	O
+large	O
+amount	O
+of	O
+arabinose	B-chemical
+,	O
+which	O
+was	O
+not	O
+produced	O
+by	O
+the	O
+other	O
+arabinoxylanases	B-protein_type
+.	O
+
+Given	O
+that	O
+GH43_10	B-protein_type
+is	O
+predominantly	O
+an	O
+arabinofuranosidase	B-protein_type
+subfamily	O
+of	O
+GH43	B-protein_type
+,	O
+the	O
+arabinose	B-chemical
+generated	O
+by	O
+VbGH5	B-protein
+is	O
+likely	O
+mediated	O
+by	O
+the	O
+N	O
+-	O
+terminal	O
+catalytic	B-structure_element
+module	I-structure_element
+(	O
+see	O
+below	O
+).	O
+
+Kinetic	O
+analysis	O
+showed	O
+that	O
+AcGH5	B-protein
+displayed	O
+similar	O
+activity	O
+to	O
+CtXyl5A	B-protein
+against	O
+both	O
+WAX	B-chemical
+and	O
+RAX	B-chemical
+and	O
+was	O
+2	O
+-	O
+fold	O
+less	O
+active	O
+against	O
+CX	B-chemical
+.	O
+
+When	O
+initially	O
+measuring	O
+the	O
+activity	O
+of	O
+wild	B-protein_state
+type	I-protein_state
+VbGH5	B-protein
+against	O
+the	O
+different	O
+substrates	O
+,	O
+no	O
+clear	O
+data	O
+could	O
+be	O
+obtained	O
+,	O
+regardless	O
+of	O
+the	O
+concentration	O
+of	O
+enzyme	O
+used	O
+the	O
+reaction	O
+appeared	O
+to	O
+cease	O
+after	O
+a	O
+few	O
+minutes	O
+.	O
+
+We	O
+hypothesized	O
+that	O
+the	O
+N	O
+-	O
+terminal	O
+GH43_10	B-protein_type
+rapidly	O
+removed	O
+single	O
+arabinose	B-chemical
+decorations	O
+from	O
+the	O
+arabinoxylans	B-chemical
+depleting	O
+the	O
+substrate	O
+available	O
+to	O
+the	O
+arabinoxylanase	B-protein_type
+,	O
+explaining	O
+why	O
+this	O
+activity	O
+was	O
+short	O
+lived	O
+.	O
+
+To	O
+test	O
+this	O
+hypothesis	O
+,	O
+the	O
+conserved	B-protein_state
+catalytic	O
+base	O
+(	O
+Asp45	B-residue_name_number
+)	O
+of	O
+the	O
+GH43_10	B-structure_element
+module	O
+of	O
+VbGH5	B-protein
+was	O
+substituted	B-experimental_method
+with	I-experimental_method
+alanine	B-residue_name
+,	O
+which	O
+is	O
+predicted	O
+to	O
+inactivate	O
+this	O
+catalytic	B-structure_element
+module	I-structure_element
+.	O
+
+The	O
+D45A	B-mutant
+mutant	B-protein_state
+did	O
+not	O
+produce	O
+arabinose	B-chemical
+consistent	O
+with	O
+the	O
+arabinofuranosidase	B-protein_type
+activity	O
+displayed	O
+by	O
+the	O
+GH43_10	B-structure_element
+module	O
+in	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+enzyme	O
+(	O
+Fig	O
+.	O
+6	O
+).	O
+
+The	O
+kinetics	B-evidence
+of	O
+the	O
+GH5_34	B-protein_type
+arabinoxylanase	B-protein_type
+catalytic	B-structure_element
+module	I-structure_element
+was	O
+now	O
+measurable	O
+,	O
+and	O
+activities	O
+were	O
+determined	O
+to	O
+be	O
+between	O
+∼	O
+6	O
+-	O
+and	O
+10	O
+-	O
+fold	O
+lower	O
+than	O
+that	O
+of	O
+CtXyl5A	B-protein
+.	O
+
+Interestingly	O
+,	O
+the	O
+fungal	B-taxonomy_domain
+arabinoxylanase	B-protein_type
+displays	O
+the	O
+highest	O
+activities	O
+against	O
+WAX	B-chemical
+and	O
+RAX	B-chemical
+,	O
+∼	O
+4	O
+-	O
+and	O
+6	O
+-	O
+fold	O
+higher	O
+,	O
+respectively	O
+,	O
+than	O
+CtXyl5A	B-protein
+;	O
+however	O
+,	O
+there	O
+is	O
+very	O
+little	O
+difference	O
+in	O
+the	O
+activity	O
+between	O
+the	O
+eukaryotic	B-taxonomy_domain
+and	O
+prokaryotic	B-taxonomy_domain
+enzymes	O
+against	O
+CX	B-chemical
+.	O
+
+Attempts	O
+to	O
+express	O
+individual	O
+modules	O
+of	O
+a	O
+variety	O
+of	O
+truncations	O
+of	O
+AcGH5	B-protein
+and	O
+VbGH5	B-protein
+were	O
+unsuccessful	O
+.	O
+
+This	O
+may	O
+indicate	O
+that	O
+the	O
+individual	O
+modules	O
+can	O
+only	O
+fold	O
+correctly	O
+when	O
+incorporated	O
+into	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+enzyme	O
+,	O
+demonstrating	O
+the	O
+importance	O
+of	O
+intermodule	O
+interactions	O
+to	O
+maintain	O
+the	O
+structural	O
+integrity	O
+of	O
+these	O
+enzymes	O
+.	O
+
+Products	O
+profile	O
+generated	O
+of	O
+GH5_34	B-protein_type
+enzymes	O
+.	O
+
+The	O
+enzymes	O
+at	O
+1	O
+μm	O
+were	O
+incubated	B-experimental_method
+with	O
+the	O
+four	O
+different	O
+xylans	B-chemical
+at	O
+1	O
+%	O
+in	O
+50	O
+mm	O
+sodium	O
+phosphate	O
+buffer	O
+for	O
+16	O
+h	O
+at	O
+37	O
+°	O
+C	O
+(	O
+GpGH5	B-protein
+,	O
+VbGH5	B-protein
+,	O
+and	O
+AcGH5	B-protein
+)	O
+or	O
+60	O
+°	O
+C	O
+.	O
+
+The	O
+limit	O
+products	O
+were	O
+separated	O
+by	O
+TLC	B-experimental_method
+.	O
+
+The	O
+xylooligosaccharide	B-chemical
+standards	O
+(	O
+X	O
+)	O
+are	O
+indicated	O
+by	O
+their	O
+degrees	O
+of	O
+polymerization	O
+.	O
+
+A	O
+characteristic	O
+feature	O
+of	O
+enzymes	O
+that	O
+attack	O
+the	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+is	O
+their	O
+complex	O
+molecular	O
+architecture	O
+.	O
+
+The	O
+CBMs	B-structure_element
+in	O
+these	O
+enzymes	O
+generally	O
+play	O
+a	O
+role	O
+in	O
+substrate	O
+targeting	O
+and	O
+are	O
+appended	O
+to	O
+the	O
+catalytic	B-structure_element
+modules	I-structure_element
+through	O
+flexible	B-structure_element
+linker	I-structure_element
+sequences	I-structure_element
+.	O
+
+CtXyl5A	B-protein
+provides	O
+a	O
+rare	O
+visualization	O
+of	O
+the	O
+structure	B-evidence
+of	O
+multiple	O
+modules	O
+within	O
+a	O
+single	O
+enzyme	O
+.	O
+
+The	O
+central	O
+feature	O
+of	O
+these	O
+data	O
+is	O
+the	O
+structural	O
+role	O
+played	O
+by	O
+two	O
+of	O
+the	O
+CBMs	B-structure_element
+,	O
+CtCBM6	B-structure_element
+and	O
+CtCBM13	B-structure_element
+,	O
+in	O
+maintaining	O
+the	O
+active	B-protein_state
+conformation	O
+of	O
+the	O
+catalytic	B-structure_element
+module	I-structure_element
+,	O
+CtGH5	B-structure_element
+.	O
+
+The	O
+crystallographic	B-evidence
+data	I-evidence
+described	O
+here	O
+are	O
+supported	O
+by	O
+biochemical	O
+data	O
+showing	O
+either	O
+that	O
+these	O
+two	O
+modules	O
+do	O
+not	O
+bind	O
+to	O
+glycans	B-chemical
+(	O
+CtCBM13	B-structure_element
+)	O
+or	O
+that	O
+the	O
+recognition	O
+of	O
+the	O
+non	O
+-	O
+reducing	O
+end	O
+of	O
+xylan	B-chemical
+or	O
+cellulose	B-chemical
+chains	O
+(	O
+CtCBM6	B-structure_element
+)	O
+is	O
+unlikely	O
+to	O
+be	O
+biologically	O
+significant	O
+.	O
+
+It	O
+should	O
+be	O
+emphasized	O
+,	O
+however	O
+,	O
+that	O
+glycan	B-chemical
+binding	O
+and	O
+substrate	O
+targeting	O
+may	O
+only	O
+be	O
+evident	O
+in	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+enzyme	O
+acting	O
+on	O
+highly	O
+complex	O
+structures	O
+such	O
+as	O
+the	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+,	O
+as	O
+observed	O
+recently	O
+by	O
+a	O
+CBM46	B-structure_element
+module	O
+in	O
+the	O
+Bacillus	B-taxonomy_domain
+xyloglucanase	B-protein_type
+/	O
+mixed	B-protein_type
+linked	I-protein_type
+glucanase	I-protein_type
+BhCel5B	B-protein
+.	O
+
+CtXyl5A	B-protein
+is	O
+a	O
+member	O
+of	O
+GH5	B-protein_type
+that	O
+contains	O
+6644	O
+members	O
+.	O
+
+CtXyl5A	B-protein
+is	O
+a	O
+member	O
+of	O
+subfamily	O
+GH5_34	B-protein_type
+.	O
+
+Despite	O
+differences	O
+in	O
+sequence	O
+identity	O
+all	O
+of	O
+the	O
+homologs	O
+were	O
+shown	O
+to	O
+be	O
+arabinoxylanases	B-protein_type
+.	O
+
+Consistent	O
+with	O
+the	O
+conserved	O
+substrate	O
+specificity	O
+,	O
+all	O
+members	O
+of	O
+GH5_34	B-protein_type
+contained	O
+the	O
+specificity	B-site
+determinants	I-site
+Glu68	B-residue_name_number
+,	O
+Tyr92	B-residue_name_number
+,	O
+and	O
+Asn139	B-residue_name_number
+,	O
+which	O
+make	O
+critical	O
+interactions	O
+with	O
+the	O
+xylose	B-chemical
+or	O
+arabinose	B-chemical
+in	O
+the	O
+−	B-site
+2	I-site
+*	I-site
+subsite	I-site
+,	O
+which	O
+are	O
+1	O
+,	O
+3	O
+-	O
+linked	O
+to	O
+the	O
+xylose	B-chemical
+positioned	O
+in	O
+the	O
+active	B-site
+site	I-site
+.	O
+
+The	O
+presence	O
+of	O
+a	O
+CBM62	B-structure_element
+in	O
+CtXyl5A	B-protein
+and	O
+AcGH5	B-protein
+suggests	O
+that	O
+these	O
+enzymes	O
+target	O
+highly	O
+complex	O
+xylans	B-chemical
+that	O
+contain	O
+d	B-chemical
+-	I-chemical
+galactose	I-chemical
+in	O
+their	O
+side	O
+chains	O
+.	O
+
+The	O
+absence	B-protein_state
+of	I-protein_state
+a	O
+“	O
+non	O
+-	O
+structural	O
+”	O
+CBM	B-structure_element
+in	O
+GpGH5	B-protein
+may	O
+indicate	O
+that	O
+this	O
+arabinoxylanase	B-protein_type
+is	O
+designed	O
+to	O
+target	O
+simpler	O
+arabinoxylans	B-chemical
+present	O
+in	O
+the	O
+endosperm	O
+of	O
+cereals	B-taxonomy_domain
+.	O
+
+Although	O
+the	O
+characterization	O
+of	O
+all	O
+members	O
+of	O
+GH5_34	B-protein_type
+suggests	O
+that	O
+this	O
+subfamily	O
+is	O
+monospecific	O
+,	O
+differences	O
+in	O
+specificity	O
+are	O
+observed	O
+in	O
+other	O
+subfamilies	O
+of	O
+GHs	B-protein_type
+including	O
+GH43	B-protein_type
+and	O
+GH5	B-protein_type
+.	O
+
+Thus	O
+,	O
+as	O
+new	O
+members	O
+of	O
+GH5_34	B-protein_type
+are	O
+identified	O
+from	O
+genomic	O
+sequence	O
+data	O
+and	O
+subsequently	O
+characterized	O
+,	O
+the	O
+specificity	O
+of	O
+this	O
+family	O
+may	O
+require	O
+reinterpretation	O
+.	O
+
+An	O
+intriguing	O
+feature	O
+of	O
+VbGH5	B-protein
+is	O
+that	O
+the	O
+limited	O
+products	O
+generated	O
+by	O
+this	O
+enzymes	O
+are	O
+much	O
+larger	O
+than	O
+those	O
+produced	O
+by	O
+the	O
+other	O
+arabinoxylanases	B-protein_type
+.	O
+
+This	O
+suggests	O
+that	O
+although	O
+arabinose	B-chemical
+decorations	O
+contribute	O
+to	O
+enzyme	O
+specificity	O
+(	O
+VbGH5	B-protein
+is	O
+not	O
+active	O
+on	O
+xylans	B-chemical
+lacking	O
+arabinose	B-chemical
+side	O
+chains	O
+),	O
+the	O
+enzyme	O
+requires	O
+other	O
+specificity	O
+determinants	O
+that	O
+occur	O
+less	O
+frequently	O
+in	O
+arabinoxylans	B-chemical
+.	O
+
+This	O
+has	O
+some	O
+resonance	O
+with	O
+a	O
+recently	O
+described	O
+GH98	B-protein_type
+xylanase	B-protein_type
+that	O
+also	O
+exploits	O
+specificity	O
+determinants	O
+that	O
+occur	O
+infrequently	O
+and	O
+are	O
+only	O
+evident	O
+in	O
+highly	O
+complex	O
+xylans	B-chemical
+(	O
+e	O
+.	O
+g	O
+.	O
+CX	B-chemical
+).	O
+
+To	O
+conclude	O
+,	O
+this	O
+study	O
+provides	O
+the	O
+molecular	O
+basis	O
+for	O
+the	O
+specificity	O
+displayed	O
+by	O
+arabinoxylanases	B-protein_type
+.	O
+
+Substrate	O
+specificity	O
+is	O
+dominated	O
+by	O
+the	O
+pocket	B-site
+that	O
+binds	O
+single	O
+arabinose	B-chemical
+or	O
+xylose	B-chemical
+side	O
+chains	O
+.	O
+
+The	O
+open	B-protein_state
+xylan	B-site
+binding	I-site
+cleft	I-site
+explains	O
+why	O
+the	O
+enzyme	O
+is	O
+able	O
+to	O
+attack	O
+highly	O
+decorated	O
+forms	O
+of	O
+the	O
+hemicellulose	B-chemical
+.	O
+
+It	O
+is	O
+also	O
+evident	O
+that	O
+appending	O
+additional	O
+catalytic	B-structure_element
+modules	I-structure_element
+and	O
+CBMs	B-structure_element
+onto	O
+the	O
+core	O
+components	O
+of	O
+these	O
+enzymes	O
+generates	O
+bespoke	O
+arabinoxylanases	B-protein_type
+with	O
+activities	O
+optimized	O
+for	O
+specific	O
+functions	O
+.	O
+
+The	O
+specificities	O
+of	O
+the	O
+arabinoxylanases	B-protein_type
+described	O
+here	O
+are	O
+distinct	O
+from	O
+the	O
+classical	O
+endo	B-protein_type
+-	I-protein_type
+xylanases	I-protein_type
+and	O
+thus	O
+have	O
+the	O
+potential	O
+to	O
+contribute	O
+to	O
+the	O
+toolbox	O
+of	O
+biocatalysts	O
+required	O
+by	O
+industries	O
+that	O
+exploit	O
+the	O
+plant	B-taxonomy_domain
+cell	O
+wall	O
+as	O
+a	O
+sustainable	O
+substrate	O
+.	O
+
+Data	B-evidence
+collection	I-evidence
+and	I-evidence
+refinement	I-evidence
+statistics	I-evidence
+
+CtXyl5A	B-mutant
+-	I-mutant
+D	I-mutant
+GH5	B-complex_assembly
+-	I-complex_assembly
+CBM6	I-complex_assembly
+-	I-complex_assembly
+Arap	I-complex_assembly
+GH5	B-complex_assembly
+-	I-complex_assembly
+CBM6	I-complex_assembly
+-	I-complex_assembly
+Xylp	I-complex_assembly
+GH5	B-complex_assembly
+-	I-complex_assembly
+CBM6	I-complex_assembly
+-	I-complex_assembly
+(	I-complex_assembly
+Araf	I-complex_assembly
+-	I-complex_assembly
+Xylp4	I-complex_assembly
+)	I-complex_assembly
+Data	O
+collection	O
+Source	O
+ESRF	O
+-	O
+ID14	O
+-	O
+1	O
+Diamond	O
+I04	O
+–	O
+1	O
+Diamond	O
+I24	O
+Diamond	O
+I02	O
+Wavelength	O
+(	O
+Å	O
+)	O
+0	O
+.	O
+9334	O
+0	O
+.	O
+9173	O
+0	O
+.	O
+9772	O
+0	O
+.	O
+9791	O
+Space	O
+group	O
+P21212	O
+P212121	O
+P212121	O
+P212121	O
+Cell	O
+dimensions	O
+a	O
+,	O
+b	O
+,	O
+c	O
+(	O
+Å	O
+)	O
+147	O
+.	O
+4	O
+,	O
+191	O
+.	O
+7	O
+,	O
+50	O
+.	O
+7	O
+67	O
+.	O
+1	O
+,	O
+72	O
+.	O
+4	O
+,	O
+109	O
+.	O
+1	O
+67	O
+.	O
+9	O
+,	O
+72	O
+.	O
+5	O
+,	O
+109	O
+.	O
+5	O
+76	O
+.	O
+3	O
+,	O
+123	O
+.	O
+2	O
+,	O
+125	O
+.	O
+4	O
+α	O
+,	O
+β	O
+,	O
+γ	O
+(°)	O
+90	O
+,	O
+90	O
+,	O
+90	O
+90	O
+,	O
+90	O
+,	O
+90	O
+90	O
+,	O
+90	O
+,	O
+90	O
+90	O
+,	O
+90	O
+,	O
+90	O
+No	O
+.	O
+of	O
+measured	O
+reflections	O
+244	O
+,	O
+475	O
+(	O
+29	O
+,	O
+324	O
+)	O
+224	O
+,	O
+842	O
+(	O
+11	O
+,	O
+281	O
+)	O
+152	O
+,	O
+004	O
+(	O
+4	O
+,	O
+996	O
+)	O
+463	O
+,	O
+237	O
+(	O
+23	O
+,	O
+068	O
+)	O
+No	O
+.	O
+of	O
+independent	O
+reflections	O
+42246	O
+(	O
+5	O
+,	O
+920	O
+)	O
+63	O
+,	O
+523	O
+(	O
+3	O
+,	O
+175	O
+)	O
+42	O
+,	O
+716	O
+(	O
+2	O
+,	O
+334	O
+)	O
+140	O
+,	O
+288	O
+(	O
+6	O
+,	O
+879	O
+)	O
+Resolution	O
+(	O
+Å	O
+)	O
+50	O
+.	O
+70	O
+–	O
+2	O
+.	O
+64	O
+(	O
+2	O
+.	O
+78	O
+–	O
+2	O
+.	O
+64	O
+)	O
+44	O
+.	O
+85	O
+–	O
+1	O
+.	O
+65	O
+(	O
+1	O
+.	O
+68	O
+–	O
+1	O
+.	O
+65	O
+)	O
+45	O
+.	O
+16	O
+–	O
+1	O
+.	O
+90	O
+(	O
+1	O
+.	O
+94	O
+–	O
+1	O
+.	O
+90	O
+)	O
+48	O
+.	O
+43	O
+–	O
+1	O
+.	O
+65	O
+(	O
+1	O
+.	O
+68	O
+–	O
+1	O
+.	O
+65	O
+)	O
+Rmerge	O
+(%)	O
+16	O
+.	O
+5	O
+(	O
+69	O
+.	O
+5	O
+)	O
+6	O
+.	O
+7	O
+(	O
+65	O
+.	O
+1	O
+)	O
+2	O
+.	O
+8	O
+(	O
+8	O
+.	O
+4	O
+)	O
+5	O
+.	O
+7	O
+(	O
+74	O
+.	O
+9	O
+)	O
+CC1	O
+/	O
+2	O
+0	O
+.	O
+985	O
+(	O
+0	O
+.	O
+478	O
+)	O
+0	O
+.	O
+998	O
+(	O
+0	O
+.	O
+594	O
+)	O
+0	O
+.	O
+999	O
+(	O
+0	O
+.	O
+982	O
+)	O
+0	O
+.	O
+998	O
+(	O
+0	O
+.	O
+484	O
+)	O
+I	O
+/	O
+σI	O
+8	O
+.	O
+0	O
+(	O
+2	O
+.	O
+0	O
+)	O
+13	O
+(	O
+1	O
+.	O
+6	O
+)	O
+26	O
+.	O
+6	O
+(	O
+8	O
+.	O
+0	O
+)	O
+11	O
+.	O
+2	O
+(	O
+1	O
+.	O
+6	O
+)	O
+Completeness	O
+(%)	O
+98	O
+.	O
+5	O
+(	O
+96	O
+.	O
+4	O
+)	O
+98	O
+.	O
+5	O
+(	O
+99	O
+.	O
+4	O
+)	O
+98	O
+.	O
+6	O
+(	O
+85	O
+.	O
+0	O
+)	O
+98	O
+.	O
+8	O
+(	O
+99	O
+.	O
+4	O
+)	O
+Redundancy	O
+5	O
+.	O
+8	O
+(	O
+5	O
+.	O
+0	O
+)	O
+3	O
+.	O
+5	O
+(	O
+3	O
+.	O
+6	O
+)	O
+3	O
+.	O
+6	O
+(	O
+2	O
+.	O
+1	O
+)	O
+3	O
+.	O
+3	O
+(	O
+3	O
+.	O
+4	O
+)	O
+Refinement	O
+Rwork	B-evidence
+/	O
+Rfree	B-evidence
+23	O
+.	O
+7	O
+/	O
+27	O
+.	O
+8	O
+12	O
+.	O
+2	O
+/	O
+17	O
+.	O
+0	O
+12	O
+.	O
+9	O
+/	O
+16	O
+.	O
+1	O
+14	O
+.	O
+5	O
+/	O
+19	O
+.	O
+9	O
+No	O
+.	O
+atoms	O
+Protein	O
+5446	O
+3790	O
+3729	O
+7333	O
+Ligand	O
+19	O
+20	O
+20	O
+92	O
+Water	O
+227	O
+579	O
+601	O
+923	O
+B	O
+-	O
+factors	O
+Protein	O
+41	O
+.	O
+6	O
+17	O
+.	O
+8	O
+15	O
+.	O
+8	O
+21	O
+.	O
+0	O
+Ligand	O
+65	O
+.	O
+0	O
+19	O
+.	O
+4	O
+24	O
+.	O
+2	O
+39	O
+.	O
+5	O
+Water	O
+35	O
+.	O
+4	O
+38	O
+.	O
+5	O
+32	O
+.	O
+2	O
+37	O
+.	O
+6	O
+R	O
+.	O
+m	O
+.	O
+s	O
+deviations	O
+Bond	O
+lengths	O
+(	O
+Å	O
+)	O
+0	O
+.	O
+008	O
+0	O
+.	O
+015	O
+0	O
+.	O
+012	O
+0	O
+.	O
+012	O
+Bond	O
+angles	O
+(°)	O
+1	O
+.	O
+233	O
+1	O
+.	O
+502	O
+1	O
+.	O
+624	O
+1	O
+.	O
+554	O
+Protein	O
+Data	O
+Bank	O
+code	O
+5G56	O
+5LA0	O
+5LA1	O
+2LA2	O
+
diff --git a/annotation_IOB/PMC5173035.tsv b/annotation_IOB/PMC5173035.tsv
new file mode 100644
index 0000000000000000000000000000000000000000..d8b8c96941f5651316092a6248a6b55333941646
--- /dev/null
+++ b/annotation_IOB/PMC5173035.tsv
@@ -0,0 +1,6195 @@
+Biochemical	B-experimental_method
+and	I-experimental_method
+structural	I-experimental_method
+characterization	I-experimental_method
+of	O
+a	O
+DNA	B-protein_type
+N6	I-protein_type
+-	I-protein_type
+adenine	I-protein_type
+methyltransferase	I-protein_type
+from	O
+Helicobacter	B-species
+pylori	I-species
+
+DNA	B-ptm
+N6	I-ptm
+-	I-ptm
+methyladenine	I-ptm
+modification	O
+plays	O
+an	O
+important	O
+role	O
+in	O
+regulating	O
+a	O
+variety	O
+of	O
+biological	O
+functions	O
+in	O
+bacteria	B-taxonomy_domain
+.	O
+
+However	O
+,	O
+the	O
+mechanism	O
+of	O
+sequence	O
+-	O
+specific	O
+recognition	O
+in	O
+N6	B-ptm
+-	I-ptm
+methyladenine	I-ptm
+modification	O
+remains	O
+elusive	O
+.	O
+
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+,	O
+a	O
+DNA	B-protein_type
+N6	I-protein_type
+-	I-protein_type
+adenine	I-protein_type
+methyltransferase	I-protein_type
+from	O
+Helicobacter	B-species
+pylori	I-species
+,	O
+shows	O
+more	O
+promiscuous	O
+substrate	O
+specificity	O
+than	O
+other	O
+enzymes	O
+.	O
+
+Here	O
+,	O
+we	O
+present	O
+the	O
+crystal	B-evidence
+structures	I-evidence
+of	O
+cofactor	B-protein_state
+-	I-protein_state
+free	I-protein_state
+and	O
+AdoMet	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+structures	B-evidence
+of	O
+this	O
+enzyme	O
+,	O
+which	O
+were	O
+determined	O
+at	O
+resolutions	O
+of	O
+3	O
+.	O
+0	O
+Å	O
+and	O
+3	O
+.	O
+1	O
+Å	O
+,	O
+respectively	O
+.	O
+
+The	O
+core	O
+structure	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+resembles	O
+the	O
+canonical	O
+AdoMet	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+MTase	I-protein_type
+fold	O
+,	O
+while	O
+the	O
+putative	O
+DNA	B-site
+binding	I-site
+regions	I-site
+considerably	O
+differ	O
+from	O
+those	O
+of	O
+the	O
+other	O
+MTases	B-protein_type
+,	O
+which	O
+may	O
+account	O
+for	O
+the	O
+substrate	O
+promiscuity	O
+of	O
+this	O
+enzyme	O
+.	O
+
+Site	B-experimental_method
+-	I-experimental_method
+directed	I-experimental_method
+mutagenesis	I-experimental_method
+experiments	O
+identified	O
+residues	O
+D29	B-residue_name_number
+and	O
+E216	B-residue_name_number
+as	O
+crucial	O
+amino	O
+acids	O
+for	O
+cofactor	O
+binding	O
+and	O
+the	O
+methyl	B-chemical
+transfer	O
+activity	O
+of	O
+the	O
+enzyme	O
+,	O
+while	O
+P41	B-residue_name_number
+,	O
+located	O
+in	O
+a	O
+highly	B-protein_state
+flexible	I-protein_state
+loop	B-structure_element
+,	O
+playing	O
+a	O
+determinant	O
+role	O
+for	O
+substrate	O
+specificity	O
+.	O
+
+Taken	O
+together	O
+,	O
+our	O
+data	O
+revealed	O
+the	O
+structural	O
+basis	O
+underlying	O
+DNA	B-protein_type
+N6	I-protein_type
+-	I-protein_type
+adenine	I-protein_type
+methyltransferase	I-protein_type
+substrate	O
+promiscuity	O
+.	O
+
+DNA	B-ptm
+methylation	I-ptm
+is	O
+a	O
+common	O
+form	O
+of	O
+modification	O
+on	O
+nucleic	O
+acids	O
+occurring	O
+in	O
+both	O
+prokaryotes	B-taxonomy_domain
+and	O
+eukaryotes	B-taxonomy_domain
+.	O
+
+Such	O
+a	O
+modification	O
+creates	O
+a	O
+signature	O
+motif	O
+recognized	O
+by	O
+DNA	B-chemical
+-	O
+interacting	O
+proteins	O
+and	O
+functions	O
+as	O
+a	O
+mechanism	O
+to	O
+regulate	O
+gene	O
+expression	O
+.	O
+
+DNA	B-ptm
+methylation	I-ptm
+is	O
+mediated	O
+by	O
+DNA	B-protein_type
+methyltransferases	I-protein_type
+(	O
+MTases	B-protein_type
+),	O
+which	O
+catalyze	O
+the	O
+transfer	O
+of	O
+a	O
+methyl	B-chemical
+group	O
+from	O
+S	B-chemical
+-	I-chemical
+adenosyl	I-chemical
+-	I-chemical
+L	I-chemical
+-	I-chemical
+methionine	I-chemical
+(	O
+AdoMet	B-chemical
+)	O
+to	O
+a	O
+given	O
+position	O
+of	O
+a	O
+particular	O
+DNA	B-chemical
+base	O
+within	O
+a	O
+specific	O
+DNA	B-chemical
+sequence	O
+.	O
+
+Three	O
+classes	O
+of	O
+DNA	B-protein_type
+MTases	I-protein_type
+have	O
+been	O
+identified	O
+to	O
+transfer	O
+a	O
+methyl	B-chemical
+group	O
+to	O
+different	O
+positions	O
+of	O
+DNA	B-chemical
+bases	O
+.	O
+
+C5	B-protein_type
+-	I-protein_type
+cytosine	I-protein_type
+MTases	I-protein_type
+,	O
+for	O
+example	O
+,	O
+methylate	O
+C5	O
+of	O
+cytosine	B-residue_name
+(	O
+m5C	B-ptm
+).	O
+
+In	O
+eukaryotes	B-taxonomy_domain
+,	O
+m5C	B-ptm
+plays	O
+an	O
+important	O
+role	O
+in	O
+gene	O
+expression	O
+,	O
+chromatin	O
+organization	O
+,	O
+genome	O
+maintenance	O
+and	O
+parental	O
+imprinting	O
+,	O
+and	O
+is	O
+involved	O
+in	O
+a	O
+variety	O
+of	O
+human	B-species
+diseases	O
+including	O
+cancer	O
+.	O
+
+By	O
+contrast	O
+,	O
+the	O
+functions	O
+of	O
+the	O
+prokaryotic	B-taxonomy_domain
+DNA	B-protein_type
+cytosine	I-protein_type
+MTase	I-protein_type
+remain	O
+unknown	O
+.	O
+
+N4	B-protein_type
+-	I-protein_type
+cytosine	I-protein_type
+MTases	I-protein_type
+,	O
+which	O
+are	O
+frequently	O
+present	O
+in	O
+thermophilic	B-taxonomy_domain
+or	O
+mesophilic	B-taxonomy_domain
+bacteria	B-taxonomy_domain
+,	O
+transfer	O
+a	O
+methyl	B-chemical
+group	O
+to	O
+the	O
+exocyclic	O
+amino	O
+group	O
+of	O
+cytosine	B-residue_name
+(	O
+4mC	B-ptm
+).	O
+
+N4	B-ptm
+methylation	I-ptm
+seems	O
+to	O
+be	O
+primarily	O
+a	O
+component	O
+of	O
+bacterial	B-taxonomy_domain
+immune	O
+system	O
+against	O
+invasion	O
+by	O
+foreign	O
+DNA	B-chemical
+,	O
+such	O
+as	O
+conjugative	O
+plasmids	O
+and	O
+bacteriophages	B-taxonomy_domain
+.	O
+
+The	O
+third	O
+group	O
+,	O
+N6	B-protein_type
+-	I-protein_type
+adenine	I-protein_type
+MTases	I-protein_type
+methylate	O
+the	O
+exocyclic	O
+amino	O
+groups	O
+of	O
+adenine	B-residue_name
+(	O
+6mA	B-ptm
+),	O
+which	O
+exists	O
+in	O
+prokaryotes	B-taxonomy_domain
+as	O
+a	O
+signal	O
+for	O
+genome	O
+defense	O
+,	O
+DNA	B-chemical
+replication	O
+and	O
+repair	O
+,	O
+regulation	O
+of	O
+gene	O
+expression	O
+,	O
+control	O
+of	O
+transposition	O
+and	O
+host	O
+-	O
+pathogen	O
+interactions	O
+.	O
+
+Recent	O
+studies	O
+utilizing	O
+new	O
+sequencing	O
+approaches	O
+have	O
+showed	O
+the	O
+existence	O
+of	O
+6mA	B-ptm
+in	O
+several	O
+eukaryotic	B-taxonomy_domain
+species	O
+.	O
+
+DNA	B-chemical
+6mA	B-ptm
+modification	O
+is	O
+associated	O
+with	O
+important	O
+biological	O
+processes	O
+including	O
+nucleosome	O
+distribution	O
+close	O
+to	O
+the	O
+transcription	O
+start	O
+sites	O
+in	O
+Chlamydomonas	B-taxonomy_domain
+,	O
+carrying	O
+heritable	O
+epigenetic	O
+information	O
+in	O
+C	B-species
+.	I-species
+elegans	I-species
+or	O
+controlling	O
+development	O
+of	O
+Drosophila	B-taxonomy_domain
+.	O
+
+All	O
+the	O
+three	O
+types	O
+of	O
+methylation	B-ptm
+exist	O
+in	O
+prokaryotes	B-taxonomy_domain
+,	O
+and	O
+most	O
+DNA	B-protein_type
+MTases	I-protein_type
+are	O
+components	O
+of	O
+the	O
+restriction	O
+-	O
+modification	O
+(	O
+R	O
+-	O
+M	O
+)	O
+systems	O
+.	O
+
+“	O
+R	O
+”	O
+stands	O
+for	O
+a	O
+restriction	B-protein_type
+endonuclease	I-protein_type
+cleaving	O
+specific	O
+DNA	B-chemical
+sequences	O
+,	O
+while	O
+“	O
+M	O
+”	O
+symbolizes	O
+a	O
+modification	B-protein_type
+methyltransferase	I-protein_type
+rendering	O
+these	O
+sequences	O
+resistant	O
+to	O
+cleavage	O
+.	O
+
+The	O
+cooperation	O
+of	O
+these	O
+two	O
+enzymes	O
+provides	O
+a	O
+defensive	O
+mechanism	O
+to	O
+protect	O
+bacteria	B-taxonomy_domain
+from	O
+infection	O
+by	O
+bacteriophages	B-taxonomy_domain
+.	O
+
+The	O
+R	O
+-	O
+M	O
+systems	O
+are	O
+classified	O
+into	O
+three	O
+types	O
+based	O
+on	O
+specific	O
+structural	O
+features	O
+,	O
+position	O
+of	O
+DNA	B-chemical
+cleavage	O
+and	O
+cofactor	O
+requirements	O
+.	O
+
+In	O
+types	O
+I	O
+and	O
+III	O
+,	O
+the	O
+DNA	B-protein_type
+adenine	I-protein_type
+or	I-protein_type
+cytosine	I-protein_type
+methyltransferase	I-protein_type
+is	O
+part	O
+of	O
+a	O
+multi	O
+-	O
+subunit	O
+enzyme	O
+that	O
+catalyzes	O
+both	O
+restriction	O
+and	O
+modification	O
+.	O
+
+By	O
+contrast	O
+,	O
+two	O
+separate	O
+enzymes	O
+exist	O
+in	O
+type	O
+II	O
+systems	O
+,	O
+where	O
+a	O
+restriction	B-protein_type
+endonuclease	I-protein_type
+and	O
+a	O
+DNA	B-protein_type
+adenine	I-protein_type
+or	I-protein_type
+cytosine	I-protein_type
+methyltransferase	I-protein_type
+recognize	O
+the	O
+same	O
+targets	O
+.	O
+
+To	O
+date	O
+,	O
+a	O
+number	O
+of	O
+bacterial	B-taxonomy_domain
+DNA	B-protein_type
+MTases	I-protein_type
+have	O
+been	O
+structurally	B-experimental_method
+characterized	I-experimental_method
+,	O
+covering	O
+enzymes	O
+from	O
+all	O
+the	O
+three	O
+classes	O
+.	O
+
+All	O
+these	O
+MTases	B-protein_type
+exhibit	O
+high	O
+similarity	O
+in	O
+their	O
+overall	O
+architectures	O
+,	O
+which	O
+are	O
+generally	O
+folded	O
+into	O
+two	O
+domains	O
+:	O
+a	O
+conserved	B-protein_state
+larger	O
+catalytic	B-structure_element
+domain	I-structure_element
+comprising	O
+an	O
+active	B-site
+site	I-site
+for	O
+methyl	B-chemical
+transfer	O
+and	O
+a	O
+site	O
+for	O
+AdoMet	B-chemical
+-	O
+binding	O
+,	O
+and	O
+a	O
+smaller	O
+target	B-structure_element
+(	I-structure_element
+DNA	I-structure_element
+)-	I-structure_element
+recognition	I-structure_element
+domain	I-structure_element
+(	O
+TRD	B-structure_element
+)	O
+containing	O
+variable	O
+regions	O
+implicated	O
+in	O
+sequence	O
+-	O
+specific	O
+DNA	B-chemical
+recognition	O
+and	O
+the	O
+infiltration	O
+of	O
+the	O
+DNA	B-chemical
+to	O
+flip	O
+the	O
+target	O
+base	O
+.	O
+
+Conserved	B-protein_state
+amino	O
+acid	O
+motifs	O
+have	O
+been	O
+identified	O
+from	O
+reported	O
+structures	B-evidence
+,	O
+including	O
+ten	O
+motifs	O
+(	O
+I	B-structure_element
+-	I-structure_element
+X	I-structure_element
+)	O
+in	O
+cytosine	B-protein_type
+MTases	I-protein_type
+and	O
+nine	O
+motifs	O
+(	O
+I	B-structure_element
+-	I-structure_element
+VIII	I-structure_element
+and	O
+X	B-structure_element
+)	O
+in	O
+adenine	B-protein_type
+MTases	I-protein_type
+,	O
+all	O
+of	O
+which	O
+are	O
+arranged	O
+in	O
+an	O
+almost	O
+constant	O
+order	O
+.	O
+
+According	O
+to	O
+the	O
+linear	O
+arrangement	O
+of	O
+three	O
+conserved	B-protein_state
+domains	O
+,	O
+exocyclic	B-protein_type
+amino	I-protein_type
+MTases	I-protein_type
+are	O
+subdivided	O
+into	O
+six	O
+groups	O
+(	O
+namely	O
+α	B-protein_type
+,	O
+β	B-protein_type
+,	O
+γ	B-protein_type
+,	O
+ζ	B-protein_type
+,	O
+δ	B-protein_type
+and	O
+ε	B-protein_type
+).	O
+
+N6	B-protein_type
+-	I-protein_type
+adenine	I-protein_type
+and	I-protein_type
+N4	I-protein_type
+-	I-protein_type
+cytosine	I-protein_type
+MTases	I-protein_type
+,	O
+in	O
+particular	O
+,	O
+are	O
+closely	O
+related	O
+by	O
+sharing	O
+common	O
+structural	O
+features	O
+.	O
+
+Despite	O
+the	O
+considerable	O
+similarity	O
+among	O
+bacterial	B-taxonomy_domain
+MTases	B-protein_type
+,	O
+some	O
+differences	O
+were	O
+observed	O
+among	O
+the	O
+enzymes	O
+from	O
+various	O
+species	O
+.	O
+
+For	O
+example	O
+,	O
+the	O
+structural	O
+regions	O
+of	O
+MTases	B-protein_type
+beyond	O
+the	O
+catalytic	B-structure_element
+domain	I-structure_element
+are	O
+rather	O
+variable	O
+,	O
+such	O
+as	O
+the	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+of	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+,	O
+the	O
+extended	O
+arm	O
+of	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+and	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+,	O
+the	O
+helix	B-structure_element
+bundle	I-structure_element
+of	O
+EcoDam	B-protein
+,	O
+and	O
+so	O
+on	O
+.	O
+
+DNA	B-ptm
+methylation	I-ptm
+is	O
+thought	O
+to	O
+influence	O
+bacterial	B-taxonomy_domain
+virulence	O
+.	O
+
+DNA	B-protein_type
+adenine	I-protein_type
+methyltransferase	I-protein_type
+has	O
+been	O
+shown	O
+to	O
+play	O
+a	O
+crucial	O
+role	O
+in	O
+colonization	O
+of	O
+deep	O
+tissue	O
+sites	O
+in	O
+Salmonella	B-species
+typhimurium	I-species
+and	O
+Aeromonas	B-species
+hydrophila	I-species
+.	O
+
+Importantly	O
+,	O
+DNA	B-ptm
+adenine	I-ptm
+methylation	I-ptm
+is	O
+a	O
+global	O
+regulator	O
+of	O
+genes	O
+expressed	O
+during	O
+infection	O
+and	O
+inhibitors	O
+of	O
+DNA	B-ptm
+adenine	I-ptm
+methylation	I-ptm
+are	O
+likely	O
+to	O
+have	O
+a	O
+broad	O
+antimicrobial	O
+action	O
+.	O
+
+Dam	B-protein_type
+was	O
+considered	O
+a	O
+promising	O
+target	O
+for	O
+antimicrobial	O
+drug	O
+development	O
+.	O
+
+Helicobacter	B-species
+pylori	I-species
+is	O
+a	O
+Gram	B-taxonomy_domain
+-	I-taxonomy_domain
+negative	I-taxonomy_domain
+bacterium	I-taxonomy_domain
+that	O
+persistently	O
+colonizes	O
+in	O
+human	B-species
+stomach	O
+worldwide	O
+.	O
+
+H	B-species
+.	I-species
+pylori	I-species
+is	O
+involved	O
+in	O
+90	O
+%	O
+of	O
+all	O
+gastric	O
+malignancies	O
+,	O
+infecting	O
+nearly	O
+50	O
+%	O
+of	O
+the	O
+world	O
+'	O
+s	O
+population	O
+and	O
+is	O
+the	O
+most	O
+crucial	O
+etiologic	O
+agent	O
+for	O
+gastric	O
+adenocarcinoma	O
+.	O
+
+H	B-species
+.	I-species
+pylori	I-species
+strains	O
+possess	O
+a	O
+few	O
+R	O
+-	O
+M	O
+systems	O
+like	O
+other	O
+bacteria	B-taxonomy_domain
+to	O
+function	O
+as	O
+defensive	O
+systems	O
+.	O
+
+H	B-species
+.	I-species
+pylori	I-species
+26695	I-species
+,	O
+for	O
+example	O
+,	O
+has	O
+23	O
+R	O
+-	O
+M	O
+systems	O
+.	O
+
+Methyltransferases	B-protein_type
+were	O
+suggested	O
+to	O
+be	O
+involved	O
+in	O
+H	B-species
+.	I-species
+pylori	I-species
+pathogenicity	O
+.	O
+
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+is	O
+a	O
+DNA	B-protein_type
+adenine	I-protein_type
+MTase	I-protein_type
+that	O
+belongs	O
+to	O
+the	O
+type	O
+II	O
+R	O
+-	O
+M	O
+system	O
+.	O
+
+This	O
+system	O
+contains	O
+two	O
+DNA	B-protein_type
+MTases	I-protein_type
+named	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+and	O
+M2	B-protein
+.	I-protein
+HpyAVI	I-protein
+,	O
+and	O
+a	O
+putative	O
+restriction	B-protein_type
+enzyme	I-protein_type
+.	O
+
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+encoded	O
+by	O
+ORF	O
+hp0050	B-gene
+is	O
+an	O
+N6	B-protein_type
+-	I-protein_type
+adenine	I-protein_type
+methyltransferase	I-protein_type
+belonging	O
+to	O
+the	O
+β	B-protein_type
+-	I-protein_type
+class	I-protein_type
+MTase	I-protein_type
+.	O
+
+It	O
+has	O
+been	O
+reported	O
+recently	O
+that	O
+this	O
+enzyme	O
+recognizes	O
+the	O
+sequence	O
+of	O
+5	B-chemical
+′-	I-chemical
+GAGG	I-chemical
+-	I-chemical
+3	I-chemical
+′,	I-chemical
+5	B-chemical
+′-	I-chemical
+GGAG	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+or	O
+5	B-chemical
+′-	I-chemical
+GAAG	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+and	O
+methylates	O
+adenines	B-residue_name
+in	O
+these	O
+sequences	O
+.	O
+
+Given	O
+that	O
+methylation	B-ptm
+of	O
+two	O
+adjacent	O
+adenines	B-residue_name
+on	O
+the	O
+same	O
+strand	O
+have	O
+never	O
+been	O
+observed	O
+for	O
+other	O
+N6	B-protein_type
+-	I-protein_type
+adenine	I-protein_type
+MTases	I-protein_type
+,	O
+the	O
+methylation	B-ptm
+activity	O
+on	O
+5	B-chemical
+′-	I-chemical
+GAAG	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+seems	O
+to	O
+be	O
+a	O
+unique	O
+feature	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+,	O
+compared	O
+with	O
+the	O
+homologs	O
+from	O
+other	O
+strains	O
+of	O
+H	B-species
+.	I-species
+pylori	I-species
+which	O
+is	O
+able	O
+to	O
+methylate	O
+only	O
+5	B-chemical
+′-	I-chemical
+GAGG	I-chemical
+-	I-chemical
+3	I-chemical
+′.	I-chemical
+The	O
+structural	O
+basis	O
+and	O
+the	O
+catalytic	O
+mechanism	O
+underlying	O
+such	O
+a	O
+distinct	O
+activity	O
+are	O
+not	O
+well	O
+understood	O
+due	O
+to	O
+the	O
+lack	O
+of	O
+an	O
+available	O
+3D	O
+structure	B-evidence
+of	O
+this	O
+enzyme	O
+.	O
+
+Here	O
+,	O
+we	O
+report	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+from	O
+H	B-species
+.	I-species
+pylori	I-species
+26695	I-species
+,	O
+which	O
+is	O
+the	O
+first	O
+determined	O
+N6	B-protein_type
+-	I-protein_type
+adenine	I-protein_type
+MTase	I-protein_type
+structure	B-evidence
+in	O
+H	B-species
+.	I-species
+pylori	I-species
+.	O
+
+The	O
+structure	B-evidence
+reveals	O
+a	O
+similar	O
+architecture	O
+as	O
+the	O
+canonical	O
+fold	O
+of	O
+homologous	O
+proteins	O
+,	O
+but	O
+displays	O
+several	O
+differences	O
+in	O
+the	O
+loop	B-structure_element
+regions	O
+and	O
+TRD	B-structure_element
+.	O
+
+Based	O
+on	O
+structural	B-experimental_method
+and	I-experimental_method
+biochemical	I-experimental_method
+analyses	I-experimental_method
+,	O
+we	O
+then	O
+identified	O
+two	O
+conserved	B-protein_state
+amino	O
+acids	O
+,	O
+D29	B-residue_name_number
+at	O
+the	O
+catalytic	B-site
+site	I-site
+and	O
+E216	B-residue_name_number
+close	O
+to	O
+the	O
+C	O
+-	O
+terminus	O
+,	O
+as	O
+crucial	O
+residues	O
+for	O
+cofactor	O
+binding	O
+and	O
+methyltransferase	B-protein_type
+activity	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+.	O
+
+In	O
+addition	O
+,	O
+a	O
+non	B-protein_state
+-	I-protein_state
+conserved	I-protein_state
+amino	O
+acid	O
+,	O
+P41	B-residue_name_number
+,	O
+seems	O
+to	O
+play	O
+a	O
+key	O
+role	O
+in	O
+substrate	O
+recognition	O
+.	O
+
+Overall	O
+structure	B-evidence
+
+Recombinant	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+was	O
+produced	O
+as	O
+a	O
+soluble	O
+protein	O
+in	O
+Escherichia	B-species
+coli	I-species
+,	O
+but	O
+was	O
+quite	O
+unstable	O
+and	O
+tended	O
+to	O
+aggregate	O
+in	O
+low	O
+salt	O
+environment	O
+.	O
+
+The	O
+protein	O
+,	O
+however	O
+,	O
+remained	O
+fully	O
+soluble	O
+in	O
+a	O
+buffer	O
+containing	O
+higher	O
+concentration	O
+of	O
+sodium	B-chemical
+chloride	I-chemical
+(>	O
+300	O
+mM	O
+),	O
+which	O
+prompted	O
+that	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+is	O
+likely	O
+a	O
+halophilic	B-protein_state
+protein	O
+.	O
+
+The	O
+cofactor	B-protein_state
+-	I-protein_state
+free	I-protein_state
+and	O
+AdoMet	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+proteins	O
+were	O
+crystallized	B-experimental_method
+at	O
+different	O
+conditions	O
+.	O
+
+Both	O
+structures	B-evidence
+were	O
+determined	O
+by	O
+means	O
+of	O
+molecular	B-experimental_method
+replacement	I-experimental_method
+,	O
+and	O
+refined	O
+to	O
+3	O
+.	O
+0	O
+Å	O
+and	O
+3	O
+.	O
+1	O
+Å	O
+,	O
+respectively	O
+.	O
+
+Statistics	O
+of	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+data	I-experimental_method
+collection	I-experimental_method
+and	O
+structure	B-experimental_method
+refinement	I-experimental_method
+were	O
+summarized	O
+in	O
+Table	O
+1	O
+.	O
+
+Data	O
+collection	O
+and	O
+structure	B-evidence
+refinement	I-evidence
+statistics	I-evidence
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+M1	B-complex_assembly
+.	I-complex_assembly
+HpyAVI	I-complex_assembly
+-	I-complex_assembly
+AdoMet	I-complex_assembly
+complex	O
+Data	O
+collection	O
+Wavelength	O
+(	O
+Å	O
+)	O
+1	O
+.	O
+0000	O
+0	O
+.	O
+97772	O
+Space	O
+group	O
+P43212	O
+P65	O
+Unit	O
+-	O
+cell	O
+parameters	O
+(	O
+Å	O
+,	O
+˚)	O
+a	O
+=	O
+b	O
+=	O
+69	O
+.	O
+73	O
+,	O
+c	O
+=	O
+532	O
+.	O
+75α	O
+=	O
+β	O
+=	O
+γ	O
+=	O
+90	O
+a	O
+=	O
+b	O
+=	O
+135	O
+.	O
+60	O
+,	O
+c	O
+=	O
+265	O
+.	O
+15α	O
+=	O
+β	O
+=	O
+90	O
+,	O
+γ	O
+=	O
+120	O
+Resolution	O
+range	O
+(	O
+Å	O
+)	O
+a	O
+49	O
+.	O
+09	O
+-	O
+3	O
+.	O
+00	O
+(	O
+3	O
+.	O
+09	O
+-	O
+3	O
+.	O
+00	O
+)	O
+48	O
+.	O
+91	O
+-	O
+3	O
+.	O
+10	O
+(	O
+3	O
+.	O
+18	O
+-	O
+3	O
+.	O
+10	O
+)	O
+Unique	O
+reflections	O
+a	O
+27243	O
+49833	O
+Multiplicity	O
+a	O
+3	O
+.	O
+7	O
+(	O
+3	O
+.	O
+8	O
+)	O
+5	O
+.	O
+6	O
+(	O
+4	O
+.	O
+0	O
+)	O
+Completeness	O
+(%)	O
+a	O
+98	O
+.	O
+7	O
+(	O
+98	O
+.	O
+9	O
+)	O
+99	O
+.	O
+7	O
+(	O
+97	O
+.	O
+8	O
+)	O
+Mean	O
+I	O
+/	O
+δ	O
+(	O
+I	O
+)	O
+a	O
+12	O
+.	O
+1	O
+(	O
+3	O
+.	O
+4	O
+)	O
+14	O
+.	O
+0	O
+(	O
+1	O
+.	O
+9	O
+)	O
+Solvent	O
+content	O
+(%)	O
+58	O
+.	O
+67	O
+61	O
+.	O
+96	O
+Rmergea	O
+0	O
+.	O
+073	O
+(	O
+0	O
+.	O
+378	O
+)	O
+0	O
+.	O
+106	O
+(	O
+0	O
+.	O
+769	O
+)	O
+Structure	O
+refinement	O
+Rwork	O
+0	O
+.	O
+251	O
+0	O
+.	O
+221	O
+Rfree	O
+0	O
+.	O
+308	O
+0	O
+.	O
+276	O
+R	B-evidence
+.	I-evidence
+m	I-evidence
+.	I-evidence
+s	I-evidence
+.	I-evidence
+d	I-evidence
+.,	O
+bond	O
+lengths	O
+(	O
+Å	O
+)	O
+0	O
+.	O
+007	O
+0	O
+.	O
+007	O
+R	B-evidence
+.	I-evidence
+m	I-evidence
+.	I-evidence
+s	I-evidence
+.	I-evidence
+d	I-evidence
+.,	O
+bond	O
+angles	O
+(˚)	O
+1	O
+.	O
+408	O
+1	O
+.	O
+651	O
+Ramachandran	O
+plot	O
+Favoured	O
+region	O
+(%)	O
+89	O
+.	O
+44	O
+91	O
+.	O
+44	O
+Allowed	O
+region	O
+(%)	O
+9	O
+.	O
+58	O
+7	O
+.	O
+11	O
+Outliers	O
+(%)	O
+0	O
+.	O
+99	O
+1	O
+.	O
+45	O
+
+Four	O
+and	O
+eight	O
+protein	O
+monomers	B-oligomeric_state
+resided	O
+in	O
+the	O
+asymmetric	O
+units	O
+of	O
+the	O
+two	O
+crystal	B-evidence
+structures	I-evidence
+.	O
+
+Some	O
+amino	O
+acids	O
+,	O
+particularly	O
+those	O
+within	O
+two	O
+loops	B-structure_element
+(	O
+residues	O
+32	B-residue_range
+-	I-residue_range
+61	I-residue_range
+and	O
+152	B-residue_range
+-	I-residue_range
+172	I-residue_range
+)	O
+in	O
+both	O
+structures	B-evidence
+,	O
+were	O
+poorly	O
+defined	O
+in	O
+electron	B-evidence
+density	I-evidence
+and	O
+had	O
+to	O
+be	O
+omitted	O
+from	O
+the	O
+refined	O
+models	O
+.	O
+
+The	O
+two	O
+structures	B-evidence
+are	O
+very	O
+similar	O
+to	O
+each	O
+other	O
+(	O
+Figure	O
+1	O
+)	O
+and	O
+could	O
+be	O
+well	O
+overlaid	O
+with	O
+an	O
+RMSD	B-evidence
+of	O
+0	O
+.	O
+76	O
+Å	O
+on	O
+191	O
+Cα	O
+atoms	O
+.	O
+
+The	O
+overall	O
+architecture	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+revealed	O
+in	O
+these	O
+structures	B-evidence
+resembles	O
+the	O
+AdoMet	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+MTase	I-protein_type
+fold	O
+in	O
+which	O
+a	O
+twisted	O
+seven	O
+-	O
+stranded	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+flanked	O
+by	O
+six	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+forms	O
+the	O
+structural	O
+core	O
+.	O
+
+Like	O
+the	O
+reported	O
+structures	B-evidence
+of	O
+the	O
+larger	O
+domain	O
+of	O
+MTases	B-protein_type
+,	O
+three	O
+helices	B-structure_element
+(	O
+αA	B-structure_element
+,	O
+αB	B-structure_element
+and	O
+αZ	B-structure_element
+)	O
+are	O
+located	O
+at	O
+one	O
+face	O
+of	O
+the	O
+central	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+,	O
+while	O
+the	O
+other	O
+three	O
+αD	B-structure_element
+,	O
+αE	B-structure_element
+and	O
+αC	B-structure_element
+sit	O
+at	O
+the	O
+other	O
+side	O
+.	O
+
+All	O
+these	O
+conserved	B-protein_state
+structural	O
+motifs	O
+form	O
+a	O
+typical	O
+α	B-structure_element
+/	I-structure_element
+β	I-structure_element
+Rossmann	I-structure_element
+fold	I-structure_element
+.	O
+
+The	O
+catalytic	B-structure_element
+motif	I-structure_element
+DPPY	B-structure_element
+lies	O
+in	O
+a	O
+loop	B-structure_element
+connecting	O
+αD	B-structure_element
+and	O
+β4	B-structure_element
+,	O
+and	O
+the	O
+cofactor	O
+AdoMet	B-chemical
+binds	O
+in	O
+a	O
+neighboring	O
+cavity	B-site
+.	O
+
+The	O
+loop	B-structure_element
+(	O
+residues	O
+136	B-residue_range
+-	I-residue_range
+166	I-residue_range
+)	O
+located	O
+between	O
+β7	B-structure_element
+and	O
+αZ	B-structure_element
+corresponds	O
+to	O
+a	O
+highly	B-protein_state
+diverse	I-protein_state
+region	O
+in	O
+other	O
+MTases	B-protein_type
+that	O
+is	O
+involved	O
+in	O
+target	O
+DNA	B-chemical
+recognition	O
+.	O
+
+The	O
+hairpin	B-structure_element
+loop	I-structure_element
+(	O
+residues	O
+101	B-residue_range
+-	I-residue_range
+133	I-residue_range
+)	O
+bridging	O
+β6	B-structure_element
+and	O
+β7	B-structure_element
+,	O
+which	O
+is	O
+proposed	O
+to	O
+bind	O
+DNA	B-chemical
+in	O
+the	O
+minor	B-structure_element
+groove	I-structure_element
+,	O
+displays	O
+a	O
+similar	O
+conformation	O
+as	O
+those	O
+observed	O
+in	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+,	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+and	O
+M	B-protein
+.	I-protein
+pvuII	I-protein
+.	O
+
+The	O
+missing	B-protein_state
+loop	B-structure_element
+(	O
+residues	O
+33	B-residue_range
+-	I-residue_range
+58	I-residue_range
+)	O
+in	O
+the	O
+structure	B-evidence
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+corresponds	O
+to	O
+loop	B-structure_element
+I	I-structure_element
+in	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+,	O
+which	O
+was	O
+also	O
+invisible	O
+in	O
+a	O
+structure	B-evidence
+without	B-protein_state
+DNA	I-protein_state
+.	O
+
+This	O
+loop	B-structure_element
+,	O
+however	O
+,	O
+was	O
+well	B-protein_state
+ordered	I-protein_state
+in	O
+an	O
+M	B-evidence
+.	I-evidence
+TaqI	I-evidence
+-	I-evidence
+DNA	I-evidence
+complex	I-evidence
+structure	I-evidence
+and	O
+was	O
+shown	O
+to	O
+play	O
+a	O
+crucial	O
+role	O
+in	O
+DNA	B-ptm
+methylation	I-ptm
+by	O
+contacting	O
+the	O
+flipping	O
+adenine	B-residue_name
+and	O
+recognizing	O
+specific	O
+DNA	B-chemical
+sequence	O
+.	O
+
+Overall	O
+structure	B-evidence
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+
+A	O
+.	O
+Free	B-protein_state
+form	O
+B	O
+.	O
+AdoMet	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+form	O
+.	O
+
+Ribbon	O
+diagram	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+resembles	O
+an	O
+“	O
+AdoMet	B-protein_type
+-	I-protein_type
+dependent	I-protein_type
+MTase	I-protein_type
+fold	O
+”,	O
+a	O
+mixed	O
+seven	O
+-	O
+stranded	O
+β	B-structure_element
+-	I-structure_element
+sheet	I-structure_element
+flanked	O
+by	O
+six	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+,	O
+αA	B-structure_element
+,	O
+αB	B-structure_element
+,	O
+αZ	B-structure_element
+on	O
+one	O
+side	O
+and	O
+αD	B-structure_element
+,	O
+αE	B-structure_element
+,	O
+αC	B-structure_element
+on	O
+the	O
+other	O
+side	O
+,	O
+the	O
+cofactor	O
+AdoMet	B-chemical
+is	O
+bound	B-protein_state
+in	I-protein_state
+a	O
+cavity	B-site
+near	O
+the	O
+conserved	B-protein_state
+enzyme	O
+activity	O
+motif	O
+DPPY	B-structure_element
+.	O
+
+The	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+and	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+are	O
+labelled	O
+and	O
+numbered	O
+according	O
+to	O
+the	O
+commonly	O
+numbering	O
+rule	O
+for	O
+the	O
+known	O
+MTases	B-protein_type
+.	O
+
+The	O
+AdoMet	B-chemical
+molecule	O
+is	O
+shown	O
+in	O
+green	O
+.	O
+
+Dimeric	B-oligomeric_state
+state	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+in	O
+crystal	B-evidence
+and	O
+solution	B-experimental_method
+
+Previous	O
+studies	O
+showed	O
+that	O
+some	O
+DNA	B-protein_type
+MTases	I-protein_type
+,	O
+e	O
+.	O
+g	O
+.	O
+M	B-protein
+.	I-protein
+BamHI	I-protein
+and	O
+M	B-protein
+.	I-protein
+EcoRI	I-protein
+,	O
+exist	O
+as	O
+monomer	B-oligomeric_state
+in	O
+solution	O
+,	O
+in	O
+agreement	O
+with	O
+the	O
+fact	O
+that	O
+a	O
+DNA	B-chemical
+substrate	O
+for	O
+a	O
+typical	O
+MTase	B-protein_type
+is	O
+hemimethylated	B-protein_state
+and	O
+therefore	O
+needs	O
+only	O
+a	O
+single	O
+methylation	B-ptm
+event	O
+to	O
+convert	O
+it	O
+into	O
+a	O
+fully	B-protein_state
+methylated	I-protein_state
+state	O
+.	O
+
+Increasing	O
+number	O
+of	O
+dimeric	B-oligomeric_state
+DNA	B-protein_type
+MTases	I-protein_type
+,	O
+however	O
+,	O
+has	O
+been	O
+identified	O
+from	O
+later	O
+studies	O
+.	O
+
+For	O
+instance	O
+,	O
+M	B-protein
+.	I-protein
+DpnII	I-protein
+,	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+,	O
+M	B-protein
+.	I-protein
+KpnI	I-protein
+,	O
+and	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+have	O
+been	O
+found	O
+as	O
+dimers	B-oligomeric_state
+in	O
+solution	O
+.	O
+
+In	O
+addition	O
+,	O
+several	O
+MTases	B-protein_type
+including	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+,	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+and	O
+TTH0409	B-protein
+form	O
+tightly	O
+associated	O
+dimers	B-oligomeric_state
+in	O
+crystal	B-evidence
+structures	I-evidence
+.	O
+
+Nonetheless	O
+,	O
+some	O
+DNA	B-protein_type
+MTases	I-protein_type
+such	O
+as	O
+M	B-protein
+.	I-protein
+CcrMI	I-protein
+and	O
+the	O
+Bacillus	B-species
+amyloliquefaciens	I-species
+MTase	B-protein_type
+dissociate	O
+from	O
+dimer	B-oligomeric_state
+into	O
+monomer	B-oligomeric_state
+upon	O
+DNA	B-chemical
+-	O
+binding	O
+.	O
+
+According	O
+to	O
+the	O
+arrangement	O
+of	O
+the	O
+three	O
+conserved	B-protein_state
+domains	O
+,	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+belongs	O
+to	O
+the	O
+β	B-protein_type
+-	I-protein_type
+subgroup	I-protein_type
+,	O
+in	O
+which	O
+a	O
+conserved	B-protein_state
+motif	O
+NXXTX9	B-structure_element
+−	I-structure_element
+11AXRXFSXXHX4WX6	I-structure_element
+−	I-structure_element
+9	I-structure_element
+YXFXLX3RX9	I-structure_element
+−	I-structure_element
+26NPX1	I-structure_element
+−	I-structure_element
+6NVWX29	I-structure_element
+−	I-structure_element
+34A	I-structure_element
+has	O
+been	O
+identified	O
+at	O
+the	O
+dimerization	B-site
+interface	I-site
+in	O
+crystal	B-evidence
+structures	I-evidence
+.	O
+
+Most	O
+of	O
+conserved	B-protein_state
+amino	O
+acids	O
+within	O
+that	O
+motif	O
+are	O
+present	O
+in	O
+the	O
+sequence	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+(	O
+Figure	O
+2A	O
+),	O
+implying	O
+dimerization	B-oligomeric_state
+of	O
+this	O
+protein	O
+.	O
+
+In	O
+agreement	O
+,	O
+a	O
+dimer	B-oligomeric_state
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+was	O
+observed	O
+in	O
+our	O
+crystal	B-evidence
+structures	I-evidence
+with	O
+the	O
+two	O
+monomers	B-oligomeric_state
+related	O
+by	O
+a	O
+two	O
+-	O
+fold	O
+axis	O
+(	O
+Figure	O
+2B	O
+and	O
+2C	O
+).	O
+
+An	O
+area	O
+of	O
+~	O
+1900	O
+Å2	O
+was	O
+buried	O
+at	O
+the	O
+dimeric	B-site
+interface	I-site
+,	O
+taking	O
+up	O
+ca	O
+17	O
+%	O
+of	O
+the	O
+total	O
+area	O
+.	O
+
+The	O
+dimeric	B-oligomeric_state
+architecture	O
+was	O
+greatly	O
+stabilized	O
+by	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+and	O
+salt	B-bond_interaction
+bridges	I-bond_interaction
+formed	O
+among	O
+residues	O
+R86	B-residue_name_number
+,	O
+D93	B-residue_name_number
+and	O
+E96	B-residue_name_number
+.	O
+
+In	O
+addition	O
+,	O
+comparison	O
+of	O
+the	O
+dimer	B-oligomeric_state
+structure	B-evidence
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+with	O
+some	O
+other	O
+β	B-protein_type
+-	I-protein_type
+class	I-protein_type
+MTases	I-protein_type
+(	O
+M1	B-protein
+.	I-protein
+MboIIA	I-protein
+,	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+and	O
+TTHA0409	B-protein
+)	O
+suggested	O
+that	O
+the	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+dimer	B-oligomeric_state
+organized	O
+in	O
+a	O
+similar	O
+form	O
+as	O
+others	O
+(	O
+Figure	O
+S3	O
+).	O
+
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+exists	O
+as	O
+dimer	B-oligomeric_state
+in	O
+crystal	B-evidence
+and	O
+solution	O
+
+A	O
+.	O
+A	O
+conserved	B-protein_state
+interface	B-site
+area	I-site
+of	O
+β	B-protein_type
+-	I-protein_type
+class	I-protein_type
+MTases	I-protein_type
+is	O
+defined	O
+in	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+.	O
+
+Residues	O
+that	O
+involved	O
+are	O
+signed	O
+in	O
+red	O
+color	O
+;	O
+Dimerization	B-oligomeric_state
+of	O
+free	B-protein_state
+-	O
+form	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+B	O
+.	O
+and	O
+cofactor	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+C	O
+.	O
+The	O
+two	O
+monomers	B-oligomeric_state
+are	O
+marked	O
+in	O
+green	O
+and	O
+blue	O
+,	O
+AdoMet	B-chemical
+molecules	O
+are	O
+marked	O
+in	O
+magenta	O
+.	O
+
+D	O
+.	O
+Gel	B-experimental_method
+-	I-experimental_method
+filtration	I-experimental_method
+analysis	I-experimental_method
+revealed	O
+that	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+exist	O
+as	O
+a	O
+dimer	B-oligomeric_state
+in	O
+solution	O
+.	O
+
+FPLC	B-experimental_method
+system	O
+coupled	O
+to	O
+a	O
+Superdex	O
+75	O
+10	O
+/	O
+300	O
+column	O
+.	O
+
+Elution	B-evidence
+profiles	I-evidence
+at	O
+280	O
+nm	O
+(	O
+blue	O
+)	O
+and	O
+260	O
+nm	O
+(	O
+red	O
+)	O
+are	O
+:	O
+different	O
+concentration	O
+(	O
+0	O
+.	O
+05	O
+,	O
+0	O
+.	O
+1	O
+,	O
+0	O
+.	O
+2	O
+,	O
+0	O
+.	O
+5	O
+mg	O
+/	O
+ml	O
+)	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+protein	O
+.	O
+
+To	O
+probe	O
+the	O
+oligomeric	O
+form	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+in	O
+solution	O
+,	O
+different	O
+concentrations	O
+of	O
+purified	O
+enzyme	O
+was	O
+loaded	O
+onto	O
+a	O
+Superdex	O
+75	O
+10	O
+/	O
+300	O
+column	O
+.	O
+
+The	O
+protein	O
+was	O
+eluted	O
+at	O
+~	O
+10	O
+ml	O
+regardless	O
+of	O
+the	O
+protein	O
+concentrations	O
+,	O
+corresponding	O
+to	O
+a	O
+dimeric	B-oligomeric_state
+molecular	B-evidence
+mass	I-evidence
+of	O
+54	O
+kDa	O
+(	O
+Figure	O
+2D	O
+).	O
+
+Our	O
+results	O
+clearly	O
+showed	O
+that	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+forms	O
+a	O
+dimer	B-oligomeric_state
+in	O
+both	O
+crystal	B-evidence
+and	O
+solution	O
+as	O
+other	O
+β	B-protein_type
+-	I-protein_type
+class	I-protein_type
+MTases	I-protein_type
+,	O
+which	O
+however	O
+disagrees	O
+with	O
+a	O
+previous	O
+investigation	O
+using	O
+dynamic	B-experimental_method
+light	I-experimental_method
+scattering	I-experimental_method
+(	O
+DLS	B-experimental_method
+)	O
+measurement	O
+and	O
+gel	B-experimental_method
+-	I-experimental_method
+filtration	I-experimental_method
+chromatography	I-experimental_method
+,	O
+suggesting	O
+that	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+is	O
+taking	O
+a	O
+monomeric	B-oligomeric_state
+state	O
+in	O
+solution	O
+.	O
+
+This	O
+variance	O
+might	O
+be	O
+caused	O
+by	O
+an	O
+addition	O
+of	O
+100	O
+mM	O
+arginine	B-chemical
+before	O
+cell	O
+lysis	O
+to	O
+keep	O
+protein	O
+solubility	O
+and	O
+also	O
+by	O
+later	O
+replacement	O
+of	O
+arginine	B-chemical
+with	O
+30	O
+%	O
+glycerol	B-chemical
+by	O
+dialysis	O
+.	O
+
+Structure	B-experimental_method
+comparisons	I-experimental_method
+
+As	O
+a	O
+β	B-protein_type
+-	I-protein_type
+class	I-protein_type
+N6	I-protein_type
+adenine	I-protein_type
+MTase	I-protein_type
+,	O
+the	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+structure	B-evidence
+displayed	O
+a	O
+good	O
+similarity	O
+with	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+(	O
+PDB	O
+ID	O
+1G60	O
+)	O
+and	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+(	O
+PDB	O
+ID	O
+1NW7	O
+),	O
+which	O
+are	O
+falling	O
+into	O
+the	O
+same	O
+subgroup	O
+.	O
+
+Superimposition	B-experimental_method
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+onto	O
+them	O
+gave	O
+RMSDs	B-evidence
+of	O
+1	O
+.	O
+63	O
+Å	O
+and	O
+1	O
+.	O
+9	O
+Å	O
+on	O
+168	O
+and	O
+190	O
+Cα	O
+atoms	O
+,	O
+respectively	O
+.	O
+
+The	O
+most	O
+striking	O
+structural	O
+difference	O
+was	O
+found	O
+to	O
+locate	O
+on	O
+the	O
+TRD	B-structure_element
+region	O
+(	O
+residues	O
+133	B-residue_range
+-	I-residue_range
+163	I-residue_range
+in	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+)	O
+(	O
+Figure	O
+3A	O
+–	O
+3C	O
+),	O
+where	O
+the	O
+secondary	O
+structures	O
+vary	O
+among	O
+these	O
+structures	O
+.	O
+
+By	O
+comparison	O
+with	O
+the	O
+other	O
+two	O
+enzymes	O
+that	O
+possess	O
+protruding	O
+arms	O
+containing	O
+several	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+and	O
+/	O
+or	O
+β	B-structure_element
+-	I-structure_element
+strands	I-structure_element
+,	O
+the	O
+TRD	B-structure_element
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+is	O
+much	O
+shorter	O
+in	O
+length	O
+(	O
+Figure	O
+S1	O
+),	O
+wrapping	O
+more	O
+closely	O
+around	O
+the	O
+structural	O
+core	O
+and	O
+lacking	B-protein_state
+apparent	O
+secondary	O
+structures	O
+.	O
+
+Given	O
+the	O
+proposed	O
+role	O
+of	O
+the	O
+TRD	B-structure_element
+for	O
+DNA	B-chemical
+interaction	O
+at	O
+the	O
+major	B-structure_element
+groove	I-structure_element
+,	O
+some	O
+differences	O
+of	O
+DNA	B-chemical
+recognition	O
+mode	O
+can	O
+be	O
+expected	O
+.	O
+
+Another	O
+difference	O
+locates	O
+at	O
+the	O
+highly	B-protein_state
+flexible	I-protein_state
+loop	B-structure_element
+between	O
+β4	B-structure_element
+and	O
+αD	B-structure_element
+(	O
+residues	O
+33	B-residue_range
+-	I-residue_range
+58	I-residue_range
+)	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+,	O
+which	O
+was	O
+invisible	O
+in	O
+our	O
+structures	B-evidence
+but	O
+present	O
+in	O
+the	O
+structures	B-evidence
+of	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+and	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+.	O
+Sequence	B-experimental_method
+alignment	I-experimental_method
+revealed	O
+that	O
+this	O
+region	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+was	O
+longer	O
+than	O
+its	O
+counterparts	O
+by	O
+13	O
+and	O
+16	O
+amino	O
+acids	O
+respectively	O
+,	O
+which	O
+likely	O
+renders	O
+the	O
+H	B-species
+.	I-species
+pylori	I-species
+enzyme	O
+more	O
+flexible	B-protein_state
+.	O
+
+Structural	B-experimental_method
+comparisons	I-experimental_method
+between	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+and	O
+other	O
+DNA	B-protein_type
+MTases	I-protein_type
+
+A	O
+.	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+;	O
+B	O
+.	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+;	O
+C	O
+.	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+;	O
+D	O
+.	O
+TTHA0409	B-protein
+;	O
+E	O
+.	O
+DpnM	B-protein
+;	O
+F	O
+.	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+.	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+possesses	O
+only	O
+a	O
+long	B-protein_state
+disorder	I-protein_state
+TRD	B-structure_element
+region	O
+,	O
+compared	O
+with	O
+the	O
+structure	B-protein_state
+-	I-protein_state
+rich	I-protein_state
+TRD	B-structure_element
+of	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+,	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+and	O
+TTHA0409	B-protein
+,	O
+or	O
+the	O
+extra	O
+DNA	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+of	O
+DpnM	B-protein
+and	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+.	O
+The	O
+core	O
+structure	O
+is	O
+in	O
+cyan	O
+;	O
+TRD	B-structure_element
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+,	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+,	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+and	O
+TTHA0409	B-protein
+is	O
+in	O
+red	O
+;	O
+The	O
+region	O
+between	O
+β4	B-structure_element
+and	O
+αD	B-structure_element
+of	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+and	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+is	O
+in	O
+green	O
+;	O
+DNA	B-structure_element
+-	I-structure_element
+binding	I-structure_element
+domain	I-structure_element
+of	O
+DpnM	B-protein
+is	O
+in	O
+magenta	O
+;	O
+The	O
+C	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+of	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+is	O
+in	O
+orange	O
+.	O
+
+Structural	B-experimental_method
+comparison	I-experimental_method
+between	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+and	O
+a	O
+putative	O
+β	B-protein_type
+-	I-protein_type
+class	I-protein_type
+N4	I-protein_type
+cytosine	I-protein_type
+MTase	I-protein_type
+named	O
+TTHA0409	B-protein
+(	O
+PDB	O
+ID	O
+2ZIF	O
+)	O
+showed	O
+a	O
+good	O
+similarity	O
+as	O
+well	O
+,	O
+giving	O
+an	O
+RMSD	B-evidence
+of	O
+1	O
+.	O
+73	O
+Å	O
+on	O
+164	O
+Cα	O
+atoms	O
+(	O
+Figure	O
+3D	O
+).	O
+
+Exactly	O
+like	O
+the	O
+above	O
+comparison	O
+,	O
+the	O
+most	O
+significant	O
+difference	O
+exists	O
+in	O
+the	O
+TRD	B-structure_element
+,	O
+where	O
+the	O
+structures	B-evidence
+vary	O
+in	O
+terms	O
+of	O
+length	O
+and	O
+presence	O
+of	O
+α	B-structure_element
+-	I-structure_element
+helices	I-structure_element
+(	O
+Figure	O
+S1	O
+).	O
+
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+displayed	O
+a	O
+considerable	O
+structural	O
+dissimilarity	O
+in	O
+comparison	O
+with	O
+N6	B-protein_type
+-	I-protein_type
+adenine	I-protein_type
+MTases	I-protein_type
+from	O
+other	O
+subgroups	O
+including	O
+the	O
+α	B-protein_type
+-	I-protein_type
+class	I-protein_type
+DpnM	B-protein
+(	O
+PDB	O
+ID	O
+2DPM	O
+)	O
+and	O
+the	O
+γ	B-protein_type
+-	I-protein_type
+class	I-protein_type
+M	B-protein
+.	I-protein
+TaqI	I-protein
+(	O
+PDB	O
+ID	O
+2ADM	O
+).	O
+
+Both	O
+comparisons	O
+gave	O
+RMSDs	B-evidence
+above	O
+3	O
+.	O
+0	O
+Å	O
+(	O
+Figure	O
+3E	O
+and	O
+3F	O
+).	O
+
+These	O
+two	O
+enzymes	O
+lack	B-protein_state
+a	O
+counterpart	B-structure_element
+loop	I-structure_element
+present	O
+in	O
+the	O
+TRD	B-structure_element
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+,	O
+but	O
+instead	O
+rely	O
+on	O
+an	O
+extra	O
+domain	O
+for	O
+DNA	B-chemical
+binding	O
+and	O
+sequence	O
+recognition	O
+.	O
+
+Collectively	O
+,	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+possesses	O
+a	O
+long	B-protein_state
+disordered	I-protein_state
+TRD	B-structure_element
+,	O
+which	O
+is	O
+in	O
+sharp	O
+contrast	O
+to	O
+the	O
+secondary	B-protein_state
+structure	I-protein_state
+-	I-protein_state
+rich	I-protein_state
+TRD	B-structure_element
+in	O
+other	O
+β	B-protein_type
+-	I-protein_type
+class	I-protein_type
+N6	I-protein_type
+adenine	I-protein_type
+or	I-protein_type
+N4	I-protein_type
+cytosine	I-protein_type
+MTases	I-protein_type
+or	O
+the	O
+extra	O
+DNA	O
+binding	O
+domain	O
+present	O
+in	O
+DNA	B-protein_type
+MTases	I-protein_type
+from	O
+other	O
+subgroups	O
+.	O
+
+This	O
+striking	O
+difference	O
+may	O
+be	O
+a	O
+significant	O
+determinant	O
+of	O
+the	O
+wider	O
+substrate	O
+spectrum	O
+of	O
+this	O
+H	B-species
+.	I-species
+pylori	I-species
+enzyme	O
+.	O
+
+AdoMet	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+
+The	O
+cofactor	B-site
+binding	I-site
+pocket	I-site
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+is	O
+surrounded	O
+by	O
+residues	O
+7	B-residue_range
+-	I-residue_range
+9	I-residue_range
+,	O
+29	B-residue_range
+-	I-residue_range
+31	I-residue_range
+,	O
+165	B-residue_range
+-	I-residue_range
+167	I-residue_range
+,	O
+216	B-residue_range
+-	I-residue_range
+218	I-residue_range
+and	O
+221	B-residue_number
+(	O
+Figure	O
+4A	O
+),	O
+which	O
+are	O
+conserved	B-protein_state
+among	O
+most	O
+of	O
+DNA	B-protein_type
+MTases	I-protein_type
+.	O
+
+A	O
+hydrogen	B-bond_interaction
+bond	I-bond_interaction
+between	O
+D29	B-residue_name_number
+in	O
+the	O
+catalytic	B-structure_element
+motif	I-structure_element
+DPPY	B-structure_element
+and	O
+the	O
+amino	O
+group	O
+of	O
+bound	B-protein_state
+AdoMet	B-chemical
+is	O
+preserved	O
+as	O
+other	O
+MTase	B-protein_type
+structures	B-evidence
+.	O
+
+Residues	O
+D8	B-residue_name_number
+and	O
+A9	B-residue_name_number
+from	O
+hydrogen	B-bond_interaction
+-	I-bond_interaction
+bonds	I-bond_interaction
+with	O
+N6	O
+and	O
+N1	O
+of	O
+the	O
+purine	B-chemical
+ring	O
+,	O
+respectively	O
+,	O
+and	O
+E216	B-residue_name_number
+also	O
+locates	O
+at	O
+hydrogen	B-bond_interaction
+bonding	I-bond_interaction
+distance	O
+with	O
+O2	O
+′	O
+and	O
+O3	O
+′	O
+of	O
+the	O
+ribose	B-chemical
+.	O
+
+In	O
+addition	O
+,	O
+H168	B-residue_name_number
+,	O
+T200	B-residue_name_number
+and	O
+S198	B-residue_name_number
+contact	O
+the	O
+terminal	O
+carboxyl	O
+of	O
+AdoMet	B-chemical
+.	O
+
+Superposition	B-experimental_method
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+with	O
+the	O
+five	O
+structures	B-evidence
+shown	O
+in	O
+Figure	O
+3	O
+reveals	O
+that	O
+the	O
+orientation	O
+of	O
+cofactor	O
+is	O
+rather	B-protein_state
+conserved	I-protein_state
+except	O
+for	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+(	O
+Figure	O
+4B	O
+).	O
+
+The	O
+different	O
+conformation	O
+of	O
+the	O
+bound	B-protein_state
+cofactor	O
+observed	O
+in	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+might	O
+be	O
+attributable	O
+to	O
+the	O
+absence	B-protein_state
+of	I-protein_state
+corresponding	O
+residues	O
+of	O
+the	O
+conserved	B-protein_state
+AdoMet	B-chemical
+-	O
+binding	O
+motif	O
+FXGXG	B-structure_element
+in	O
+that	O
+structure	B-evidence
+.	O
+
+Structural	B-experimental_method
+and	I-experimental_method
+biochemical	I-experimental_method
+analyses	I-experimental_method
+define	O
+two	O
+conserved	B-protein_state
+residues	O
+D29	B-residue_name_number
+and	O
+E216	B-residue_name_number
+to	O
+be	O
+the	O
+key	O
+sites	O
+for	O
+AdoMet	B-chemical
+binding	O
+
+A	O
+.	O
+The	O
+cofactor	B-site
+-	I-site
+binding	I-site
+cavity	I-site
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+.	O
+
+Residues	O
+(	O
+yellow	O
+)	O
+that	O
+form	O
+direct	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+AdoMet	B-chemical
+(	O
+green	O
+)	O
+are	O
+indicated	O
+,	O
+distance	O
+of	O
+the	O
+hydrogen	B-bond_interaction
+bond	I-bond_interaction
+is	O
+marked	O
+.	O
+
+B	O
+.	O
+Superposition	B-experimental_method
+of	O
+AdoMet	B-chemical
+in	O
+the	O
+structures	B-evidence
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+(	O
+green	O
+),	O
+DpnM	B-protein
+(	O
+yellow	O
+)	O
+and	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+(	O
+orange	O
+).	O
+
+The	O
+AdoMet	B-chemical
+terminal	O
+carboxyl	O
+of	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+reveals	O
+different	O
+orientations	O
+.	O
+
+C	O
+.	O
+Cofactor	B-evidence
+binding	I-evidence
+affinity	I-evidence
+of	O
+wt	B-protein_state
+-/	O
+mutants	B-protein_state
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+proteins	O
+analyzed	O
+by	O
+microscale	B-experimental_method
+thermophoresis	I-experimental_method
+(	O
+MST	B-experimental_method
+).	O
+
+The	O
+binding	B-evidence
+affinity	I-evidence
+was	O
+determined	O
+between	O
+fluorescently	O
+labelled	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+protein	O
+and	O
+unlabeled	B-protein_state
+AdoMet	B-chemical
+.	O
+
+AdoMet	B-chemical
+(	O
+15	O
+nM	O
+to	O
+1	O
+mM	O
+)	O
+was	O
+titrated	B-experimental_method
+into	O
+a	O
+fixed	O
+concentration	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+wt	B-protein_state
+/	O
+mutant	B-protein_state
+proteins	O
+(	O
+800	O
+nM	O
+).	O
+
+The	O
+dissociation	B-evidence
+constant	I-evidence
+(	O
+KD	B-evidence
+)	O
+is	O
+yielded	O
+according	O
+to	O
+the	O
+law	O
+of	O
+mass	O
+action	O
+from	O
+the	O
+isotherm	B-evidence
+derived	O
+of	O
+the	O
+raw	O
+data	O
+:	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+-	O
+wt	B-protein_state
+:	O
+41	O
+±	O
+6	O
+μM	O
+;	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+D8A	I-mutant
+:	O
+212	O
+±	O
+11	O
+μM	O
+;	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+D29A	I-mutant
+:	O
+0	O
+μM	O
+;	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+H168A	I-mutant
+:	O
+471	O
+±	O
+51	O
+μM	O
+;	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+S198A	I-mutant
+:	O
+242	O
+±	O
+32	O
+μM	O
+;	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+T200A	I-mutant
+:	O
+252	O
+±	O
+28	O
+μM	O
+;	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+E216A	I-mutant
+:	O
+0	O
+μM	O
+.	O
+Standard	O
+for	O
+three	O
+replicates	O
+is	O
+indicated	O
+.	O
+
+D	O
+.	O
+DNA	B-protein_type
+methyltransferase	I-protein_type
+activity	O
+of	O
+wide	B-protein_state
+type	I-protein_state
+protein	O
+and	O
+the	O
+mutants	B-protein_state
+is	O
+quantified	O
+using	O
+radioactive	B-experimental_method
+assay	I-experimental_method
+.	O
+
+[	B-chemical
+3H	I-chemical
+]-	I-chemical
+methyl	I-chemical
+transferred	O
+to	O
+duplex	O
+DNA	B-chemical
+containing	O
+5	B-chemical
+′-	I-chemical
+GAGG	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+was	O
+quantified	O
+by	O
+Beckman	O
+LS6500	O
+for	O
+10	O
+min	O
+,	O
+experiments	O
+were	O
+repeated	O
+for	O
+three	O
+times	O
+and	O
+data	O
+were	O
+corrected	O
+by	O
+subtraction	O
+of	O
+the	O
+background	O
+.	O
+
+E	O
+.	O
+Superposition	B-experimental_method
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+(	O
+green	O
+)	O
+with	O
+M	B-protein
+.	I-protein
+MboIIA	I-protein
+(	O
+cyan	O
+)	O
+and	O
+M	B-protein
+.	I-protein
+RsrI	I-protein
+(	O
+magenta	O
+).	O
+
+Residues	O
+D29	B-residue_name_number
+and	O
+E216	B-residue_name_number
+are	O
+conserved	B-protein_state
+through	O
+all	O
+the	O
+DNA	B-protein_type
+MTases	I-protein_type
+mentioned	O
+in	O
+Figure	O
+3	O
+(	O
+not	O
+shown	O
+in	O
+Figure	O
+4	O
+).	O
+
+To	O
+confirm	O
+the	O
+key	O
+residues	O
+for	O
+ligand	O
+binding	O
+,	O
+we	O
+prepared	O
+a	O
+series	O
+of	O
+single	B-experimental_method
+mutants	I-experimental_method
+by	O
+replacing	B-experimental_method
+D8	B-residue_name_number
+,	O
+D29	B-residue_name_number
+,	O
+H168	B-residue_name_number
+,	O
+S198	B-residue_name_number
+,	O
+T200	B-residue_name_number
+,	O
+E216	B-residue_name_number
+with	O
+alanine	B-residue_name
+and	O
+investigated	O
+their	O
+ligand	B-evidence
+binding	I-evidence
+affinity	I-evidence
+using	O
+microscale	B-experimental_method
+thermophoresis	I-experimental_method
+(	O
+MST	B-experimental_method
+)	O
+assay	O
+.	O
+
+As	O
+shown	O
+in	O
+Figure	O
+4C	O
+,	O
+by	O
+contrast	O
+to	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+enzyme	O
+,	O
+most	O
+mutants	B-protein_state
+displayed	O
+variable	O
+reduction	O
+of	O
+KD	B-evidence
+value	O
+,	O
+among	O
+them	O
+the	O
+D29A	B-mutant
+and	O
+E216A	B-mutant
+mutants	B-protein_state
+displayed	O
+no	O
+protein	B-evidence
+-	I-evidence
+AdoMet	I-evidence
+affinity	I-evidence
+at	O
+all	O
+.	O
+
+The	O
+results	O
+suggested	O
+that	O
+the	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+formed	O
+by	O
+D29	B-residue_name_number
+and	O
+E216	B-residue_name_number
+with	O
+AdoMet	B-chemical
+were	O
+most	O
+crucial	O
+interactions	O
+for	O
+cofactor	O
+binding	O
+.	O
+
+Mutation	B-experimental_method
+of	O
+the	O
+two	O
+residues	O
+may	O
+directly	O
+prevent	O
+the	O
+methyl	B-chemical
+transfer	O
+reaction	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+.	O
+
+The	O
+importance	O
+of	O
+D29	B-residue_name_number
+is	O
+preserved	O
+because	O
+it	O
+belongs	O
+to	O
+the	O
+catalytic	B-site
+active	I-site
+site	I-site
+DPPY	B-structure_element
+,	O
+but	O
+the	O
+residue	O
+E216	B-residue_name_number
+has	O
+not	O
+been	O
+fully	O
+investigated	O
+even	O
+being	O
+a	O
+conserved	B-protein_state
+amino	B-chemical
+acid	I-chemical
+throughout	O
+MTases	B-protein_type
+(	O
+Figure	O
+4E	O
+).	O
+
+E216	B-residue_name_number
+is	O
+the	O
+last	O
+residue	O
+of	O
+β2	B-structure_element
+,	O
+which	O
+contacts	O
+the	O
+two	O
+hydroxyls	O
+of	O
+the	O
+ribose	B-chemical
+of	O
+AdoMet	B-chemical
+.	O
+
+Replacement	B-experimental_method
+of	O
+this	O
+residue	O
+by	O
+alanine	B-residue_name
+completely	O
+abolishes	O
+the	O
+key	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+for	O
+AdoMet	B-chemical
+-	O
+binding	O
+,	O
+and	O
+very	O
+likely	O
+blocks	O
+the	O
+methyl	B-chemical
+transfer	O
+reaction	O
+.	O
+
+To	O
+confirm	O
+this	O
+notion	O
+,	O
+[	B-experimental_method
+3H	I-experimental_method
+]	I-experimental_method
+AdoMet	I-experimental_method
+radiological	I-experimental_method
+assay	I-experimental_method
+was	O
+applied	O
+to	O
+quantify	O
+the	O
+methyl	B-chemical
+transfer	O
+activity	O
+of	O
+the	O
+mutants	B-protein_state
+.	O
+
+As	O
+shown	O
+in	O
+Figure	O
+4D	O
+,	O
+the	O
+result	O
+of	O
+radiological	B-experimental_method
+assay	I-experimental_method
+agreed	O
+well	O
+with	O
+the	O
+MST	B-experimental_method
+measurement	O
+.	O
+
+The	O
+D29A	B-mutant
+and	O
+E216A	B-mutant
+mutants	B-protein_state
+showed	O
+little	O
+or	O
+no	O
+methyl	B-chemical
+transfer	O
+activity	O
+,	O
+while	O
+other	O
+mutants	B-protein_state
+exhibited	O
+reduced	O
+methyltransferase	B-protein_type
+activity	O
+.	O
+
+As	O
+mentioned	O
+previously	O
+,	O
+FXGXG	B-structure_element
+is	O
+a	O
+conserved	B-protein_state
+AdoMet	B-chemical
+-	O
+binding	O
+motif	O
+of	O
+DNA	B-protein_type
+MTases	I-protein_type
+.	O
+
+We	O
+also	O
+made	O
+mutants	B-protein_state
+of	O
+“	O
+FMGSG	B-structure_element
+”	O
+to	O
+alanine	B-residue_name
+for	O
+every	O
+amino	B-chemical
+acid	I-chemical
+,	O
+and	O
+found	O
+that	O
+the	O
+F195A	B-mutant
+mutant	B-protein_state
+was	O
+insoluble	O
+probably	O
+due	O
+to	O
+decreasing	O
+the	O
+local	O
+hydrophobicity	O
+upon	O
+this	O
+mutation	O
+.	O
+
+We	O
+subsequently	O
+investigated	O
+the	O
+ligand	B-evidence
+binding	I-evidence
+affinity	I-evidence
+and	O
+methyl	B-chemical
+transfer	O
+reaction	O
+of	O
+the	O
+other	O
+mutants	B-protein_state
+using	O
+MST	B-experimental_method
+and	O
+a	O
+radiological	B-experimental_method
+assay	I-experimental_method
+.	O
+
+We	O
+found	O
+that	O
+G197	B-residue_name_number
+played	O
+a	O
+crucial	O
+role	O
+in	O
+AdoMet	B-chemical
+-	O
+binding	O
+,	O
+while	O
+mutagenesis	B-experimental_method
+of	O
+M196	B-residue_name_number
+and	O
+G199	B-residue_name_number
+did	O
+not	O
+influence	O
+cofactor	O
+binding	O
+and	O
+catalytic	O
+activity	O
+(	O
+Figure	O
+S2A	O
+and	O
+B	O
+).	O
+
+G197	B-residue_name_number
+is	O
+a	O
+conserved	B-protein_state
+residue	O
+throughout	O
+the	O
+DNA	B-protein_type
+MTases	I-protein_type
+,	O
+and	O
+replacing	B-experimental_method
+by	O
+alanine	B-residue_name
+at	O
+this	O
+site	O
+likely	O
+change	O
+the	O
+local	O
+conformation	O
+of	O
+cofactor	B-site
+-	I-site
+binding	I-site
+pocket	I-site
+.	O
+
+Mutagenesis	B-experimental_method
+on	O
+this	O
+glycine	B-residue_name
+residue	O
+in	O
+M	B-protein
+.	I-protein
+EcoKI	I-protein
+or	O
+M	B-protein
+.	I-protein
+EcoP15I	I-protein
+also	O
+abolished	O
+the	O
+AdoMet	B-chemical
+-	O
+binding	O
+activity	O
+.	O
+
+Although	O
+mutational	B-experimental_method
+study	I-experimental_method
+could	O
+not	O
+tell	O
+the	O
+role	O
+of	O
+F195	B-residue_name_number
+in	O
+ligand	O
+binding	O
+due	O
+to	O
+the	O
+insolubility	O
+of	O
+the	O
+F195A	B-mutant
+mutant	B-protein_state
+,	O
+structural	B-experimental_method
+analysis	I-experimental_method
+suggested	O
+the	O
+importance	O
+of	O
+this	O
+residue	O
+in	O
+AdoMet	B-chemical
+-	O
+binding	O
+.	O
+
+The	O
+phenyl	O
+ring	O
+of	O
+F195	B-residue_name_number
+forms	O
+a	O
+perpendicular	O
+π	B-bond_interaction
+-	I-bond_interaction
+stacking	I-bond_interaction
+interaction	I-bond_interaction
+with	O
+the	O
+purine	O
+ring	O
+of	O
+AdoMet	B-chemical
+,	O
+which	O
+stabilizes	O
+the	O
+orientation	O
+of	O
+AdoMet	B-chemical
+bound	B-protein_state
+in	I-protein_state
+the	O
+pocket	B-site
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+(	O
+Figure	O
+S2C	O
+).	O
+
+In	O
+a	O
+separate	O
+scenario	O
+,	O
+mutagenesis	B-experimental_method
+of	O
+this	O
+residue	O
+in	O
+M	B-protein
+.	I-protein
+EcoRV	I-protein
+has	O
+been	O
+proven	O
+to	O
+play	O
+an	O
+important	O
+role	O
+in	O
+AdoMet	B-chemical
+binding	O
+.	O
+
+Potential	O
+DNA	B-site
+-	I-site
+binding	I-site
+sites	I-site
+
+The	O
+putative	O
+DNA	B-site
+binding	I-site
+region	I-site
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+involves	O
+the	O
+hairpin	B-structure_element
+loop	I-structure_element
+(	O
+residue	O
+101	B-residue_range
+-	I-residue_range
+133	I-residue_range
+),	O
+the	O
+TRD	B-structure_element
+(	O
+residues	O
+136	B-residue_range
+-	I-residue_range
+166	I-residue_range
+),	O
+and	O
+a	O
+highly	B-protein_state
+flexible	I-protein_state
+loop	B-structure_element
+(	O
+residues	O
+33	B-residue_range
+-	I-residue_range
+58	I-residue_range
+).	O
+
+The	O
+hairpin	B-structure_element
+loop	I-structure_element
+between	O
+β6	B-structure_element
+and	O
+β7	B-structure_element
+strands	O
+that	O
+carries	O
+a	O
+conserved	B-protein_state
+HRRY	B-structure_element
+sequence	O
+signature	O
+in	O
+the	O
+middle	O
+is	O
+proposed	O
+to	O
+insert	O
+into	O
+the	O
+minor	B-structure_element
+groove	I-structure_element
+of	O
+the	O
+bound	B-protein_state
+DNA	B-chemical
+.	O
+
+As	O
+aforementioned	O
+,	O
+the	O
+TRD	B-structure_element
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+shows	O
+striking	O
+difference	O
+from	O
+the	O
+other	O
+DNA	B-protein_type
+MTases	I-protein_type
+,	O
+and	O
+the	O
+relaxed	O
+specificity	O
+of	O
+substrate	O
+recognition	O
+may	O
+be	O
+at	O
+least	O
+partially	O
+attributable	O
+to	O
+the	O
+disordered	B-protein_state
+TRD	B-structure_element
+.	O
+
+In	O
+addition	O
+,	O
+the	O
+highly	B-protein_state
+flexible	I-protein_state
+loop	B-structure_element
+immediately	O
+following	O
+the	O
+DPPY	B-structure_element
+motif	O
+in	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+was	O
+poorly	O
+defined	O
+in	O
+electron	B-evidence
+density	I-evidence
+,	O
+exactly	O
+like	O
+the	O
+corresponding	O
+loops	B-structure_element
+in	O
+the	O
+AdoMet	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+structures	B-evidence
+of	O
+M	B-protein
+.	I-protein
+PvuII	I-protein
+,	O
+DpnM	B-protein
+or	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+that	O
+were	O
+invisible	O
+either	O
+.	O
+
+This	O
+loop	B-structure_element
+,	O
+however	O
+,	O
+was	O
+largely	O
+stabilized	O
+upon	O
+DNA	B-chemical
+binding	O
+,	O
+as	O
+observed	O
+in	O
+the	O
+protein	B-evidence
+-	I-evidence
+DNA	I-evidence
+complex	I-evidence
+structures	I-evidence
+of	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+(	O
+PDB	O
+ID	O
+2IBS	O
+),	O
+M	B-protein
+.	I-protein
+HhaI	I-protein
+(	O
+PDB	O
+ID	O
+1MHT	O
+)	O
+and	O
+M	B-protein
+.	I-protein
+HaeIII	I-protein
+(	O
+PDB	O
+ID	O
+1DCT	O
+).	O
+
+The	O
+well	B-protein_state
+-	I-protein_state
+ordered	I-protein_state
+loop	B-structure_element
+in	O
+those	O
+structures	B-evidence
+directly	O
+contacts	O
+the	O
+flipping	O
+adenine	B-residue_name
+and	O
+forms	O
+hydrogen	B-bond_interaction
+bond	I-bond_interaction
+with	O
+neighboring	O
+bases	O
+.	O
+
+These	O
+observations	O
+implied	O
+that	O
+the	O
+corresponding	O
+loop	B-structure_element
+in	O
+other	O
+MTases	B-protein_type
+,	O
+e	O
+.	O
+g	O
+.	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+,	O
+is	O
+likely	O
+responsible	O
+for	O
+reducing	O
+sequence	O
+recognition	O
+specificity	O
+and	O
+thus	O
+plays	O
+crucial	O
+roles	O
+in	O
+catalysis	O
+.	O
+
+Previous	O
+research	O
+suggested	O
+that	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+from	O
+strain	O
+26695	O
+was	O
+the	O
+first	O
+N6	B-protein_type
+adenine	I-protein_type
+MTase	I-protein_type
+that	O
+can	O
+methylate	O
+the	O
+adenine	B-residue_name
+of	O
+5	B-chemical
+′-	I-chemical
+GAGG	I-chemical
+-	I-chemical
+3	I-chemical
+′/	I-chemical
+5	B-chemical
+′-	I-chemical
+GGAG	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+or	O
+both	O
+two	O
+adenines	B-residue_name
+of	O
+5	B-chemical
+′-	I-chemical
+GAAG	I-chemical
+-	I-chemical
+3	I-chemical
+′,	I-chemical
+compared	O
+with	O
+the	O
+homologs	O
+from	O
+other	O
+strains	O
+that	O
+can	O
+methylate	O
+only	O
+one	O
+adenine	B-residue_name
+of	O
+5	B-chemical
+′-	I-chemical
+GAGG	I-chemical
+-	I-chemical
+3	I-chemical
+′.	I-chemical
+To	O
+answer	O
+why	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+displayed	O
+a	O
+wider	O
+specificity	O
+for	O
+DNA	B-chemical
+recognition	O
+,	O
+we	O
+randomly	O
+choose	O
+fifty	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+sequences	O
+from	O
+hundreds	O
+of	O
+H	B-species
+.	I-species
+pylori	I-species
+strains	O
+for	O
+multiple	B-experimental_method
+sequence	I-experimental_method
+alignment	I-experimental_method
+.	O
+
+Based	O
+on	O
+sequence	B-experimental_method
+comparison	I-experimental_method
+and	O
+structural	B-experimental_method
+analysis	I-experimental_method
+,	O
+four	O
+residues	O
+including	O
+P41	B-residue_name_number
+,	O
+N111	B-residue_name_number
+,	O
+K165	B-residue_name_number
+and	O
+T166	B-residue_name_number
+were	O
+selected	O
+and	O
+replaced	B-experimental_method
+by	O
+serine	B-residue_name
+,	O
+threonine	B-residue_name
+,	O
+threonine	B-residue_name
+and	O
+valine	B-residue_name
+,	O
+respectively	O
+(	O
+Figure	O
+5A	O
+).	O
+
+Then	O
+,	O
+a	O
+[	B-experimental_method
+3H	I-experimental_method
+]	I-experimental_method
+AdoMet	I-experimental_method
+radiological	I-experimental_method
+assay	I-experimental_method
+was	O
+applied	O
+to	O
+quantify	O
+the	O
+methyl	B-chemical
+transfer	O
+activity	O
+of	O
+the	O
+wide	B-protein_state
+type	I-protein_state
+protein	O
+and	O
+the	O
+mutants	B-protein_state
+.	O
+
+As	O
+shown	O
+in	O
+Figure	O
+5	O
+,	O
+when	O
+the	O
+substrate	O
+DNA	B-chemical
+contains	O
+5	B-chemical
+′-	I-chemical
+GAGG	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+or	O
+5	B-chemical
+′-	I-chemical
+GAAG	I-chemical
+-	I-chemical
+3	I-chemical
+′,	I-chemical
+all	O
+the	O
+mutants	B-protein_state
+showed	O
+no	O
+apparent	O
+difference	O
+of	O
+methyl	B-chemical
+transfer	O
+activity	O
+compared	O
+to	O
+the	O
+wt	B-protein_state
+-	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+;	O
+but	O
+when	O
+the	O
+recognition	O
+sequence	O
+was	O
+5	B-chemical
+′-	I-chemical
+GGAG	I-chemical
+-	I-chemical
+3	I-chemical
+′,	I-chemical
+the	O
+methyl	B-chemical
+transfer	O
+activity	O
+of	O
+the	O
+P41S	B-mutant
+mutant	B-protein_state
+was	O
+significantly	O
+reduced	O
+compared	O
+to	O
+the	O
+wild	B-protein_state
+type	I-protein_state
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+.	O
+
+Sequence	B-experimental_method
+alignment	I-experimental_method
+,	O
+structural	B-experimental_method
+analysis	I-experimental_method
+and	O
+radioactive	B-experimental_method
+methyl	I-experimental_method
+transfer	I-experimental_method
+activity	I-experimental_method
+define	O
+the	O
+key	O
+residue	O
+for	O
+wider	O
+substrate	O
+specificity	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+
+A	O
+.	O
+Sequence	B-experimental_method
+alignment	I-experimental_method
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+from	O
+50	O
+H	B-species
+.	I-species
+pylori	I-species
+strains	O
+including	O
+26695	O
+revealed	O
+several	O
+variant	O
+residues	O
+.	O
+
+Residues	O
+P41	B-residue_name_number
+,	O
+N111	B-residue_name_number
+,	O
+K165	B-residue_name_number
+and	O
+T166	B-residue_name_number
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+from	O
+strain	O
+26695	B-species
+were	O
+chosen	O
+based	O
+on	O
+structural	B-experimental_method
+analysis	I-experimental_method
+and	O
+sequence	B-experimental_method
+alignment	I-experimental_method
+(	O
+shown	O
+in	O
+red	O
+arrow	O
+).	O
+
+Amino	O
+-	O
+acid	O
+conservation	O
+is	O
+depicted	O
+using	O
+WebLogo	B-experimental_method
+(	O
+Crooks	O
+et	O
+al	O
+,	O
+2004	O
+).	O
+
+B	O
+.,	O
+C	O
+.,	O
+D	O
+.	O
+Methyl	B-chemical
+transfer	O
+reactions	O
+were	O
+performed	O
+using	O
+wt	B-protein_state
+-	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+,	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+P41S	I-mutant
+,	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+N111T	I-mutant
+,	O
+and	O
+M1	B-mutant
+.	I-mutant
+HpyAVI	I-mutant
+-	I-mutant
+K165R	I-mutant
+T166V	I-mutant
+,	O
+respectively	O
+.	O
+
+Radioactivity	O
+incorporated	O
+into	O
+the	O
+duplex	O
+DNA	B-chemical
+containing	O
+5	B-chemical
+′-	I-chemical
+GAGG	I-chemical
+-	I-chemical
+3	I-chemical
+′,	I-chemical
+5	B-chemical
+′-	I-chemical
+GAAG	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+or	O
+5	B-chemical
+′-	I-chemical
+GGAG	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+was	O
+quantified	O
+by	O
+Beckman	O
+LS6500	O
+for	O
+10	O
+min	O
+.	O
+
+Our	O
+experimental	O
+data	O
+identified	O
+P41	B-residue_name_number
+as	O
+a	O
+key	O
+residue	O
+determining	O
+the	O
+recognition	O
+of	O
+GGAG	B-structure_element
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+.	O
+
+This	O
+amino	O
+acid	O
+locates	O
+in	O
+the	O
+highly	B-protein_state
+flexible	I-protein_state
+loop	B-structure_element
+between	O
+residues	O
+33	B-residue_range
+and	I-residue_range
+58	I-residue_range
+,	O
+which	O
+is	O
+involved	O
+in	O
+DNA	B-chemical
+binding	O
+and	O
+substrate	O
+recognition	O
+as	O
+shown	O
+above	O
+.	O
+
+Replacement	B-experimental_method
+by	O
+serine	B-residue_name
+at	O
+this	O
+position	O
+definitely	O
+changes	O
+the	O
+local	O
+conformation	O
+and	O
+hydrophobicity	O
+,	O
+and	O
+probably	O
+some	O
+structural	O
+properties	O
+of	O
+the	O
+whole	O
+loop	B-structure_element
+,	O
+which	O
+may	O
+in	O
+turn	O
+result	O
+in	O
+reduced	O
+specificity	O
+for	O
+sequence	O
+recognition	O
+of	O
+the	O
+enzyme	O
+from	O
+strain	O
+26695	B-species
+.	O
+
+Although	O
+the	O
+DNA	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+structure	B-evidence
+of	O
+previous	O
+investigation	O
+on	O
+a	O
+γ	B-protein_type
+-	I-protein_type
+class	I-protein_type
+N6	I-protein_type
+-	I-protein_type
+adenine	I-protein_type
+MTase	I-protein_type
+revealed	O
+that	O
+the	O
+target	O
+adenine	B-residue_name
+was	O
+rotated	O
+out	O
+of	O
+DNA	B-chemical
+helix	O
+,	O
+details	O
+of	O
+the	O
+methyl	B-chemical
+transfer	O
+process	O
+were	O
+still	O
+unclear	O
+.	O
+
+Additionally	O
+,	O
+recent	O
+studies	O
+reported	O
+the	O
+importance	O
+of	O
+N6	B-ptm
+-	I-ptm
+methyladenine	I-ptm
+in	O
+some	O
+eukaryotic	B-taxonomy_domain
+species	O
+,	O
+but	O
+until	O
+now	O
+there	O
+has	O
+not	O
+been	O
+any	O
+N6	B-protein_type
+-	I-protein_type
+adenine	I-protein_type
+MTases	I-protein_type
+being	O
+identified	O
+in	O
+eukaryotes	B-taxonomy_domain
+.	O
+
+Biochemical	B-experimental_method
+and	I-experimental_method
+structural	I-experimental_method
+characterization	I-experimental_method
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+provides	O
+a	O
+new	O
+model	O
+for	O
+uncovering	O
+the	O
+methyl	B-chemical
+transfer	O
+mechanism	O
+and	O
+for	O
+investigating	O
+the	O
+N6	B-ptm
+-	I-ptm
+methyladenine	I-ptm
+in	O
+eukaryotes	B-taxonomy_domain
+.	O
+
+Oligomeric	O
+state	O
+of	O
+DNA	B-protein_type
+MTases	I-protein_type
+was	O
+long	O
+accepted	O
+as	O
+monomer	B-oligomeric_state
+,	O
+but	O
+our	O
+study	O
+indicated	O
+here	O
+that	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+exists	O
+as	O
+a	O
+dimer	B-oligomeric_state
+both	O
+in	O
+crystal	B-evidence
+and	O
+solution	O
+.	O
+
+Interestingly	O
+,	O
+some	O
+other	O
+β	B-protein_type
+-	I-protein_type
+class	I-protein_type
+DNA	I-protein_type
+exocyclic	I-protein_type
+MTases	I-protein_type
+showed	O
+similar	O
+oligomeric	O
+state	O
+in	O
+crystal	B-evidence
+and	O
+in	O
+solution	O
+,	O
+indicating	O
+that	O
+dimer	B-oligomeric_state
+may	O
+be	O
+the	O
+functional	O
+state	O
+shared	O
+by	O
+a	O
+subgroup	O
+of	O
+DNA	B-protein_type
+MTases	I-protein_type
+.	O
+
+The	O
+highly	B-protein_state
+flexible	I-protein_state
+region	O
+(	O
+residues	O
+33	B-residue_range
+-	I-residue_range
+58	I-residue_range
+)	O
+and	O
+TRD	B-structure_element
+(	O
+residues	O
+133	B-residue_range
+-	I-residue_range
+163	I-residue_range
+)	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+are	O
+supposed	O
+to	O
+interact	O
+with	O
+DNA	B-chemical
+at	O
+minor	B-structure_element
+and	I-structure_element
+major	I-structure_element
+grooves	I-structure_element
+,	O
+respectively	O
+.	O
+
+And	O
+residue	O
+P41	B-residue_name_number
+might	O
+be	O
+a	O
+key	O
+residue	O
+partially	O
+determining	O
+the	O
+substrate	O
+spectrum	O
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+.	O
+
+The	O
+missing	B-protein_state
+loop	B-structure_element
+between	O
+residues	O
+33	B-residue_range
+and	I-residue_range
+58	I-residue_range
+may	O
+need	O
+DNA	B-chemical
+binding	O
+so	O
+as	O
+to	O
+form	O
+a	O
+stable	B-protein_state
+conformation	O
+,	O
+which	O
+is	O
+similar	O
+to	O
+the	O
+condition	O
+of	O
+M	B-protein
+.	I-protein
+TaqI	I-protein
+.	O
+Crystallization	B-experimental_method
+of	O
+M1	B-complex_assembly
+.	I-complex_assembly
+HpyAVI	I-complex_assembly
+-	I-complex_assembly
+DNA	I-complex_assembly
+complex	O
+warrants	O
+future	O
+investigations	O
+,	O
+with	O
+the	O
+purpose	O
+of	O
+revealing	O
+the	O
+mechanism	O
+behind	O
+the	O
+wider	O
+substrate	O
+specificity	O
+of	O
+this	O
+enzyme	O
+.	O
+
+DNA	B-ptm
+methylation	I-ptm
+plays	O
+an	O
+important	O
+role	O
+in	O
+bacterial	B-taxonomy_domain
+pathogenicity	O
+.	O
+
+DNA	B-ptm
+adenine	I-ptm
+methylation	I-ptm
+was	O
+known	O
+to	O
+regulate	O
+the	O
+expression	O
+of	O
+some	O
+virulence	O
+genes	O
+in	O
+bacteria	B-taxonomy_domain
+including	O
+H	B-species
+.	I-species
+pylori	I-species
+.	O
+
+Inhibitors	O
+of	O
+DNA	B-ptm
+adenine	I-ptm
+methylation	I-ptm
+may	O
+have	O
+a	O
+broad	O
+antimicrobial	O
+action	O
+by	O
+targeting	O
+DNA	B-protein_type
+adenine	I-protein_type
+methyltransferase	I-protein_type
+.	O
+
+As	O
+an	O
+important	O
+biological	O
+modification	O
+,	O
+DNA	B-ptm
+methylation	I-ptm
+directly	O
+influences	O
+bacterial	B-taxonomy_domain
+survival	O
+.	O
+
+Knockout	B-experimental_method
+of	I-experimental_method
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+largely	O
+prevents	O
+the	O
+growth	O
+of	O
+H	B-species
+.	I-species
+pylori	I-species
+.	O
+
+Importantly	O
+,	O
+H	B-species
+.	I-species
+pylori	I-species
+is	O
+involved	O
+in	O
+90	O
+%	O
+of	O
+all	O
+gastric	O
+malignancies	O
+.	O
+
+Appropriate	O
+antibiotic	O
+regimens	O
+could	O
+successfully	O
+cure	O
+gastric	O
+diseases	O
+caused	O
+by	O
+H	B-species
+.	I-species
+pylori	I-species
+infection	O
+.	O
+
+However	O
+,	O
+eradication	O
+of	O
+H	B-species
+.	I-species
+pylori	I-species
+infection	O
+remains	O
+a	O
+big	O
+challenge	O
+for	O
+the	O
+significantly	O
+increasing	O
+prevalence	O
+of	O
+its	O
+resistance	O
+to	O
+antibiotics	O
+.	O
+
+The	O
+development	O
+of	O
+new	O
+drugs	O
+targeting	O
+adenine	B-protein_type
+MTases	I-protein_type
+such	O
+as	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+offers	O
+a	O
+new	O
+opportunity	O
+for	O
+inhibition	O
+of	O
+H	B-species
+.	I-species
+pylori	I-species
+infection	O
+.	O
+
+Residues	O
+that	O
+play	O
+crucial	O
+roles	O
+for	O
+catalytic	O
+activity	O
+like	O
+D29	B-residue_name_number
+or	O
+E216	B-residue_name_number
+may	O
+influence	O
+the	O
+H	B-species
+.	I-species
+pylori	I-species
+survival	O
+.	O
+
+Small	O
+molecules	O
+targeting	O
+these	O
+highly	B-protein_state
+conserved	I-protein_state
+residues	O
+are	O
+likely	O
+to	O
+emerge	O
+less	O
+drug	O
+resistance	O
+.	O
+
+In	O
+summary	O
+,	O
+the	O
+structure	B-evidence
+of	O
+M1	B-protein
+.	I-protein
+HpyAVI	I-protein
+is	O
+featured	O
+with	O
+a	O
+disordered	B-protein_state
+TRD	B-structure_element
+and	O
+a	O
+key	O
+residue	O
+P41that	B-residue_name_number
+located	O
+in	O
+the	O
+putative	O
+DNA	B-site
+binding	I-site
+region	I-site
+that	O
+may	O
+associate	O
+with	O
+the	O
+wider	O
+substrate	O
+specificity	O
+.	O
+
+Residues	O
+D29	B-residue_name_number
+and	O
+E216	B-residue_name_number
+were	O
+identified	O
+to	O
+play	O
+a	O
+crucial	O
+role	O
+in	O
+cofactor	O
+binding	O
+.	O
+
+As	O
+the	O
+first	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+N6	B-protein_type
+-	I-protein_type
+adenine	I-protein_type
+MTase	I-protein_type
+in	O
+H	B-species
+.	I-species
+pylori	I-species
+,	O
+this	O
+model	O
+may	O
+shed	O
+light	O
+on	O
+design	O
+of	O
+new	O
+antibiotics	O
+to	O
+interfere	O
+the	O
+growth	O
+and	O
+pathogenesis	O
+of	O
+H	B-species
+.	I-species
+pylori	I-species
+in	O
+human	B-species
+.	O
+
diff --git a/annotation_IOB/PMC5603727.tsv b/annotation_IOB/PMC5603727.tsv
new file mode 100644
index 0000000000000000000000000000000000000000..e78d72e0e3c2f1e1321c632cc21bfa2291d86e72
--- /dev/null
+++ b/annotation_IOB/PMC5603727.tsv
@@ -0,0 +1,8414 @@
+Roquin	B-protein
+recognizes	O
+a	O
+non	O
+-	O
+canonical	O
+hexaloop	B-structure_element
+structure	O
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+Ox40	B-protein
+
+The	O
+RNA	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+protein	I-protein_type
+Roquin	B-protein
+is	O
+required	O
+to	O
+prevent	O
+autoimmunity	O
+.	O
+
+Roquin	B-protein
+controls	O
+T	O
+-	O
+helper	O
+cell	O
+activation	O
+and	O
+differentiation	O
+by	O
+limiting	O
+the	O
+induced	O
+expression	O
+of	O
+costimulatory	B-protein_type
+receptors	I-protein_type
+such	O
+as	O
+tumor	B-protein
+necrosis	I-protein
+factor	I-protein
+receptor	I-protein
+superfamily	I-protein
+4	I-protein
+(	O
+Tnfrs4	B-protein
+or	O
+Ox40	B-protein
+).	O
+
+A	O
+constitutive	B-structure_element
+decay	I-structure_element
+element	I-structure_element
+(	O
+CDE	B-structure_element
+)	O
+with	O
+a	O
+characteristic	O
+triloop	B-structure_element
+hairpin	I-structure_element
+was	O
+previously	O
+shown	O
+to	O
+be	O
+recognized	O
+by	O
+Roquin	B-protein
+.	O
+
+Here	O
+we	O
+use	O
+SELEX	B-experimental_method
+assays	I-experimental_method
+to	O
+identify	O
+a	O
+novel	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+hexaloop	I-structure_element
+motif	I-structure_element
+,	O
+representing	O
+an	O
+alternative	B-structure_element
+decay	I-structure_element
+element	I-structure_element
+(	O
+ADE	B-structure_element
+).	O
+
+Crystal	B-evidence
+structures	I-evidence
+and	O
+NMR	B-experimental_method
+data	O
+show	O
+that	O
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+ROQ	B-structure_element
+domain	O
+recognizes	O
+hexaloops	B-structure_element
+in	O
+the	O
+SELEX	B-experimental_method
+-	O
+derived	O
+ADE	B-structure_element
+and	O
+in	O
+an	O
+ADE	B-structure_element
+-	O
+like	O
+variant	O
+present	O
+in	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+with	O
+identical	O
+binding	O
+modes	O
+.	O
+
+In	O
+cells	O
+,	O
+ADE	B-structure_element
+-	O
+like	O
+and	O
+CDE	B-structure_element
+-	O
+like	O
+motifs	O
+cooperate	O
+in	O
+the	O
+repression	O
+of	O
+Ox40	B-protein
+by	O
+Roquin	B-protein
+.	O
+
+Our	O
+data	O
+reveal	O
+an	O
+unexpected	O
+recognition	O
+of	O
+hexaloop	B-structure_element
+cis	I-structure_element
+elements	I-structure_element
+for	O
+the	O
+posttranscriptional	O
+regulation	O
+of	O
+target	O
+messenger	B-chemical
+RNAs	I-chemical
+by	O
+Roquin	B-protein
+.	O
+
+Roquin	B-protein
+is	O
+an	O
+RNA	B-protein_type
+-	I-protein_type
+binding	I-protein_type
+protein	I-protein_type
+that	O
+prevents	O
+autoimmunity	O
+by	O
+limiting	O
+expression	O
+of	O
+receptors	O
+such	O
+as	O
+Ox40	B-protein
+.	O
+
+Here	O
+,	O
+the	O
+authors	O
+identify	O
+an	O
+RNA	B-chemical
+structure	B-evidence
+that	O
+they	O
+describe	O
+as	O
+an	O
+alternative	B-structure_element
+decay	I-structure_element
+element	I-structure_element
+,	O
+and	O
+they	O
+characterise	O
+its	O
+interaction	O
+with	O
+Roquin	B-protein
+using	O
+structural	B-experimental_method
+and	I-experimental_method
+biochemical	I-experimental_method
+techniques	I-experimental_method
+.	O
+
+The	O
+Roquin	B-protein
+protein	O
+is	O
+essential	O
+in	O
+T	O
+cells	O
+for	O
+the	O
+prevention	O
+of	O
+autoimmune	O
+disease	O
+.	O
+
+This	O
+is	O
+evident	O
+from	O
+the	O
+so	O
+-	O
+called	O
+sanroque	O
+mutation	O
+in	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+,	O
+a	O
+single	O
+amino	O
+acid	O
+exchange	O
+from	O
+Met199	B-residue_name_number
+to	O
+Arg	B-residue_name
+that	O
+causes	O
+the	O
+development	O
+of	O
+systemic	O
+lupus	O
+erythematosus	O
+-	O
+like	O
+symptoms	O
+in	O
+homozygous	O
+mice	B-taxonomy_domain
+.	O
+
+The	O
+Rc3h1	B-gene
+and	O
+Rc3h2	B-gene
+genes	O
+,	O
+encoding	O
+for	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+and	O
+Roquin	B-protein
+-	I-protein
+2	I-protein
+proteins	O
+in	O
+vertebrates	B-taxonomy_domain
+,	O
+respectively	O
+,	O
+have	O
+both	O
+been	O
+shown	O
+to	O
+be	O
+essential	O
+for	O
+the	O
+survival	O
+of	O
+mice	B-taxonomy_domain
+,	O
+but	O
+apparently	O
+serve	O
+redundant	O
+functions	O
+in	O
+T	O
+cells	O
+.	O
+
+Consistently	O
+,	O
+CD4	O
++	O
+and	O
+CD8	O
++	O
+T	O
+cells	O
+with	O
+the	O
+combined	O
+deletion	B-experimental_method
+of	I-experimental_method
+Roquin	B-protein
+-	O
+encoding	O
+genes	O
+are	O
+spontaneously	O
+activated	O
+and	O
+CD4	O
++	O
+T	O
+-	O
+helper	O
+cells	O
+preferentially	O
+differentiate	O
+into	O
+the	O
+Th1	O
+,	O
+Tfh	O
+or	O
+Th17	O
+subsets	O
+.	O
+
+Roquin	B-protein
+-	I-protein
+1	I-protein
+was	O
+shown	O
+to	O
+negatively	O
+regulate	O
+expression	O
+of	O
+transcripts	O
+encoding	O
+for	O
+co	B-protein_type
+-	I-protein_type
+stimulatory	I-protein_type
+receptors	I-protein_type
+such	O
+as	O
+Icos	B-protein
+,	O
+Ox40	B-protein
+and	O
+CTLA	B-protein
+-	I-protein
+4	I-protein
+,	O
+for	O
+cytokines	B-protein_type
+such	O
+as	O
+interleukin	B-protein
+(	I-protein
+IL	I-protein
+)-	I-protein
+6	I-protein
+and	O
+tumour	B-protein
+necrosis	I-protein
+factor	I-protein
+or	O
+for	O
+transcription	B-protein_type
+factors	I-protein_type
+such	O
+as	O
+IRF4	B-protein
+,	O
+IκBNS	B-protein
+and	O
+IκBζ	B-protein
+(	O
+refs	O
+).	O
+
+We	O
+have	O
+recently	O
+reported	O
+structural	B-evidence
+and	I-evidence
+functional	I-evidence
+data	I-evidence
+of	O
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+ROQ	B-structure_element
+domain	O
+bound	B-protein_state
+to	I-protein_state
+a	O
+canonical	O
+constitutive	B-structure_element
+decay	I-structure_element
+element	I-structure_element
+(	O
+CDE	B-structure_element
+),	O
+a	O
+short	B-structure_element
+stem	I-structure_element
+loop	I-structure_element
+(	O
+SL	B-structure_element
+)	O
+that	O
+acts	O
+as	O
+a	O
+cis	O
+-	O
+regulatory	O
+RNA	B-chemical
+element	O
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+untranslated	I-structure_element
+regions	I-structure_element
+(	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+)	O
+of	O
+target	O
+genes	O
+such	O
+as	O
+Tnf	B-protein
+(	O
+ref	O
+).	O
+
+The	O
+ROQ	B-structure_element
+domain	O
+adopts	O
+an	O
+extended	B-structure_element
+winged	I-structure_element
+helix	I-structure_element
+fold	I-structure_element
+that	O
+engages	O
+predominantly	O
+non	O
+-	O
+sequence	O
+-	O
+specific	O
+protein	O
+–	O
+RNA	B-chemical
+contacts	O
+and	O
+mainly	O
+recognizes	O
+the	O
+shape	O
+of	O
+the	O
+canonical	O
+Tnf	B-protein
+CDE	B-structure_element
+RNA	B-chemical
+.	O
+
+The	O
+structural	B-evidence
+data	I-evidence
+and	O
+mutational	B-experimental_method
+analysis	I-experimental_method
+indicated	O
+that	O
+a	O
+broader	O
+,	O
+extended	O
+range	O
+of	O
+sequence	O
+variations	O
+in	O
+both	O
+the	O
+loop	B-structure_element
+and	O
+stem	B-structure_element
+of	O
+the	O
+CDE	B-structure_element
+element	O
+is	O
+recognized	O
+and	O
+regulated	O
+by	O
+Roquin	B-protein
+.	O
+
+At	O
+the	O
+same	O
+time	O
+,	O
+Tan	O
+et	O
+al	O
+.	O
+described	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+and	O
+supporting	O
+functional	O
+data	O
+of	O
+a	O
+similar	O
+interaction	O
+with	O
+a	O
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+,	O
+and	O
+reported	O
+a	O
+second	B-site
+binding	I-site
+site	I-site
+for	O
+a	O
+double	B-chemical
+-	I-chemical
+stranded	I-chemical
+RNA	I-chemical
+(	O
+dsRNA	B-chemical
+)	O
+within	O
+an	O
+extended	B-protein_state
+ROQ	B-structure_element
+domain	O
+.	O
+
+The	O
+structural	O
+basis	O
+for	O
+CDE	B-structure_element
+recognition	O
+by	O
+the	O
+Roquin	B-protein
+-	I-protein
+2	I-protein
+ROQ	B-structure_element
+domain	O
+has	O
+also	O
+been	O
+recently	O
+reported	O
+.	O
+
+We	O
+found	O
+that	O
+the	O
+posttranscriptional	O
+activity	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+and	O
+Roquin	B-protein
+-	I-protein
+2	I-protein
+is	O
+regulated	O
+through	O
+cleavage	O
+by	O
+the	O
+paracaspase	B-protein_type
+MALT1	B-protein
+(	O
+refs	O
+).	O
+
+Enhanced	O
+MALT1	B-protein
+-	O
+dependent	O
+cleavage	O
+and	O
+inactivation	O
+of	O
+Roquin	B-protein
+,	O
+and	O
+thus	O
+less	O
+effective	O
+repression	O
+of	O
+target	O
+genes	O
+,	O
+result	O
+from	O
+increased	O
+strength	O
+of	O
+antigen	O
+recognition	O
+in	O
+T	O
+cells	O
+.	O
+
+These	O
+findings	O
+suggest	O
+that	O
+dependent	O
+on	O
+the	O
+strength	O
+of	O
+cognate	O
+antigen	O
+recognition	O
+differential	O
+gene	O
+expression	O
+and	O
+cell	O
+fate	O
+decisions	O
+can	O
+be	O
+established	O
+in	O
+naive	O
+T	O
+cells	O
+by	O
+a	O
+graded	O
+cleavage	O
+and	O
+inactivation	O
+of	O
+Roquin	B-protein
+.	O
+
+In	O
+addition	O
+to	O
+this	O
+mechanism	O
+,	O
+the	O
+composition	O
+and	O
+binding	B-evidence
+affinity	I-evidence
+of	O
+cis	O
+-	O
+regulatory	O
+SL	B-structure_element
+elements	O
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+of	O
+target	O
+mRNAs	B-chemical
+may	O
+determine	O
+the	O
+sensitivity	O
+to	O
+repression	O
+by	O
+the	O
+trans	O
+-	O
+acting	O
+factor	O
+Roquin	B-protein
+.	O
+Defining	O
+the	O
+SL	B-structure_element
+RNA	B-chemical
+structures	O
+that	O
+are	O
+recognized	O
+by	O
+Roquin	B-protein
+is	O
+therefore	O
+essential	O
+for	O
+our	O
+understanding	O
+of	O
+posttranscriptional	O
+gene	O
+regulation	O
+by	O
+Roquin	B-protein
+and	O
+its	O
+involvement	O
+in	O
+T	O
+-	O
+cell	O
+biology	O
+and	O
+T	O
+-	O
+cell	O
+-	O
+driven	O
+pathology	O
+.	O
+
+Here	O
+we	O
+present	O
+structural	O
+and	O
+functional	O
+evidence	O
+for	O
+a	O
+greatly	O
+expanded	O
+repertoire	O
+of	O
+RNA	B-chemical
+elements	O
+that	O
+are	O
+regulated	O
+by	O
+Roquin	B-protein
+as	O
+demonstrated	O
+with	O
+a	O
+novel	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+hexaloop	I-structure_element
+SL	B-structure_element
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+Ox40	B-protein
+bound	B-protein_state
+to	I-protein_state
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+ROQ	B-structure_element
+domain	O
+.	O
+
+We	O
+find	O
+an	O
+additive	O
+regulation	O
+of	O
+Ox40	B-protein
+gene	O
+expression	O
+based	O
+on	O
+both	O
+its	O
+CDE	B-structure_element
+-	O
+like	O
+and	O
+hexaloop	B-structure_element
+SL	B-structure_element
+RNAs	B-chemical
+that	O
+we	O
+identified	O
+using	O
+Systematic	B-experimental_method
+Evolution	I-experimental_method
+of	I-experimental_method
+Ligands	I-experimental_method
+by	I-experimental_method
+Exponential	I-experimental_method
+Enrichment	I-experimental_method
+(	O
+SELEX	B-experimental_method
+)	O
+experiments	O
+.	O
+
+Our	O
+X	B-experimental_method
+-	I-experimental_method
+ray	I-experimental_method
+crystallographic	I-experimental_method
+,	O
+NMR	B-experimental_method
+,	O
+biochemical	B-evidence
+and	I-evidence
+functional	I-evidence
+data	I-evidence
+combined	O
+with	O
+mutational	B-experimental_method
+analysis	I-experimental_method
+demonstrate	O
+that	O
+both	O
+triloop	B-structure_element
+and	O
+hexaloop	B-structure_element
+SL	B-structure_element
+RNAs	B-chemical
+contribute	O
+to	O
+the	O
+functional	O
+activity	O
+of	O
+Roquin	B-protein
+in	O
+T	O
+cells	O
+.	O
+
+SELEX	B-experimental_method
+identifies	O
+novel	O
+RNA	B-chemical
+ligands	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+
+We	O
+set	O
+out	O
+to	O
+identify	O
+Roquin	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+RNA	B-chemical
+motifs	O
+in	O
+an	O
+unbiased	O
+manner	O
+by	O
+performing	O
+SELEX	B-experimental_method
+experiments	O
+.	O
+
+A	O
+biotinylated	B-protein_state
+amino	O
+-	O
+terminal	O
+protein	O
+fragment	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+(	O
+residues	O
+2	B-residue_range
+–	I-residue_range
+440	I-residue_range
+)	O
+was	O
+used	O
+to	O
+enrich	O
+RNAs	B-chemical
+from	O
+a	O
+library	O
+containing	O
+47	O
+random	O
+nucleotides	O
+over	O
+three	O
+sequential	O
+selection	O
+rounds	O
+.	O
+
+Next	B-experimental_method
+-	I-experimental_method
+generation	I-experimental_method
+sequencing	I-experimental_method
+(	O
+NGS	B-experimental_method
+)	O
+of	O
+the	O
+RNA	B-chemical
+before	O
+and	O
+after	O
+each	O
+selection	O
+round	O
+revealed	O
+that	O
+the	O
+starting	O
+pool	O
+represented	O
+about	O
+99	O
+.	O
+6	O
+%	O
+unique	O
+reads	O
+in	O
+∼	O
+4	O
+.	O
+2	O
+×	O
+106	O
+sequences	O
+.	O
+
+Bioinformatic	B-experimental_method
+analysis	I-experimental_method
+of	O
+NGS	B-experimental_method
+data	O
+sets	O
+derived	O
+from	O
+the	O
+starting	O
+pool	O
+and	O
+enriched	O
+selection	O
+rounds	O
+revealed	O
+that	O
+the	O
+complexity	O
+was	O
+reduced	O
+to	O
+78	O
+.	O
+6	O
+%	O
+unique	O
+reads	O
+in	O
+3	O
+.	O
+7	O
+×	O
+106	O
+sequences	O
+that	O
+were	O
+analysed	O
+after	O
+3	O
+rounds	O
+of	O
+selection	O
+and	O
+enrichment	O
+.	O
+
+For	O
+NGS	B-experimental_method
+data	O
+analysis	O
+,	O
+the	O
+COMPAS	O
+software	O
+(	O
+AptaIT	O
+,	O
+Munich	O
+,	O
+Germany	O
+)	O
+was	O
+applied	O
+.	O
+
+Enriched	O
+sequences	B-experimental_method
+were	I-experimental_method
+clustered	I-experimental_method
+into	O
+so	O
+-	O
+called	O
+patterns	O
+with	O
+highly	O
+homologous	O
+sequences	O
+.	O
+
+Based	O
+on	O
+this	O
+so	O
+-	O
+called	O
+co	B-experimental_method
+-	I-experimental_method
+occurrence	I-experimental_method
+approach	I-experimental_method
+,	O
+patterns	O
+on	O
+the	O
+basis	O
+of	O
+frequent	O
+motifs	O
+were	O
+generated	O
+and	O
+were	O
+searched	O
+for	O
+prominent	O
+hexamer	O
+sequences	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1a	O
+).	O
+
+We	O
+identified	O
+5	B-chemical
+′-	I-chemical
+CGTTTT	I-chemical
+-	I-chemical
+3	I-chemical
+′,	I-chemical
+5	B-chemical
+′-	I-chemical
+GCGTTT	I-chemical
+-	I-chemical
+3	I-chemical
+′,	I-chemical
+5	B-chemical
+′-	I-chemical
+TGCGTT	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+and	O
+5	B-chemical
+′-	I-chemical
+GTTTTA	I-chemical
+-	I-chemical
+3	I-chemical
+′	I-chemical
+motifs	O
+that	O
+were	O
+also	O
+reconfirmed	O
+in	O
+an	O
+independent	O
+experiment	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1a	O
+)	O
+and	O
+are	O
+located	O
+within	O
+highly	O
+similar	O
+sequences	O
+(	O
+Fig	O
+.	O
+1a	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1b	O
+).	O
+
+Consistent	O
+with	O
+previous	O
+findings	O
+showing	O
+that	O
+the	O
+sanroque	B-mutant
+mutation	I-mutant
+does	O
+not	O
+impair	O
+RNA	B-chemical
+binding	O
+of	O
+Roquin	B-protein
+,	O
+we	O
+found	O
+similarly	O
+enriched	O
+sequences	O
+in	O
+SELEX	B-experimental_method
+approaches	O
+using	O
+a	O
+corresponding	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+fragment	O
+harbouring	O
+the	O
+M199R	B-mutant
+mutation	O
+(	O
+Fig	O
+.	O
+1a	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+1b	O
+).	O
+
+Notably	O
+,	O
+our	O
+SELEX	B-experimental_method
+approach	O
+did	O
+not	O
+reveal	O
+the	O
+previously	O
+identified	O
+CDE	B-structure_element
+sequence	O
+.	O
+
+We	O
+assume	O
+that	O
+the	O
+region	O
+of	O
+sequence	O
+identity	O
+in	O
+the	O
+CDE	B-structure_element
+is	O
+too	O
+short	O
+for	O
+our	O
+sequence	B-experimental_method
+clustering	I-experimental_method
+algorithm	I-experimental_method
+.	O
+
+Evaluation	O
+of	O
+the	O
+structural	O
+context	O
+for	O
+the	O
+SELEX	B-experimental_method
+-	O
+derived	O
+motif	O
+suggested	O
+a	O
+putative	O
+SL	B-structure_element
+formation	O
+with	O
+six	O
+unpaired	O
+nucleotides	O
+in	O
+a	O
+loop	B-structure_element
+followed	O
+by	O
+a	O
+5	O
+–	O
+8	O
+nt	O
+stem	B-structure_element
+,	O
+with	O
+one	O
+base	O
+in	O
+the	O
+stem	B-structure_element
+not	O
+being	O
+paired	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+1c	O
+).	O
+
+Searching	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+of	O
+known	O
+Roquin	B-protein
+targets	O
+with	O
+the	O
+consensus	O
+5	B-chemical
+′-	I-chemical
+TGCGTTTTAGGA	I-chemical
+-	I-chemical
+3	I-chemical
+′,	I-chemical
+obtained	O
+by	O
+Motif	B-experimental_method
+-	I-experimental_method
+based	I-experimental_method
+sequence	I-experimental_method
+analysis	I-experimental_method
+(	O
+MEME	B-experimental_method
+),	O
+revealed	O
+a	O
+homologous	O
+sequence	O
+with	O
+the	O
+potential	O
+to	O
+form	O
+a	O
+hexaloop	B-structure_element
+structure	O
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+Ox40	B-protein
+(	O
+Fig	O
+.	O
+1b	O
+).	O
+
+Importantly	O
+,	O
+this	O
+motif	O
+is	O
+present	O
+across	O
+species	O
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+of	O
+respective	O
+mRNAs	B-chemical
+and	O
+showed	O
+highest	O
+conservation	O
+in	O
+the	O
+loop	B-structure_element
+and	O
+the	O
+upper	O
+stem	B-structure_element
+sequences	O
+with	O
+a	O
+drop	O
+of	O
+conservation	O
+towards	O
+the	O
+boundaries	O
+of	O
+the	O
+motif	O
+(	O
+Fig	O
+.	O
+1c	O
+,	O
+d	O
+).	O
+
+The	O
+predicted	O
+SL	B-structure_element
+for	O
+the	O
+consensus	O
+SELEX	B-experimental_method
+-	O
+derived	O
+motif	O
+(	O
+from	O
+here	O
+on	O
+referred	O
+to	O
+as	O
+alternative	B-structure_element
+decay	I-structure_element
+element	I-structure_element
+SL	B-structure_element
+,	O
+ADE	B-structure_element
+SL	B-structure_element
+),	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+,	O
+is	O
+positioned	O
+5	O
+′	O
+to	O
+another	O
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+Ox40	B-protein
+mRNA	B-chemical
+.	O
+
+This	O
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+differs	O
+in	O
+the	O
+sequence	O
+of	O
+the	O
+upper	O
+stem	O
+from	O
+the	O
+canonical	O
+CDE	B-structure_element
+from	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+Tnf	B-protein
+mRNA	B-chemical
+(	O
+CDE	B-structure_element
+SL	B-structure_element
+)	O
+(	O
+Fig	O
+.	O
+1d	O
+).	O
+
+NMR	B-experimental_method
+analysis	O
+of	O
+Roquin	B-protein_state
+-	I-protein_state
+bound	I-protein_state
+SL	B-structure_element
+RNAs	B-chemical
+
+We	O
+used	O
+NMR	B-experimental_method
+to	O
+analyse	O
+the	O
+secondary	O
+structure	O
+of	O
+Roquin	B-structure_element
+-	I-structure_element
+1	I-structure_element
+-	I-structure_element
+binding	I-structure_element
+motifs	I-structure_element
+derived	O
+from	O
+SELEX	B-experimental_method
+.	O
+
+Imino	B-experimental_method
+one	I-experimental_method
+-	I-experimental_method
+and	I-experimental_method
+two	I-experimental_method
+-	I-experimental_method
+dimensional	I-experimental_method
+nuclear	I-experimental_method
+Overhauser	I-experimental_method
+enhancement	I-experimental_method
+spectroscopy	I-experimental_method
+(	O
+NOESY	B-experimental_method
+)	O
+NMR	B-experimental_method
+spectra	B-evidence
+of	O
+the	O
+free	B-protein_state
+RNA	B-chemical
+and	O
+when	O
+bound	B-protein_state
+to	I-protein_state
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+ROQ	B-structure_element
+domain	O
+were	O
+recorded	O
+for	O
+the	O
+ADE	B-structure_element
+SL	B-structure_element
+,	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+Ox40	B-protein
+and	O
+the	O
+previously	O
+identified	O
+Ox40	B-protein
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+(	O
+Fig	O
+.	O
+2	O
+).	O
+
+The	O
+NMR	B-experimental_method
+data	O
+of	O
+the	O
+free	B-protein_state
+RNAs	B-chemical
+show	O
+that	O
+almost	O
+all	O
+predicted	O
+base	O
+pairs	O
+in	O
+the	O
+stem	B-structure_element
+regions	I-structure_element
+of	O
+the	O
+hexa	B-structure_element
+-	I-structure_element
+and	I-structure_element
+triloop	I-structure_element
+SL	B-structure_element
+including	O
+the	O
+closing	O
+base	O
+pairs	O
+are	O
+formed	O
+in	O
+all	O
+three	O
+RNAs	B-chemical
+.	O
+
+Notably	O
+,	O
+we	O
+also	O
+found	O
+an	O
+unambiguous	O
+imino	O
+proton	O
+signal	O
+for	O
+G15	B-residue_name_number
+,	O
+but	O
+not	O
+G6	B-residue_name_number
+,	O
+in	O
+the	O
+ADE	B-structure_element
+SL	B-structure_element
+,	O
+indicating	O
+a	O
+non	B-bond_interaction
+-	I-bond_interaction
+Watson	I-bond_interaction
+–	I-bond_interaction
+Crick	I-bond_interaction
+G	I-bond_interaction
+–	I-bond_interaction
+G	I-bond_interaction
+base	I-bond_interaction
+pair	I-bond_interaction
+at	O
+this	O
+position	O
+(	O
+Fig	O
+.	O
+2a	O
+).	O
+
+Significant	O
+chemical	B-evidence
+shift	I-evidence
+perturbations	I-evidence
+(	O
+CSPs	B-evidence
+)	O
+are	O
+observed	O
+for	O
+imino	O
+proton	O
+signals	O
+on	O
+binding	O
+to	O
+the	O
+ROQ	B-structure_element
+domain	O
+,	O
+demonstrating	O
+that	O
+formation	O
+of	O
+protein	O
+–	O
+RNA	B-chemical
+complexes	O
+involves	O
+contacts	O
+of	O
+the	O
+ROQ	B-structure_element
+domain	O
+to	O
+the	O
+stem	B-structure_element
+region	I-structure_element
+of	O
+the	O
+RNA	B-chemical
+ligands	O
+(	O
+Fig	O
+.	O
+2	O
+,	O
+bases	O
+coloured	O
+red	O
+).	O
+
+No	O
+imino	O
+correlations	O
+are	O
+observed	O
+for	O
+the	O
+predicted	O
+Watson	B-bond_interaction
+–	I-bond_interaction
+Crick	I-bond_interaction
+base	I-bond_interaction
+pairs	I-bond_interaction
+at	O
+the	O
+bottom	O
+of	O
+the	O
+ADE	B-structure_element
+SL	B-structure_element
+and	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+,	O
+as	O
+well	O
+as	O
+for	O
+the	O
+A	B-residue_name
+–	O
+U	B-residue_name
+base	O
+pair	O
+flanking	O
+the	O
+bulge	B-structure_element
+in	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+(	O
+Fig	O
+.	O
+2a	O
+,	O
+b	O
+),	O
+suggesting	O
+that	O
+these	O
+base	O
+pairs	O
+are	O
+dynamic	O
+.	O
+
+In	O
+contrast	O
+,	O
+all	O
+expected	O
+base	O
+pairs	O
+are	O
+observed	O
+for	O
+the	O
+Ox40	B-protein
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+(	O
+Fig	O
+.	O
+2c	O
+;	O
+see	O
+also	O
+Supplementary	O
+Notes	O
+).	O
+
+Structures	B-evidence
+of	O
+ROQ	B-structure_element
+bound	B-protein_state
+to	I-protein_state
+ADE	B-structure_element
+SL	B-structure_element
+RNAs	B-chemical
+
+To	O
+elucidate	O
+how	O
+Roquin	B-protein
+can	O
+recognize	O
+the	O
+novel	O
+SL	B-structure_element
+elements	O
+identified	O
+in	O
+the	O
+SELEX	B-experimental_method
+approach	O
+,	O
+we	O
+solved	B-experimental_method
+crystal	B-evidence
+structures	I-evidence
+of	O
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+ROQ	B-structure_element
+domain	O
+bound	B-protein_state
+to	I-protein_state
+these	O
+non	O
+-	O
+canonical	O
+RNA	B-chemical
+elements	O
+.	O
+
+The	O
+structures	B-evidence
+of	O
+ROQ	B-structure_element
+bound	B-protein_state
+to	I-protein_state
+the	O
+20	O
+-	O
+mer	O
+ADE	B-structure_element
+SL	B-structure_element
+(	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+)	O
+and	O
+to	O
+the	O
+22	O
+-	O
+mer	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+(	O
+Fig	O
+.	O
+3a	O
+)	O
+were	O
+refined	O
+to	O
+a	O
+resolution	O
+of	O
+3	O
+.	O
+0	O
+and	O
+2	O
+.	O
+2	O
+Å	O
+,	O
+respectively	O
+.	O
+
+In	O
+both	O
+structures	B-evidence
+the	O
+RNA	B-chemical
+adopts	O
+an	O
+SL	B-structure_element
+fold	O
+,	O
+where	O
+the	O
+hexaloop	B-structure_element
+is	O
+located	O
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+carboxy	O
+-	O
+terminal	O
+end	O
+of	O
+ROQ	B-structure_element
+helix	B-structure_element
+α4	B-structure_element
+and	O
+the	O
+N	O
+-	O
+terminal	O
+part	O
+of	O
+β3	B-structure_element
+(	O
+Fig	O
+.	O
+3a	O
+,	O
+b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+,	O
+b	O
+).	O
+
+The	O
+dsRNA	B-chemical
+stem	B-structure_element
+is	O
+recognized	O
+in	O
+the	O
+same	O
+way	O
+as	O
+previously	O
+reported	O
+for	O
+the	O
+Tnf	B-protein
+CDE	B-structure_element
+SL	B-structure_element
+RNA	B-chemical
+(	O
+Supplementary	O
+Fig	O
+.	O
+2c	O
+–	O
+e	O
+).	O
+
+As	O
+may	O
+be	O
+expected	O
+,	O
+the	O
+recognition	O
+of	O
+the	O
+hexaloop	B-structure_element
+is	O
+significantly	O
+different	O
+from	O
+the	O
+triloop	B-structure_element
+in	O
+the	O
+CDE	B-structure_element
+RNA	B-chemical
+(	O
+Fig	O
+.	O
+3b	O
+,	O
+c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2b	O
+).	O
+
+Interestingly	O
+,	O
+although	O
+the	O
+sequences	O
+of	O
+the	O
+ADE	B-structure_element
+SL	B-structure_element
+and	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+are	O
+different	O
+,	O
+the	O
+overall	O
+structures	B-evidence
+and	O
+protein	O
+–	O
+RNA	B-chemical
+contacts	O
+are	O
+virtually	O
+identical	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+,	O
+d	O
+,	O
+e	O
+).	O
+
+The	O
+only	O
+differences	O
+are	O
+a	O
+C19	B-residue_name_number
+bulge	B-structure_element
+,	O
+the	O
+non	B-bond_interaction
+-	I-bond_interaction
+Watson	I-bond_interaction
+–	I-bond_interaction
+Crick	I-bond_interaction
+G6	B-residue_name_number
+–	O
+G15	B-residue_name_number
+base	B-bond_interaction
+pair	I-bond_interaction
+and	O
+the	O
+interaction	O
+of	O
+U1	B-residue_name_number
+with	O
+Trp184	B-residue_name_number
+and	O
+Phe194	B-residue_name_number
+in	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+(	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+,	O
+e	O
+–	O
+g	O
+).	O
+
+Given	O
+their	O
+highly	O
+similar	O
+binding	O
+modes	O
+we	O
+focus	O
+the	O
+following	O
+discussion	O
+on	O
+the	O
+structure	B-evidence
+of	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+,	O
+as	O
+it	O
+naturally	O
+exists	O
+in	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+and	O
+was	O
+solved	O
+at	O
+higher	O
+resolution	O
+.	O
+
+The	O
+overall	O
+orientation	O
+and	O
+recognition	O
+of	O
+the	O
+double	B-structure_element
+-	I-structure_element
+stranded	I-structure_element
+stem	I-structure_element
+in	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+is	O
+similar	O
+to	O
+the	O
+CDE	B-structure_element
+triloop	B-structure_element
+.	O
+
+Notably	O
+,	O
+the	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+hexaloop	I-structure_element
+in	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+binds	O
+to	O
+an	O
+extended	O
+surface	B-site
+on	O
+the	O
+ROQ	B-structure_element
+domain	O
+that	O
+cannot	O
+be	O
+accessed	O
+by	O
+the	O
+CDE	B-structure_element
+triloop	B-structure_element
+(	O
+Fig	O
+.	O
+3b	O
+,	O
+c	O
+)	O
+and	O
+includes	O
+a	O
+few	O
+pyrimidine	O
+-	O
+specific	O
+contacts	O
+.	O
+
+For	O
+example	O
+,	O
+the	O
+main	O
+chain	O
+atoms	O
+of	O
+Phe255	B-residue_name_number
+form	O
+two	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+the	O
+Watson	O
+–	O
+Crick	O
+face	O
+of	O
+the	O
+U11	B-residue_name_number
+base	O
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+
+Although	O
+in	O
+the	O
+structure	B-evidence
+of	O
+the	O
+Tnf	B-protein
+CDE	B-structure_element
+triloop	B-structure_element
+the	O
+Tyr250	B-residue_name_number
+side	O
+chain	O
+engages	O
+only	O
+one	O
+hydrogen	B-bond_interaction
+bond	I-bond_interaction
+to	O
+the	O
+phosphate	O
+group	O
+of	O
+G12	B-residue_name_number
+(	O
+ref	O
+.),	O
+a	O
+number	O
+of	O
+contacts	O
+are	O
+observed	O
+with	O
+the	O
+hexaloop	B-structure_element
+(	O
+Fig	O
+.	O
+3d	O
+–	O
+f	O
+):	O
+the	O
+side	O
+chain	O
+hydroxyl	O
+of	O
+Tyr250	B-residue_name_number
+contacts	O
+the	O
+phosphate	O
+group	O
+of	O
+U11	B-residue_name_number
+,	O
+while	O
+the	O
+aromatic	O
+ring	O
+is	O
+positioned	O
+by	O
+parallel	O
+and	O
+orthogonal	O
+stacking	B-bond_interaction
+interactions	I-bond_interaction
+with	O
+the	O
+U10	B-residue_name_number
+and	O
+U11	B-residue_name_number
+bases	O
+,	O
+on	O
+either	O
+side	O
+,	O
+respectively	O
+(	O
+Fig	O
+.	O
+3e	O
+).	O
+
+In	O
+addition	O
+,	O
+the	O
+Tyr250	B-residue_name_number
+main	O
+-	O
+chain	O
+carbonyl	O
+interacts	O
+with	O
+U13	B-residue_name_number
+imino	O
+proton	O
+(	O
+Fig	O
+.	O
+3d	O
+,	O
+e	O
+).	O
+
+Val257	B-residue_name_number
+and	O
+Lys259	B-residue_name_number
+in	O
+strand	B-structure_element
+β3	B-structure_element
+are	O
+too	O
+far	O
+to	O
+contact	O
+the	O
+UGU	B-structure_element
+triloop	B-structure_element
+in	O
+the	O
+Tnf	B-protein
+CDE	B-structure_element
+RNA	B-chemical
+,	O
+but	O
+mediate	O
+a	O
+number	O
+of	O
+contacts	O
+with	O
+the	O
+longer	O
+hexaloop	B-structure_element
+.	O
+
+The	O
+side	O
+chain	O
+of	O
+Lys259	B-residue_name_number
+forms	O
+hydrogen	B-bond_interaction
+bonds	I-bond_interaction
+with	O
+the	O
+phosphate	O
+groups	O
+of	O
+U10	B-residue_name_number
+and	O
+U11	B-residue_name_number
+(	O
+Fig	O
+.	O
+3e	O
+,	O
+f	O
+)	O
+and	O
+the	O
+hydrophobic	O
+side	O
+chain	O
+of	O
+Val257	B-residue_name_number
+stacks	B-bond_interaction
+with	O
+the	O
+U11	B-residue_name_number
+base	O
+(	O
+Fig	O
+.	O
+3d	O
+,	O
+f	O
+).	O
+
+The	O
+RNA	B-chemical
+stem	B-structure_element
+is	O
+closed	O
+by	O
+a	O
+Watson	B-bond_interaction
+–	I-bond_interaction
+Crick	I-bond_interaction
+base	I-bond_interaction
+pair	I-bond_interaction
+(	O
+C8	B-residue_name_number
+–	O
+G15	B-residue_name_number
+in	O
+the	O
+hexaloop	B-structure_element
+SL	B-structure_element
+RNA	B-chemical
+).	O
+
+Interestingly	O
+,	O
+the	O
+G9	B-residue_name_number
+base	O
+stacks	B-bond_interaction
+on	O
+top	O
+of	O
+this	O
+closing	O
+base	O
+pair	O
+and	O
+takes	O
+a	O
+position	O
+that	O
+is	O
+very	O
+similar	O
+to	O
+the	O
+purine	O
+base	O
+of	O
+G12	B-residue_name_number
+in	O
+the	O
+CDE	B-structure_element
+triloop	B-structure_element
+(	O
+Fig	O
+.	O
+3b	O
+,	O
+c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2b	O
+).	O
+
+The	O
+G9	B-residue_name_number
+base	O
+does	O
+not	O
+form	O
+a	O
+base	O
+pair	O
+with	O
+A14	B-residue_name_number
+but	O
+rather	O
+the	O
+A14	B-residue_name_number
+base	O
+packs	O
+into	O
+the	O
+minor	B-site
+groove	I-site
+of	O
+the	O
+RNA	B-chemical
+duplex	O
+.	O
+
+This	O
+arrangement	O
+provides	O
+an	O
+extended	O
+stacking	B-bond_interaction
+interaction	I-bond_interaction
+of	O
+G9	B-residue_name_number
+,	O
+U10	B-residue_name_number
+and	O
+Tyr250	B-residue_name_number
+in	O
+the	O
+ROQ	B-structure_element
+domain	O
+at	O
+the	O
+5	O
+′-	O
+side	O
+of	O
+the	O
+RNA	B-chemical
+stem	B-structure_element
+(	O
+Fig	O
+.	O
+3e	O
+).	O
+
+The	O
+U11	B-residue_name_number
+and	O
+U13	B-residue_name_number
+bases	O
+stack	B-bond_interaction
+with	O
+each	O
+other	O
+in	O
+the	O
+vicinity	O
+of	O
+the	O
+ROQ	B-structure_element
+domain	O
+wing	B-structure_element
+(	O
+Fig	O
+.	O
+3b	O
+,	O
+d	O
+,	O
+f	O
+).	O
+
+This	O
+is	O
+possible	O
+by	O
+exposing	O
+the	O
+base	O
+C12	B-residue_name_number
+of	O
+the	O
+Ox	B-protein
+-	I-protein
+40	I-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+towards	O
+the	O
+solvent	O
+,	O
+which	O
+accordingly	O
+does	O
+not	O
+show	O
+any	O
+contacts	O
+to	O
+the	O
+protein	O
+.	O
+
+In	O
+summary	O
+,	O
+similar	O
+to	O
+the	O
+CDE	B-structure_element
+SL	B-structure_element
+,	O
+both	O
+the	O
+ADE	B-structure_element
+SL	B-structure_element
+and	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+are	O
+recognized	O
+mainly	O
+by	O
+non	O
+-	O
+sequence	O
+-	O
+specific	O
+contacts	O
+.	O
+
+However	O
+,	O
+these	O
+involve	O
+an	O
+extended	O
+binding	O
+surface	O
+on	O
+the	O
+ROQ	B-structure_element
+domain	O
+with	O
+a	O
+number	O
+of	O
+additional	O
+residues	O
+compared	O
+with	O
+the	O
+triloop	O
+RNA	B-chemical
+.	O
+
+NMR	B-experimental_method
+analysis	O
+of	O
+ROQ	B-structure_element
+interactions	O
+with	O
+ADE	B-structure_element
+SLs	B-structure_element
+
+We	O
+next	O
+used	O
+NMR	B-experimental_method
+spectroscopy	I-experimental_method
+to	O
+compare	O
+the	O
+ROQ	B-structure_element
+domain	O
+interaction	O
+of	O
+ADE	B-structure_element
+-	O
+like	O
+and	O
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+in	O
+solution	O
+.	O
+
+CSPs	B-evidence
+observed	O
+for	O
+amides	O
+in	O
+the	O
+ROQ	B-structure_element
+domain	O
+on	O
+binding	O
+to	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+b	O
+)	O
+map	O
+to	O
+residues	O
+that	O
+also	O
+mediate	O
+key	O
+interactions	O
+with	O
+CDE	B-structure_element
+SLs	B-structure_element
+,	O
+such	O
+as	O
+Lys220	B-residue_name_number
+,	O
+Lys239	B-residue_name_number
+/	O
+Thr240	B-residue_name_number
+and	O
+Lys259	B-residue_name_number
+/	O
+Arg260	B-residue_name_number
+(	O
+Fig	O
+.	O
+4b	O
+).	O
+
+This	O
+is	O
+fully	O
+consistent	O
+with	O
+the	O
+interactions	O
+observed	O
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+(	O
+Supplementary	O
+Fig	O
+.	O
+2c	O
+–	O
+e	O
+)	O
+and	O
+indicates	O
+a	O
+similar	O
+binding	B-site
+surface	I-site
+.	O
+
+However	O
+,	O
+there	O
+are	O
+also	O
+notable	O
+CSP	B-evidence
+differences	I-evidence
+when	O
+comparing	O
+binding	O
+of	O
+the	O
+ROQ	B-structure_element
+domain	O
+to	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+and	O
+to	O
+the	O
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+in	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+(	O
+Fig	O
+.	O
+4c	O
+),	O
+or	O
+to	O
+the	O
+Tnf	B-protein
+CDE	B-structure_element
+SL	B-structure_element
+RNA	B-chemical
+(	O
+Supplementary	O
+Fig	O
+.	O
+3	O
+and	O
+Supplementary	O
+Notes	O
+).	O
+
+For	O
+example	O
+,	O
+Ser253	B-residue_name_number
+is	O
+strongly	O
+affected	O
+only	O
+on	O
+binding	O
+to	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+b	O
+)	O
+in	O
+line	O
+with	O
+tight	O
+interactions	O
+with	O
+the	O
+hexaloop	B-structure_element
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+
+On	O
+the	O
+other	O
+hand	O
+,	O
+comparison	O
+of	O
+ROQ	B-structure_element
+domain	O
+binding	O
+with	O
+the	O
+ADE	B-structure_element
+and	O
+with	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+indicates	O
+almost	O
+identical	O
+NMR	B-experimental_method
+spectra	B-evidence
+and	O
+CSPs	B-evidence
+.	O
+
+This	O
+is	O
+consistent	O
+with	O
+the	O
+very	O
+similar	O
+structural	O
+features	O
+and	O
+mode	O
+of	O
+RNA	B-chemical
+recognition	O
+of	O
+the	O
+ROQ	B-structure_element
+domain	O
+with	O
+these	O
+RNAs	B-chemical
+(	O
+Supplementary	O
+Fig	O
+.	O
+2a	O
+,	O
+d	O
+,	O
+e	O
+).	O
+
+Mutational	B-experimental_method
+analysis	I-experimental_method
+of	O
+the	O
+ROQ	B-structure_element
+-	O
+ADE	B-structure_element
+interaction	O
+
+To	O
+examine	O
+the	O
+individual	O
+contributions	O
+of	O
+ROQ	B-structure_element
+–	O
+hexaloop	O
+interactions	O
+for	O
+complex	O
+formation	O
+,	O
+we	O
+performed	O
+electrophoretic	B-experimental_method
+mobility	I-experimental_method
+shift	I-experimental_method
+assays	I-experimental_method
+(	O
+EMSAs	B-experimental_method
+)	O
+with	O
+variants	O
+of	O
+the	O
+ROQ	B-structure_element
+domain	O
+and	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+RNA	B-chemical
+(	O
+Fig	O
+.	O
+5a	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+).	O
+
+Analysis	O
+of	O
+the	O
+interaction	O
+with	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+ROQ	B-structure_element
+revealed	O
+an	O
+apparent	O
+affinity	B-evidence
+in	O
+a	O
+similar	O
+range	O
+as	O
+for	O
+the	O
+Tnf	B-protein
+CDE	B-structure_element
+(	O
+Fig	O
+.	O
+5a	O
+and	O
+)	O
+Table	O
+2	O
+).	O
+
+We	O
+next	O
+tested	O
+a	O
+set	O
+of	O
+mutants	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+),	O
+which	O
+were	O
+designed	O
+based	O
+on	O
+contacts	O
+observed	O
+in	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+(	O
+Fig	O
+.	O
+3	O
+)	O
+and	O
+the	O
+NMR	B-experimental_method
+CSPs	B-evidence
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+b	O
+).	O
+
+In	O
+line	O
+with	O
+expectations	O
+from	O
+ROQ	B-complex_assembly
+-	I-complex_assembly
+Tnf	I-complex_assembly
+CDE	I-complex_assembly
+binding	O
+(	O
+see	O
+comparison	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+)	O
+and	O
+based	O
+on	O
+our	O
+structural	B-experimental_method
+analysis	I-experimental_method
+,	O
+the	O
+key	O
+residues	O
+Lys220	B-residue_name_number
+,	O
+Lys239	B-residue_name_number
+,	O
+Lys259	B-residue_name_number
+and	O
+Arg260	B-residue_name_number
+strongly	O
+reduce	O
+or	O
+abolish	O
+binding	O
+after	O
+replacement	B-experimental_method
+by	O
+alanine	B-residue_name
+.	O
+
+We	O
+also	O
+observe	O
+an	O
+almost	O
+complete	O
+loss	O
+of	O
+binding	O
+in	O
+the	O
+Y250A	B-mutant
+mutant	B-protein_state
+to	O
+the	O
+hexaloop	B-structure_element
+SL	B-structure_element
+RNA	B-chemical
+,	O
+which	O
+had	O
+not	O
+been	O
+seen	O
+for	O
+the	O
+Tnf	B-protein
+CDE	B-structure_element
+previously	O
+(	O
+Fig	O
+.	O
+5a	O
+).	O
+
+This	O
+underlines	O
+the	O
+central	O
+role	O
+of	O
+Tyr250	B-residue_name_number
+for	O
+stabilization	O
+of	O
+the	O
+hexaloop	B-structure_element
+structure	O
+and	O
+recognition	O
+by	O
+stacking	B-bond_interaction
+interactions	I-bond_interaction
+(	O
+Fig	O
+.	O
+3b	O
+,	O
+e	O
+).	O
+
+Mutation	B-experimental_method
+of	O
+Ser253	B-residue_name_number
+,	O
+which	O
+shows	O
+large	O
+CSPs	B-evidence
+in	O
+the	O
+NMR	B-experimental_method
+titrations	I-experimental_method
+(	O
+Fig	O
+.	O
+4a	O
+,	O
+b	O
+),	O
+does	O
+not	O
+significantly	O
+impair	O
+complex	O
+formation	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+).	O
+
+The	O
+large	O
+chemical	B-evidence
+shift	I-evidence
+change	I-evidence
+is	O
+probably	O
+caused	O
+by	O
+ring	O
+current	O
+effects	O
+induced	O
+by	O
+the	O
+close	O
+proximity	O
+of	O
+the	O
+U11	B-residue_name_number
+and	O
+U13	B-residue_name_number
+bases	O
+.	O
+
+Finally	O
+,	O
+a	O
+mutant	B-protein_state
+in	O
+the	O
+wing	B-structure_element
+of	O
+the	O
+ROQ	B-structure_element
+domain	O
+(	O
+S265Y	B-mutant
+)	O
+does	O
+only	O
+slightly	O
+impair	O
+binding	O
+,	O
+as	O
+has	O
+been	O
+previously	O
+observed	O
+for	O
+the	O
+interaction	O
+with	O
+the	O
+Tnf	B-protein
+CDE	B-structure_element
+(	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+).	O
+
+This	O
+indicates	O
+that	O
+replacement	B-experimental_method
+by	O
+Tyr	B-residue_name
+does	O
+not	O
+strongly	O
+affect	O
+the	O
+RNA	B-chemical
+interaction	O
+,	O
+and	O
+that	O
+some	O
+conformational	O
+variations	O
+are	O
+tolerated	O
+.	O
+
+Thus	O
+,	O
+the	O
+mutational	B-experimental_method
+analysis	I-experimental_method
+is	O
+fully	O
+consistent	O
+with	O
+the	O
+recognition	O
+of	O
+the	O
+hexaloop	B-structure_element
+observed	O
+in	O
+our	O
+crystal	B-evidence
+structures	I-evidence
+.	O
+
+To	O
+prove	O
+the	O
+contribution	O
+of	O
+the	O
+key	O
+residue	O
+Tyr250	B-residue_name_number
+in	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+to	O
+Ox40	B-protein
+mRNA	B-chemical
+recognition	O
+and	O
+regulation	O
+,	O
+we	O
+set	O
+up	O
+a	O
+retroviral	B-experimental_method
+reconstitution	I-experimental_method
+system	I-experimental_method
+in	O
+Roquin	B-protein
+-	O
+deficient	O
+CD4	O
++	O
+T	O
+cells	O
+.	O
+
+Isolated	O
+CD4	O
++	O
+T	O
+cells	O
+from	O
+Rc3h1	B-gene
+/	O
+2fl	B-gene
+/	O
+fl	B-gene
+;	O
+Cd4	O
+-	O
+Cre	O
+-	O
+ERT2	O
+;	O
+rtTA	O
+mice	B-taxonomy_domain
+harbouring	O
+floxed	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+/	O
+2	B-protein
+encoding	O
+alleles	O
+,	O
+a	O
+tamoxifen	B-chemical
+-	O
+inducible	O
+Cre	O
+recombinase	O
+and	O
+the	O
+reverse	B-protein_type
+tetracycline	I-protein_type
+-	I-protein_type
+controlled	I-protein_type
+transactivator	I-protein_type
+rtTA	B-protein
+were	O
+treated	O
+in	O
+vitro	O
+with	O
+4	B-chemical
+-	I-chemical
+hydroxy	I-chemical
+tamoxifen	I-chemical
+,	O
+to	O
+induce	O
+deletion	O
+.	O
+
+The	O
+cells	O
+were	O
+then	O
+transduced	O
+with	O
+doxycycline	B-chemical
+-	O
+inducible	O
+retroviral	O
+vectors	O
+to	O
+reconstitute	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+expression	O
+(	O
+Fig	O
+.	O
+5b	O
+).	O
+
+Depletion	O
+of	O
+Roquin	B-protein
+proteins	O
+on	O
+tamoxifen	B-chemical
+treatment	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+5a	O
+)	O
+strongly	O
+increased	O
+surface	O
+expression	O
+of	O
+Ox40	B-protein
+and	O
+Icos	B-protein
+(	O
+Fig	O
+.	O
+5c	O
+).	O
+
+This	O
+increase	O
+in	O
+surface	O
+expression	O
+of	O
+both	O
+costimulatory	B-protein_type
+receptors	I-protein_type
+was	O
+partially	O
+corrected	O
+by	O
+the	O
+doxycycline	B-chemical
+-	O
+induced	O
+reconstitution	O
+with	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+WT	B-protein_state
+protein	O
+(	O
+Fig	O
+.	O
+5c	O
+left	O
+panels	O
+).	O
+
+Importantly	O
+,	O
+no	O
+effect	O
+was	O
+observed	O
+on	O
+expression	O
+of	O
+the	O
+Y250A	B-mutant
+mutant	B-protein_state
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+or	O
+the	O
+K220A	B-mutant
+,	O
+K239A	B-mutant
+and	O
+R260	B-mutant
+mutant	B-protein_state
+,	O
+which	O
+is	O
+strongly	O
+impaired	O
+in	O
+CDE	B-structure_element
+SL	B-structure_element
+interactions	O
+(	O
+Fig	O
+.	O
+5c	O
+middle	O
+and	O
+right	O
+panels	O
+).	O
+
+However	O
+,	O
+it	O
+is	O
+also	O
+possible	O
+that	O
+continuous	O
+overexpression	B-experimental_method
+of	O
+targets	O
+following	O
+Roquin	B-protein
+deletion	O
+induces	O
+a	O
+hyperactivated	O
+state	O
+in	O
+the	O
+T	O
+cells	O
+.	O
+
+This	O
+hyperactivation	O
+,	O
+compared	O
+with	O
+the	O
+actual	O
+posttranscriptional	O
+derepression	O
+,	O
+may	O
+contribute	O
+even	O
+stronger	O
+to	O
+the	O
+increased	O
+Icos	B-protein
+and	O
+Ox40	B-protein
+expression	O
+levels	O
+.	O
+
+Hence	O
+,	O
+our	O
+structure	B-experimental_method
+–	I-experimental_method
+function	I-experimental_method
+analyses	I-experimental_method
+conclusively	O
+show	O
+that	O
+the	O
+Y250	B-residue_name_number
+residue	O
+is	O
+essential	O
+for	O
+Roquin	B-protein
+interaction	O
+and	O
+regulation	O
+of	O
+Ox40	B-protein
+,	O
+and	O
+potentially	O
+also	O
+for	O
+other	O
+Roquin	B-protein
+targets	O
+such	O
+as	O
+Icos	B-protein
+.	O
+
+We	O
+also	O
+investigated	O
+the	O
+role	O
+of	O
+individual	O
+nucleotides	O
+in	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+for	O
+complex	O
+formation	O
+with	O
+the	O
+ROQ	B-structure_element
+domain	O
+.	O
+
+We	O
+designed	O
+four	O
+mutants	O
+(	O
+Mut1	O
+–	O
+4	O
+,	O
+see	O
+Supplementary	O
+Fig	O
+.	O
+6	O
+)	O
+that	O
+were	O
+expected	O
+to	O
+disrupt	O
+key	O
+interactions	O
+with	O
+the	O
+protein	O
+according	O
+to	O
+our	O
+co	B-evidence
+-	I-evidence
+crystal	I-evidence
+structure	I-evidence
+(	O
+Fig	O
+.	O
+3d	O
+–	O
+f	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+2	O
+).	O
+
+NMR	B-experimental_method
+analysis	O
+confirmed	O
+that	O
+all	O
+mutant	B-protein_state
+RNAs	B-chemical
+formed	O
+the	O
+same	O
+base	O
+pairs	O
+in	O
+the	O
+stem	B-structure_element
+region	I-structure_element
+,	O
+identical	O
+to	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+(	O
+Fig	O
+.	O
+2b	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+6	O
+).	O
+
+We	O
+next	O
+used	O
+surface	B-experimental_method
+plasmon	I-experimental_method
+resonance	I-experimental_method
+experiments	O
+to	O
+determine	O
+dissociation	B-evidence
+constants	I-evidence
+for	O
+the	O
+ROQ	B-structure_element
+-	O
+RNA	B-chemical
+interaction	O
+(	O
+Table	O
+2	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+7	O
+).	O
+
+Although	O
+the	O
+replacement	B-experimental_method
+of	O
+a	O
+C8	B-residue_name_number
+–	O
+G15	B-residue_name_number
+closing	O
+base	O
+pair	O
+by	O
+A	B-residue_name
+-	O
+U	B-residue_name
+(	O
+Mut	B-mutant
+4	I-mutant
+)	O
+only	O
+reduces	O
+the	O
+affinity	B-evidence
+threefold	O
+,	O
+reduction	O
+of	O
+loop	B-structure_element
+size	O
+in	O
+the	O
+A14C	B-mutant
+mutant	B-protein_state
+(	O
+Mut	B-mutant
+1	I-mutant
+,	O
+see	O
+Table	O
+2	O
+)	O
+reduces	O
+the	O
+affinity	B-evidence
+and	O
+binding	O
+is	O
+not	O
+detected	O
+by	O
+surface	B-experimental_method
+plasmon	I-experimental_method
+resonance	I-experimental_method
+.	O
+
+As	O
+intended	O
+,	O
+the	O
+mutation	O
+Mut	B-mutant
+1	I-mutant
+allows	O
+the	O
+formation	O
+of	O
+an	O
+additional	O
+base	O
+pair	O
+and	O
+thus	O
+leads	O
+to	O
+the	O
+formation	O
+of	O
+a	O
+tetraloop	B-structure_element
+with	O
+a	O
+new	O
+G	B-residue_name
+-	O
+C	B-residue_name
+closing	O
+base	O
+pair	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+6a	O
+).	O
+
+Consistent	O
+with	O
+the	O
+structural	B-experimental_method
+analysis	I-experimental_method
+,	O
+we	O
+assume	O
+that	O
+this	O
+variant	O
+alters	O
+the	O
+hexaloop	B-structure_element
+conformation	O
+and	O
+thus	O
+reduces	O
+the	O
+interaction	O
+with	O
+ROQ	B-structure_element
+.	O
+
+Disruption	O
+of	O
+stacking	B-bond_interaction
+interactions	I-bond_interaction
+between	O
+G15	B-residue_name_number
+,	O
+G9	B-residue_name_number
+and	O
+Y250	B-residue_name_number
+in	O
+the	O
+G9C	B-mutant
+mutant	B-protein_state
+(	O
+Mut	B-mutant
+2	I-mutant
+)	O
+completely	O
+abolished	O
+binding	O
+of	O
+ROQ	B-structure_element
+to	O
+the	O
+SL	B-structure_element
+RNA	B-chemical
+(	O
+Table	O
+2	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+7	O
+).	O
+
+No	O
+binding	O
+is	O
+also	O
+observed	O
+for	O
+the	O
+U11AU13G	B-mutant
+double	B-protein_state
+mutant	I-protein_state
+(	O
+Mut	B-mutant
+3	I-mutant
+)	O
+(	O
+Table	O
+2	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+7	O
+),	O
+which	O
+abolishes	O
+specific	O
+interactions	O
+mediated	O
+by	O
+U11	B-residue_name_number
+and	O
+U13	B-residue_name_number
+in	O
+the	O
+hexaloop	B-structure_element
+with	O
+ROQ	B-structure_element
+(	O
+Fig	O
+.	O
+3d	O
+).	O
+
+Consistent	O
+with	O
+the	O
+SELEX	B-experimental_method
+consensus	O
+(	O
+Fig	O
+.	O
+1b	O
+),	O
+all	O
+of	O
+the	O
+tested	O
+mutations	B-experimental_method
+of	O
+conserved	B-protein_state
+nucleotides	B-chemical
+in	O
+the	O
+loop	B-structure_element
+reduce	O
+or	O
+abolish	O
+the	O
+interaction	O
+with	O
+ROQ	B-structure_element
+.	O
+
+Interestingly	O
+,	O
+the	O
+affinity	B-evidence
+of	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+Tnf	B-protein
+CDE	B-structure_element
+and	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SLs	B-structure_element
+to	O
+ROQ	B-structure_element
+are	O
+very	O
+similar	O
+(	O
+42	O
+and	O
+81	O
+nM	O
+,	O
+respectively	O
+,	O
+Table	O
+2	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+7	O
+).	O
+
+Roquin	B-protein
+binding	O
+to	O
+different	O
+SLs	B-structure_element
+in	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+
+We	O
+have	O
+recently	O
+shown	O
+that	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+binds	O
+to	O
+a	O
+CDE	B-structure_element
+-	O
+like	O
+motif	O
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+Ox40	B-protein
+mRNA	B-chemical
+(	O
+Figs	O
+1d	O
+and	O
+4c	O
+).	O
+
+We	O
+therefore	O
+investigated	O
+whether	O
+the	O
+interactions	O
+with	O
+the	O
+CDE	B-structure_element
+-	O
+like	O
+and	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+both	O
+contribute	O
+to	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+binding	O
+in	O
+the	O
+context	O
+of	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+.	O
+
+The	O
+binding	B-evidence
+affinities	I-evidence
+of	O
+either	O
+motif	O
+for	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+domain	I-structure_element
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+(	O
+residues	O
+2	B-residue_range
+–	I-residue_range
+440	I-residue_range
+)	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+8a	O
+,	O
+b	O
+)	O
+or	O
+the	O
+ROQ	B-structure_element
+domain	O
+alone	B-protein_state
+are	O
+in	O
+a	O
+similar	O
+range	O
+(	O
+Table	O
+2	O
+).	O
+
+The	O
+dissociation	B-evidence
+constants	I-evidence
+for	O
+the	O
+ROQ	B-structure_element
+interaction	O
+with	O
+the	O
+Ox40	B-protein
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+and	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+are	O
+1	O
+,	O
+460	O
+and	O
+81	O
+nM	O
+,	O
+respectively	O
+(	O
+Table	O
+2	O
+).	O
+
+This	O
+is	O
+consistent	O
+with	O
+the	O
+extended	O
+binding	B-site
+interface	I-site
+and	O
+additional	O
+interactions	O
+observed	O
+with	O
+the	O
+hexaloop	B-structure_element
+,	O
+and	O
+suggests	O
+a	O
+preferential	O
+binding	O
+to	O
+the	O
+hexaloop	B-structure_element
+SL	B-structure_element
+RNA	B-chemical
+in	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+.	O
+
+We	O
+designed	O
+different	O
+variants	O
+of	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+by	O
+point	B-experimental_method
+mutagenesis	I-experimental_method
+abrogating	O
+base	O
+pairing	O
+in	O
+the	O
+stem	B-structure_element
+region	I-structure_element
+,	O
+where	O
+none	O
+,	O
+individual	O
+,	O
+or	O
+both	O
+SL	B-structure_element
+RNA	B-chemical
+motifs	O
+were	O
+mutated	B-experimental_method
+to	O
+impair	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+binding	O
+(	O
+Fig	O
+.	O
+6a	O
+).	O
+
+These	O
+RNAs	B-chemical
+were	O
+then	O
+tested	O
+in	O
+EMSAs	B-experimental_method
+with	O
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+N	O
+terminus	O
+(	O
+residues	O
+2	B-residue_range
+–	I-residue_range
+440	I-residue_range
+)	O
+(	O
+Fig	O
+.	O
+6b	O
+).	O
+
+Gel	B-experimental_method
+shift	I-experimental_method
+assays	I-experimental_method
+show	O
+that	O
+binding	O
+to	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+construct	O
+leads	O
+to	O
+two	O
+distinct	O
+bands	O
+during	O
+the	O
+titrations	B-experimental_method
+,	O
+which	O
+should	O
+reflect	O
+binding	O
+to	O
+one	O
+and	O
+both	O
+RNA	B-chemical
+motifs	O
+,	O
+respectively	O
+.	O
+
+Consistent	O
+with	O
+this	O
+,	O
+both	O
+bands	O
+are	O
+strongly	O
+reduced	O
+when	O
+mutations	O
+are	O
+introduced	O
+that	O
+interfere	O
+with	O
+the	O
+formation	O
+of	O
+both	O
+SLs	B-structure_element
+.	O
+
+Notably	O
+,	O
+among	O
+these	O
+,	O
+the	O
+slower	O
+migrating	O
+band	O
+disappears	O
+when	O
+either	O
+of	O
+the	O
+two	O
+SL	B-structure_element
+RNA	B-chemical
+motifs	O
+is	O
+altered	O
+to	O
+impair	O
+Roquin	B-protein
+binding	O
+,	O
+indicating	O
+an	O
+interaction	O
+with	O
+the	O
+remaining	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+SL	B-structure_element
+.	O
+
+We	O
+thus	O
+conclude	O
+that	O
+Roquin	B-protein
+is	O
+able	O
+to	O
+bind	O
+to	O
+both	O
+SL	B-structure_element
+RNA	B-chemical
+motifs	O
+in	O
+the	O
+context	O
+of	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+.	O
+
+Regulation	O
+of	O
+Ox40	B-protein
+expression	O
+via	O
+two	O
+motifs	O
+in	O
+its	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+
+To	O
+investigate	O
+the	O
+role	O
+of	O
+the	O
+new	O
+ADE	B-structure_element
+-	O
+like	O
+motif	O
+in	O
+target	O
+mRNA	B-chemical
+regulation	O
+,	O
+we	O
+introduced	B-experimental_method
+Ox40	B-protein
+mRNA	B-chemical
+variants	O
+harbouring	O
+altered	B-protein_state
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+in	O
+cells	O
+.	O
+
+Considering	O
+the	O
+close	O
+proximity	O
+of	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+and	O
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+(	O
+Fig	O
+.	O
+6a	O
+),	O
+which	O
+is	O
+essential	O
+for	O
+Roquin	B-protein
+-	O
+mediated	O
+posttranscriptional	O
+regulation	O
+of	O
+Ox40	B-protein
+(	O
+ref	O
+.)	O
+we	O
+tested	O
+individual	O
+contributions	O
+and	O
+the	O
+functional	O
+cooperation	O
+of	O
+the	O
+two	O
+RNA	B-chemical
+elements	O
+by	O
+deletion	B-experimental_method
+and	I-experimental_method
+point	I-experimental_method
+mutagenesis	I-experimental_method
+abrogating	B-protein_state
+base	B-bond_interaction
+pairing	I-bond_interaction
+in	O
+the	O
+stem	B-structure_element
+region	I-structure_element
+(	O
+Fig	O
+.	O
+6a	O
+,	O
+c	O
+and	O
+Supplementary	O
+Fig	O
+.	O
+8c	O
+).	O
+
+Specifically	O
+,	O
+using	O
+retroviruses	B-taxonomy_domain
+we	O
+introduced	O
+Ox40	B-protein
+expression	O
+constructs	O
+placed	O
+under	O
+the	O
+control	O
+of	O
+different	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+into	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+/	O
+2	B-protein
+-	O
+deficient	O
+mouse	B-taxonomy_domain
+embryonic	O
+fibroblasts	O
+.	O
+
+Doxycycline	B-chemical
+treatment	O
+of	O
+cells	O
+from	O
+this	O
+cell	O
+line	O
+enabled	O
+ectopic	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+and	O
+co	O
+-	O
+translational	O
+mCherry	O
+expression	O
+due	O
+to	O
+the	O
+stable	O
+integration	O
+of	O
+an	O
+inducible	O
+lentiviral	B-taxonomy_domain
+vector	O
+(	O
+Supplementary	O
+Fig	O
+.	O
+8c	O
+).	O
+
+The	O
+expression	O
+of	O
+Ox40	B-protein
+in	O
+cells	O
+with	O
+and	O
+without	O
+doxycycline	B-chemical
+treatment	O
+was	O
+then	O
+quantified	O
+by	O
+flow	B-experimental_method
+cytometry	I-experimental_method
+(	O
+Supplementary	O
+Fig	O
+.	O
+8c	O
+).	O
+
+Comparing	O
+the	O
+ratio	O
+of	O
+Ox40	B-protein
+mean	B-evidence
+fluorescence	I-evidence
+intensities	I-evidence
+in	O
+cells	O
+with	O
+and	O
+without	O
+doxycycline	B-chemical
+treatment	O
+normalized	O
+to	O
+the	O
+values	O
+from	O
+cells	O
+that	O
+expressed	O
+Ox40	B-protein
+constructs	O
+without	B-protein_state
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+revealed	O
+a	O
+comparable	O
+importance	O
+of	O
+both	O
+structural	O
+elements	O
+(	O
+Fig	O
+.	O
+6c	O
+).	O
+
+In	O
+fact	O
+,	O
+only	O
+deletion	B-experimental_method
+or	I-experimental_method
+point	I-experimental_method
+mutagenesis	I-experimental_method
+of	O
+the	O
+sequences	O
+encoding	O
+both	O
+structures	O
+at	O
+the	O
+same	O
+time	O
+(	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+1	B-residue_range
+–	I-residue_range
+80	I-residue_range
+and	O
+double	B-protein_state
+mut	I-protein_state
+)	O
+neutralized	O
+Roquin	B-protein
+-	O
+dependent	O
+repression	O
+of	O
+Ox40	B-protein
+.	O
+
+In	O
+contrast	O
+,	O
+individual	O
+mutations	B-experimental_method
+that	O
+left	O
+the	O
+hexaloop	B-structure_element
+(	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+1	B-residue_range
+–	I-residue_range
+120	I-residue_range
+or	O
+CDE	B-mutant
+mut	I-mutant
+)	O
+or	O
+the	O
+CDE	B-structure_element
+-	O
+like	O
+triloop	B-structure_element
+intact	B-protein_state
+still	O
+enabled	O
+Roquin	B-protein
+-	O
+dependent	O
+repression	O
+,	O
+which	O
+occurred	O
+in	O
+an	O
+attenuated	O
+manner	O
+compared	O
+with	O
+the	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+(	O
+Fig	O
+.	O
+6c	O
+).	O
+
+To	O
+further	O
+analyse	O
+the	O
+functional	O
+consequences	O
+of	O
+Roquin	B-protein
+binding	O
+to	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+,	O
+we	O
+also	O
+measured	O
+mRNA	B-evidence
+decay	I-evidence
+rates	I-evidence
+after	O
+introducing	O
+the	O
+different	O
+Ox40	B-protein
+constructs	O
+into	O
+HeLa	O
+tet	O
+-	O
+off	O
+cells	O
+that	O
+allow	O
+to	O
+turn	O
+off	O
+transcription	O
+from	O
+the	O
+tetracycline	O
+-	O
+repressed	O
+vectors	O
+by	O
+addition	O
+of	O
+doxycycline	B-chemical
+(	O
+Fig	O
+.	O
+6d	O
+).	O
+
+Quantitative	B-experimental_method
+reverse	I-experimental_method
+transcriptase	I-experimental_method
+–	I-experimental_method
+PCR	I-experimental_method
+revealed	O
+a	O
+strong	O
+stabilization	O
+of	O
+the	O
+Ox40	B-protein
+mRNA	B-chemical
+by	O
+deletion	B-experimental_method
+of	I-experimental_method
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+(	O
+CDS	B-structure_element
+t1	B-evidence
+/	I-evidence
+2	I-evidence
+=	O
+311	O
+min	O
+vs	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+t1	B-evidence
+/	I-evidence
+2	I-evidence
+=	O
+96	O
+min	O
+).	O
+
+A	O
+comparable	O
+stabilization	O
+was	O
+achieved	O
+by	O
+combined	B-experimental_method
+mutation	I-experimental_method
+of	O
+the	O
+CDE	B-structure_element
+-	O
+like	O
+and	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+SLs	B-structure_element
+(	O
+ADE	B-structure_element
+/	O
+CDE	B-structure_element
+-	O
+like	O
+mut	B-protein_state
+t1	B-evidence
+/	I-evidence
+2	I-evidence
+=	O
+255	O
+min	O
+).	O
+
+Individual	O
+mutations	B-experimental_method
+of	O
+either	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+or	O
+the	O
+CDE	B-structure_element
+-	O
+like	O
+SLs	B-structure_element
+showed	O
+intermediate	O
+effects	O
+(	O
+ADE	B-structure_element
+-	O
+like	O
+mut	B-protein_state
+t1	B-evidence
+/	I-evidence
+2	I-evidence
+=	O
+170	O
+min	O
+,	O
+CDE	B-structure_element
+-	O
+like	O
+mut	B-protein_state
+t1	B-evidence
+/	I-evidence
+2	I-evidence
+=	O
+167	O
+min	O
+),	O
+respectively	O
+.	O
+
+These	O
+findings	O
+underscore	O
+the	O
+importance	O
+of	O
+both	O
+structural	O
+motifs	O
+and	O
+reveal	O
+that	O
+they	O
+have	O
+an	O
+additive	O
+effect	O
+on	O
+the	O
+regulation	O
+of	O
+Ox40	B-protein
+mRNA	B-chemical
+expression	O
+in	O
+cells	O
+.	O
+
+Recent	O
+structural	B-experimental_method
+and	I-experimental_method
+functional	I-experimental_method
+studies	I-experimental_method
+have	O
+provided	O
+first	O
+insight	O
+into	O
+the	O
+RNA	B-chemical
+binding	O
+of	O
+Roquin	B-protein
+.	O
+
+Structures	B-evidence
+of	O
+Roquin	B-protein
+bound	B-protein_state
+to	I-protein_state
+CDE	B-structure_element
+SL	B-structure_element
+RNAs	B-chemical
+indicated	O
+mainly	O
+shape	O
+recognition	O
+of	O
+the	O
+SL	B-structure_element
+RNA	B-chemical
+in	O
+the	O
+so	O
+-	O
+called	O
+A	B-site
+-	I-site
+site	I-site
+of	O
+the	O
+N	B-structure_element
+-	I-structure_element
+terminal	I-structure_element
+region	I-structure_element
+of	O
+the	O
+Roquin	B-protein
+protein	O
+with	O
+no	O
+sequence	O
+specificity	O
+,	O
+except	O
+the	O
+requirement	O
+for	O
+a	O
+pyrimidine	B-structure_element
+–	I-structure_element
+purine	I-structure_element
+–	I-structure_element
+pyrimidine	I-structure_element
+triloop	I-structure_element
+.	O
+
+Considering	O
+that	O
+the	O
+CDE	B-structure_element
+RNA	B-chemical
+recognition	O
+is	O
+mostly	O
+structure	O
+specific	O
+and	O
+not	O
+sequence	O
+dependent	O
+,	O
+a	O
+wide	O
+spectrum	O
+of	O
+target	O
+mRNA	B-chemical
+might	O
+be	O
+recognized	O
+by	O
+Roquin	B-protein
+.	O
+
+Here	O
+we	O
+have	O
+used	O
+SELEX	B-experimental_method
+assays	I-experimental_method
+to	O
+identify	O
+a	O
+novel	O
+RNA	B-structure_element
+recognition	I-structure_element
+motif	I-structure_element
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+,	O
+which	O
+is	O
+present	O
+in	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+and	O
+variations	O
+of	O
+which	O
+may	O
+be	O
+found	O
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+of	O
+many	O
+other	O
+genes	O
+.	O
+
+Our	O
+experiments	O
+show	O
+that	O
+this	O
+SELEX	B-experimental_method
+-	O
+derived	O
+ADE	B-structure_element
+shows	O
+functional	O
+activity	O
+comparable	O
+to	O
+the	O
+previously	O
+established	O
+CDE	B-structure_element
+motif	O
+.	O
+
+The	O
+ADE	B-structure_element
+and	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+adopt	O
+SL	B-structure_element
+folds	O
+with	O
+a	O
+hexaloop	B-structure_element
+instead	O
+of	O
+a	O
+triloop	B-structure_element
+.	O
+
+Notably	O
+,	O
+the	O
+recognition	O
+of	O
+the	O
+respective	O
+RNA	B-structure_element
+-	I-structure_element
+helical	I-structure_element
+stem	I-structure_element
+regions	I-structure_element
+by	O
+the	O
+ROQ	B-structure_element
+domain	O
+is	O
+identical	O
+for	O
+the	O
+triloop	B-structure_element
+and	O
+hexaloop	B-structure_element
+motifs	O
+.	O
+
+However	O
+,	O
+the	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+hexaloops	I-structure_element
+in	O
+the	O
+ADE	B-structure_element
+and	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNAs	B-chemical
+mediate	O
+a	O
+number	O
+of	O
+additional	O
+contacts	O
+with	O
+the	O
+helix	B-structure_element
+α4	B-structure_element
+and	O
+strand	B-structure_element
+β3	B-structure_element
+in	O
+the	O
+ROQ	B-structure_element
+domain	O
+that	O
+are	O
+absent	O
+in	O
+the	O
+triloop	B-structure_element
+CDE	B-structure_element
+(	O
+Fig	O
+.	O
+3b	O
+–	O
+f	O
+).	O
+
+Of	O
+particular	O
+importance	O
+for	O
+the	O
+hexaloop	B-structure_element
+recognition	O
+is	O
+Tyr250	B-residue_name_number
+,	O
+which	O
+acts	O
+as	O
+a	O
+stabilizing	O
+element	O
+for	O
+the	O
+integrity	O
+of	O
+a	O
+defined	O
+loop	B-structure_element
+conformation	O
+.	O
+
+It	O
+stacks	B-bond_interaction
+with	O
+nucleotides	O
+in	O
+the	O
+hexaloop	B-structure_element
+but	O
+not	O
+the	O
+CDE	B-structure_element
+triloop	B-structure_element
+(	O
+Fig	O
+.	O
+3b	O
+,	O
+c	O
+).	O
+
+The	O
+functional	O
+role	O
+of	O
+Tyr250	B-residue_name_number
+for	O
+ADE	B-structure_element
+-	O
+mediated	O
+mRNA	B-chemical
+regulation	O
+by	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+is	O
+thus	O
+explained	O
+by	O
+our	O
+experiments	O
+(	O
+Fig	O
+.	O
+5b	O
+,	O
+c	O
+).	O
+
+The	O
+preference	O
+for	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+hexaloops	I-structure_element
+depends	O
+on	O
+nucleotide	O
+-	O
+specific	O
+interactions	O
+of	O
+ROQ	B-structure_element
+with	O
+U10	B-residue_name_number
+,	O
+U11	B-residue_name_number
+and	O
+U13	B-residue_name_number
+in	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+.	O
+
+Consistent	O
+with	O
+this	O
+,	O
+loss	O
+of	O
+ROQ	B-structure_element
+binding	O
+is	O
+observed	O
+on	O
+replacement	B-experimental_method
+of	O
+U11	B-residue_name_number
+and	O
+U13	B-residue_name_number
+by	O
+other	O
+bases	O
+(	O
+Table	O
+2	O
+).	O
+
+In	O
+spite	O
+of	O
+these	O
+differences	O
+in	O
+some	O
+aspects	O
+of	O
+the	O
+RNA	B-chemical
+recognition	O
+,	O
+overall	O
+features	O
+of	O
+Roquin	B-protein
+targets	O
+are	O
+conserved	O
+in	O
+ADE	B-structure_element
+and	O
+CDE	B-structure_element
+-	O
+like	O
+RNAs	B-chemical
+,	O
+namely	O
+,	O
+a	O
+crucial	O
+role	O
+of	O
+non	O
+-	O
+sequence	O
+-	O
+specific	O
+contacts	O
+to	O
+the	O
+RNA	B-chemical
+stem	B-structure_element
+and	O
+mainly	O
+shape	O
+recognition	O
+of	O
+the	O
+hexa	B-structure_element
+-	I-structure_element
+and	I-structure_element
+triloops	I-structure_element
+,	O
+respectively	O
+.	O
+
+A	O
+unique	O
+feature	O
+of	O
+the	O
+bound	B-protein_state
+RNA	B-chemical
+structure	B-evidence
+,	O
+common	O
+to	O
+both	O
+tri	B-structure_element
+-	I-structure_element
+and	I-structure_element
+hexaloops	I-structure_element
+,	O
+is	O
+the	O
+stacking	B-bond_interaction
+of	O
+a	O
+purine	O
+base	O
+onto	O
+the	O
+closing	O
+base	O
+pair	O
+(	O
+Fig	O
+.	O
+3b	O
+,	O
+c	O
+).	O
+
+Previous	O
+structural	B-evidence
+data	I-evidence
+and	O
+the	O
+results	O
+presented	O
+here	O
+therefore	O
+suggest	O
+that	O
+Roquin	B-protein
+may	O
+recognize	O
+additional	O
+SL	B-structure_element
+RNA	B-chemical
+motifs	O
+,	O
+potentially	O
+with	O
+larger	O
+loops	B-structure_element
+.	O
+
+Interestingly	O
+,	O
+the	O
+SELEX	B-experimental_method
+-	O
+derived	O
+motif	O
+resembles	O
+the	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+motifs	I-structure_element
+that	O
+were	O
+identified	O
+recently	O
+by	O
+Murakawa	O
+et	O
+al	O
+..	O
+In	O
+their	O
+study	O
+,	O
+several	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+loops	I-structure_element
+of	O
+various	O
+sizes	O
+were	O
+identified	O
+by	O
+crosslinking	B-experimental_method
+and	I-experimental_method
+immunoprecipitation	I-experimental_method
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+using	O
+PAR	B-experimental_method
+-	I-experimental_method
+CLIP	I-experimental_method
+and	O
+the	O
+data	O
+also	O
+included	O
+sequences	O
+comprising	O
+the	O
+U	B-structure_element
+-	I-structure_element
+rich	I-structure_element
+hexaloop	I-structure_element
+identified	O
+in	O
+our	O
+present	O
+work	O
+.	O
+
+Most	O
+probably	O
+,	O
+the	O
+experimental	O
+setup	O
+of	O
+Murakawa	O
+et	O
+al	O
+.	O
+revealed	O
+both	O
+high	O
+-	O
+and	O
+low	O
+-	O
+affinity	O
+target	O
+motifs	O
+for	O
+Roquin	B-protein
+,	O
+whereas	O
+our	O
+structural	B-experimental_method
+study	I-experimental_method
+reports	O
+on	O
+a	O
+high	O
+-	O
+affinity	O
+binding	O
+motif	O
+.	O
+
+Notably	O
+,	O
+Murakawa	O
+et	O
+al	O
+.	O
+neither	O
+found	O
+the	O
+Roquin	B-protein
+-	O
+regulated	O
+Ox40	B-protein
+nor	O
+the	O
+Tnf	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+,	O
+as	O
+both	O
+genes	O
+are	O
+not	O
+expressed	O
+in	O
+HEK	O
+293	O
+cells	O
+.	O
+
+However	O
+,	O
+their	O
+newly	O
+identified	O
+U	O
+-	O
+rich	O
+target	O
+SL	B-structure_element
+within	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+A20	B-protein
+mRNA	B-chemical
+supports	O
+our	O
+conclusion	O
+that	O
+Roquin	B-protein
+can	O
+accept	O
+alternative	O
+target	O
+motifs	O
+apart	O
+from	O
+the	O
+classical	O
+CDE	B-structure_element
+triloop	B-structure_element
+arrangement	O
+.	O
+
+It	O
+remains	O
+to	O
+be	O
+seen	O
+which	O
+exact	O
+features	O
+govern	O
+the	O
+recognition	O
+of	O
+the	O
+A20	B-protein
+SL	B-structure_element
+by	O
+Roquin	B-protein
+.	O
+
+The	O
+regulatory	O
+cis	B-structure_element
+RNA	I-structure_element
+elements	I-structure_element
+in	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+may	O
+also	O
+be	O
+targeted	O
+by	O
+additional	O
+trans	O
+-	O
+acting	O
+factors	O
+.	O
+
+We	O
+have	O
+recently	O
+identified	O
+the	O
+endonuclease	B-protein_type
+Regnase	B-protein
+-	I-protein
+1	I-protein
+as	O
+a	O
+cofactor	O
+of	O
+Roquin	B-protein
+function	O
+that	O
+shares	O
+an	O
+overlapping	O
+set	O
+of	O
+target	O
+mRNAs	B-chemical
+.	O
+
+In	O
+another	O
+study	O
+,	O
+the	O
+overlap	O
+in	O
+targets	O
+was	O
+confirmed	O
+,	O
+but	O
+a	O
+mutually	O
+exclusive	O
+regulation	O
+was	O
+proposed	O
+based	O
+on	O
+studies	O
+in	O
+lipopolysaccharide	B-chemical
+(	O
+LPS	B-chemical
+)-	O
+stimulated	O
+myeloid	O
+cells	O
+.	O
+
+In	O
+these	O
+cells	O
+,	O
+Roquin	B-protein
+induced	O
+mRNA	B-chemical
+decay	O
+only	O
+for	O
+translationally	O
+inactive	B-protein_state
+mRNAs	B-chemical
+,	O
+while	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+-	O
+induced	O
+mRNA	B-chemical
+decay	O
+depended	O
+on	O
+active	O
+translation	O
+of	O
+the	O
+target	O
+.	O
+
+In	O
+CD4	O
++	O
+T	O
+cells	O
+,	O
+Ox40	B-protein
+does	O
+not	O
+show	O
+derepression	O
+in	O
+individual	O
+knockouts	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+or	O
+Roquin	B-protein
+-	I-protein
+2	I-protein
+encoding	O
+genes	O
+,	O
+but	O
+is	O
+strongly	O
+induced	O
+upon	O
+combined	O
+deficiency	B-experimental_method
+of	O
+both	O
+genes	O
+.	O
+
+In	O
+addition	O
+,	O
+conditional	O
+deletion	B-experimental_method
+of	I-experimental_method
+the	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+-	O
+encoding	O
+gene	O
+induced	O
+Ox40	B-protein
+expression	O
+in	O
+these	O
+cells	O
+.	O
+
+Whether	O
+induced	O
+decay	O
+of	O
+Ox40	B-protein
+mRNA	B-chemical
+by	O
+Roquin	B-protein
+or	O
+Regnase	B-protein_type
+proteins	O
+occurs	O
+in	O
+a	O
+mutually	O
+exclusive	O
+manner	O
+at	O
+different	O
+points	O
+during	O
+T	O
+-	O
+cell	O
+activation	O
+or	O
+shows	O
+cooperative	O
+regulation	O
+will	O
+have	O
+to	O
+await	O
+a	O
+direct	O
+comparison	O
+of	O
+T	O
+cells	O
+with	O
+single	O
+,	O
+double	B-experimental_method
+and	I-experimental_method
+triple	I-experimental_method
+knockouts	I-experimental_method
+of	O
+these	O
+genes	O
+.	O
+
+However	O
+,	O
+in	O
+cultures	O
+of	O
+CD4	O
++	O
+T	O
+cells	O
+,	O
+Ox40	B-protein
+is	O
+translated	O
+on	O
+day	O
+4	O
+–	O
+5	O
+and	O
+is	O
+expressed	O
+much	O
+higher	O
+in	O
+T	O
+cells	O
+with	O
+combined	O
+deficiency	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+and	O
+Roquin	B-protein
+-	I-protein
+2	I-protein
+.	O
+
+At	O
+this	O
+time	O
+point	O
+,	O
+the	O
+short	O
+-	O
+term	O
+inducible	O
+reconstitution	B-experimental_method
+with	O
+WT	B-protein_state
+Roquin	B-protein
+-	I-protein
+1	I-protein
+was	O
+effective	O
+to	O
+reduced	O
+Ox40	B-protein
+expression	O
+,	O
+demonstrating	O
+the	O
+regulation	O
+of	O
+a	O
+translationally	O
+active	B-protein_state
+mRNA	B-chemical
+by	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+in	O
+T	O
+cells	O
+(	O
+Fig	O
+.	O
+5c	O
+).	O
+
+Recombinant	O
+N	O
+-	O
+terminal	O
+protein	O
+fragments	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+or	O
+Roquin	B-protein
+-	I-protein
+2	I-protein
+bind	O
+with	O
+comparable	O
+affinity	O
+to	O
+Ox40	B-protein
+mRNA	B-chemical
+in	O
+EMSAs	B-experimental_method
+and	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+Ox40	B-protein
+is	O
+similarly	O
+retained	O
+by	O
+the	O
+two	O
+recombinant	O
+proteins	O
+in	O
+filter	B-experimental_method
+binding	I-experimental_method
+assays	I-experimental_method
+.	O
+
+Given	O
+the	O
+almost	O
+identical	O
+RNA	B-chemical
+contacts	O
+in	O
+both	O
+paralogues	O
+,	O
+we	O
+assume	O
+a	O
+similar	O
+recognition	O
+of	O
+ADE	B-structure_element
+and	O
+CDE	B-structure_element
+motifs	O
+in	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+by	O
+both	O
+proteins	O
+.	O
+
+In	O
+contrast	O
+,	O
+structural	O
+details	O
+on	O
+how	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+can	O
+interact	O
+with	O
+these	O
+SL	B-structure_element
+RNAs	B-chemical
+are	O
+currently	O
+missing	O
+.	O
+
+Surprisingly	O
+,	O
+transcriptome	O
+-	O
+wide	O
+mapping	O
+of	O
+Regnase	B-site
+-	I-site
+1	I-site
+-	I-site
+binding	I-site
+sites	I-site
+in	O
+crosslinking	B-experimental_method
+and	I-experimental_method
+immunoprecipitation	I-experimental_method
+experiments	I-experimental_method
+identified	O
+specific	O
+triloop	B-structure_element
+structures	O
+with	O
+pyrimidine	B-structure_element
+–	I-structure_element
+purine	I-structure_element
+–	I-structure_element
+pyrimidine	I-structure_element
+loops	I-structure_element
+in	O
+3	O
+-	O
+to	O
+7	O
+-	O
+nt	O
+-	O
+long	O
+stems	B-structure_element
+,	O
+as	O
+well	O
+as	O
+a	O
+novel	O
+hexaloop	B-structure_element
+structure	O
+in	O
+the	O
+Ptgs2	B-gene
+gene	O
+.	O
+
+Both	O
+were	O
+required	O
+for	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+-	O
+mediated	O
+repression	O
+.	O
+
+These	O
+findings	O
+therefore	O
+raise	O
+the	O
+possibility	O
+that	O
+Regnase	B-protein
+-	I-protein
+1	I-protein
+interacts	O
+with	O
+ADE	B-structure_element
+-	O
+like	O
+hexaloop	B-structure_element
+structures	O
+either	O
+in	O
+a	O
+direct	O
+or	O
+indirect	O
+manner	O
+.	O
+
+Nevertheless	O
+,	O
+it	O
+becomes	O
+clear	O
+that	O
+composite	O
+cis	B-structure_element
+-	I-structure_element
+elements	I-structure_element
+,	O
+that	O
+is	O
+,	O
+the	O
+presence	O
+of	O
+several	O
+SLs	B-structure_element
+as	O
+in	O
+Ox40	B-protein
+or	O
+Icos	B-protein
+,	O
+could	O
+attract	O
+multiple	O
+trans	O
+-	O
+acting	O
+factors	O
+that	O
+may	O
+potentially	O
+co	O
+-	O
+regulate	O
+or	O
+even	O
+act	O
+cooperatively	O
+to	O
+control	O
+mRNA	B-chemical
+expression	O
+through	O
+posttranscriptional	O
+pathways	O
+of	O
+gene	O
+regulation	O
+.	O
+
+The	O
+novel	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+loop	B-structure_element
+motif	I-structure_element
+that	O
+we	O
+have	O
+identified	O
+as	O
+a	O
+bona	O
+fide	O
+target	O
+of	O
+Roquin	B-protein
+now	O
+expands	O
+this	O
+multilayer	O
+mode	O
+of	O
+co	O
+-	O
+regulation	O
+.	O
+
+We	O
+suggest	O
+that	O
+differential	O
+regulation	O
+of	O
+mRNA	B-chemical
+expression	O
+is	O
+not	O
+only	O
+achieved	O
+through	O
+multiple	O
+regulators	O
+with	O
+individual	O
+preferences	O
+for	O
+a	O
+given	O
+motif	O
+or	O
+variants	O
+thereof	O
+,	O
+but	O
+that	O
+regulators	O
+may	O
+also	O
+identify	O
+and	O
+use	O
+distinct	O
+motifs	O
+,	O
+as	O
+long	O
+as	O
+they	O
+exhibit	O
+some	O
+basic	O
+features	O
+regarding	O
+shape	O
+,	O
+size	O
+and	O
+sequence	O
+.	O
+
+The	O
+presence	O
+of	O
+distinct	O
+motifs	O
+in	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+offers	O
+a	O
+broader	O
+variability	O
+for	O
+gene	O
+regulation	O
+by	O
+RNA	B-chemical
+cis	B-structure_element
+elements	I-structure_element
+.	O
+
+Their	O
+accessibility	O
+can	O
+be	O
+modulated	O
+by	O
+trans	O
+-	O
+acting	O
+factors	O
+that	O
+may	O
+bind	O
+regulatory	O
+motifs	O
+,	O
+unfold	O
+higher	O
+-	O
+order	O
+structures	O
+in	O
+the	O
+RNA	B-chemical
+or	O
+maintain	O
+a	O
+preference	O
+for	O
+duplex	O
+structures	O
+as	O
+was	O
+shown	O
+recently	O
+for	O
+mRNAs	B-chemical
+that	O
+are	O
+recognized	O
+by	O
+Staufen	B-protein
+-	I-protein
+1	I-protein
+(	O
+ref	O
+.).	O
+
+In	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+the	O
+Ox40	B-protein
+mRNA	B-chemical
+,	O
+we	O
+find	O
+one	O
+ADE	B-structure_element
+-	O
+like	O
+and	O
+one	O
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+,	O
+with	O
+similar	O
+binding	O
+to	O
+the	O
+ROQ	B-structure_element
+domain	O
+.	O
+
+The	O
+exact	O
+stoichiometry	O
+of	O
+Roquin	B-protein
+bound	B-protein_state
+to	I-protein_state
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+is	O
+unknown	O
+.	O
+
+The	O
+recently	O
+identified	O
+secondary	B-site
+binding	I-site
+site	I-site
+for	O
+dsRNA	B-chemical
+in	O
+Roquin	B-protein
+(	O
+B	B-site
+-	I-site
+site	I-site
+)	O
+could	O
+potentially	O
+allow	O
+for	O
+simultaneous	O
+binding	O
+of	O
+dsRNA	B-chemical
+and	O
+thereby	O
+promote	O
+engagement	O
+of	O
+Roquin	B-protein
+and	O
+target	O
+RNAs	B-chemical
+before	O
+recognition	O
+of	O
+high	O
+-	O
+affinity	B-evidence
+SLs	B-structure_element
+.	O
+
+In	O
+this	O
+respect	O
+,	O
+it	O
+is	O
+interesting	O
+to	O
+note	O
+that	O
+symmetry	O
+-	O
+related	O
+RNA	B-chemical
+molecules	O
+of	O
+both	O
+Tnf	B-protein
+CDE	B-structure_element
+and	O
+ADE	B-structure_element
+SL	B-structure_element
+RNAs	B-chemical
+are	O
+found	O
+in	O
+the	O
+respective	O
+crystal	B-evidence
+lattice	I-evidence
+in	O
+a	O
+position	O
+that	O
+corresponds	O
+to	O
+the	O
+recognition	O
+of	O
+dsRNA	B-chemical
+in	O
+the	O
+B	B-site
+site	I-site
+.	O
+
+This	O
+opens	O
+the	O
+possibility	O
+that	O
+one	O
+Roquin	B-protein
+molecule	O
+may	O
+cluster	O
+two	O
+motifs	O
+in	O
+a	O
+given	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+and	O
+/	O
+or	O
+cluster	O
+motifs	O
+from	O
+distinct	O
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+to	O
+enhance	O
+downstream	O
+processing	O
+.	O
+
+Interestingly	O
+,	O
+two	O
+SL	B-structure_element
+RNA	B-chemical
+elements	O
+that	O
+resemble	O
+bona	O
+fide	O
+ligands	O
+of	O
+Roquin	B-protein
+have	O
+also	O
+been	O
+identified	O
+in	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+of	O
+the	O
+Nfkbid	B-protein
+mRNA	B-chemical
+.	O
+
+We	O
+therefore	O
+hypothesize	O
+that	O
+the	O
+combination	O
+of	O
+multiple	O
+binding	B-site
+sites	I-site
+may	O
+be	O
+more	O
+commonly	O
+used	O
+to	O
+enhance	O
+the	O
+functional	O
+activity	O
+of	O
+Roquin	B-protein
+.	O
+
+At	O
+the	O
+same	O
+time	O
+,	O
+the	O
+combination	O
+of	O
+cis	B-structure_element
+elements	I-structure_element
+may	O
+be	O
+important	O
+for	O
+differential	O
+gene	O
+regulation	O
+,	O
+as	O
+composite	O
+cis	B-structure_element
+elements	I-structure_element
+with	O
+lower	O
+affinity	B-evidence
+may	O
+be	O
+less	O
+sensitive	O
+to	O
+Roquin	B-protein
+.	O
+
+This	O
+will	O
+lead	O
+to	O
+less	O
+effective	O
+repression	O
+in	O
+T	O
+cells	O
+when	O
+antigen	O
+recognition	O
+is	O
+of	O
+moderate	O
+signal	O
+strength	O
+and	O
+only	O
+incomplete	O
+cleavage	O
+of	O
+Roquin	B-protein
+by	O
+MALT1	B-protein
+occurs	O
+.	O
+
+For	O
+understanding	O
+the	O
+intricate	O
+complexity	O
+of	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+regulation	O
+,	O
+future	O
+work	O
+will	O
+be	O
+necessary	O
+by	O
+combining	O
+large	O
+-	O
+scale	O
+approaches	O
+,	O
+such	O
+as	O
+cross	B-experimental_method
+-	I-experimental_method
+linking	I-experimental_method
+and	I-experimental_method
+immunoprecipitation	I-experimental_method
+experiments	I-experimental_method
+to	O
+identify	O
+RNA	B-site
+-	I-site
+binding	I-site
+sites	I-site
+,	O
+and	O
+structural	B-experimental_method
+biology	I-experimental_method
+to	O
+dissect	O
+the	O
+underlying	O
+molecular	O
+mechanisms	O
+.	O
+
+SELEX	B-experimental_method
+identifies	O
+a	O
+novel	O
+SL	B-structure_element
+RNA	B-chemical
+ligand	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+.	O
+
+(	O
+a	O
+)	O
+Enriched	O
+hexamers	O
+that	O
+were	O
+found	O
+by	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+N	O
+terminus	O
+(	O
+residues	O
+2	B-residue_range
+–	I-residue_range
+440	I-residue_range
+)	O
+or	O
+Roquin	B-mutant
+-	I-mutant
+1	I-mutant
+M199R	I-mutant
+N	O
+terminus	O
+(	O
+residues	O
+2	B-residue_range
+–	I-residue_range
+440	I-residue_range
+)	O
+(	O
+see	O
+also	O
+Supplementary	O
+Fig	O
+.	O
+1	O
+).	O
+(	O
+b	O
+)	O
+An	O
+ADE	B-structure_element
+sequence	O
+motif	O
+in	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+closely	O
+resembles	O
+the	O
+MEME	B-experimental_method
+motif	O
+found	O
+in	O
+SELEX	B-experimental_method
+-	O
+enriched	O
+RNA	B-chemical
+sequences	O
+.	O
+
+(	O
+c	O
+)	O
+Conservation	O
+of	O
+the	O
+motif	O
+found	O
+in	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTRs	I-structure_element
+for	O
+various	O
+species	O
+as	O
+indicated	O
+.	O
+
+rn5	B-gene
+is	O
+the	O
+fifth	O
+assembly	O
+version	O
+of	O
+the	O
+rat	B-taxonomy_domain
+(	O
+Rattus	B-species
+novegicus	I-species
+).	O
+(	O
+d	O
+)	O
+Schematic	O
+representation	O
+of	O
+the	O
+predicted	O
+SELEX	B-experimental_method
+-	O
+derived	O
+consensus	O
+SL	B-structure_element
+,	O
+ADE	B-structure_element
+and	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+hexaloop	B-structure_element
+SL	B-structure_element
+.	O
+
+The	O
+broken	O
+line	O
+between	O
+the	O
+G	O
+–	O
+G	O
+base	O
+pair	O
+in	O
+the	O
+ADE	B-structure_element
+SL	B-structure_element
+indicates	O
+a	O
+putative	O
+non	B-bond_interaction
+-	I-bond_interaction
+Watson	I-bond_interaction
+–	I-bond_interaction
+Crick	I-bond_interaction
+pairing	I-bond_interaction
+.	O
+
+The	O
+Ox40	B-protein
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+and	O
+the	O
+Tnf	B-protein
+CDE	B-structure_element
+SL	B-structure_element
+are	O
+shown	O
+for	O
+comparison	O
+.	O
+
+NMR	B-experimental_method
+analysis	O
+of	O
+the	O
+SL	B-structure_element
+RNAs	B-chemical
+used	O
+in	O
+this	O
+study	O
+.	O
+
+Imino	O
+proton	O
+regions	O
+of	O
+one	O
+-	O
+dimensional	O
+1H	B-experimental_method
+NMR	I-experimental_method
+spectra	B-evidence
+of	O
+(	O
+a	O
+)	O
+the	O
+ADE	B-structure_element
+SL	B-structure_element
+(	O
+b	O
+),	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+and	O
+(	O
+c	O
+)	O
+the	O
+Ox40	B-protein
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+are	O
+shown	O
+for	O
+free	B-protein_state
+RNAs	B-chemical
+(	O
+black	O
+)	O
+and	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+ROQ	B-structure_element
+domain	O
+(	O
+red	O
+).	O
+
+The	O
+respective	O
+SL	B-structure_element
+RNAs	B-chemical
+and	O
+their	O
+base	O
+pairs	O
+are	O
+indicated	O
+.	O
+
+Red	O
+asterisks	O
+indicate	O
+NMR	B-experimental_method
+signals	O
+of	O
+the	O
+protein	O
+.	O
+
+Green	O
+lines	O
+in	O
+the	O
+secondary	O
+structure	O
+schemes	O
+on	O
+the	O
+left	O
+refer	O
+to	O
+visible	O
+imino	O
+NMR	B-experimental_method
+signals	B-evidence
+and	O
+thus	O
+experimental	O
+confirmation	O
+of	O
+the	O
+base	O
+pairs	O
+indicated	O
+.	O
+
+The	O
+dotted	O
+green	O
+line	O
+between	O
+G6	B-residue_name_number
+and	O
+G15	B-residue_name_number
+in	O
+a	O
+highlights	O
+a	O
+G	B-residue_name
+–	O
+G	B-residue_name
+base	O
+pair	O
+.	O
+
+Structure	B-evidence
+of	O
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+ROQ	B-structure_element
+domain	O
+bound	B-protein_state
+to	I-protein_state
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+RNA	B-chemical
+.	O
+
+(	O
+a	O
+)	O
+Cartoon	O
+presentation	O
+of	O
+the	O
+crystal	B-evidence
+structure	I-evidence
+of	O
+the	O
+ROQ	B-structure_element
+domain	O
+(	O
+residues	O
+174	B-residue_range
+–	I-residue_range
+325	I-residue_range
+;	O
+blue	O
+)	O
+and	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+(	O
+magenta	O
+).	O
+
+Selected	O
+RNA	B-chemical
+bases	O
+and	O
+protein	O
+secondary	O
+structure	O
+elements	O
+are	O
+labelled	O
+.	O
+
+(	O
+b	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+(	O
+bases	O
+in	O
+the	O
+RNA	B-chemical
+hexaloop	B-structure_element
+are	O
+shown	O
+in	O
+magenta	O
+)	O
+and	O
+(	O
+c	O
+)	O
+the	O
+previously	O
+reported	O
+structure	B-evidence
+of	O
+the	O
+ROQ	B-complex_assembly
+-	I-complex_assembly
+Tnf	I-complex_assembly
+CDE	I-complex_assembly
+complex	O
+(	O
+bases	O
+of	O
+the	O
+triloop	O
+RNA	B-chemical
+are	O
+shown	O
+in	O
+green	O
+).	O
+
+Only	O
+RNA	B-site
+-	I-site
+interacting	I-site
+residues	I-site
+that	O
+are	O
+different	O
+in	O
+both	O
+structures	B-evidence
+are	O
+shown	O
+.	O
+
+Both	O
+protein	O
+chains	O
+and	O
+remaining	O
+parts	O
+of	O
+both	O
+RNAs	B-chemical
+are	O
+shown	O
+in	O
+grey	O
+and	O
+protein	O
+residue	O
+side	O
+chains	O
+are	O
+shown	O
+in	O
+turquoise	O
+.	O
+(	O
+d	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+contacts	O
+between	O
+the	O
+ROQ	B-structure_element
+domain	O
+and	O
+nucleotides	O
+U11	B-residue_name_number
+and	O
+U13	B-residue_name_number
+of	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+RNA	B-chemical
+.	O
+
+The	O
+nucleotides	O
+interact	O
+with	O
+the	O
+C	O
+-	O
+terminal	O
+end	O
+of	O
+helix	B-structure_element
+α4	B-structure_element
+(	O
+Tyr250	B-residue_name_number
+and	O
+Ser253	B-residue_name_number
+)	O
+and	O
+the	O
+N	O
+-	O
+terminal	O
+part	O
+of	O
+strand	B-structure_element
+β3	B-structure_element
+(	O
+Phe255	B-residue_name_number
+and	O
+Val257	B-residue_name_number
+).	O
+
+The	O
+protein	O
+chain	O
+is	O
+shown	O
+in	O
+turquoise	O
+and	O
+the	O
+RNA	B-chemical
+is	O
+shown	O
+in	O
+grey	O
+.	O
+
+(	O
+e	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+contacts	O
+between	O
+the	O
+ROQ	B-structure_element
+domain	O
+and	O
+nucleotides	O
+U10	B-residue_name_number
+,	O
+U11	B-residue_name_number
+and	O
+U13	B-residue_name_number
+in	O
+the	O
+RNA	B-chemical
+hexaloop	B-structure_element
+.	O
+
+U11	B-residue_name_number
+and	O
+U13	B-residue_name_number
+contact	O
+the	O
+C	O
+-	O
+terminal	O
+end	O
+of	O
+helix	B-structure_element
+α4	B-structure_element
+:	O
+residues	O
+Tyr250	B-residue_name_number
+and	O
+Gln247	B-residue_name_number
+.	O
+
+The	O
+side	O
+chain	O
+of	O
+Tyr250	B-residue_name_number
+makes	O
+hydrophobic	B-bond_interaction
+interactions	I-bond_interaction
+with	O
+the	O
+pyrimidine	O
+side	O
+chain	O
+of	O
+U10	B-residue_name_number
+on	O
+one	O
+side	O
+and	O
+U11	B-residue_name_number
+on	O
+the	O
+other	O
+side	O
+.	O
+
+Lys259	B-residue_name_number
+interacts	O
+with	O
+the	O
+phosphate	O
+groups	O
+of	O
+U10	B-residue_name_number
+and	O
+U11	B-residue_name_number
+.	O
+
+(	O
+f	O
+)	O
+Close	O
+-	O
+up	O
+view	O
+of	O
+the	O
+hydrophobic	B-bond_interaction
+interaction	I-bond_interaction
+between	O
+Val257	B-residue_name_number
+and	O
+U11	B-residue_name_number
+,	O
+as	O
+well	O
+as	O
+the	O
+double	O
+hydrogen	B-bond_interaction
+bond	I-bond_interaction
+of	O
+Lys259	B-residue_name_number
+with	O
+phosphate	O
+groups	O
+of	O
+U10	B-residue_name_number
+and	O
+U11	B-residue_name_number
+.	O
+
+NMR	B-experimental_method
+analysis	O
+of	O
+ROQ	B-structure_element
+domain	O
+interactions	O
+with	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+hexaloop	B-structure_element
+RNA	B-chemical
+.	O
+
+(	O
+a	O
+)	O
+Overlay	B-experimental_method
+of	O
+1H	B-experimental_method
+,	I-experimental_method
+15N	I-experimental_method
+HSQC	I-experimental_method
+spectra	B-evidence
+of	O
+either	O
+the	O
+free	B-protein_state
+ROQ	B-structure_element
+domain	O
+(	O
+171	B-residue_range
+–	I-residue_range
+326	I-residue_range
+,	O
+black	O
+)	O
+or	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+stoichiometric	O
+amounts	O
+of	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+(	O
+red	O
+).	O
+
+(	O
+b	O
+)	O
+Plot	O
+of	O
+chemical	B-evidence
+shift	I-evidence
+change	I-evidence
+versus	O
+residue	O
+number	O
+in	O
+the	O
+ROQ	B-structure_element
+domain	O
+(	O
+residues	O
+171	B-residue_range
+–	I-residue_range
+326	I-residue_range
+)	O
+from	O
+a	O
+.	O
+Grey	O
+negative	O
+bars	O
+indicate	O
+missing	O
+assignments	O
+in	O
+one	O
+of	O
+the	O
+spectra	B-evidence
+.	O
+
+Gaps	O
+indicate	O
+prolines	B-residue_name
+.	O
+
+(	O
+c	O
+)	O
+Overlay	B-experimental_method
+of	O
+the	O
+ROQ	B-structure_element
+domain	O
+alone	B-protein_state
+(	O
+black	O
+)	O
+or	O
+in	B-protein_state
+complex	I-protein_state
+with	I-protein_state
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+(	O
+red	O
+)	O
+or	O
+the	O
+Ox40	B-protein
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+(	O
+green	O
+).	O
+
+Mutational	B-experimental_method
+analysis	I-experimental_method
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+-	O
+interactions	O
+with	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+and	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+.	O
+
+(	O
+a	O
+)	O
+EMSA	B-experimental_method
+assay	I-experimental_method
+comparing	O
+binding	O
+of	O
+the	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+and	O
+of	O
+the	O
+Y250A	B-mutant
+mutant	B-protein_state
+ROQ	B-structure_element
+domain	O
+for	O
+binding	O
+to	O
+the	O
+Ox40	B-protein
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+(	O
+left	O
+)	O
+or	O
+the	O
+previously	O
+described	O
+Tnf	B-protein
+CDE	B-structure_element
+SL	B-structure_element
+(	O
+right	O
+).	O
+
+A	O
+comparison	O
+of	O
+further	O
+mutants	O
+is	O
+shown	O
+in	O
+Supplementary	O
+Fig	O
+.	O
+4	O
+.	O
+(	O
+b	O
+)	O
+Schematic	O
+overview	O
+of	O
+the	O
+timeline	O
+used	O
+for	O
+the	O
+reconstitution	O
+experiment	O
+shown	O
+in	O
+c	O
+.	O
+(	O
+c	O
+)	O
+Flow	B-experimental_method
+cytometry	I-experimental_method
+of	O
+Ox40	B-protein
+and	O
+Icos	B-protein
+surface	O
+expression	O
+on	O
+CD4	O
++	O
+Th1	O
+cells	O
+from	O
+Rc3h1	B-gene
+/	O
+2fl	B-gene
+/	O
+fl	B-gene
+;	O
+Cd4	O
+-	O
+Cre	O
+-	O
+ERT2	O
+;	O
+rtTA	O
+mice	B-taxonomy_domain
+treated	O
+with	O
+tamoxifen	B-chemical
+(+	O
+tam	O
+)	O
+to	O
+induce	O
+Rc3h1	B-gene
+/	O
+2fl	B-gene
+/	O
+fl	B-gene
+deletion	B-experimental_method
+or	O
+left	O
+untreated	O
+(−	O
+tam	O
+).	O
+
+The	O
+cells	O
+were	O
+then	O
+either	O
+left	O
+untransduced	O
+(	O
+UT	O
+)	O
+or	O
+were	O
+transduced	O
+with	O
+retrovirus	B-taxonomy_domain
+containing	O
+a	O
+doxycycline	B-chemical
+-	O
+inducible	O
+cassette	O
+,	O
+to	O
+express	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+WT	B-protein_state
+,	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+Y250A	B-mutant
+or	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+K220A	B-mutant
+,	O
+K239A	B-mutant
+and	O
+R260A	B-mutant
+mutants	B-protein_state
+(	O
+see	O
+also	O
+Supplementary	O
+Fig	O
+.	O
+5	O
+).	O
+
+Functional	O
+importance	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+target	O
+motifs	O
+in	O
+cells	O
+.	O
+
+(	O
+a	O
+)	O
+Overview	O
+of	O
+the	O
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+and	O
+truncated	B-protein_state
+/	O
+mutated	B-protein_state
+versions	O
+thereof	O
+as	O
+used	O
+for	O
+EMSA	B-experimental_method
+assays	O
+in	O
+b	O
+and	O
+the	O
+expression	O
+experiments	O
+of	O
+Ox40	B-protein
+in	O
+c	O
+and	O
+d	O
+.	O
+(	O
+b	O
+)	O
+EMSA	B-experimental_method
+experiments	O
+probing	O
+the	O
+interaction	O
+between	O
+the	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+N	O
+-	O
+terminal	O
+region	O
+(	O
+residues	O
+2	B-residue_range
+–	I-residue_range
+440	I-residue_range
+)	O
+and	O
+either	O
+the	O
+complete	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+Ox40	B-protein
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+or	O
+versions	O
+with	O
+mutations	B-experimental_method
+of	O
+the	O
+CDE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+,	O
+the	O
+ADE	B-structure_element
+-	O
+like	O
+SL	B-structure_element
+or	O
+both	O
+SLs	B-structure_element
+(	O
+see	O
+a	O
+).	O
+
+It	O
+is	O
+noteworthy	O
+that	O
+the	O
+higher	O
+bands	O
+observed	O
+at	O
+large	O
+protein	O
+concentrations	O
+are	O
+probably	O
+additional	O
+nonspecific	O
+,	O
+lower	O
+-	O
+affinity	O
+interactions	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+with	O
+the	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+or	O
+protein	O
+aggregates	O
+.	O
+
+(	O
+c	O
+)	O
+Relative	O
+Ox40	B-protein
+MFI	B-evidence
+normalized	I-evidence
+to	I-evidence
+expression	I-evidence
+levels	I-evidence
+from	O
+the	O
+Ox40	B-protein
+CDS	B-structure_element
+construct	O
+.	O
+
+Error	O
+bars	O
+show	O
+s	O
+.	O
+d	O
+.	O
+of	O
+seven	O
+(	O
+CDS	B-structure_element
+,	O
+1	B-residue_range
+–	I-residue_range
+40	I-residue_range
+,	O
+1	B-residue_range
+–	I-residue_range
+80	I-residue_range
+,	O
+1	B-residue_range
+–	I-residue_range
+120	I-residue_range
+and	O
+full	B-protein_state
+-	I-protein_state
+length	I-protein_state
+),	O
+six	O
+(	O
+ADE	B-structure_element
+-	O
+like	O
+mut	B-protein_state
+and	O
+CDE	B-structure_element
+mut	B-protein_state
+)	O
+or	O
+three	O
+(	O
+double	B-protein_state
+mut	I-protein_state
+)	O
+independent	O
+experiments	O
+.	O
+
+Statistical	O
+significance	O
+was	O
+calculated	O
+by	O
+one	B-experimental_method
+-	I-experimental_method
+way	I-experimental_method
+analysis	I-experimental_method
+of	I-experimental_method
+variance	I-experimental_method
+(	O
+ANOVA	B-experimental_method
+)	O
+Kruskal	B-experimental_method
+–	I-experimental_method
+Wallis	I-experimental_method
+test	I-experimental_method
+followed	O
+by	O
+Dunn	B-experimental_method
+’	I-experimental_method
+s	I-experimental_method
+multiple	I-experimental_method
+comparison	I-experimental_method
+test	I-experimental_method
+(**	O
+P	O
+<	O
+0	O
+.	O
+01	O
+).	O
+
+(	O
+d	O
+)	O
+mRNA	B-evidence
+decay	I-evidence
+curves	I-evidence
+of	O
+Hela	O
+Tet	O
+-	O
+Off	O
+cells	O
+stably	O
+transduced	O
+with	O
+retroviruses	B-taxonomy_domain
+expressing	O
+Ox40	B-protein
+CDS	B-structure_element
+without	O
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+(	O
+CDS	B-structure_element
+,	O
+red	O
+line	O
+),	O
+Ox40	B-protein
+CDS	B-structure_element
+with	O
+its	O
+wild	B-protein_state
+-	I-protein_state
+type	I-protein_state
+3	B-structure_element
+′-	I-structure_element
+UTR	I-structure_element
+(	O
+full	B-protein_state
+length	I-protein_state
+,	O
+black	O
+line	O
+),	O
+Ox40	B-protein
+full	B-protein_state
+length	I-protein_state
+with	O
+mutated	B-protein_state
+ADE	B-structure_element
+-	O
+like	O
+motif	O
+(	O
+ADE	B-structure_element
+-	O
+like	O
+mut	B-protein_state
+,	O
+grey	O
+line	O
+),	O
+Ox40	B-protein
+full	B-protein_state
+length	I-protein_state
+with	O
+mutated	B-protein_state
+CDE	B-structure_element
+-	O
+like	O
+motif	O
+(	O
+CDE	B-structure_element
+-	O
+like	O
+mut	B-protein_state
+,	O
+green	O
+line	O
+)	O
+or	O
+Ox40	B-protein
+full	B-protein_state
+length	I-protein_state
+with	O
+mutated	B-protein_state
+ADE	B-structure_element
+and	O
+CDE	B-structure_element
+motifs	O
+(	O
+Double	B-protein_state
+mut	I-protein_state
+,	O
+blue	O
+line	O
+).	O
+
+mRNA	B-evidence
+half	I-evidence
+-	I-evidence
+life	I-evidence
+times	I-evidence
+were	O
+calculated	O
+with	O
+Graph	O
+Pad	O
+Prism	O
+.	O
+
+Data	B-evidence
+collection	I-evidence
+and	I-evidence
+refinement	I-evidence
+statistics	I-evidence
+.	O
+
+ROQ	B-structure_element
+-	O
+Ox40ADE	B-protein
+-	O
+like	O
+SL	B-structure_element
+ROQ	B-structure_element
+-	O
+ADE	B-structure_element
+SL	B-structure_element
+Data	O
+collection	O
+space	O
+group	O
+P21212	O
+P212121	O
+Cell	O
+dimensions	O
+a	O
+,	O
+b	O
+,	O
+c	O
+(	O
+Å	O
+)	O
+89	O
+.	O
+66	O
+,	O
+115	O
+.	O
+79	O
+,	O
+42	O
+.	O
+61	O
+72	O
+.	O
+90	O
+,	O
+89	O
+.	O
+30	O
+,	O
+144	O
+.	O
+70	O
+α	O
+,	O
+β	O
+,	O
+γ	O
+(°)	O
+90	O
+,	O
+90	O
+,	O
+90	O
+90	O
+,	O
+90	O
+,	O
+90	O
+Resolution	O
+(	O
+Å	O
+)	O
+50	O
+–	O
+2	O
+.	O
+23	O
+(	O
+2	O
+.	O
+29	O
+–	O
+2	O
+.	O
+23	O
+)	O
+50	O
+–	O
+3	O
+.	O
+0	O
+(	O
+3	O
+.	O
+08	O
+–	O
+3	O
+.	O
+00	O
+)	O
+Rmerge	O
+5	O
+.	O
+9	O
+(	O
+68	O
+.	O
+3	O
+)	O
+14	O
+.	O
+8	O
+(	O
+93	O
+.	O
+8	O
+)	O
+I	O
+/	O
+σI	O
+14	O
+.	O
+9	O
+(	O
+2	O
+.	O
+1	O
+)	O
+16	O
+.	O
+7	O
+(	O
+3	O
+.	O
+1	O
+)	O
+Completeness	O
+(%)	O
+98	O
+.	O
+7	O
+(	O
+97	O
+.	O
+7	O
+)	O
+99	O
+.	O
+9	O
+(	O
+99	O
+.	O
+9	O
+)	O
+Redundancy	O
+3	O
+.	O
+9	O
+(	O
+3	O
+.	O
+7	O
+)	O
+13	O
+.	O
+2	O
+(	O
+12	O
+.	O
+7	O
+)	O
+Refinement	O
+Resolution	O
+(	O
+Å	O
+)	O
+2	O
+.	O
+23	O
+3	O
+.	O
+00	O
+No	O
+.	O
+reflections	O
+21	O
+,	O
+018	O
+18	O
+,	O
+598	O
+Rwork	O
+/	O
+Rfree	O
+21	O
+.	O
+8	O
+/	O
+25	O
+.	O
+7	O
+18	O
+.	O
+6	O
+/	O
+23	O
+.	O
+4	O
+No	O
+.	O
+atoms	O
+Protein	O
+2	O
+,	O
+404	O
+4	O
+,	O
+820	O
+Ligand	O
+/	O
+ion	O
+894	O
+1	O
+,	O
+708	O
+Water	O
+99	O
+49	O
+B	O
+-	O
+factor	O
+overall	O
+47	O
+.	O
+2	O
+60	O
+.	O
+4	O
+Root	B-evidence
+mean	I-evidence
+squared	I-evidence
+deviations	I-evidence
+Bond	O
+lengths	O
+(	O
+Å	O
+)	O
+0	O
+.	O
+006	O
+0	O
+.	O
+014	O
+Bond	O
+angles	O
+(°)	O
+1	O
+.	O
+07	O
+1	O
+.	O
+77	O
+Ramachandran	O
+plot	O
+Most	O
+favoured	O
+(%)	O
+98	O
+.	O
+6	O
+99	O
+.	O
+8	O
+Additional	O
+allowed	O
+(%)	O
+1	O
+.	O
+4	O
+0	O
+.	O
+2	O
+
+ADE	B-structure_element
+,	O
+alternative	B-structure_element
+decay	I-structure_element
+element	I-structure_element
+;	O
+CDE	B-structure_element
+,	O
+constitutive	B-structure_element
+decay	I-structure_element
+element	I-structure_element
+;	O
+SL	B-structure_element
+,	O
+stem	B-structure_element
+loop	I-structure_element
+.	O
+
+For	O
+each	O
+data	O
+set	O
+,	O
+only	O
+one	O
+crystal	B-evidence
+has	O
+been	O
+used	O
+.	O
+
+KD	B-evidence
+for	O
+selected	O
+RNAs	B-chemical
+obtained	O
+from	O
+SPR	B-experimental_method
+measurements	I-experimental_method
+with	O
+immobilized	O
+ROQ	B-structure_element
+domain	O
+of	O
+Roquin	B-protein
+-	I-protein
+1	I-protein
+.	O
+
diff --git a/annotation_JSON/annotations.json b/annotation_JSON/annotations.json
new file mode 100644
index 0000000000000000000000000000000000000000..2aa2eedc327143637e415ad328f987b7f477d7a9
--- /dev/null
+++ b/annotation_JSON/annotations.json
@@ -0,0 +1,75403 @@
+{
+    "PMC5173035": {
+        "annotations": [
+            {
+                "sid": 0,
+                "sent": "Biochemical and structural characterization of a DNA N6-adenine methyltransferase from Helicobacter pylori",
+                "section": "TITLE",
+                "ner": [
+                    [
+                        0,
+                        43,
+                        "Biochemical and structural characterization",
+                        "experimental_method"
+                    ],
+                    [
+                        49,
+                        81,
+                        "DNA N6-adenine methyltransferase",
+                        "protein_type"
+                    ],
+                    [
+                        87,
+                        106,
+                        "Helicobacter pylori",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 1,
+                "sent": "DNA N6-methyladenine modification plays an important role in regulating a variety of biological functions in bacteria.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        20,
+                        "DNA N6-methyladenine",
+                        "ptm"
+                    ],
+                    [
+                        109,
+                        117,
+                        "bacteria",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 2,
+                "sent": "However, the mechanism of sequence-specific recognition in N6-methyladenine modification remains elusive.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        59,
+                        75,
+                        "N6-methyladenine",
+                        "ptm"
+                    ]
+                ]
+            },
+            {
+                "sid": 3,
+                "sent": "M1.HpyAVI, a DNA N6-adenine methyltransferase from Helicobacter pylori, shows more promiscuous substrate specificity than other enzymes.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        13,
+                        45,
+                        "DNA N6-adenine methyltransferase",
+                        "protein_type"
+                    ],
+                    [
+                        51,
+                        70,
+                        "Helicobacter pylori",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 4,
+                "sent": "Here, we present the crystal structures of cofactor-free and AdoMet-bound structures of this enzyme, which were determined at resolutions of 3.0 \u00c5 and 3.1 \u00c5, respectively.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        21,
+                        39,
+                        "crystal structures",
+                        "evidence"
+                    ],
+                    [
+                        43,
+                        56,
+                        "cofactor-free",
+                        "protein_state"
+                    ],
+                    [
+                        61,
+                        73,
+                        "AdoMet-bound",
+                        "protein_state"
+                    ],
+                    [
+                        74,
+                        84,
+                        "structures",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 5,
+                "sent": "The core structure of M1.HpyAVI resembles the canonical AdoMet-dependent MTase fold, while the putative DNA binding regions considerably differ from those of the other MTases, which may account for the substrate promiscuity of this enzyme.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        22,
+                        31,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        56,
+                        78,
+                        "AdoMet-dependent MTase",
+                        "protein_type"
+                    ],
+                    [
+                        104,
+                        123,
+                        "DNA binding regions",
+                        "site"
+                    ],
+                    [
+                        168,
+                        174,
+                        "MTases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 6,
+                "sent": "Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        25,
+                        "Site-directed mutagenesis",
+                        "experimental_method"
+                    ],
+                    [
+                        58,
+                        61,
+                        "D29",
+                        "residue_name_number"
+                    ],
+                    [
+                        66,
+                        70,
+                        "E216",
+                        "residue_name_number"
+                    ],
+                    [
+                        123,
+                        129,
+                        "methyl",
+                        "chemical"
+                    ],
+                    [
+                        169,
+                        172,
+                        "P41",
+                        "residue_name_number"
+                    ],
+                    [
+                        187,
+                        202,
+                        "highly flexible",
+                        "protein_state"
+                    ],
+                    [
+                        203,
+                        207,
+                        "loop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 7,
+                "sent": "Taken together, our data revealed the structural basis underlying DNA N6-adenine methyltransferase substrate promiscuity.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        66,
+                        98,
+                        "DNA N6-adenine methyltransferase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 8,
+                "sent": "DNA methylation is a common form of modification on nucleic acids occurring in both prokaryotes and eukaryotes.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        15,
+                        "DNA methylation",
+                        "ptm"
+                    ],
+                    [
+                        84,
+                        95,
+                        "prokaryotes",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        100,
+                        110,
+                        "eukaryotes",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 9,
+                "sent": "Such a modification creates a signature motif recognized by DNA-interacting proteins and functions as a mechanism to regulate gene expression.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        60,
+                        63,
+                        "DNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 10,
+                "sent": "DNA methylation is mediated by DNA methyltransferases (MTases), which catalyze the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to a given position of a particular DNA base within a specific DNA sequence.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        15,
+                        "DNA methylation",
+                        "ptm"
+                    ],
+                    [
+                        31,
+                        53,
+                        "DNA methyltransferases",
+                        "protein_type"
+                    ],
+                    [
+                        55,
+                        61,
+                        "MTases",
+                        "protein_type"
+                    ],
+                    [
+                        97,
+                        103,
+                        "methyl",
+                        "chemical"
+                    ],
+                    [
+                        115,
+                        139,
+                        "S-adenosyl-L- methionine",
+                        "chemical"
+                    ],
+                    [
+                        141,
+                        147,
+                        "AdoMet",
+                        "chemical"
+                    ],
+                    [
+                        185,
+                        188,
+                        "DNA",
+                        "chemical"
+                    ],
+                    [
+                        212,
+                        215,
+                        "DNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 11,
+                "sent": "Three classes of DNA MTases have been identified to transfer a methyl group to different positions of DNA bases.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        17,
+                        27,
+                        "DNA MTases",
+                        "protein_type"
+                    ],
+                    [
+                        63,
+                        69,
+                        "methyl",
+                        "chemical"
+                    ],
+                    [
+                        102,
+                        105,
+                        "DNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 12,
+                "sent": "C5-cytosine MTases, for example, methylate C5 of cytosine (m5C).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        18,
+                        "C5-cytosine MTases",
+                        "protein_type"
+                    ],
+                    [
+                        49,
+                        57,
+                        "cytosine",
+                        "residue_name"
+                    ],
+                    [
+                        59,
+                        62,
+                        "m5C",
+                        "ptm"
+                    ]
+                ]
+            },
+            {
+                "sid": 13,
+                "sent": "In eukaryotes, m5C plays an important role in gene expression, chromatin organization, genome maintenance and parental imprinting, and is involved in a variety of human diseases including cancer.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        3,
+                        13,
+                        "eukaryotes",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        15,
+                        18,
+                        "m5C",
+                        "ptm"
+                    ],
+                    [
+                        163,
+                        168,
+                        "human",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 14,
+                "sent": "By contrast, the functions of the prokaryotic DNA cytosine MTase remain unknown.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        34,
+                        45,
+                        "prokaryotic",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        46,
+                        64,
+                        "DNA cytosine MTase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 15,
+                "sent": "N4-cytosine MTases, which are frequently present in thermophilic or mesophilic bacteria, transfer a methyl group to the exocyclic amino group of cytosine (4mC).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        18,
+                        "N4-cytosine MTases",
+                        "protein_type"
+                    ],
+                    [
+                        52,
+                        64,
+                        "thermophilic",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        68,
+                        78,
+                        "mesophilic",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        79,
+                        87,
+                        "bacteria",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        100,
+                        106,
+                        "methyl",
+                        "chemical"
+                    ],
+                    [
+                        145,
+                        153,
+                        "cytosine",
+                        "residue_name"
+                    ],
+                    [
+                        155,
+                        158,
+                        "4mC",
+                        "ptm"
+                    ]
+                ]
+            },
+            {
+                "sid": 16,
+                "sent": "N4 methylation seems to be primarily a component of bacterial immune system against invasion by foreign DNA, such as conjugative plasmids and bacteriophages.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        14,
+                        "N4 methylation",
+                        "ptm"
+                    ],
+                    [
+                        52,
+                        61,
+                        "bacterial",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        104,
+                        107,
+                        "DNA",
+                        "chemical"
+                    ],
+                    [
+                        142,
+                        156,
+                        "bacteriophages",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 17,
+                "sent": "The third group, N6-adenine MTases methylate the exocyclic amino groups of adenine (6mA), which exists in prokaryotes as a signal for genome defense, DNA replication and repair, regulation of gene expression, control of transposition and host-pathogen interactions.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        17,
+                        34,
+                        "N6-adenine MTases",
+                        "protein_type"
+                    ],
+                    [
+                        75,
+                        82,
+                        "adenine",
+                        "residue_name"
+                    ],
+                    [
+                        84,
+                        87,
+                        "6mA",
+                        "ptm"
+                    ],
+                    [
+                        106,
+                        117,
+                        "prokaryotes",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        150,
+                        153,
+                        "DNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 18,
+                "sent": "Recent studies utilizing new sequencing approaches have showed the existence of 6mA in several eukaryotic species.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        80,
+                        83,
+                        "6mA",
+                        "ptm"
+                    ],
+                    [
+                        95,
+                        105,
+                        "eukaryotic",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 19,
+                "sent": "DNA 6mA modification is associated with important biological processes including nucleosome distribution close to the transcription start sites in Chlamydomonas, carrying heritable epigenetic information in C.elegans or controlling development of Drosophila.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "DNA",
+                        "chemical"
+                    ],
+                    [
+                        4,
+                        7,
+                        "6mA",
+                        "ptm"
+                    ],
+                    [
+                        147,
+                        160,
+                        "Chlamydomonas",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        207,
+                        216,
+                        "C.elegans",
+                        "species"
+                    ],
+                    [
+                        247,
+                        257,
+                        "Drosophila",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 20,
+                "sent": "All the three types of methylation exist in prokaryotes, and most DNA MTases are components of the restriction-modification (R-M) systems.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        23,
+                        34,
+                        "methylation",
+                        "ptm"
+                    ],
+                    [
+                        44,
+                        55,
+                        "prokaryotes",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        66,
+                        76,
+                        "DNA MTases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 21,
+                "sent": "\u201cR\u201d stands for a restriction endonuclease cleaving specific DNA sequences, while \u201cM\u201d symbolizes a modification methyltransferase rendering these sequences resistant to cleavage.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        17,
+                        41,
+                        "restriction endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        60,
+                        63,
+                        "DNA",
+                        "chemical"
+                    ],
+                    [
+                        98,
+                        128,
+                        "modification methyltransferase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 22,
+                "sent": "The cooperation of these two enzymes provides a defensive mechanism to protect bacteria from infection by bacteriophages.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        79,
+                        87,
+                        "bacteria",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        106,
+                        120,
+                        "bacteriophages",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 23,
+                "sent": "The R-M systems are classified into three types based on specific structural features, position of DNA cleavage and cofactor requirements.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        99,
+                        102,
+                        "DNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 24,
+                "sent": "In types I and III, the DNA adenine or cytosine methyltransferase is part of a multi-subunit enzyme that catalyzes both restriction and modification.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        24,
+                        65,
+                        "DNA adenine or cytosine methyltransferase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 25,
+                "sent": "By contrast, two separate enzymes exist in type II systems, where a restriction endonuclease and a DNA adenine or cytosine methyltransferase recognize the same targets.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        68,
+                        92,
+                        "restriction endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        99,
+                        140,
+                        "DNA adenine or cytosine methyltransferase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 26,
+                "sent": "To date, a number of bacterial DNA MTases have been structurally characterized, covering enzymes from all the three classes.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        21,
+                        30,
+                        "bacterial",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        31,
+                        41,
+                        "DNA MTases",
+                        "protein_type"
+                    ],
+                    [
+                        52,
+                        78,
+                        "structurally characterized",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 27,
+                "sent": "All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        10,
+                        16,
+                        "MTases",
+                        "protein_type"
+                    ],
+                    [
+                        120,
+                        129,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        137,
+                        153,
+                        "catalytic domain",
+                        "structure_element"
+                    ],
+                    [
+                        168,
+                        179,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        184,
+                        190,
+                        "methyl",
+                        "chemical"
+                    ],
+                    [
+                        215,
+                        221,
+                        "AdoMet",
+                        "chemical"
+                    ],
+                    [
+                        245,
+                        276,
+                        "target (DNA)-recognition domain",
+                        "structure_element"
+                    ],
+                    [
+                        278,
+                        281,
+                        "TRD",
+                        "structure_element"
+                    ],
+                    [
+                        343,
+                        346,
+                        "DNA",
+                        "chemical"
+                    ],
+                    [
+                        387,
+                        390,
+                        "DNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 28,
+                "sent": "Conserved amino acid motifs have been identified from reported structures, including ten motifs (I-X) in cytosine MTases and nine motifs (I-VIII and X) in adenine MTases, all of which are arranged in an almost constant order.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Conserved",
+                        "protein_state"
+                    ],
+                    [
+                        63,
+                        73,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        97,
+                        100,
+                        "I-X",
+                        "structure_element"
+                    ],
+                    [
+                        105,
+                        120,
+                        "cytosine MTases",
+                        "protein_type"
+                    ],
+                    [
+                        138,
+                        144,
+                        "I-VIII",
+                        "structure_element"
+                    ],
+                    [
+                        149,
+                        150,
+                        "X",
+                        "structure_element"
+                    ],
+                    [
+                        155,
+                        169,
+                        "adenine MTases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 29,
+                "sent": "According to the linear arrangement of three conserved domains, exocyclic amino MTases are subdivided into six groups (namely \u03b1, \u03b2, \u03b3, \u03b6, \u03b4 and \u03b5).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        45,
+                        54,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        64,
+                        86,
+                        "exocyclic amino MTases",
+                        "protein_type"
+                    ],
+                    [
+                        126,
+                        127,
+                        "\u03b1",
+                        "protein_type"
+                    ],
+                    [
+                        129,
+                        130,
+                        "\u03b2",
+                        "protein_type"
+                    ],
+                    [
+                        132,
+                        133,
+                        "\u03b3",
+                        "protein_type"
+                    ],
+                    [
+                        135,
+                        136,
+                        "\u03b6",
+                        "protein_type"
+                    ],
+                    [
+                        138,
+                        139,
+                        "\u03b4",
+                        "protein_type"
+                    ],
+                    [
+                        144,
+                        145,
+                        "\u03b5",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 30,
+                "sent": "N6-adenine and N4-cytosine MTases, in particular, are closely related by sharing common structural features.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        33,
+                        "N6-adenine and N4-cytosine MTases",
+                        "protein_type"
+                    ],
+                    [
+                        0,
+                        33,
+                        "N6-adenine and N4-cytosine MTases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 31,
+                "sent": "Despite the considerable similarity among bacterial MTases, some differences were observed among the enzymes from various species.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        42,
+                        51,
+                        "bacterial",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        52,
+                        58,
+                        "MTases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 32,
+                "sent": "For example, the structural regions of MTases beyond the catalytic domain are rather variable, such as the C-terminal domain of M.TaqI, the extended arm of M.MboIIA and M.RsrI, the helix bundle of EcoDam, and so on.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        39,
+                        45,
+                        "MTases",
+                        "protein_type"
+                    ],
+                    [
+                        57,
+                        73,
+                        "catalytic domain",
+                        "structure_element"
+                    ],
+                    [
+                        107,
+                        124,
+                        "C-terminal domain",
+                        "structure_element"
+                    ],
+                    [
+                        128,
+                        134,
+                        "M.TaqI",
+                        "protein"
+                    ],
+                    [
+                        156,
+                        164,
+                        "M.MboIIA",
+                        "protein"
+                    ],
+                    [
+                        169,
+                        175,
+                        "M.RsrI",
+                        "protein"
+                    ],
+                    [
+                        181,
+                        193,
+                        "helix bundle",
+                        "structure_element"
+                    ],
+                    [
+                        197,
+                        203,
+                        "EcoDam",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 33,
+                "sent": "DNA methylation is thought to influence bacterial virulence.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        15,
+                        "DNA methylation",
+                        "ptm"
+                    ],
+                    [
+                        40,
+                        49,
+                        "bacterial",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 34,
+                "sent": "DNA adenine methyltransferase has been shown to play a crucial role in colonization of deep tissue sites in Salmonella typhimurium and Aeromonas hydrophila.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        29,
+                        "DNA adenine methyltransferase",
+                        "protein_type"
+                    ],
+                    [
+                        108,
+                        130,
+                        "Salmonella typhimurium",
+                        "species"
+                    ],
+                    [
+                        135,
+                        155,
+                        "Aeromonas hydrophila",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 35,
+                "sent": "Importantly, DNA adenine methylation is a global regulator of genes expressed during infection and inhibitors of DNA adenine methylation are likely to have a broad antimicrobial action.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        13,
+                        36,
+                        "DNA adenine methylation",
+                        "ptm"
+                    ],
+                    [
+                        113,
+                        136,
+                        "DNA adenine methylation",
+                        "ptm"
+                    ]
+                ]
+            },
+            {
+                "sid": 36,
+                "sent": "Dam was considered a promising target for antimicrobial drug development.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "Dam",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 37,
+                "sent": "Helicobacter pylori is a Gram-negative bacterium that persistently colonizes in human stomach worldwide.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        19,
+                        "Helicobacter pylori",
+                        "species"
+                    ],
+                    [
+                        25,
+                        48,
+                        "Gram-negative bacterium",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        80,
+                        85,
+                        "human",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 38,
+                "sent": "H. pylori is involved in 90% of all gastric malignancies, infecting nearly 50% of the world's population and is the most crucial etiologic agent for gastric adenocarcinoma.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "H. pylori",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 39,
+                "sent": "H. pylori strains possess a few R-M systems like other bacteria to function as defensive systems.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "H. pylori",
+                        "species"
+                    ],
+                    [
+                        55,
+                        63,
+                        "bacteria",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 40,
+                "sent": "H. pylori 26695, for example, has 23 R-M systems.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        15,
+                        "H. pylori 26695",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 41,
+                "sent": "Methyltransferases were suggested to be involved in H. pylori pathogenicity.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        18,
+                        "Methyltransferases",
+                        "protein_type"
+                    ],
+                    [
+                        52,
+                        61,
+                        "H. pylori",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 42,
+                "sent": "M1.HpyAVI is a DNA adenine MTase that belongs to the type II R-M system.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        15,
+                        32,
+                        "DNA adenine MTase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 43,
+                "sent": "This system contains two DNA MTases named M1.HpyAVI and M2.HpyAVI, and a putative restriction enzyme.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        25,
+                        35,
+                        "DNA MTases",
+                        "protein_type"
+                    ],
+                    [
+                        42,
+                        51,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        56,
+                        65,
+                        "M2.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        82,
+                        100,
+                        "restriction enzyme",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 44,
+                "sent": "M1.HpyAVI encoded by ORF hp0050 is an N6-adenine methyltransferase belonging to the \u03b2-class MTase.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        25,
+                        31,
+                        "hp0050",
+                        "gene"
+                    ],
+                    [
+                        38,
+                        66,
+                        "N6-adenine methyltransferase",
+                        "protein_type"
+                    ],
+                    [
+                        84,
+                        97,
+                        "\u03b2-class MTase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 45,
+                "sent": "It has been reported recently that this enzyme recognizes the sequence of 5\u2032-GAGG-3\u2032, 5\u2032-GGAG-3\u2032 or 5\u2032-GAAG-3\u2032 and methylates adenines in these sequences.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        74,
+                        85,
+                        "5\u2032-GAGG-3\u2032,",
+                        "chemical"
+                    ],
+                    [
+                        86,
+                        96,
+                        "5\u2032-GGAG-3\u2032",
+                        "chemical"
+                    ],
+                    [
+                        100,
+                        110,
+                        "5\u2032-GAAG-3\u2032",
+                        "chemical"
+                    ],
+                    [
+                        126,
+                        134,
+                        "adenines",
+                        "residue_name"
+                    ]
+                ]
+            },
+            {
+                "sid": 46,
+                "sent": "Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5\u2032-GAAG-3\u2032 seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5\u2032-GAGG-3\u2032. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        11,
+                        22,
+                        "methylation",
+                        "ptm"
+                    ],
+                    [
+                        39,
+                        47,
+                        "adenines",
+                        "residue_name"
+                    ],
+                    [
+                        102,
+                        119,
+                        "N6-adenine MTases",
+                        "protein_type"
+                    ],
+                    [
+                        125,
+                        136,
+                        "methylation",
+                        "ptm"
+                    ],
+                    [
+                        149,
+                        159,
+                        "5\u2032-GAAG-3\u2032",
+                        "chemical"
+                    ],
+                    [
+                        192,
+                        201,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        252,
+                        260,
+                        "H.pylori",
+                        "species"
+                    ],
+                    [
+                        293,
+                        303,
+                        "5\u2032-GAGG-3\u2032",
+                        "chemical"
+                    ],
+                    [
+                        449,
+                        458,
+                        "structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 47,
+                "sent": "Here, we report the crystal structure of M1.HpyAVI from H. pylori 26695, which is the first determined N6-adenine MTase structure in H. pylori.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        20,
+                        37,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        41,
+                        50,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        56,
+                        71,
+                        "H. pylori 26695",
+                        "species"
+                    ],
+                    [
+                        103,
+                        119,
+                        "N6-adenine MTase",
+                        "protein_type"
+                    ],
+                    [
+                        120,
+                        129,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        133,
+                        142,
+                        "H. pylori",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 48,
+                "sent": "The structure reveals a similar architecture as the canonical fold of homologous proteins, but displays several differences in the loop regions and TRD.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        131,
+                        135,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        148,
+                        151,
+                        "TRD",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 49,
+                "sent": "Based on structural and biochemical analyses, we then identified two conserved amino acids, D29 at the catalytic site and E216 close to the C-terminus, as crucial residues for cofactor binding and methyltransferase activity of M1.HpyAVI.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        9,
+                        44,
+                        "structural and biochemical analyses",
+                        "experimental_method"
+                    ],
+                    [
+                        69,
+                        78,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        92,
+                        95,
+                        "D29",
+                        "residue_name_number"
+                    ],
+                    [
+                        103,
+                        117,
+                        "catalytic site",
+                        "site"
+                    ],
+                    [
+                        122,
+                        126,
+                        "E216",
+                        "residue_name_number"
+                    ],
+                    [
+                        197,
+                        214,
+                        "methyltransferase",
+                        "protein_type"
+                    ],
+                    [
+                        227,
+                        236,
+                        "M1.HpyAVI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 50,
+                "sent": "In addition, a non-conserved amino acid, P41, seems to play a key role in substrate recognition.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        15,
+                        28,
+                        "non-conserved",
+                        "protein_state"
+                    ],
+                    [
+                        41,
+                        44,
+                        "P41",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 51,
+                "sent": "Overall structure",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        17,
+                        "structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 52,
+                "sent": "Recombinant full-length M1.HpyAVI was produced as a soluble protein in Escherichia coli, but was quite unstable and tended to aggregate in low salt environment.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        12,
+                        23,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        24,
+                        33,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        71,
+                        87,
+                        "Escherichia coli",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 53,
+                "sent": "The protein, however, remained fully soluble in a buffer containing higher concentration of sodium chloride (>300 mM), which prompted that M1.HpyAVI is likely a halophilic protein.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        92,
+                        107,
+                        "sodium chloride",
+                        "chemical"
+                    ],
+                    [
+                        139,
+                        148,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        161,
+                        171,
+                        "halophilic",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 54,
+                "sent": "The cofactor-free and AdoMet-bound proteins were crystallized at different conditions.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        17,
+                        "cofactor-free",
+                        "protein_state"
+                    ],
+                    [
+                        22,
+                        34,
+                        "AdoMet-bound",
+                        "protein_state"
+                    ],
+                    [
+                        49,
+                        61,
+                        "crystallized",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 55,
+                "sent": "Both structures were determined by means of molecular replacement, and refined to 3.0 \u00c5 and 3.1 \u00c5, respectively.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        15,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        44,
+                        65,
+                        "molecular replacement",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 56,
+                "sent": "Statistics of X-ray data collection and structure refinement were summarized in Table 1.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        35,
+                        "X-ray data collection",
+                        "experimental_method"
+                    ],
+                    [
+                        40,
+                        60,
+                        "structure refinement",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 57,
+                "sent": "Data collection and structure refinement statistics of M1.HpyAVI",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        20,
+                        51,
+                        "structure refinement statistics",
+                        "evidence"
+                    ],
+                    [
+                        55,
+                        64,
+                        "M1.HpyAVI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 58,
+                "sent": "\tM1.HpyAVI\tM1.HpyAVI-AdoMet complex\t \tData collection\t\t\t \tWavelength (\u00c5)\t1.0000\t0.97772\t \tSpace group\tP43212\tP65\t \tUnit-cell parameters (\u00c5, \u02da)\ta = b = 69.73, c = 532.75\u03b1 = \u03b2 = \u03b3 = 90\ta = b = 135.60, c = 265.15\u03b1 = \u03b2 = 90, \u03b3 = 120\t \tResolution range (\u00c5) a\t49.09-3.00 (3.09-3.00)\t48.91-3.10 (3.18-3.10)\t \tUnique reflections a\t27243\t49833\t \tMultiplicity a\t3.7 (3.8)\t5.6 (4.0)\t \tCompleteness (%)a\t98.7 (98.9)\t99.7 (97.8)\t \tMean I/\u03b4 (I) a\t12.1 (3.4)\t14.0 (1.9)\t \tSolvent content (%)\t58.67\t61.96\t \tRmergea\t0.073 (0.378)\t0.106 (0.769)\t \tStructure refinement\t\t\t \tRwork\t0.251\t0.221\t \tRfree\t0.308\t0.276\t \tR.m.s.d., bond lengths (\u00c5)\t0.007\t0.007\t \tR.m.s.d., bond angles (\u02da)\t1.408\t1.651\t \tRamachandran plot\t\t\t \tFavoured region (%)\t89.44\t91.44\t \tAllowed region (%)\t9.58\t7.11\t \tOutliers (%)\t0.99\t1.45\t \t",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        1,
+                        10,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        11,
+                        27,
+                        "M1.HpyAVI-AdoMet",
+                        "complex_assembly"
+                    ],
+                    [
+                        594,
+                        601,
+                        "R.m.s.d",
+                        "evidence"
+                    ],
+                    [
+                        635,
+                        642,
+                        "R.m.s.d",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 59,
+                "sent": "Four and eight protein monomers resided in the asymmetric units of the two crystal structures.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        23,
+                        31,
+                        "monomers",
+                        "oligomeric_state"
+                    ],
+                    [
+                        75,
+                        93,
+                        "crystal structures",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 60,
+                "sent": "Some amino acids, particularly those within two loops (residues 32-61 and 152-172) in both structures, were poorly defined in electron density and had to be omitted from the refined models.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        48,
+                        53,
+                        "loops",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        69,
+                        "32-61",
+                        "residue_range"
+                    ],
+                    [
+                        74,
+                        81,
+                        "152-172",
+                        "residue_range"
+                    ],
+                    [
+                        91,
+                        101,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        126,
+                        142,
+                        "electron density",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 61,
+                "sent": "The two structures are very similar to each other (Figure 1) and could be well overlaid with an RMSD of 0.76 \u00c5 on 191 C\u03b1 atoms.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        18,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        96,
+                        100,
+                        "RMSD",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 62,
+                "sent": "The overall architecture of M1.HpyAVI revealed in these structures resembles the AdoMet-dependent MTase fold in which a twisted seven-stranded \u03b2-sheet flanked by six \u03b1-helices forms the structural core.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        28,
+                        37,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        56,
+                        66,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        81,
+                        103,
+                        "AdoMet-dependent MTase",
+                        "protein_type"
+                    ],
+                    [
+                        143,
+                        150,
+                        "\u03b2-sheet",
+                        "structure_element"
+                    ],
+                    [
+                        166,
+                        175,
+                        "\u03b1-helices",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 63,
+                "sent": "Like the reported structures of the larger domain of MTases, three helices (\u03b1A, \u03b1B and \u03b1Z) are located at one face of the central \u03b2-sheet, while the other three \u03b1D, \u03b1E and \u03b1C sit at the other side.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        28,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        53,
+                        59,
+                        "MTases",
+                        "protein_type"
+                    ],
+                    [
+                        67,
+                        74,
+                        "helices",
+                        "structure_element"
+                    ],
+                    [
+                        76,
+                        78,
+                        "\u03b1A",
+                        "structure_element"
+                    ],
+                    [
+                        80,
+                        82,
+                        "\u03b1B",
+                        "structure_element"
+                    ],
+                    [
+                        87,
+                        89,
+                        "\u03b1Z",
+                        "structure_element"
+                    ],
+                    [
+                        130,
+                        137,
+                        "\u03b2-sheet",
+                        "structure_element"
+                    ],
+                    [
+                        161,
+                        163,
+                        "\u03b1D",
+                        "structure_element"
+                    ],
+                    [
+                        165,
+                        167,
+                        "\u03b1E",
+                        "structure_element"
+                    ],
+                    [
+                        172,
+                        174,
+                        "\u03b1C",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 64,
+                "sent": "All these conserved structural motifs form a typical \u03b1/\u03b2 Rossmann fold.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        10,
+                        19,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        53,
+                        70,
+                        "\u03b1/\u03b2 Rossmann fold",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 65,
+                "sent": "The catalytic motif DPPY lies in a loop connecting \u03b1D and \u03b24, and the cofactor AdoMet binds in a neighboring cavity.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        19,
+                        "catalytic motif",
+                        "structure_element"
+                    ],
+                    [
+                        20,
+                        24,
+                        "DPPY",
+                        "structure_element"
+                    ],
+                    [
+                        35,
+                        39,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        51,
+                        53,
+                        "\u03b1D",
+                        "structure_element"
+                    ],
+                    [
+                        58,
+                        60,
+                        "\u03b24",
+                        "structure_element"
+                    ],
+                    [
+                        79,
+                        85,
+                        "AdoMet",
+                        "chemical"
+                    ],
+                    [
+                        109,
+                        115,
+                        "cavity",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 66,
+                "sent": "The loop (residues 136-166) located between \u03b27 and \u03b1Z corresponds to a highly diverse region in other MTases that is involved in target DNA recognition.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        19,
+                        26,
+                        "136-166",
+                        "residue_range"
+                    ],
+                    [
+                        44,
+                        46,
+                        "\u03b27",
+                        "structure_element"
+                    ],
+                    [
+                        51,
+                        53,
+                        "\u03b1Z",
+                        "structure_element"
+                    ],
+                    [
+                        71,
+                        85,
+                        "highly diverse",
+                        "protein_state"
+                    ],
+                    [
+                        102,
+                        108,
+                        "MTases",
+                        "protein_type"
+                    ],
+                    [
+                        136,
+                        139,
+                        "DNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 67,
+                "sent": "The hairpin loop (residues 101-133) bridging \u03b26 and \u03b27, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        16,
+                        "hairpin loop",
+                        "structure_element"
+                    ],
+                    [
+                        27,
+                        34,
+                        "101-133",
+                        "residue_range"
+                    ],
+                    [
+                        45,
+                        47,
+                        "\u03b26",
+                        "structure_element"
+                    ],
+                    [
+                        52,
+                        54,
+                        "\u03b27",
+                        "structure_element"
+                    ],
+                    [
+                        82,
+                        85,
+                        "DNA",
+                        "chemical"
+                    ],
+                    [
+                        93,
+                        105,
+                        "minor groove",
+                        "structure_element"
+                    ],
+                    [
+                        160,
+                        168,
+                        "M.MboIIA",
+                        "protein"
+                    ],
+                    [
+                        170,
+                        176,
+                        "M.RsrI",
+                        "protein"
+                    ],
+                    [
+                        181,
+                        188,
+                        "M.pvuII",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 68,
+                "sent": "The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "missing",
+                        "protein_state"
+                    ],
+                    [
+                        12,
+                        16,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        27,
+                        32,
+                        "33-58",
+                        "residue_range"
+                    ],
+                    [
+                        41,
+                        50,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        54,
+                        63,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        79,
+                        85,
+                        "loop I",
+                        "structure_element"
+                    ],
+                    [
+                        89,
+                        95,
+                        "M.TaqI",
+                        "protein"
+                    ],
+                    [
+                        127,
+                        136,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        137,
+                        148,
+                        "without DNA",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 69,
+                "sent": "This loop, however, was well ordered in an M.TaqI-DNA complex structure and was shown to play a crucial role in DNA methylation by contacting the flipping adenine and recognizing specific DNA sequence.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        9,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        24,
+                        36,
+                        "well ordered",
+                        "protein_state"
+                    ],
+                    [
+                        43,
+                        71,
+                        "M.TaqI-DNA complex structure",
+                        "evidence"
+                    ],
+                    [
+                        112,
+                        127,
+                        "DNA methylation",
+                        "ptm"
+                    ],
+                    [
+                        155,
+                        162,
+                        "adenine",
+                        "residue_name"
+                    ],
+                    [
+                        188,
+                        191,
+                        "DNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 70,
+                "sent": "Overall structure of M1.HpyAVI",
+                "section": "FIG",
+                "ner": [
+                    [
+                        8,
+                        17,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        30,
+                        "M1.HpyAVI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 71,
+                "sent": "A. Free form B. AdoMet-bound form.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        3,
+                        7,
+                        "Free",
+                        "protein_state"
+                    ],
+                    [
+                        16,
+                        28,
+                        "AdoMet-bound",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 72,
+                "sent": "Ribbon diagram of M1.HpyAVI resembles an \u201cAdoMet-dependent MTase fold\u201d, a mixed seven-stranded \u03b2-sheet flanked by six \u03b1-helices, \u03b1A, \u03b1B, \u03b1Z on one side and \u03b1D, \u03b1E, \u03b1C on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        18,
+                        27,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        42,
+                        64,
+                        "AdoMet-dependent MTase",
+                        "protein_type"
+                    ],
+                    [
+                        95,
+                        102,
+                        "\u03b2-sheet",
+                        "structure_element"
+                    ],
+                    [
+                        118,
+                        127,
+                        "\u03b1-helices",
+                        "structure_element"
+                    ],
+                    [
+                        129,
+                        131,
+                        "\u03b1A",
+                        "structure_element"
+                    ],
+                    [
+                        133,
+                        135,
+                        "\u03b1B",
+                        "structure_element"
+                    ],
+                    [
+                        137,
+                        139,
+                        "\u03b1Z",
+                        "structure_element"
+                    ],
+                    [
+                        156,
+                        158,
+                        "\u03b1D",
+                        "structure_element"
+                    ],
+                    [
+                        160,
+                        162,
+                        "\u03b1E",
+                        "structure_element"
+                    ],
+                    [
+                        164,
+                        166,
+                        "\u03b1C",
+                        "structure_element"
+                    ],
+                    [
+                        199,
+                        205,
+                        "AdoMet",
+                        "chemical"
+                    ],
+                    [
+                        209,
+                        217,
+                        "bound in",
+                        "protein_state"
+                    ],
+                    [
+                        220,
+                        226,
+                        "cavity",
+                        "site"
+                    ],
+                    [
+                        236,
+                        245,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        268,
+                        272,
+                        "DPPY",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 73,
+                "sent": "The \u03b1-helices and \u03b2-strands are labelled and numbered according to the commonly numbering rule for the known MTases.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "\u03b1-helices",
+                        "structure_element"
+                    ],
+                    [
+                        18,
+                        27,
+                        "\u03b2-strands",
+                        "structure_element"
+                    ],
+                    [
+                        109,
+                        115,
+                        "MTases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 74,
+                "sent": "The AdoMet molecule is shown in green.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        10,
+                        "AdoMet",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 75,
+                "sent": "Dimeric state of M1.HpyAVI in crystal and solution",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "Dimeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        17,
+                        26,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        30,
+                        37,
+                        "crystal",
+                        "evidence"
+                    ],
+                    [
+                        42,
+                        50,
+                        "solution",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 76,
+                "sent": "Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        44,
+                        "DNA MTases",
+                        "protein_type"
+                    ],
+                    [
+                        51,
+                        58,
+                        "M.BamHI",
+                        "protein"
+                    ],
+                    [
+                        63,
+                        70,
+                        "M.EcoRI",
+                        "protein"
+                    ],
+                    [
+                        81,
+                        88,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        136,
+                        139,
+                        "DNA",
+                        "chemical"
+                    ],
+                    [
+                        164,
+                        169,
+                        "MTase",
+                        "protein_type"
+                    ],
+                    [
+                        173,
+                        187,
+                        "hemimethylated",
+                        "protein_state"
+                    ],
+                    [
+                        222,
+                        233,
+                        "methylation",
+                        "ptm"
+                    ],
+                    [
+                        261,
+                        277,
+                        "fully methylated",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 77,
+                "sent": "Increasing number of dimeric DNA MTases, however, has been identified from later studies.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        21,
+                        28,
+                        "dimeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        29,
+                        39,
+                        "DNA MTases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 78,
+                "sent": "For instance, M.DpnII, M.RsrI, M.KpnI, and M.MboIIA have been found as dimers in solution.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        21,
+                        "M.DpnII",
+                        "protein"
+                    ],
+                    [
+                        23,
+                        29,
+                        "M.RsrI",
+                        "protein"
+                    ],
+                    [
+                        31,
+                        37,
+                        "M.KpnI",
+                        "protein"
+                    ],
+                    [
+                        43,
+                        51,
+                        "M.MboIIA",
+                        "protein"
+                    ],
+                    [
+                        71,
+                        77,
+                        "dimers",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 79,
+                "sent": "In addition, several MTases including M.MboIIA, M.RsrI and TTH0409 form tightly associated dimers in crystal structures.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        21,
+                        27,
+                        "MTases",
+                        "protein_type"
+                    ],
+                    [
+                        38,
+                        46,
+                        "M.MboIIA",
+                        "protein"
+                    ],
+                    [
+                        48,
+                        54,
+                        "M.RsrI",
+                        "protein"
+                    ],
+                    [
+                        59,
+                        66,
+                        "TTH0409",
+                        "protein"
+                    ],
+                    [
+                        91,
+                        97,
+                        "dimers",
+                        "oligomeric_state"
+                    ],
+                    [
+                        101,
+                        119,
+                        "crystal structures",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 80,
+                "sent": "Nonetheless, some DNA MTases such as M.CcrMI and the Bacillus amyloliquefaciens MTase dissociate from dimer into monomer upon DNA-binding.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        28,
+                        "DNA MTases",
+                        "protein_type"
+                    ],
+                    [
+                        37,
+                        44,
+                        "M.CcrMI",
+                        "protein"
+                    ],
+                    [
+                        53,
+                        79,
+                        "Bacillus amyloliquefaciens",
+                        "species"
+                    ],
+                    [
+                        80,
+                        85,
+                        "MTase",
+                        "protein_type"
+                    ],
+                    [
+                        102,
+                        107,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        113,
+                        120,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        126,
+                        129,
+                        "DNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 81,
+                "sent": "According to the arrangement of the three conserved domains, M1.HpyAVI belongs to the \u03b2-subgroup, in which a conserved motif NXXTX9\u221211AXRXFSXXHX4WX6\u22129 YXFXLX3RX9\u221226NPX1\u22126NVWX29\u221234A has been identified at the dimerization interface in crystal structures.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        42,
+                        51,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        61,
+                        70,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        86,
+                        96,
+                        "\u03b2-subgroup",
+                        "protein_type"
+                    ],
+                    [
+                        109,
+                        118,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        125,
+                        180,
+                        "NXXTX9\u221211AXRXFSXXHX4WX6\u22129 YXFXLX3RX9\u221226NPX1\u22126NVWX29\u221234A",
+                        "structure_element"
+                    ],
+                    [
+                        208,
+                        230,
+                        "dimerization interface",
+                        "site"
+                    ],
+                    [
+                        234,
+                        252,
+                        "crystal structures",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 82,
+                "sent": "Most of conserved amino acids within that motif are present in the sequence of M1.HpyAVI (Figure 2A), implying dimerization of this protein.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        17,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        79,
+                        88,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        111,
+                        123,
+                        "dimerization",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 83,
+                "sent": "In agreement, a dimer of M1.HpyAVI was observed in our crystal structures with the two monomers related by a two-fold axis (Figure 2B and 2C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        16,
+                        21,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        25,
+                        34,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        55,
+                        73,
+                        "crystal structures",
+                        "evidence"
+                    ],
+                    [
+                        87,
+                        95,
+                        "monomers",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 84,
+                "sent": "An area of ~1900 \u00c52 was buried at the dimeric interface, taking up ca 17% of the total area.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        38,
+                        55,
+                        "dimeric interface",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 85,
+                "sent": "The dimeric architecture was greatly stabilized by hydrogen bonds and salt bridges formed among residues R86, D93 and E96.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "dimeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        51,
+                        65,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        70,
+                        82,
+                        "salt bridges",
+                        "bond_interaction"
+                    ],
+                    [
+                        105,
+                        108,
+                        "R86",
+                        "residue_name_number"
+                    ],
+                    [
+                        110,
+                        113,
+                        "D93",
+                        "residue_name_number"
+                    ],
+                    [
+                        118,
+                        121,
+                        "E96",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 86,
+                "sent": "In addition, comparison of the dimer structure of M1.HpyAVI with some other \u03b2-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        31,
+                        36,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        37,
+                        46,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        50,
+                        59,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        76,
+                        90,
+                        "\u03b2-class MTases",
+                        "protein_type"
+                    ],
+                    [
+                        92,
+                        101,
+                        "M1.MboIIA",
+                        "protein"
+                    ],
+                    [
+                        103,
+                        109,
+                        "M.RsrI",
+                        "protein"
+                    ],
+                    [
+                        114,
+                        122,
+                        "TTHA0409",
+                        "protein"
+                    ],
+                    [
+                        143,
+                        152,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        153,
+                        158,
+                        "dimer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 87,
+                "sent": "M1.HpyAVI exists as dimer in crystal and solution",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        20,
+                        25,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        29,
+                        36,
+                        "crystal",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 88,
+                "sent": "A. A conserved interface area of \u03b2-class MTases is defined in M1.HpyAVI.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        5,
+                        14,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        15,
+                        29,
+                        "interface area",
+                        "site"
+                    ],
+                    [
+                        33,
+                        47,
+                        "\u03b2-class MTases",
+                        "protein_type"
+                    ],
+                    [
+                        62,
+                        71,
+                        "M1.HpyAVI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 89,
+                "sent": "Residues that involved are signed in red color; Dimerization of free-form M1.HpyAVI B. and cofactor-bound M1.HpyAVI C. The two monomers are marked in green and blue, AdoMet molecules are marked in magenta.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        48,
+                        60,
+                        "Dimerization",
+                        "oligomeric_state"
+                    ],
+                    [
+                        64,
+                        68,
+                        "free",
+                        "protein_state"
+                    ],
+                    [
+                        74,
+                        83,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        91,
+                        105,
+                        "cofactor-bound",
+                        "protein_state"
+                    ],
+                    [
+                        106,
+                        115,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        127,
+                        135,
+                        "monomers",
+                        "oligomeric_state"
+                    ],
+                    [
+                        166,
+                        172,
+                        "AdoMet",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 90,
+                "sent": "D. Gel-filtration analysis revealed that M1.HpyAVI exist as a dimer in solution.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        3,
+                        26,
+                        "Gel-filtration analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        41,
+                        50,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        62,
+                        67,
+                        "dimer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 91,
+                "sent": "FPLC system coupled to a Superdex 75 10/300 column.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "FPLC",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 92,
+                "sent": "Elution profiles at 280 nm (blue) and 260 nm (red) are: different concentration (0.05, 0.1, 0.2, 0.5 mg/ml) of M1.HpyAVI protein.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        16,
+                        "Elution profiles",
+                        "evidence"
+                    ],
+                    [
+                        111,
+                        120,
+                        "M1.HpyAVI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 93,
+                "sent": "To probe the oligomeric form of M1.HpyAVI in solution, different concentrations of purified enzyme was loaded onto a Superdex 75 10/300 column.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        32,
+                        41,
+                        "M1.HpyAVI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 94,
+                "sent": "The protein was eluted at ~10 ml regardless of the protein concentrations, corresponding to a dimeric molecular mass of 54 kDa (Figure 2D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        94,
+                        101,
+                        "dimeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        102,
+                        116,
+                        "molecular mass",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 95,
+                "sent": "Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other \u03b2-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        32,
+                        41,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        50,
+                        55,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        64,
+                        71,
+                        "crystal",
+                        "evidence"
+                    ],
+                    [
+                        94,
+                        108,
+                        "\u03b2-class MTases",
+                        "protein_type"
+                    ],
+                    [
+                        170,
+                        194,
+                        "dynamic light scattering",
+                        "experimental_method"
+                    ],
+                    [
+                        196,
+                        199,
+                        "DLS",
+                        "experimental_method"
+                    ],
+                    [
+                        217,
+                        246,
+                        "gel-filtration chromatography",
+                        "experimental_method"
+                    ],
+                    [
+                        264,
+                        273,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        286,
+                        295,
+                        "monomeric",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 96,
+                "sent": "This variance might be caused by an addition of 100 mM arginine before cell lysis to keep protein solubility and also by later replacement of arginine with 30% glycerol by dialysis.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        55,
+                        63,
+                        "arginine",
+                        "chemical"
+                    ],
+                    [
+                        142,
+                        150,
+                        "arginine",
+                        "chemical"
+                    ],
+                    [
+                        160,
+                        168,
+                        "glycerol",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 97,
+                "sent": "Structure comparisons",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        21,
+                        "Structure comparisons",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 98,
+                "sent": "As a \u03b2-class N6 adenine MTase, the M1.HpyAVI structure displayed a good similarity with M.MboIIA (PDB ID 1G60) and M.RsrI (PDB ID 1NW7), which are falling into the same subgroup.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        29,
+                        "\u03b2-class N6 adenine MTase",
+                        "protein_type"
+                    ],
+                    [
+                        35,
+                        44,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        45,
+                        54,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        88,
+                        96,
+                        "M.MboIIA",
+                        "protein"
+                    ],
+                    [
+                        115,
+                        121,
+                        "M.RsrI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 99,
+                "sent": "Superimposition of M1.HpyAVI onto them gave RMSDs of 1.63 \u00c5 and 1.9 \u00c5 on 168 and 190 C\u03b1 atoms, respectively.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        15,
+                        "Superimposition",
+                        "experimental_method"
+                    ],
+                    [
+                        19,
+                        28,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        44,
+                        49,
+                        "RMSDs",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 100,
+                "sent": "The most striking structural difference was found to locate on the TRD region (residues 133-163 in M1.HpyAVI) (Figure 3A\u20133C), where the secondary structures vary among these structures.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        67,
+                        70,
+                        "TRD",
+                        "structure_element"
+                    ],
+                    [
+                        88,
+                        95,
+                        "133-163",
+                        "residue_range"
+                    ],
+                    [
+                        99,
+                        108,
+                        "M1.HpyAVI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 101,
+                "sent": "By comparison with the other two enzymes that possess protruding arms containing several \u03b1-helices and/or \u03b2-strands, the TRD of M1.HpyAVI is much shorter in length (Figure S1), wrapping more closely around the structural core and lacking apparent secondary structures.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        89,
+                        98,
+                        "\u03b1-helices",
+                        "structure_element"
+                    ],
+                    [
+                        106,
+                        115,
+                        "\u03b2-strands",
+                        "structure_element"
+                    ],
+                    [
+                        121,
+                        124,
+                        "TRD",
+                        "structure_element"
+                    ],
+                    [
+                        128,
+                        137,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        230,
+                        237,
+                        "lacking",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 102,
+                "sent": "Given the proposed role of the TRD for DNA interaction at the major groove, some differences of DNA recognition mode can be expected.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        31,
+                        34,
+                        "TRD",
+                        "structure_element"
+                    ],
+                    [
+                        39,
+                        42,
+                        "DNA",
+                        "chemical"
+                    ],
+                    [
+                        62,
+                        74,
+                        "major groove",
+                        "structure_element"
+                    ],
+                    [
+                        96,
+                        99,
+                        "DNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 103,
+                "sent": "Another difference locates at the highly flexible loop between \u03b24 and \u03b1D (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        49,
+                        "highly flexible",
+                        "protein_state"
+                    ],
+                    [
+                        50,
+                        54,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        63,
+                        65,
+                        "\u03b24",
+                        "structure_element"
+                    ],
+                    [
+                        70,
+                        72,
+                        "\u03b1D",
+                        "structure_element"
+                    ],
+                    [
+                        83,
+                        88,
+                        "33-58",
+                        "residue_range"
+                    ],
+                    [
+                        93,
+                        102,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        131,
+                        141,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        161,
+                        171,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        175,
+                        183,
+                        "M.MboIIA",
+                        "protein"
+                    ],
+                    [
+                        188,
+                        194,
+                        "M.RsrI",
+                        "protein"
+                    ],
+                    [
+                        196,
+                        214,
+                        "Sequence alignment",
+                        "experimental_method"
+                    ],
+                    [
+                        244,
+                        253,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        351,
+                        360,
+                        "H. pylori",
+                        "species"
+                    ],
+                    [
+                        373,
+                        381,
+                        "flexible",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 104,
+                "sent": "Structural comparisons between M1.HpyAVI and other DNA MTases",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        22,
+                        "Structural comparisons",
+                        "experimental_method"
+                    ],
+                    [
+                        31,
+                        40,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        51,
+                        61,
+                        "DNA MTases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 105,
+                "sent": "A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between \u03b24 and \u03b1D of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        3,
+                        12,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        17,
+                        25,
+                        "M.MboIIA",
+                        "protein"
+                    ],
+                    [
+                        30,
+                        36,
+                        "M.RsrI",
+                        "protein"
+                    ],
+                    [
+                        41,
+                        49,
+                        "TTHA0409",
+                        "protein"
+                    ],
+                    [
+                        54,
+                        58,
+                        "DpnM",
+                        "protein"
+                    ],
+                    [
+                        63,
+                        69,
+                        "M.TaqI",
+                        "protein"
+                    ],
+                    [
+                        71,
+                        80,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        98,
+                        111,
+                        "long disorder",
+                        "protein_state"
+                    ],
+                    [
+                        112,
+                        115,
+                        "TRD",
+                        "structure_element"
+                    ],
+                    [
+                        142,
+                        156,
+                        "structure-rich",
+                        "protein_state"
+                    ],
+                    [
+                        157,
+                        160,
+                        "TRD",
+                        "structure_element"
+                    ],
+                    [
+                        164,
+                        172,
+                        "M.MboIIA",
+                        "protein"
+                    ],
+                    [
+                        174,
+                        180,
+                        "M.RsrI",
+                        "protein"
+                    ],
+                    [
+                        185,
+                        193,
+                        "TTHA0409",
+                        "protein"
+                    ],
+                    [
+                        208,
+                        226,
+                        "DNA-binding domain",
+                        "structure_element"
+                    ],
+                    [
+                        230,
+                        234,
+                        "DpnM",
+                        "protein"
+                    ],
+                    [
+                        239,
+                        245,
+                        "M.TaqI",
+                        "protein"
+                    ],
+                    [
+                        278,
+                        281,
+                        "TRD",
+                        "structure_element"
+                    ],
+                    [
+                        285,
+                        294,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        296,
+                        304,
+                        "M.MboIIA",
+                        "protein"
+                    ],
+                    [
+                        306,
+                        312,
+                        "M.RsrI",
+                        "protein"
+                    ],
+                    [
+                        317,
+                        325,
+                        "TTHA0409",
+                        "protein"
+                    ],
+                    [
+                        356,
+                        358,
+                        "\u03b24",
+                        "structure_element"
+                    ],
+                    [
+                        363,
+                        365,
+                        "\u03b1D",
+                        "structure_element"
+                    ],
+                    [
+                        369,
+                        377,
+                        "M.MboIIA",
+                        "protein"
+                    ],
+                    [
+                        382,
+                        388,
+                        "M.RsrI",
+                        "protein"
+                    ],
+                    [
+                        402,
+                        420,
+                        "DNA-binding domain",
+                        "structure_element"
+                    ],
+                    [
+                        424,
+                        428,
+                        "DpnM",
+                        "protein"
+                    ],
+                    [
+                        448,
+                        465,
+                        "C-terminal domain",
+                        "structure_element"
+                    ],
+                    [
+                        469,
+                        475,
+                        "M.TaqI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 106,
+                "sent": "Structural comparison between M1.HpyAVI and a putative \u03b2-class N4 cytosine MTase named TTHA0409 (PDB ID 2ZIF) showed a good similarity as well, giving an RMSD of 1.73 \u00c5 on 164 C\u03b1 atoms (Figure 3D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        21,
+                        "Structural comparison",
+                        "experimental_method"
+                    ],
+                    [
+                        30,
+                        39,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        55,
+                        80,
+                        "\u03b2-class N4 cytosine MTase",
+                        "protein_type"
+                    ],
+                    [
+                        87,
+                        95,
+                        "TTHA0409",
+                        "protein"
+                    ],
+                    [
+                        154,
+                        158,
+                        "RMSD",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 107,
+                "sent": "Exactly like the above comparison, the most significant difference exists in the TRD, where the structures vary in terms of length and presence of \u03b1-helices (Figure S1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        81,
+                        84,
+                        "TRD",
+                        "structure_element"
+                    ],
+                    [
+                        96,
+                        106,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        147,
+                        156,
+                        "\u03b1-helices",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 108,
+                "sent": "M1.HpyAVI displayed a considerable structural dissimilarity in comparison with N6-adenine MTases from other subgroups including the \u03b1-class DpnM (PDB ID 2DPM) and the \u03b3-class M.TaqI (PDB ID 2ADM).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        79,
+                        96,
+                        "N6-adenine MTases",
+                        "protein_type"
+                    ],
+                    [
+                        132,
+                        139,
+                        "\u03b1-class",
+                        "protein_type"
+                    ],
+                    [
+                        140,
+                        144,
+                        "DpnM",
+                        "protein"
+                    ],
+                    [
+                        167,
+                        174,
+                        "\u03b3-class",
+                        "protein_type"
+                    ],
+                    [
+                        175,
+                        181,
+                        "M.TaqI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 109,
+                "sent": "Both comparisons gave RMSDs above 3.0 \u00c5 (Figure 3E and 3F).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        22,
+                        27,
+                        "RMSDs",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 110,
+                "sent": "These two enzymes lack a counterpart loop present in the TRD of M1.HpyAVI, but instead rely on an extra domain for DNA binding and sequence recognition.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        22,
+                        "lack",
+                        "protein_state"
+                    ],
+                    [
+                        25,
+                        41,
+                        "counterpart loop",
+                        "structure_element"
+                    ],
+                    [
+                        57,
+                        60,
+                        "TRD",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        73,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        115,
+                        118,
+                        "DNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 111,
+                "sent": "Collectively, M1.HpyAVI possesses a long disordered TRD, which is in sharp contrast to the secondary structure-rich TRD in other \u03b2-class N6 adenine or N4 cytosine MTases or the extra DNA binding domain present in DNA MTases from other subgroups.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        23,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        36,
+                        51,
+                        "long disordered",
+                        "protein_state"
+                    ],
+                    [
+                        52,
+                        55,
+                        "TRD",
+                        "structure_element"
+                    ],
+                    [
+                        91,
+                        115,
+                        "secondary structure-rich",
+                        "protein_state"
+                    ],
+                    [
+                        116,
+                        119,
+                        "TRD",
+                        "structure_element"
+                    ],
+                    [
+                        129,
+                        169,
+                        "\u03b2-class N6 adenine or N4 cytosine MTases",
+                        "protein_type"
+                    ],
+                    [
+                        213,
+                        223,
+                        "DNA MTases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 112,
+                "sent": "This striking difference may be a significant determinant of the wider substrate spectrum of this H. pylori enzyme.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        98,
+                        107,
+                        "H. pylori",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 113,
+                "sent": "AdoMet-binding pocket",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        21,
+                        "AdoMet-binding pocket",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 114,
+                "sent": "The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        27,
+                        "cofactor binding pocket",
+                        "site"
+                    ],
+                    [
+                        31,
+                        40,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        67,
+                        70,
+                        "7-9",
+                        "residue_range"
+                    ],
+                    [
+                        72,
+                        77,
+                        "29-31",
+                        "residue_range"
+                    ],
+                    [
+                        79,
+                        86,
+                        "165-167",
+                        "residue_range"
+                    ],
+                    [
+                        88,
+                        95,
+                        "216-218",
+                        "residue_range"
+                    ],
+                    [
+                        100,
+                        103,
+                        "221",
+                        "residue_number"
+                    ],
+                    [
+                        127,
+                        136,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        151,
+                        161,
+                        "DNA MTases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 115,
+                "sent": "A hydrogen bond between D29 in the catalytic motif DPPY and the amino group of bound AdoMet is preserved as other MTase structures.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        2,
+                        15,
+                        "hydrogen bond",
+                        "bond_interaction"
+                    ],
+                    [
+                        24,
+                        27,
+                        "D29",
+                        "residue_name_number"
+                    ],
+                    [
+                        35,
+                        50,
+                        "catalytic motif",
+                        "structure_element"
+                    ],
+                    [
+                        51,
+                        55,
+                        "DPPY",
+                        "structure_element"
+                    ],
+                    [
+                        79,
+                        84,
+                        "bound",
+                        "protein_state"
+                    ],
+                    [
+                        85,
+                        91,
+                        "AdoMet",
+                        "chemical"
+                    ],
+                    [
+                        114,
+                        119,
+                        "MTase",
+                        "protein_type"
+                    ],
+                    [
+                        120,
+                        130,
+                        "structures",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 116,
+                "sent": "Residues D8 and A9 from hydrogen-bonds with N6 and N1 of the purine ring, respectively, and E216 also locates at hydrogen bonding distance with O2\u2032 and O3\u2032 of the ribose.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        11,
+                        "D8",
+                        "residue_name_number"
+                    ],
+                    [
+                        16,
+                        18,
+                        "A9",
+                        "residue_name_number"
+                    ],
+                    [
+                        24,
+                        38,
+                        "hydrogen-bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        61,
+                        67,
+                        "purine",
+                        "chemical"
+                    ],
+                    [
+                        92,
+                        96,
+                        "E216",
+                        "residue_name_number"
+                    ],
+                    [
+                        113,
+                        129,
+                        "hydrogen bonding",
+                        "bond_interaction"
+                    ],
+                    [
+                        163,
+                        169,
+                        "ribose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 117,
+                "sent": "In addition, H168, T200 and S198 contact the terminal carboxyl of AdoMet.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        17,
+                        "H168",
+                        "residue_name_number"
+                    ],
+                    [
+                        19,
+                        23,
+                        "T200",
+                        "residue_name_number"
+                    ],
+                    [
+                        28,
+                        32,
+                        "S198",
+                        "residue_name_number"
+                    ],
+                    [
+                        66,
+                        72,
+                        "AdoMet",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 118,
+                "sent": "Superposition of M1.HpyAVI with the five structures shown in Figure 3 reveals that the orientation of cofactor is rather conserved except for M.TaqI (Figure 4B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        13,
+                        "Superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        17,
+                        26,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        41,
+                        51,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        114,
+                        130,
+                        "rather conserved",
+                        "protein_state"
+                    ],
+                    [
+                        142,
+                        148,
+                        "M.TaqI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 119,
+                "sent": "The different conformation of the bound cofactor observed in M.TaqI might be attributable to the absence of corresponding residues of the conserved AdoMet-binding motif FXGXG in that structure.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        39,
+                        "bound",
+                        "protein_state"
+                    ],
+                    [
+                        61,
+                        67,
+                        "M.TaqI",
+                        "protein"
+                    ],
+                    [
+                        97,
+                        107,
+                        "absence of",
+                        "protein_state"
+                    ],
+                    [
+                        138,
+                        147,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        148,
+                        154,
+                        "AdoMet",
+                        "chemical"
+                    ],
+                    [
+                        169,
+                        174,
+                        "FXGXG",
+                        "structure_element"
+                    ],
+                    [
+                        183,
+                        192,
+                        "structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 120,
+                "sent": "Structural and biochemical analyses define two conserved residues D29 and E216 to be the key sites for AdoMet binding",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        35,
+                        "Structural and biochemical analyses",
+                        "experimental_method"
+                    ],
+                    [
+                        47,
+                        56,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        66,
+                        69,
+                        "D29",
+                        "residue_name_number"
+                    ],
+                    [
+                        74,
+                        78,
+                        "E216",
+                        "residue_name_number"
+                    ],
+                    [
+                        103,
+                        109,
+                        "AdoMet",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 121,
+                "sent": "A. The cofactor-binding cavity of M1.HpyAVI.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        7,
+                        30,
+                        "cofactor-binding cavity",
+                        "site"
+                    ],
+                    [
+                        34,
+                        43,
+                        "M1.HpyAVI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 122,
+                "sent": "Residues (yellow) that form direct hydrogen bonds with AdoMet (green) are indicated, distance of the hydrogen bond is marked.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        35,
+                        49,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        55,
+                        61,
+                        "AdoMet",
+                        "chemical"
+                    ],
+                    [
+                        101,
+                        114,
+                        "hydrogen bond",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 123,
+                "sent": "B. Superposition of AdoMet in the structures of M1.HpyAVI (green), DpnM (yellow) and M.TaqI (orange).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        3,
+                        16,
+                        "Superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        20,
+                        26,
+                        "AdoMet",
+                        "chemical"
+                    ],
+                    [
+                        34,
+                        44,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        48,
+                        57,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        67,
+                        71,
+                        "DpnM",
+                        "protein"
+                    ],
+                    [
+                        85,
+                        91,
+                        "M.TaqI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 124,
+                "sent": "The AdoMet terminal carboxyl of M.TaqI reveals different orientations.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        10,
+                        "AdoMet",
+                        "chemical"
+                    ],
+                    [
+                        32,
+                        38,
+                        "M.TaqI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 125,
+                "sent": "C. Cofactor binding affinity of wt-/mutants M1.HpyAVI proteins analyzed by microscale thermophoresis (MST).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        3,
+                        28,
+                        "Cofactor binding affinity",
+                        "evidence"
+                    ],
+                    [
+                        32,
+                        34,
+                        "wt",
+                        "protein_state"
+                    ],
+                    [
+                        36,
+                        43,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        44,
+                        53,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        75,
+                        100,
+                        "microscale thermophoresis",
+                        "experimental_method"
+                    ],
+                    [
+                        102,
+                        105,
+                        "MST",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 126,
+                "sent": "The binding affinity was determined between fluorescently labelled M1.HpyAVI protein and unlabeled AdoMet.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        20,
+                        "binding affinity",
+                        "evidence"
+                    ],
+                    [
+                        67,
+                        76,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        89,
+                        98,
+                        "unlabeled",
+                        "protein_state"
+                    ],
+                    [
+                        99,
+                        105,
+                        "AdoMet",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 127,
+                "sent": "AdoMet (15 nM to 1 mM) was titrated into a fixed concentration of M1.HpyAVI wt/mutant proteins (800 nM).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        6,
+                        "AdoMet",
+                        "chemical"
+                    ],
+                    [
+                        27,
+                        35,
+                        "titrated",
+                        "experimental_method"
+                    ],
+                    [
+                        66,
+                        75,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        76,
+                        78,
+                        "wt",
+                        "protein_state"
+                    ],
+                    [
+                        79,
+                        85,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 128,
+                "sent": "The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 \u00b1 6 \u03bcM; M1.HpyAVI-D8A :212 \u00b1 11 \u03bcM; M1.HpyAVI-D29A : 0 \u03bcM; M1.HpyAVI-H168A : 471 \u00b1 51 \u03bcM; M1.HpyAVI-S198A : 242 \u00b1 32 \u03bcM; M1.HpyAVI-T200A : 252 \u00b1 28 \u03bcM; M1.HpyAVI-E216A : 0 \u03bcM. Standard for three replicates is indicated.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        25,
+                        "dissociation constant",
+                        "evidence"
+                    ],
+                    [
+                        27,
+                        29,
+                        "KD",
+                        "evidence"
+                    ],
+                    [
+                        87,
+                        95,
+                        "isotherm",
+                        "evidence"
+                    ],
+                    [
+                        121,
+                        130,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        131,
+                        133,
+                        "wt",
+                        "protein_state"
+                    ],
+                    [
+                        146,
+                        159,
+                        "M1.HpyAVI-D8A",
+                        "mutant"
+                    ],
+                    [
+                        174,
+                        188,
+                        "M1.HpyAVI-D29A",
+                        "mutant"
+                    ],
+                    [
+                        197,
+                        212,
+                        "M1.HpyAVI-H168A",
+                        "mutant"
+                    ],
+                    [
+                        228,
+                        243,
+                        "M1.HpyAVI-S198A",
+                        "mutant"
+                    ],
+                    [
+                        259,
+                        274,
+                        "M1.HpyAVI-T200A",
+                        "mutant"
+                    ],
+                    [
+                        290,
+                        305,
+                        "M1.HpyAVI-E216A",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 129,
+                "sent": "D. DNA methyltransferase activity of wide type protein and the mutants is quantified using radioactive assay.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        3,
+                        24,
+                        "DNA methyltransferase",
+                        "protein_type"
+                    ],
+                    [
+                        37,
+                        46,
+                        "wide type",
+                        "protein_state"
+                    ],
+                    [
+                        63,
+                        70,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        91,
+                        108,
+                        "radioactive assay",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 130,
+                "sent": "[3H]-methyl transferred to duplex DNA containing 5\u2032-GAGG-3\u2032 was quantified by Beckman LS6500 for 10 min, experiments were repeated for three times and data were corrected by subtraction of the background.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "[3H]-methyl",
+                        "chemical"
+                    ],
+                    [
+                        34,
+                        37,
+                        "DNA",
+                        "chemical"
+                    ],
+                    [
+                        49,
+                        59,
+                        "5\u2032-GAGG-3\u2032",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 131,
+                "sent": "E. Superposition of M1.HpyAVI (green) with M.MboIIA (cyan) and M.RsrI (magenta).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        3,
+                        16,
+                        "Superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        20,
+                        29,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        43,
+                        51,
+                        "M.MboIIA",
+                        "protein"
+                    ],
+                    [
+                        63,
+                        69,
+                        "M.RsrI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 132,
+                "sent": "Residues D29 and E216 are conserved through all the DNA MTases mentioned in Figure 3 (not shown in Figure 4).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        9,
+                        12,
+                        "D29",
+                        "residue_name_number"
+                    ],
+                    [
+                        17,
+                        21,
+                        "E216",
+                        "residue_name_number"
+                    ],
+                    [
+                        26,
+                        35,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        52,
+                        62,
+                        "DNA MTases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 133,
+                "sent": "To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        72,
+                        86,
+                        "single mutants",
+                        "experimental_method"
+                    ],
+                    [
+                        90,
+                        99,
+                        "replacing",
+                        "experimental_method"
+                    ],
+                    [
+                        100,
+                        102,
+                        "D8",
+                        "residue_name_number"
+                    ],
+                    [
+                        104,
+                        107,
+                        "D29",
+                        "residue_name_number"
+                    ],
+                    [
+                        109,
+                        113,
+                        "H168",
+                        "residue_name_number"
+                    ],
+                    [
+                        115,
+                        119,
+                        "S198",
+                        "residue_name_number"
+                    ],
+                    [
+                        121,
+                        125,
+                        "T200",
+                        "residue_name_number"
+                    ],
+                    [
+                        127,
+                        131,
+                        "E216",
+                        "residue_name_number"
+                    ],
+                    [
+                        137,
+                        144,
+                        "alanine",
+                        "residue_name"
+                    ],
+                    [
+                        168,
+                        191,
+                        "ligand binding affinity",
+                        "evidence"
+                    ],
+                    [
+                        198,
+                        223,
+                        "microscale thermophoresis",
+                        "experimental_method"
+                    ],
+                    [
+                        225,
+                        228,
+                        "MST",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 134,
+                "sent": "As shown in Figure 4C, by contrast to the wild type enzyme, most mutants displayed variable reduction of KD value, among them the D29A and E216A mutants displayed no protein-AdoMet affinity at all.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        42,
+                        51,
+                        "wild type",
+                        "protein_state"
+                    ],
+                    [
+                        65,
+                        72,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        105,
+                        107,
+                        "KD",
+                        "evidence"
+                    ],
+                    [
+                        130,
+                        134,
+                        "D29A",
+                        "mutant"
+                    ],
+                    [
+                        139,
+                        144,
+                        "E216A",
+                        "mutant"
+                    ],
+                    [
+                        145,
+                        152,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        166,
+                        189,
+                        "protein-AdoMet affinity",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 135,
+                "sent": "The results suggested that the hydrogen bonds formed by D29 and E216 with AdoMet were most crucial interactions for cofactor binding.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        31,
+                        45,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        56,
+                        59,
+                        "D29",
+                        "residue_name_number"
+                    ],
+                    [
+                        64,
+                        68,
+                        "E216",
+                        "residue_name_number"
+                    ],
+                    [
+                        74,
+                        80,
+                        "AdoMet",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 136,
+                "sent": "Mutation of the two residues may directly prevent the methyl transfer reaction of M1.HpyAVI.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "Mutation",
+                        "experimental_method"
+                    ],
+                    [
+                        54,
+                        60,
+                        "methyl",
+                        "chemical"
+                    ],
+                    [
+                        82,
+                        91,
+                        "M1.HpyAVI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 137,
+                "sent": "The importance of D29 is preserved because it belongs to the catalytic active site DPPY, but the residue E216 has not been fully investigated even being a conserved amino acid throughout MTases (Figure 4E).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        21,
+                        "D29",
+                        "residue_name_number"
+                    ],
+                    [
+                        61,
+                        82,
+                        "catalytic active site",
+                        "site"
+                    ],
+                    [
+                        83,
+                        87,
+                        "DPPY",
+                        "structure_element"
+                    ],
+                    [
+                        105,
+                        109,
+                        "E216",
+                        "residue_name_number"
+                    ],
+                    [
+                        155,
+                        164,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        165,
+                        175,
+                        "amino acid",
+                        "chemical"
+                    ],
+                    [
+                        187,
+                        193,
+                        "MTases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 138,
+                "sent": "E216 is the last residue of \u03b22, which contacts the two hydroxyls of the ribose of AdoMet.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "E216",
+                        "residue_name_number"
+                    ],
+                    [
+                        28,
+                        30,
+                        "\u03b22",
+                        "structure_element"
+                    ],
+                    [
+                        72,
+                        78,
+                        "ribose",
+                        "chemical"
+                    ],
+                    [
+                        82,
+                        88,
+                        "AdoMet",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 139,
+                "sent": "Replacement of this residue by alanine completely abolishes the key hydrogen bonds for AdoMet-binding, and very likely blocks the methyl transfer reaction.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Replacement",
+                        "experimental_method"
+                    ],
+                    [
+                        31,
+                        38,
+                        "alanine",
+                        "residue_name"
+                    ],
+                    [
+                        68,
+                        82,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        87,
+                        93,
+                        "AdoMet",
+                        "chemical"
+                    ],
+                    [
+                        130,
+                        136,
+                        "methyl",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 140,
+                "sent": "To confirm this notion, [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the mutants.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        24,
+                        53,
+                        "[3H]AdoMet radiological assay",
+                        "experimental_method"
+                    ],
+                    [
+                        82,
+                        88,
+                        "methyl",
+                        "chemical"
+                    ],
+                    [
+                        114,
+                        121,
+                        "mutants",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 141,
+                "sent": "As shown in Figure 4D, the result of radiological assay agreed well with the MST measurement.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        37,
+                        55,
+                        "radiological assay",
+                        "experimental_method"
+                    ],
+                    [
+                        77,
+                        80,
+                        "MST",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 142,
+                "sent": "The D29A and E216A mutants showed little or no methyl transfer activity, while other mutants exhibited reduced methyltransferase activity.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "D29A",
+                        "mutant"
+                    ],
+                    [
+                        13,
+                        18,
+                        "E216A",
+                        "mutant"
+                    ],
+                    [
+                        19,
+                        26,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        47,
+                        53,
+                        "methyl",
+                        "chemical"
+                    ],
+                    [
+                        85,
+                        92,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        111,
+                        128,
+                        "methyltransferase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 143,
+                "sent": "As mentioned previously, FXGXG is a conserved AdoMet-binding motif of DNA MTases.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        30,
+                        "FXGXG",
+                        "structure_element"
+                    ],
+                    [
+                        36,
+                        45,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        46,
+                        52,
+                        "AdoMet",
+                        "chemical"
+                    ],
+                    [
+                        70,
+                        80,
+                        "DNA MTases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 144,
+                "sent": "We also made mutants of \u201cFMGSG\u201d to alanine for every amino acid, and found that the F195A mutant was insoluble probably due to decreasing the local hydrophobicity upon this mutation.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        20,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        25,
+                        30,
+                        "FMGSG",
+                        "structure_element"
+                    ],
+                    [
+                        35,
+                        42,
+                        "alanine",
+                        "residue_name"
+                    ],
+                    [
+                        53,
+                        63,
+                        "amino acid",
+                        "chemical"
+                    ],
+                    [
+                        84,
+                        89,
+                        "F195A",
+                        "mutant"
+                    ],
+                    [
+                        90,
+                        96,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 145,
+                "sent": "We subsequently investigated the ligand binding affinity and methyl transfer reaction of the other mutants using MST and a radiological assay.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        33,
+                        56,
+                        "ligand binding affinity",
+                        "evidence"
+                    ],
+                    [
+                        61,
+                        67,
+                        "methyl",
+                        "chemical"
+                    ],
+                    [
+                        99,
+                        106,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        113,
+                        116,
+                        "MST",
+                        "experimental_method"
+                    ],
+                    [
+                        123,
+                        141,
+                        "radiological assay",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 146,
+                "sent": "We found that G197 played a crucial role in AdoMet-binding, while mutagenesis of M196 and G199 did not influence cofactor binding and catalytic activity (Figure S2A and B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        18,
+                        "G197",
+                        "residue_name_number"
+                    ],
+                    [
+                        44,
+                        50,
+                        "AdoMet",
+                        "chemical"
+                    ],
+                    [
+                        66,
+                        77,
+                        "mutagenesis",
+                        "experimental_method"
+                    ],
+                    [
+                        81,
+                        85,
+                        "M196",
+                        "residue_name_number"
+                    ],
+                    [
+                        90,
+                        94,
+                        "G199",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 147,
+                "sent": "G197 is a conserved residue throughout the DNA MTases, and replacing by alanine at this site likely change the local conformation of cofactor-binding pocket.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "G197",
+                        "residue_name_number"
+                    ],
+                    [
+                        10,
+                        19,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        43,
+                        53,
+                        "DNA MTases",
+                        "protein_type"
+                    ],
+                    [
+                        59,
+                        68,
+                        "replacing",
+                        "experimental_method"
+                    ],
+                    [
+                        72,
+                        79,
+                        "alanine",
+                        "residue_name"
+                    ],
+                    [
+                        133,
+                        156,
+                        "cofactor-binding pocket",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 148,
+                "sent": "Mutagenesis on this glycine residue in M.EcoKI or M.EcoP15I also abolished the AdoMet-binding activity.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Mutagenesis",
+                        "experimental_method"
+                    ],
+                    [
+                        20,
+                        27,
+                        "glycine",
+                        "residue_name"
+                    ],
+                    [
+                        39,
+                        46,
+                        "M.EcoKI",
+                        "protein"
+                    ],
+                    [
+                        50,
+                        59,
+                        "M.EcoP15I",
+                        "protein"
+                    ],
+                    [
+                        79,
+                        85,
+                        "AdoMet",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 149,
+                "sent": "Although mutational study could not tell the role of F195 in ligand binding due to the insolubility of the F195A mutant, structural analysis suggested the importance of this residue in AdoMet-binding.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        25,
+                        "mutational study",
+                        "experimental_method"
+                    ],
+                    [
+                        53,
+                        57,
+                        "F195",
+                        "residue_name_number"
+                    ],
+                    [
+                        107,
+                        112,
+                        "F195A",
+                        "mutant"
+                    ],
+                    [
+                        113,
+                        119,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        121,
+                        140,
+                        "structural analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        185,
+                        191,
+                        "AdoMet",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 150,
+                "sent": "The phenyl ring of F195 forms a perpendicular \u03c0-stacking interaction with the purine ring of AdoMet, which stabilizes the orientation of AdoMet bound in the pocket of M1.HpyAVI (Figure S2C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        23,
+                        "F195",
+                        "residue_name_number"
+                    ],
+                    [
+                        46,
+                        68,
+                        "\u03c0-stacking interaction",
+                        "bond_interaction"
+                    ],
+                    [
+                        93,
+                        99,
+                        "AdoMet",
+                        "chemical"
+                    ],
+                    [
+                        137,
+                        143,
+                        "AdoMet",
+                        "chemical"
+                    ],
+                    [
+                        144,
+                        152,
+                        "bound in",
+                        "protein_state"
+                    ],
+                    [
+                        157,
+                        163,
+                        "pocket",
+                        "site"
+                    ],
+                    [
+                        167,
+                        176,
+                        "M1.HpyAVI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 151,
+                "sent": "In a separate scenario, mutagenesis of this residue in M.EcoRV has been proven to play an important role in AdoMet binding.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        24,
+                        35,
+                        "mutagenesis",
+                        "experimental_method"
+                    ],
+                    [
+                        55,
+                        62,
+                        "M.EcoRV",
+                        "protein"
+                    ],
+                    [
+                        108,
+                        114,
+                        "AdoMet",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 152,
+                "sent": "Potential DNA-binding sites",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        10,
+                        27,
+                        "DNA-binding sites",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 153,
+                "sent": "The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        31,
+                        "DNA binding region",
+                        "site"
+                    ],
+                    [
+                        35,
+                        44,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        58,
+                        70,
+                        "hairpin loop",
+                        "structure_element"
+                    ],
+                    [
+                        80,
+                        87,
+                        "101-133",
+                        "residue_range"
+                    ],
+                    [
+                        94,
+                        97,
+                        "TRD",
+                        "structure_element"
+                    ],
+                    [
+                        108,
+                        115,
+                        "136-166",
+                        "residue_range"
+                    ],
+                    [
+                        124,
+                        139,
+                        "highly flexible",
+                        "protein_state"
+                    ],
+                    [
+                        140,
+                        144,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        155,
+                        160,
+                        "33-58",
+                        "residue_range"
+                    ]
+                ]
+            },
+            {
+                "sid": 154,
+                "sent": "The hairpin loop between \u03b26 and \u03b27 strands that carries a conserved HRRY sequence signature in the middle is proposed to insert into the minor groove of the bound DNA.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        16,
+                        "hairpin loop",
+                        "structure_element"
+                    ],
+                    [
+                        25,
+                        27,
+                        "\u03b26",
+                        "structure_element"
+                    ],
+                    [
+                        32,
+                        34,
+                        "\u03b27",
+                        "structure_element"
+                    ],
+                    [
+                        58,
+                        67,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        68,
+                        72,
+                        "HRRY",
+                        "structure_element"
+                    ],
+                    [
+                        137,
+                        149,
+                        "minor groove",
+                        "structure_element"
+                    ],
+                    [
+                        157,
+                        162,
+                        "bound",
+                        "protein_state"
+                    ],
+                    [
+                        163,
+                        166,
+                        "DNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 155,
+                "sent": "As aforementioned, the TRD of M1.HpyAVI shows striking difference from the other DNA MTases, and the relaxed specificity of substrate recognition may be at least partially attributable to the disordered TRD.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        23,
+                        26,
+                        "TRD",
+                        "structure_element"
+                    ],
+                    [
+                        30,
+                        39,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        81,
+                        91,
+                        "DNA MTases",
+                        "protein_type"
+                    ],
+                    [
+                        192,
+                        202,
+                        "disordered",
+                        "protein_state"
+                    ],
+                    [
+                        203,
+                        206,
+                        "TRD",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 156,
+                "sent": "In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        17,
+                        32,
+                        "highly flexible",
+                        "protein_state"
+                    ],
+                    [
+                        33,
+                        37,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        68,
+                        "DPPY",
+                        "structure_element"
+                    ],
+                    [
+                        78,
+                        87,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        110,
+                        126,
+                        "electron density",
+                        "evidence"
+                    ],
+                    [
+                        159,
+                        164,
+                        "loops",
+                        "structure_element"
+                    ],
+                    [
+                        172,
+                        184,
+                        "AdoMet-bound",
+                        "protein_state"
+                    ],
+                    [
+                        185,
+                        195,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        199,
+                        206,
+                        "M.PvuII",
+                        "protein"
+                    ],
+                    [
+                        208,
+                        212,
+                        "DpnM",
+                        "protein"
+                    ],
+                    [
+                        216,
+                        222,
+                        "M.TaqI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 157,
+                "sent": "This loop, however, was largely stabilized upon DNA binding, as observed in the protein-DNA complex structures of M.TaqI (PDB ID 2IBS), M.HhaI (PDB ID 1MHT) and M.HaeIII (PDB ID 1DCT).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        9,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        48,
+                        51,
+                        "DNA",
+                        "chemical"
+                    ],
+                    [
+                        80,
+                        110,
+                        "protein-DNA complex structures",
+                        "evidence"
+                    ],
+                    [
+                        114,
+                        120,
+                        "M.TaqI",
+                        "protein"
+                    ],
+                    [
+                        136,
+                        142,
+                        "M.HhaI",
+                        "protein"
+                    ],
+                    [
+                        161,
+                        169,
+                        "M.HaeIII",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 158,
+                "sent": "The well-ordered loop in those structures directly contacts the flipping adenine and forms hydrogen bond with neighboring bases.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        16,
+                        "well-ordered",
+                        "protein_state"
+                    ],
+                    [
+                        17,
+                        21,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        31,
+                        41,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        73,
+                        80,
+                        "adenine",
+                        "residue_name"
+                    ],
+                    [
+                        91,
+                        104,
+                        "hydrogen bond",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 159,
+                "sent": "These observations implied that the corresponding loop in other MTases, e.g. M1.HpyAVI, is likely responsible for reducing sequence recognition specificity and thus plays crucial roles in catalysis.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        50,
+                        54,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        70,
+                        "MTases",
+                        "protein_type"
+                    ],
+                    [
+                        77,
+                        86,
+                        "M1.HpyAVI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 160,
+                "sent": "Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5\u2032-GAGG-3\u2032/5\u2032-GGAG-3\u2032 or both two adenines of 5\u2032-GAAG-3\u2032, compared with the homologs from other strains that can methylate only one adenine of 5\u2032-GAGG-3\u2032. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        33,
+                        42,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        75,
+                        91,
+                        "N6 adenine MTase",
+                        "protein_type"
+                    ],
+                    [
+                        115,
+                        122,
+                        "adenine",
+                        "residue_name"
+                    ],
+                    [
+                        126,
+                        136,
+                        "5\u2032-GAGG-3\u2032",
+                        "chemical"
+                    ],
+                    [
+                        137,
+                        147,
+                        "5\u2032-GGAG-3\u2032",
+                        "chemical"
+                    ],
+                    [
+                        160,
+                        168,
+                        "adenines",
+                        "residue_name"
+                    ],
+                    [
+                        172,
+                        182,
+                        "5\u2032-GAAG-3\u2032",
+                        "chemical"
+                    ],
+                    [
+                        258,
+                        265,
+                        "adenine",
+                        "residue_name"
+                    ],
+                    [
+                        269,
+                        279,
+                        "5\u2032-GAGG-3\u2032",
+                        "chemical"
+                    ],
+                    [
+                        295,
+                        304,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        339,
+                        342,
+                        "DNA",
+                        "chemical"
+                    ],
+                    [
+                        384,
+                        393,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        421,
+                        430,
+                        "H. pylori",
+                        "species"
+                    ],
+                    [
+                        443,
+                        470,
+                        "multiple sequence alignment",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 161,
+                "sent": "Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        28,
+                        "sequence comparison",
+                        "experimental_method"
+                    ],
+                    [
+                        33,
+                        52,
+                        "structural analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        78,
+                        81,
+                        "P41",
+                        "residue_name_number"
+                    ],
+                    [
+                        83,
+                        87,
+                        "N111",
+                        "residue_name_number"
+                    ],
+                    [
+                        89,
+                        93,
+                        "K165",
+                        "residue_name_number"
+                    ],
+                    [
+                        98,
+                        102,
+                        "T166",
+                        "residue_name_number"
+                    ],
+                    [
+                        121,
+                        129,
+                        "replaced",
+                        "experimental_method"
+                    ],
+                    [
+                        133,
+                        139,
+                        "serine",
+                        "residue_name"
+                    ],
+                    [
+                        141,
+                        150,
+                        "threonine",
+                        "residue_name"
+                    ],
+                    [
+                        152,
+                        161,
+                        "threonine",
+                        "residue_name"
+                    ],
+                    [
+                        166,
+                        172,
+                        "valine",
+                        "residue_name"
+                    ]
+                ]
+            },
+            {
+                "sid": 162,
+                "sent": "Then, a [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the wide type protein and the mutants.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        37,
+                        "[3H]AdoMet radiological assay",
+                        "experimental_method"
+                    ],
+                    [
+                        66,
+                        72,
+                        "methyl",
+                        "chemical"
+                    ],
+                    [
+                        98,
+                        107,
+                        "wide type",
+                        "protein_state"
+                    ],
+                    [
+                        124,
+                        131,
+                        "mutants",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 163,
+                "sent": "As shown in Figure 5, when the substrate DNA contains 5\u2032-GAGG-3\u2032 or 5\u2032-GAAG-3\u2032, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5\u2032-GGAG-3\u2032, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        41,
+                        44,
+                        "DNA",
+                        "chemical"
+                    ],
+                    [
+                        54,
+                        64,
+                        "5\u2032-GAGG-3\u2032",
+                        "chemical"
+                    ],
+                    [
+                        68,
+                        79,
+                        "5\u2032-GAAG-3\u2032,",
+                        "chemical"
+                    ],
+                    [
+                        88,
+                        95,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        129,
+                        135,
+                        "methyl",
+                        "chemical"
+                    ],
+                    [
+                        170,
+                        172,
+                        "wt",
+                        "protein_state"
+                    ],
+                    [
+                        173,
+                        182,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        222,
+                        233,
+                        "5\u2032-GGAG-3\u2032,",
+                        "chemical"
+                    ],
+                    [
+                        238,
+                        244,
+                        "methyl",
+                        "chemical"
+                    ],
+                    [
+                        270,
+                        274,
+                        "P41S",
+                        "mutant"
+                    ],
+                    [
+                        275,
+                        281,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        324,
+                        333,
+                        "wild type",
+                        "protein_state"
+                    ],
+                    [
+                        334,
+                        343,
+                        "M1.HpyAVI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 164,
+                "sent": "Sequence alignment, structural analysis and radioactive methyl transfer activity define the key residue for wider substrate specificity of M1.HpyAVI",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        18,
+                        "Sequence alignment",
+                        "experimental_method"
+                    ],
+                    [
+                        20,
+                        39,
+                        "structural analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        44,
+                        80,
+                        "radioactive methyl transfer activity",
+                        "experimental_method"
+                    ],
+                    [
+                        139,
+                        148,
+                        "M1.HpyAVI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 165,
+                "sent": "A. Sequence alignment of M1.HpyAVI from 50 H. pylori strains including 26695 revealed several variant residues.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        3,
+                        21,
+                        "Sequence alignment",
+                        "experimental_method"
+                    ],
+                    [
+                        25,
+                        34,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        43,
+                        52,
+                        "H. pylori",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 166,
+                "sent": "Residues P41, N111, K165 and T166 of M1.HpyAVI from strain 26695 were chosen based on structural analysis and sequence alignment (shown in red arrow).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        9,
+                        12,
+                        "P41",
+                        "residue_name_number"
+                    ],
+                    [
+                        14,
+                        18,
+                        "N111",
+                        "residue_name_number"
+                    ],
+                    [
+                        20,
+                        24,
+                        "K165",
+                        "residue_name_number"
+                    ],
+                    [
+                        29,
+                        33,
+                        "T166",
+                        "residue_name_number"
+                    ],
+                    [
+                        37,
+                        46,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        59,
+                        64,
+                        "26695",
+                        "species"
+                    ],
+                    [
+                        86,
+                        105,
+                        "structural analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        110,
+                        128,
+                        "sequence alignment",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 167,
+                "sent": "Amino-acid conservation is depicted using WebLogo (Crooks et al, 2004).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        42,
+                        49,
+                        "WebLogo",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 168,
+                "sent": "B., C., D. Methyl transfer reactions were performed using wt-M1.HpyAVI, M1.HpyAVI-P41S, M1.HpyAVI-N111T, and M1.HpyAVI-K165R T166V, respectively.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        11,
+                        17,
+                        "Methyl",
+                        "chemical"
+                    ],
+                    [
+                        58,
+                        60,
+                        "wt",
+                        "protein_state"
+                    ],
+                    [
+                        61,
+                        70,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        72,
+                        86,
+                        "M1.HpyAVI-P41S",
+                        "mutant"
+                    ],
+                    [
+                        88,
+                        103,
+                        "M1.HpyAVI-N111T",
+                        "mutant"
+                    ],
+                    [
+                        109,
+                        130,
+                        "M1.HpyAVI-K165R T166V",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 169,
+                "sent": "Radioactivity incorporated into the duplex DNA containing 5\u2032-GAGG-3\u2032, 5\u2032-GAAG-3\u2032 or 5\u2032-GGAG-3\u2032 was quantified by Beckman LS6500 for 10 min.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        43,
+                        46,
+                        "DNA",
+                        "chemical"
+                    ],
+                    [
+                        58,
+                        68,
+                        "5\u2032-GAGG-3\u2032",
+                        "chemical"
+                    ],
+                    [
+                        70,
+                        80,
+                        "5\u2032-GAAG-3\u2032",
+                        "chemical"
+                    ],
+                    [
+                        84,
+                        94,
+                        "5\u2032-GGAG-3\u2032",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 170,
+                "sent": "Our experimental data identified P41 as a key residue determining the recognition of GGAG of M1.HpyAVI.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        33,
+                        36,
+                        "P41",
+                        "residue_name_number"
+                    ],
+                    [
+                        85,
+                        89,
+                        "GGAG",
+                        "structure_element"
+                    ],
+                    [
+                        93,
+                        102,
+                        "M1.HpyAVI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 171,
+                "sent": "This amino acid locates in the highly flexible loop between residues 33 and 58, which is involved in DNA binding and substrate recognition as shown above.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        31,
+                        46,
+                        "highly flexible",
+                        "protein_state"
+                    ],
+                    [
+                        47,
+                        51,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        69,
+                        78,
+                        "33 and 58",
+                        "residue_range"
+                    ],
+                    [
+                        101,
+                        104,
+                        "DNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 172,
+                "sent": "Replacement by serine at this position definitely changes the local conformation and hydrophobicity, and probably some structural properties of the whole loop, which may in turn result in reduced specificity for sequence recognition of the enzyme from strain 26695.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Replacement",
+                        "experimental_method"
+                    ],
+                    [
+                        15,
+                        21,
+                        "serine",
+                        "residue_name"
+                    ],
+                    [
+                        154,
+                        158,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        259,
+                        264,
+                        "26695",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 173,
+                "sent": "Although the DNA-bound structure of previous investigation on a \u03b3-class N6-adenine MTase revealed that the target adenine was rotated out of DNA helix, details of the methyl transfer process were still unclear.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        13,
+                        22,
+                        "DNA-bound",
+                        "protein_state"
+                    ],
+                    [
+                        23,
+                        32,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        64,
+                        88,
+                        "\u03b3-class N6-adenine MTase",
+                        "protein_type"
+                    ],
+                    [
+                        114,
+                        121,
+                        "adenine",
+                        "residue_name"
+                    ],
+                    [
+                        141,
+                        144,
+                        "DNA",
+                        "chemical"
+                    ],
+                    [
+                        167,
+                        173,
+                        "methyl",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 174,
+                "sent": "Additionally, recent studies reported the importance of N6-methyladenine in some eukaryotic species, but until now there has not been any N6-adenine MTases being identified in eukaryotes.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        56,
+                        72,
+                        "N6-methyladenine",
+                        "ptm"
+                    ],
+                    [
+                        81,
+                        91,
+                        "eukaryotic",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        138,
+                        155,
+                        "N6-adenine MTases",
+                        "protein_type"
+                    ],
+                    [
+                        176,
+                        186,
+                        "eukaryotes",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 175,
+                "sent": "Biochemical and structural characterization of M1.HpyAVI provides a new model for uncovering the methyl transfer mechanism and for investigating the N6-methyladenine in eukaryotes.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        43,
+                        "Biochemical and structural characterization",
+                        "experimental_method"
+                    ],
+                    [
+                        47,
+                        56,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        97,
+                        103,
+                        "methyl",
+                        "chemical"
+                    ],
+                    [
+                        149,
+                        165,
+                        "N6-methyladenine",
+                        "ptm"
+                    ],
+                    [
+                        169,
+                        179,
+                        "eukaryotes",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 176,
+                "sent": "Oligomeric state of DNA MTases was long accepted as monomer, but our study indicated here that M1.HpyAVI exists as a dimer both in crystal and solution.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        20,
+                        30,
+                        "DNA MTases",
+                        "protein_type"
+                    ],
+                    [
+                        52,
+                        59,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        95,
+                        104,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        117,
+                        122,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        131,
+                        138,
+                        "crystal",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 177,
+                "sent": "Interestingly, some other \u03b2-class DNA exocyclic MTases showed similar oligomeric state in crystal and in solution, indicating that dimer may be the functional state shared by a subgroup of DNA MTases.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        26,
+                        54,
+                        "\u03b2-class DNA exocyclic MTases",
+                        "protein_type"
+                    ],
+                    [
+                        90,
+                        97,
+                        "crystal",
+                        "evidence"
+                    ],
+                    [
+                        131,
+                        136,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        189,
+                        199,
+                        "DNA MTases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 178,
+                "sent": "The highly flexible region (residues 33-58) and TRD (residues 133-163) of M1.HpyAVI are supposed to interact with DNA at minor and major grooves, respectively.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        19,
+                        "highly flexible",
+                        "protein_state"
+                    ],
+                    [
+                        37,
+                        42,
+                        "33-58",
+                        "residue_range"
+                    ],
+                    [
+                        48,
+                        51,
+                        "TRD",
+                        "structure_element"
+                    ],
+                    [
+                        62,
+                        69,
+                        "133-163",
+                        "residue_range"
+                    ],
+                    [
+                        74,
+                        83,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        114,
+                        117,
+                        "DNA",
+                        "chemical"
+                    ],
+                    [
+                        121,
+                        144,
+                        "minor and major grooves",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 179,
+                "sent": "And residue P41 might be a key residue partially determining the substrate spectrum of M1.HpyAVI.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        12,
+                        15,
+                        "P41",
+                        "residue_name_number"
+                    ],
+                    [
+                        87,
+                        96,
+                        "M1.HpyAVI",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 180,
+                "sent": "The missing loop between residues 33 and 58 may need DNA binding so as to form a stable conformation, which is similar to the condition of M.TaqI. Crystallization of M1.HpyAVI-DNA complex warrants future investigations, with the purpose of revealing the mechanism behind the wider substrate specificity of this enzyme.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "missing",
+                        "protein_state"
+                    ],
+                    [
+                        12,
+                        16,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        34,
+                        43,
+                        "33 and 58",
+                        "residue_range"
+                    ],
+                    [
+                        53,
+                        56,
+                        "DNA",
+                        "chemical"
+                    ],
+                    [
+                        81,
+                        87,
+                        "stable",
+                        "protein_state"
+                    ],
+                    [
+                        139,
+                        145,
+                        "M.TaqI",
+                        "protein"
+                    ],
+                    [
+                        147,
+                        162,
+                        "Crystallization",
+                        "experimental_method"
+                    ],
+                    [
+                        166,
+                        179,
+                        "M1.HpyAVI-DNA",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 181,
+                "sent": "DNA methylation plays an important role in bacterial pathogenicity.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        15,
+                        "DNA methylation",
+                        "ptm"
+                    ],
+                    [
+                        43,
+                        52,
+                        "bacterial",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 182,
+                "sent": "DNA adenine methylation was known to regulate the expression of some virulence genes in bacteria including H.pylori.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        23,
+                        "DNA adenine methylation",
+                        "ptm"
+                    ],
+                    [
+                        88,
+                        96,
+                        "bacteria",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        107,
+                        115,
+                        "H.pylori",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 183,
+                "sent": "Inhibitors of DNA adenine methylation may have a broad antimicrobial action by targeting DNA adenine methyltransferase.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        14,
+                        37,
+                        "DNA adenine methylation",
+                        "ptm"
+                    ],
+                    [
+                        89,
+                        118,
+                        "DNA adenine methyltransferase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 184,
+                "sent": "As an important biological modification, DNA methylation directly influences bacterial survival.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        41,
+                        56,
+                        "DNA methylation",
+                        "ptm"
+                    ],
+                    [
+                        77,
+                        86,
+                        "bacterial",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 185,
+                "sent": "Knockout of M1.HpyAVI largely prevents the growth of H. pylori.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Knockout of",
+                        "experimental_method"
+                    ],
+                    [
+                        12,
+                        21,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        53,
+                        62,
+                        "H. pylori",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 186,
+                "sent": "Importantly, H. pylori is involved in 90% of all gastric malignancies.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        13,
+                        22,
+                        "H. pylori",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 187,
+                "sent": "Appropriate antibiotic regimens could successfully cure gastric diseases caused by H.pylori infection.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        83,
+                        91,
+                        "H.pylori",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 188,
+                "sent": "However, eradication of H. pylori infection remains a big challenge for the significantly increasing prevalence of its resistance to antibiotics.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        24,
+                        33,
+                        "H. pylori",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 189,
+                "sent": "The development of new drugs targeting adenine MTases such as M1.HpyAVI offers a new opportunity for inhibition of H. pylori infection.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        39,
+                        53,
+                        "adenine MTases",
+                        "protein_type"
+                    ],
+                    [
+                        62,
+                        71,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        115,
+                        124,
+                        "H. pylori",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 190,
+                "sent": "Residues that play crucial roles for catalytic activity like D29 or E216 may influence the H.pylori survival.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        61,
+                        64,
+                        "D29",
+                        "residue_name_number"
+                    ],
+                    [
+                        68,
+                        72,
+                        "E216",
+                        "residue_name_number"
+                    ],
+                    [
+                        91,
+                        99,
+                        "H.pylori",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 191,
+                "sent": "Small molecules targeting these highly conserved residues are likely to emerge less drug resistance.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        32,
+                        48,
+                        "highly conserved",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 192,
+                "sent": "In summary, the structure of M1.HpyAVI is featured with a disordered TRD and a key residue P41that located in the putative DNA binding region that may associate with the wider substrate specificity.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        16,
+                        25,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        29,
+                        38,
+                        "M1.HpyAVI",
+                        "protein"
+                    ],
+                    [
+                        58,
+                        68,
+                        "disordered",
+                        "protein_state"
+                    ],
+                    [
+                        69,
+                        72,
+                        "TRD",
+                        "structure_element"
+                    ],
+                    [
+                        91,
+                        94,
+                        "P41",
+                        "residue_name_number"
+                    ],
+                    [
+                        123,
+                        141,
+                        "DNA binding region",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 193,
+                "sent": "Residues D29 and E216 were identified to play a crucial role in cofactor binding.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        9,
+                        12,
+                        "D29",
+                        "residue_name_number"
+                    ],
+                    [
+                        17,
+                        21,
+                        "E216",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 194,
+                "sent": "As the first crystal structure of N6-adenine MTase in H.pylori, this model may shed light on design of new antibiotics to interfere the growth and pathogenesis of H.pylori in human.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        13,
+                        30,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        34,
+                        50,
+                        "N6-adenine MTase",
+                        "protein_type"
+                    ],
+                    [
+                        54,
+                        62,
+                        "H.pylori",
+                        "species"
+                    ],
+                    [
+                        163,
+                        171,
+                        "H.pylori",
+                        "species"
+                    ],
+                    [
+                        175,
+                        180,
+                        "human",
+                        "species"
+                    ]
+                ]
+            }
+        ]
+    },
+    "PMC4993997": {
+        "annotations": [
+            {
+                "sid": 0,
+                "sent": "Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase",
+                "section": "TITLE",
+                "ner": [
+                    [
+                        26,
+                        31,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        32,
+                        37,
+                        "Naa60",
+                        "protein"
+                    ],
+                    [
+                        39,
+                        43,
+                        "NatF",
+                        "complex_assembly"
+                    ],
+                    [
+                        78,
+                        95,
+                        "acetyltransferase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 1,
+                "sent": "N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        22,
+                        "N-terminal acetylation",
+                        "ptm"
+                    ],
+                    [
+                        24,
+                        38,
+                        "Nt-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        56,
+                        85,
+                        "N-terminal acetyltransferases",
+                        "protein_type"
+                    ],
+                    [
+                        87,
+                        91,
+                        "NATs",
+                        "protein_type"
+                    ],
+                    [
+                        145,
+                        152,
+                        "peptide",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 2,
+                "sent": "Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "Naa60",
+                        "protein"
+                    ],
+                    [
+                        18,
+                        22,
+                        "NatF",
+                        "complex_assembly"
+                    ],
+                    [
+                        49,
+                        52,
+                        "NAT",
+                        "protein_type"
+                    ],
+                    [
+                        67,
+                        91,
+                        "multicellular eukaryotes",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 3,
+                "sent": "This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine N\u03b5-acetyltransferase (KAT) activity to catalyze the acetylation of lysine \u03b5-amine.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        80,
+                        94,
+                        "Nt-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        142,
+                        169,
+                        "lysine N\u03b5-acetyltransferase",
+                        "protein_type"
+                    ],
+                    [
+                        171,
+                        174,
+                        "KAT",
+                        "protein_type"
+                    ],
+                    [
+                        201,
+                        212,
+                        "acetylation",
+                        "ptm"
+                    ],
+                    [
+                        216,
+                        222,
+                        "lysine",
+                        "residue_name"
+                    ]
+                ]
+            },
+            {
+                "sid": 4,
+                "sent": "Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA).",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        20,
+                        38,
+                        "crystal structures",
+                        "evidence"
+                    ],
+                    [
+                        42,
+                        47,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        48,
+                        53,
+                        "Naa60",
+                        "protein"
+                    ],
+                    [
+                        55,
+                        61,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        63,
+                        78,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        79,
+                        96,
+                        "Acetyl-Coenzyme A",
+                        "chemical"
+                    ],
+                    [
+                        98,
+                        104,
+                        "Ac-CoA",
+                        "chemical"
+                    ],
+                    [
+                        109,
+                        119,
+                        "Coenzyme A",
+                        "chemical"
+                    ],
+                    [
+                        121,
+                        124,
+                        "CoA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 5,
+                "sent": "The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the \u03b27-\u03b28 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        4,
+                        10,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        31,
+                        48,
+                        "amphipathic helix",
+                        "structure_element"
+                    ],
+                    [
+                        63,
+                        74,
+                        "GNAT domain",
+                        "structure_element"
+                    ],
+                    [
+                        120,
+                        126,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        136,
+                        149,
+                        "\u03b27-\u03b28 hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        189,
+                        195,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        196,
+                        201,
+                        "1-242",
+                        "residue_range"
+                    ],
+                    [
+                        207,
+                        220,
+                        "hNaa60(1-199)",
+                        "mutant"
+                    ],
+                    [
+                        221,
+                        239,
+                        "crystal structures",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 6,
+                "sent": "Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        44,
+                        50,
+                        "Phe 34",
+                        "residue_name_number"
+                    ],
+                    [
+                        85,
+                        93,
+                        "coenzyme",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 7,
+                "sent": "Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved \u03b23-\u03b24 long loop participates in the regulation of hNaa60 activity.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        10,
+                        55,
+                        "structural comparison and biochemical studies",
+                        "experimental_method"
+                    ],
+                    [
+                        71,
+                        77,
+                        "Tyr 97",
+                        "residue_name_number"
+                    ],
+                    [
+                        82,
+                        89,
+                        "His 138",
+                        "residue_name_number"
+                    ],
+                    [
+                        141,
+                        154,
+                        "non-conserved",
+                        "protein_state"
+                    ],
+                    [
+                        155,
+                        170,
+                        "\u03b23-\u03b24 long loop",
+                        "structure_element"
+                    ],
+                    [
+                        205,
+                        211,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 8,
+                "sent": "Acetylation is one of the most ubiquitous modifications that plays a vital role in many biological processes, such as transcriptional regulation, protein-protein interaction, enzyme activity, protein stability, antibiotic resistance, biological rhythm and so on.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Acetylation",
+                        "ptm"
+                    ]
+                ]
+            },
+            {
+                "sid": 9,
+                "sent": "Protein acetylation can be grouped into lysine N\u03b5-acetylation and peptide N-terminal acetylation (Nt-acetylation).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        8,
+                        19,
+                        "acetylation",
+                        "ptm"
+                    ],
+                    [
+                        40,
+                        61,
+                        "lysine N\u03b5-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        66,
+                        73,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        74,
+                        96,
+                        "N-terminal acetylation",
+                        "ptm"
+                    ],
+                    [
+                        98,
+                        112,
+                        "Nt-acetylation",
+                        "ptm"
+                    ]
+                ]
+            },
+            {
+                "sid": 10,
+                "sent": "Generally, N\u03b5-acetylation refers to the transfer of an acetyl group from an acetyl coenzyme A (Ac-CoA) to the \u03b5-amino group of lysine.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        11,
+                        25,
+                        "N\u03b5-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        55,
+                        61,
+                        "acetyl",
+                        "chemical"
+                    ],
+                    [
+                        76,
+                        93,
+                        "acetyl coenzyme A",
+                        "chemical"
+                    ],
+                    [
+                        95,
+                        101,
+                        "Ac-CoA",
+                        "chemical"
+                    ],
+                    [
+                        127,
+                        133,
+                        "lysine",
+                        "residue_name"
+                    ]
+                ]
+            },
+            {
+                "sid": 11,
+                "sent": "This kind of modification is catalyzed by lysine acetyltransferases (KATs), some of which are named histone acetyltransferases (HATs) because early studies focused mostly on the post-transcriptional acetylation of histones.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        42,
+                        67,
+                        "lysine acetyltransferases",
+                        "protein_type"
+                    ],
+                    [
+                        69,
+                        73,
+                        "KATs",
+                        "protein_type"
+                    ],
+                    [
+                        100,
+                        126,
+                        "histone acetyltransferases",
+                        "protein_type"
+                    ],
+                    [
+                        128,
+                        132,
+                        "HATs",
+                        "protein_type"
+                    ],
+                    [
+                        199,
+                        210,
+                        "acetylation",
+                        "ptm"
+                    ],
+                    [
+                        214,
+                        222,
+                        "histones",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 12,
+                "sent": "Despite the prominent accomplishments in the field regarding N\u03b5-acetylation by KATs for over 50 years, the significance of the more evolutionarily conserved Nt-acetylation is still inconclusive.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        61,
+                        75,
+                        "N\u03b5-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        79,
+                        83,
+                        "KATs",
+                        "protein_type"
+                    ],
+                    [
+                        157,
+                        171,
+                        "Nt-acetylation",
+                        "ptm"
+                    ]
+                ]
+            },
+            {
+                "sid": 13,
+                "sent": "Nt-acetylation is an abundant and evolutionarily conserved modification occurring in bacteria, archaea and eukaryotes.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        14,
+                        "Nt-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        85,
+                        93,
+                        "bacteria",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        95,
+                        102,
+                        "archaea",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        107,
+                        117,
+                        "eukaryotes",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 14,
+                "sent": "It is estimated that about 80\u201390% of soluble human proteins and 50\u201370% of yeast proteins are subjected to Nt-acetylation, where an acetyl moiety is transferred from Ac-CoA to the \u03b1-amino group of the first residue.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        45,
+                        50,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        74,
+                        79,
+                        "yeast",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        106,
+                        120,
+                        "Nt-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        131,
+                        137,
+                        "acetyl",
+                        "chemical"
+                    ],
+                    [
+                        165,
+                        171,
+                        "Ac-CoA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 15,
+                "sent": "Recently Nt-acetylome expands the Nt-acetylation to transmembrane proteins.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        34,
+                        48,
+                        "Nt-acetylation",
+                        "ptm"
+                    ]
+                ]
+            },
+            {
+                "sid": 16,
+                "sent": "Unlike N\u03b5-acetylation that can be eliminated by deacetylases, Nt-acetylation is considered irreversible since no corresponding deacetylase is found to date.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        7,
+                        21,
+                        "N\u03b5-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        48,
+                        60,
+                        "deacetylases",
+                        "protein_type"
+                    ],
+                    [
+                        62,
+                        76,
+                        "Nt-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        91,
+                        103,
+                        "irreversible",
+                        "protein_state"
+                    ],
+                    [
+                        127,
+                        138,
+                        "deacetylase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 17,
+                "sent": "Although Nt-acetylation has been regarded as a co-translational modification traditionally, there is evidence that post-translational Nt-acetylation exists.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        9,
+                        23,
+                        "Nt-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        134,
+                        148,
+                        "Nt-acetylation",
+                        "ptm"
+                    ]
+                ]
+            },
+            {
+                "sid": 18,
+                "sent": "During the past decades, a large number of Nt-acetylome researches have shed light on the functional roles of Nt-acetylation, including protein degradation, subcellular localization, protein-protein interaction, protein-membrane interaction, plant development, stress-response and protein stability.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        110,
+                        124,
+                        "Nt-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        242,
+                        247,
+                        "plant",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 19,
+                "sent": "The Nt-acetylation is carried out by N-terminal acetyltransferases (NATs) that belong to the GNAT superfamily.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        18,
+                        "Nt-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        37,
+                        66,
+                        "N-terminal acetyltransferases",
+                        "protein_type"
+                    ],
+                    [
+                        68,
+                        72,
+                        "NATs",
+                        "protein_type"
+                    ],
+                    [
+                        93,
+                        109,
+                        "GNAT superfamily",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 20,
+                "sent": "To date, six NATs (NatA/B/C/D/E/F) have been identified in eukaryotes.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        13,
+                        17,
+                        "NATs",
+                        "protein_type"
+                    ],
+                    [
+                        19,
+                        23,
+                        "NatA",
+                        "complex_assembly"
+                    ],
+                    [
+                        24,
+                        25,
+                        "B",
+                        "complex_assembly"
+                    ],
+                    [
+                        26,
+                        27,
+                        "C",
+                        "complex_assembly"
+                    ],
+                    [
+                        28,
+                        29,
+                        "D",
+                        "complex_assembly"
+                    ],
+                    [
+                        30,
+                        31,
+                        "E",
+                        "complex_assembly"
+                    ],
+                    [
+                        32,
+                        33,
+                        "F",
+                        "complex_assembly"
+                    ],
+                    [
+                        59,
+                        69,
+                        "eukaryotes",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 21,
+                "sent": "About 40 percent of Nt-acetylation of soluble proteins in cells is catalyzed by NatA complex which is composed of the catalytic subunit Naa10p and the auxiliary subunit Naa15p.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        20,
+                        34,
+                        "Nt-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        80,
+                        84,
+                        "NatA",
+                        "complex_assembly"
+                    ],
+                    [
+                        136,
+                        142,
+                        "Naa10p",
+                        "protein"
+                    ],
+                    [
+                        169,
+                        175,
+                        "Naa15p",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 22,
+                "sent": "NatE was found to physically interact with the NatA complex without any observation of impact on NatA-activity.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "NatE",
+                        "complex_assembly"
+                    ],
+                    [
+                        47,
+                        51,
+                        "NatA",
+                        "complex_assembly"
+                    ],
+                    [
+                        97,
+                        101,
+                        "NatA",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 23,
+                "sent": "Two other multimeric complexes of NATs are NatB and NatC which contain the catalytic subunits Naa20 and Naa30 and the auxiliary subunits Naa25 and Naa35/Naa38, respectively.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        34,
+                        38,
+                        "NATs",
+                        "protein_type"
+                    ],
+                    [
+                        43,
+                        47,
+                        "NatB",
+                        "complex_assembly"
+                    ],
+                    [
+                        52,
+                        56,
+                        "NatC",
+                        "complex_assembly"
+                    ],
+                    [
+                        94,
+                        99,
+                        "Naa20",
+                        "protein"
+                    ],
+                    [
+                        104,
+                        109,
+                        "Naa30",
+                        "protein"
+                    ],
+                    [
+                        137,
+                        142,
+                        "Naa25",
+                        "protein"
+                    ],
+                    [
+                        147,
+                        152,
+                        "Naa35",
+                        "protein"
+                    ],
+                    [
+                        153,
+                        158,
+                        "Naa38",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 24,
+                "sent": "Furthermore, only the catalytic subunits Naa40 and Naa60 were found for NatD and NatF, respectively.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        41,
+                        46,
+                        "Naa40",
+                        "protein"
+                    ],
+                    [
+                        51,
+                        56,
+                        "Naa60",
+                        "protein"
+                    ],
+                    [
+                        72,
+                        76,
+                        "NatD",
+                        "complex_assembly"
+                    ],
+                    [
+                        81,
+                        85,
+                        "NatF",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 25,
+                "sent": "Besides Nt-acetylation, accumulating reports have proposed N\u03b5-acetylation carried out by NATs.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        8,
+                        22,
+                        "Nt-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        59,
+                        73,
+                        "N\u03b5-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        89,
+                        93,
+                        "NATs",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 26,
+                "sent": "There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        53,
+                        67,
+                        "Nt-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        76,
+                        81,
+                        "yeast",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        86,
+                        91,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        147,
+                        151,
+                        "NatF",
+                        "complex_assembly"
+                    ],
+                    [
+                        166,
+                        194,
+                        "N-terminal acetyltransferase",
+                        "protein_type"
+                    ],
+                    [
+                        223,
+                        227,
+                        "NatF",
+                        "complex_assembly"
+                    ],
+                    [
+                        240,
+                        245,
+                        "NAA60",
+                        "protein"
+                    ],
+                    [
+                        264,
+                        306,
+                        "Histone acetyltransferase type B protein 4",
+                        "protein"
+                    ],
+                    [
+                        308,
+                        312,
+                        "HAT4",
+                        "protein"
+                    ],
+                    [
+                        315,
+                        320,
+                        "Naa60",
+                        "protein"
+                    ],
+                    [
+                        324,
+                        346,
+                        "N-acetyltransferase 15",
+                        "protein"
+                    ],
+                    [
+                        348,
+                        353,
+                        "NAT15",
+                        "protein"
+                    ],
+                    [
+                        386,
+                        389,
+                        "NAT",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 27,
+                "sent": "Unlike other NATs that are highly conserved among lower and higher eukaryotes, NatF only exists in higher eukaryotes.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        13,
+                        17,
+                        "NATs",
+                        "protein_type"
+                    ],
+                    [
+                        27,
+                        43,
+                        "highly conserved",
+                        "protein_state"
+                    ],
+                    [
+                        50,
+                        55,
+                        "lower",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        60,
+                        77,
+                        "higher eukaryotes",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        79,
+                        83,
+                        "NatF",
+                        "complex_assembly"
+                    ],
+                    [
+                        99,
+                        116,
+                        "higher eukaryotes",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 28,
+                "sent": "Subsequent researches indicated that NatF displays its catalytic ability with both Nt-acetylation and lysine N\u03b5-acetylation.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        37,
+                        41,
+                        "NatF",
+                        "complex_assembly"
+                    ],
+                    [
+                        83,
+                        97,
+                        "Nt-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        102,
+                        123,
+                        "lysine N\u03b5-acetylation",
+                        "ptm"
+                    ]
+                ]
+            },
+            {
+                "sid": 29,
+                "sent": "As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal \u03b1-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        6,
+                        34,
+                        "N-terminal acetyltransferase",
+                        "protein_type"
+                    ],
+                    [
+                        36,
+                        40,
+                        "NatF",
+                        "complex_assembly"
+                    ],
+                    [
+                        67,
+                        78,
+                        "acetylation",
+                        "ptm"
+                    ],
+                    [
+                        187,
+                        195,
+                        "Met-Lys-",
+                        "structure_element"
+                    ],
+                    [
+                        197,
+                        205,
+                        "Met-Val-",
+                        "structure_element"
+                    ],
+                    [
+                        207,
+                        215,
+                        "Met-Ala-",
+                        "structure_element"
+                    ],
+                    [
+                        220,
+                        228,
+                        "Met-Met-",
+                        "structure_element"
+                    ],
+                    [
+                        293,
+                        297,
+                        "NatC",
+                        "complex_assembly"
+                    ],
+                    [
+                        302,
+                        306,
+                        "NatE",
+                        "complex_assembly"
+                    ],
+                    [
+                        327,
+                        331,
+                        "NatF",
+                        "complex_assembly"
+                    ],
+                    [
+                        342,
+                        366,
+                        "lysine acetyltransferase",
+                        "protein_type"
+                    ],
+                    [
+                        390,
+                        408,
+                        "lysine acetylation",
+                        "ptm"
+                    ],
+                    [
+                        417,
+                        424,
+                        "histone",
+                        "protein_type"
+                    ],
+                    [
+                        425,
+                        427,
+                        "H4",
+                        "protein_type"
+                    ],
+                    [
+                        439,
+                        441,
+                        "H4",
+                        "protein_type"
+                    ],
+                    [
+                        441,
+                        444,
+                        "K20",
+                        "residue_name_number"
+                    ],
+                    [
+                        446,
+                        448,
+                        "H4",
+                        "protein_type"
+                    ],
+                    [
+                        448,
+                        451,
+                        "K79",
+                        "residue_name_number"
+                    ],
+                    [
+                        456,
+                        458,
+                        "H4",
+                        "protein_type"
+                    ],
+                    [
+                        458,
+                        461,
+                        "K91",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 30,
+                "sent": "Another important feature of NatF is that this protein is anchored on the Golgi apparatus through its C-terminal membrane-integrating region and takes part in the maintaining of Golgi integrity.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        29,
+                        33,
+                        "NatF",
+                        "complex_assembly"
+                    ],
+                    [
+                        113,
+                        140,
+                        "membrane-integrating region",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 31,
+                "sent": "With its unique intracellular organellar localization and substrate selectivity, NatF appears to provide more evolutionary information among the NAT family members.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        81,
+                        85,
+                        "NatF",
+                        "complex_assembly"
+                    ],
+                    [
+                        145,
+                        148,
+                        "NAT",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 32,
+                "sent": "It was recently found that NatF facilitates nucleosomes assembly and that NAA60 knockdown in MCF7-cell inhibits cell proliferation, sensitizes cells to DNA damage and induces cell apoptosis.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        27,
+                        31,
+                        "NatF",
+                        "complex_assembly"
+                    ],
+                    [
+                        44,
+                        55,
+                        "nucleosomes",
+                        "complex_assembly"
+                    ],
+                    [
+                        74,
+                        79,
+                        "NAA60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 33,
+                "sent": "In Drosophila cells, NAA60 knockdown induces chromosomal segregation defects during anaphase including lagging chromosomes and chromosomal bridges.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        3,
+                        13,
+                        "Drosophila",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        21,
+                        26,
+                        "NAA60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 34,
+                "sent": "Much recent attention has also been focused on the requirement of NatF for regulation of organellar structure.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        66,
+                        70,
+                        "NatF",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 35,
+                "sent": "In HeLa cells, NAA60 knockdown causes Golgi apparatus fragmentation which can be rescued by overexpression Naa60.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        15,
+                        20,
+                        "NAA60",
+                        "protein"
+                    ],
+                    [
+                        92,
+                        106,
+                        "overexpression",
+                        "experimental_method"
+                    ],
+                    [
+                        107,
+                        112,
+                        "Naa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 36,
+                "sent": "The systematic investigation of publicly available microarray data showed that NATs share distinct tissue-specific expression patterns in Drosophila and NatF shows a higher expression level in central nervous system of Drosophila.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        79,
+                        83,
+                        "NATs",
+                        "protein_type"
+                    ],
+                    [
+                        138,
+                        148,
+                        "Drosophila",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        153,
+                        157,
+                        "NatF",
+                        "complex_assembly"
+                    ],
+                    [
+                        219,
+                        229,
+                        "Drosophila",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 37,
+                "sent": "In this study, we solved the structures of human Naa60 (NatF) in complex with coenzyme.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        18,
+                        24,
+                        "solved",
+                        "experimental_method"
+                    ],
+                    [
+                        29,
+                        39,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        43,
+                        48,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        49,
+                        54,
+                        "Naa60",
+                        "protein"
+                    ],
+                    [
+                        56,
+                        60,
+                        "NatF",
+                        "complex_assembly"
+                    ],
+                    [
+                        62,
+                        77,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        78,
+                        86,
+                        "coenzyme",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 38,
+                "sent": "The hNaa60 protein contains a unique amphipathic \u03b1-helix (\u03b15) following its GNAT domain that might account for the Golgi localization of this protein.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        10,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        37,
+                        56,
+                        "amphipathic \u03b1-helix",
+                        "structure_element"
+                    ],
+                    [
+                        58,
+                        60,
+                        "\u03b15",
+                        "structure_element"
+                    ],
+                    [
+                        76,
+                        87,
+                        "GNAT domain",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 39,
+                "sent": "Crystal structures showed that the \u03b27-\u03b28 hairpin rotated about 50 degrees upon removing the C-terminal region of the protein and this movement substantially changed the geometry of the substrate-binding pocket.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        18,
+                        "Crystal structures",
+                        "evidence"
+                    ],
+                    [
+                        35,
+                        48,
+                        "\u03b27-\u03b28 hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        92,
+                        109,
+                        "C-terminal region",
+                        "structure_element"
+                    ],
+                    [
+                        185,
+                        209,
+                        "substrate-binding pocket",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 40,
+                "sent": "Remarkably, we find that Phe 34 may participate in the proper positioning of the coenzyme for the transfer reaction to occur.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        25,
+                        31,
+                        "Phe 34",
+                        "residue_name_number"
+                    ],
+                    [
+                        81,
+                        89,
+                        "coenzyme",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 41,
+                "sent": "Further structure comparison and biochemical studies also identified other key structural elements essential for the enzyme activity of Naa60.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        8,
+                        28,
+                        "structure comparison",
+                        "experimental_method"
+                    ],
+                    [
+                        33,
+                        52,
+                        "biochemical studies",
+                        "experimental_method"
+                    ],
+                    [
+                        136,
+                        141,
+                        "Naa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 42,
+                "sent": "Overall structure of hNaa60",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        17,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        27,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 43,
+                "sent": "In the effort to prepare the protein for structural studies, we tried a large number of hNaa60 constructs but all failed due to heavy precipitation or aggregation.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        88,
+                        94,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 44,
+                "sent": "Sequence alignment of Naa60 from different species revealed a Glu-Glu-Arg (EER) versus Val-Val-Pro (VVP) sequence difference near the N-terminus of the protein in Xenopus Laevis versus Homo sapiens (Fig. 1A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        18,
+                        "Sequence alignment",
+                        "experimental_method"
+                    ],
+                    [
+                        22,
+                        27,
+                        "Naa60",
+                        "protein"
+                    ],
+                    [
+                        62,
+                        73,
+                        "Glu-Glu-Arg",
+                        "structure_element"
+                    ],
+                    [
+                        75,
+                        78,
+                        "EER",
+                        "structure_element"
+                    ],
+                    [
+                        87,
+                        98,
+                        "Val-Val-Pro",
+                        "structure_element"
+                    ],
+                    [
+                        100,
+                        103,
+                        "VVP",
+                        "structure_element"
+                    ],
+                    [
+                        163,
+                        177,
+                        "Xenopus Laevis",
+                        "species"
+                    ],
+                    [
+                        185,
+                        197,
+                        "Homo sapiens",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 45,
+                "sent": "Considering that terminal residues may lack higher-order structure and hydrophobic residues in this region may expose to solvent and hence cause protein aggregation, we mutated residues 4\u20136 from VVP to EER for the purpose of improving solubility of this protein.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        169,
+                        176,
+                        "mutated",
+                        "experimental_method"
+                    ],
+                    [
+                        186,
+                        189,
+                        "4\u20136",
+                        "residue_range"
+                    ],
+                    [
+                        195,
+                        205,
+                        "VVP to EER",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 46,
+                "sent": "According to previous studies, this N-terminal region should not interfere with hNaa60\u2019s Golgi localization.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        80,
+                        86,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 47,
+                "sent": "We tried many hNaa60 constructs with the three-residues mutation but only the truncated variant 1-199 and the full-length protein behaved well.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        20,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        56,
+                        64,
+                        "mutation",
+                        "experimental_method"
+                    ],
+                    [
+                        78,
+                        87,
+                        "truncated",
+                        "protein_state"
+                    ],
+                    [
+                        96,
+                        101,
+                        "1-199",
+                        "residue_range"
+                    ],
+                    [
+                        110,
+                        121,
+                        "full-length",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 48,
+                "sent": "We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        16,
+                        23,
+                        "crystal",
+                        "evidence"
+                    ],
+                    [
+                        31,
+                        40,
+                        "truncated",
+                        "protein_state"
+                    ],
+                    [
+                        49,
+                        54,
+                        "1-199",
+                        "residue_range"
+                    ],
+                    [
+                        55,
+                        70,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        71,
+                        74,
+                        "CoA",
+                        "chemical"
+                    ],
+                    [
+                        120,
+                        127,
+                        "crystal",
+                        "evidence"
+                    ],
+                    [
+                        135,
+                        146,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        174,
+                        179,
+                        "1-242",
+                        "residue_range"
+                    ],
+                    [
+                        181,
+                        196,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        197,
+                        203,
+                        "Ac-CoA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 49,
+                "sent": "Hereafter, all deletions or point mutants of hNaa60 we describe here are with the EER mutation.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        41,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        45,
+                        51,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        82,
+                        85,
+                        "EER",
+                        "structure_element"
+                    ],
+                    [
+                        86,
+                        94,
+                        "mutation",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 50,
+                "sent": "The crystal structures of hNaa60(1-242)/Ac-CoA and hNaa60(1-199)/CoA were determined by molecular replacement and refined to 1.38\u2009\u00c5 and 1.60\u2009\u00c5 resolution, respectively (Table 1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        22,
+                        "crystal structures",
+                        "evidence"
+                    ],
+                    [
+                        26,
+                        46,
+                        "hNaa60(1-242)/Ac-CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        51,
+                        68,
+                        "hNaa60(1-199)/CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        88,
+                        109,
+                        "molecular replacement",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 51,
+                "sent": "The electron density maps were of sufficient quality to trace residues 1-211 of hNaa60(1-242) and residues 5-199 of hNaa60(1-199).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        25,
+                        "electron density maps",
+                        "evidence"
+                    ],
+                    [
+                        71,
+                        76,
+                        "1-211",
+                        "residue_range"
+                    ],
+                    [
+                        80,
+                        86,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        87,
+                        92,
+                        "1-242",
+                        "residue_range"
+                    ],
+                    [
+                        107,
+                        112,
+                        "5-199",
+                        "residue_range"
+                    ],
+                    [
+                        116,
+                        129,
+                        "hNaa60(1-199)",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 52,
+                "sent": "The structure of hNaa60 protein contains a central domain exhibiting a classic GCN5-related N-acetyltransferase (GNAT) folding, along with the extended N- and C-terminal regions (Fig. 1B,C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        17,
+                        23,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        43,
+                        57,
+                        "central domain",
+                        "structure_element"
+                    ],
+                    [
+                        79,
+                        111,
+                        "GCN5-related N-acetyltransferase",
+                        "protein_type"
+                    ],
+                    [
+                        113,
+                        117,
+                        "GNAT",
+                        "protein_type"
+                    ],
+                    [
+                        143,
+                        151,
+                        "extended",
+                        "protein_state"
+                    ],
+                    [
+                        152,
+                        177,
+                        "N- and C-terminal regions",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 53,
+                "sent": "The central domain includes nine \u03b2 strands (\u03b21-\u03b29) and four \u03b1-helixes (\u03b11-\u03b14) and is highly similar to the known hNaa50p and other reported NATs (Fig. 1D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        18,
+                        "central domain",
+                        "structure_element"
+                    ],
+                    [
+                        33,
+                        42,
+                        "\u03b2 strands",
+                        "structure_element"
+                    ],
+                    [
+                        44,
+                        49,
+                        "\u03b21-\u03b29",
+                        "structure_element"
+                    ],
+                    [
+                        60,
+                        69,
+                        "\u03b1-helixes",
+                        "structure_element"
+                    ],
+                    [
+                        71,
+                        76,
+                        "\u03b11-\u03b14",
+                        "structure_element"
+                    ],
+                    [
+                        85,
+                        99,
+                        "highly similar",
+                        "protein_state"
+                    ],
+                    [
+                        113,
+                        120,
+                        "hNaa50p",
+                        "protein"
+                    ],
+                    [
+                        140,
+                        144,
+                        "NATs",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 54,
+                "sent": "However, in hNaa60, there is an extra 20-residue loop between \u03b23 and \u03b24 that forms a small subdomain with well-defined 3D structure (Fig. 1B\u2013D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        12,
+                        18,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        32,
+                        53,
+                        "extra 20-residue loop",
+                        "structure_element"
+                    ],
+                    [
+                        62,
+                        64,
+                        "\u03b23",
+                        "structure_element"
+                    ],
+                    [
+                        69,
+                        71,
+                        "\u03b24",
+                        "structure_element"
+                    ],
+                    [
+                        85,
+                        100,
+                        "small subdomain",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 55,
+                "sent": "Furthermore, the \u03b27-\u03b28 strands form an approximately antiparallel \u03b2-hairpin structure remarkably different from that in hNaa50p (Fig. 1D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        17,
+                        30,
+                        "\u03b27-\u03b28 strands",
+                        "structure_element"
+                    ],
+                    [
+                        39,
+                        85,
+                        "approximately antiparallel \u03b2-hairpin structure",
+                        "structure_element"
+                    ],
+                    [
+                        120,
+                        127,
+                        "hNaa50p",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 56,
+                "sent": "The N- and C-terminal regions form helical structures (\u03b10 and \u03b15) stretching out from the central GCN5-domain (Fig. 1C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        29,
+                        "N- and C-terminal regions",
+                        "structure_element"
+                    ],
+                    [
+                        35,
+                        53,
+                        "helical structures",
+                        "structure_element"
+                    ],
+                    [
+                        55,
+                        57,
+                        "\u03b10",
+                        "structure_element"
+                    ],
+                    [
+                        62,
+                        64,
+                        "\u03b15",
+                        "structure_element"
+                    ],
+                    [
+                        98,
+                        109,
+                        "GCN5-domain",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 57,
+                "sent": "Interestingly, we found that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) (Figure S1), indicating that residues 200\u2013242 may have some auto-inhibitory effect on the activity of the enzyme.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        55,
+                        61,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        62,
+                        67,
+                        "1-242",
+                        "residue_range"
+                    ],
+                    [
+                        96,
+                        109,
+                        "hNaa60(1-199)",
+                        "mutant"
+                    ],
+                    [
+                        148,
+                        155,
+                        "200\u2013242",
+                        "residue_range"
+                    ]
+                ]
+            },
+            {
+                "sid": 58,
+                "sent": "However, since this region was not visible in the hNaa60(1-242) crystal structure, we do not yet understand how this happens.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        50,
+                        56,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        57,
+                        62,
+                        "1-242",
+                        "residue_range"
+                    ],
+                    [
+                        64,
+                        81,
+                        "crystal structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 59,
+                "sent": "Another possibility is that since hNaa60 is localized on Golgi apparatus, the observed low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        40,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        107,
+                        118,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        119,
+                        125,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 60,
+                "sent": "For the convenience of studying the kinetics of mutants, the mutagenesis studies described hereafter were all based on hNaa60 (1-199).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        48,
+                        55,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        61,
+                        80,
+                        "mutagenesis studies",
+                        "experimental_method"
+                    ],
+                    [
+                        119,
+                        133,
+                        "hNaa60 (1-199)",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 61,
+                "sent": "An amphipathic \u03b1-helix in the C-terminal region may contribute to Golgi localization of hNaa60",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        3,
+                        22,
+                        "amphipathic \u03b1-helix",
+                        "structure_element"
+                    ],
+                    [
+                        30,
+                        47,
+                        "C-terminal region",
+                        "structure_element"
+                    ],
+                    [
+                        88,
+                        94,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 62,
+                "sent": "There is one hNaa60 molecule in the asymmetric unit in the hNaa60(1-242)/Ac-CoA structure.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        19,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        59,
+                        79,
+                        "hNaa60(1-242)/Ac-CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        80,
+                        89,
+                        "structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 63,
+                "sent": "The C-terminal region extended from the GCN5-domain forms an amphipathic helix (\u03b15) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between \u03b15-helix and a hydrophobic groove between the N-terminal \u03b21 and \u03b23 strands of the neighbor molecule (Fig. 2A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        21,
+                        "C-terminal region",
+                        "structure_element"
+                    ],
+                    [
+                        40,
+                        51,
+                        "GCN5-domain",
+                        "structure_element"
+                    ],
+                    [
+                        61,
+                        78,
+                        "amphipathic helix",
+                        "structure_element"
+                    ],
+                    [
+                        80,
+                        82,
+                        "\u03b15",
+                        "structure_element"
+                    ],
+                    [
+                        152,
+                        176,
+                        "hydrophobic interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        185,
+                        193,
+                        "\u03b15-helix",
+                        "structure_element"
+                    ],
+                    [
+                        200,
+                        218,
+                        "hydrophobic groove",
+                        "site"
+                    ],
+                    [
+                        242,
+                        244,
+                        "\u03b21",
+                        "structure_element"
+                    ],
+                    [
+                        249,
+                        259,
+                        "\u03b23 strands",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 64,
+                "sent": "The C-terminal extension following \u03b15-helix forms a \u03b2-turn that wraps around and interacts with the neighbor protein molecule through hydrophobic interactions, too.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        24,
+                        "C-terminal extension",
+                        "structure_element"
+                    ],
+                    [
+                        35,
+                        43,
+                        "\u03b15-helix",
+                        "structure_element"
+                    ],
+                    [
+                        52,
+                        58,
+                        "\u03b2-turn",
+                        "structure_element"
+                    ],
+                    [
+                        134,
+                        158,
+                        "hydrophobic interactions",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 65,
+                "sent": "In the hNaa60(1-199)/CoA structure, a part of the \u03b15-helix is deleted due to truncation of the C-terminal region (Fig. 1B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        7,
+                        24,
+                        "hNaa60(1-199)/CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        25,
+                        34,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        50,
+                        58,
+                        "\u03b15-helix",
+                        "structure_element"
+                    ],
+                    [
+                        95,
+                        112,
+                        "C-terminal region",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 66,
+                "sent": "Interestingly, the remaining residues in \u03b15-helix still form an amphipathic helix although the hydrophobic interaction with the N-terminal hydrophobic groove of a neighbor molecule is abolished and the helix is largely exposed in solvent due to different crystal packing (Fig. 2B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        41,
+                        49,
+                        "\u03b15-helix",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        81,
+                        "amphipathic helix",
+                        "structure_element"
+                    ],
+                    [
+                        95,
+                        118,
+                        "hydrophobic interaction",
+                        "bond_interaction"
+                    ],
+                    [
+                        139,
+                        157,
+                        "hydrophobic groove",
+                        "site"
+                    ],
+                    [
+                        202,
+                        207,
+                        "helix",
+                        "structure_element"
+                    ],
+                    [
+                        255,
+                        270,
+                        "crystal packing",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 67,
+                "sent": "A recent research showed that residues 182\u2013216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (\u03b15) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        39,
+                        46,
+                        "182\u2013216",
+                        "residue_range"
+                    ],
+                    [
+                        85,
+                        91,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        119,
+                        128,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        134,
+                        149,
+                        "solvent-exposed",
+                        "protein_state"
+                    ],
+                    [
+                        150,
+                        167,
+                        "amphipathic helix",
+                        "structure_element"
+                    ],
+                    [
+                        169,
+                        171,
+                        "\u03b15",
+                        "structure_element"
+                    ],
+                    [
+                        192,
+                        199,
+                        "190-202",
+                        "residue_range"
+                    ],
+                    [
+                        259,
+                        266,
+                        "Ile 190",
+                        "residue_name_number"
+                    ],
+                    [
+                        268,
+                        275,
+                        "Leu 191",
+                        "residue_name_number"
+                    ],
+                    [
+                        277,
+                        284,
+                        "Ile 194",
+                        "residue_name_number"
+                    ],
+                    [
+                        286,
+                        293,
+                        "Leu 197",
+                        "residue_name_number"
+                    ],
+                    [
+                        298,
+                        305,
+                        "Leu 201",
+                        "residue_name_number"
+                    ],
+                    [
+                        398,
+                        404,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        579,
+                        587,
+                        "KalSec14",
+                        "protein"
+                    ],
+                    [
+                        589,
+                        593,
+                        "Atg3",
+                        "protein"
+                    ],
+                    [
+                        595,
+                        601,
+                        "PB1-F2",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 68,
+                "sent": "The \u03b27-\u03b28 hairpin showed alternative conformations in the hNaa60 crystal structures",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        17,
+                        "\u03b27-\u03b28 hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        58,
+                        64,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        65,
+                        83,
+                        "crystal structures",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 69,
+                "sent": "Superposition of hNaa60(1-242)/Ac-CoA, hNaa60(1-199)/CoA and hNaa50/CoA/peptide (PDB 3TFY) revealed considerable difference in the \u03b27-\u03b28 hairpin region despite the overall stability and similarity of the GNAT domain (Fig. 1D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        13,
+                        "Superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        17,
+                        37,
+                        "hNaa60(1-242)/Ac-CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        39,
+                        56,
+                        "hNaa60(1-199)/CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        61,
+                        79,
+                        "hNaa50/CoA/peptide",
+                        "complex_assembly"
+                    ],
+                    [
+                        131,
+                        144,
+                        "\u03b27-\u03b28 hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        204,
+                        215,
+                        "GNAT domain",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 70,
+                "sent": "In hNaa60(1-242), the \u03b27-\u03b28 hairpin is located in close proximity to the \u03b11-\u03b12 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (\u03b26-\u03b27 loop).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        3,
+                        9,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        10,
+                        15,
+                        "1-242",
+                        "residue_range"
+                    ],
+                    [
+                        22,
+                        35,
+                        "\u03b27-\u03b28 hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        73,
+                        83,
+                        "\u03b11-\u03b12 loop",
+                        "structure_element"
+                    ],
+                    [
+                        109,
+                        131,
+                        "substrate binding site",
+                        "site"
+                    ],
+                    [
+                        145,
+                        151,
+                        "hNaa50",
+                        "protein"
+                    ],
+                    [
+                        185,
+                        193,
+                        "flexible",
+                        "protein_state"
+                    ],
+                    [
+                        194,
+                        198,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        213,
+                        223,
+                        "\u03b26-\u03b27 loop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 71,
+                "sent": "Upon removing the C-terminal region of hNaa60, we observed that hNaa60 (1-199) molecules pack in a different way involving the \u03b27-\u03b28 hairpin in the crystal, leading to about 50 degree rotation of the hairpin which moves away from the \u03b11-\u03b12 loop (Figs 1D and 2C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        13,
+                        "removing",
+                        "experimental_method"
+                    ],
+                    [
+                        18,
+                        35,
+                        "C-terminal region",
+                        "structure_element"
+                    ],
+                    [
+                        39,
+                        45,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        64,
+                        78,
+                        "hNaa60 (1-199)",
+                        "mutant"
+                    ],
+                    [
+                        127,
+                        140,
+                        "\u03b27-\u03b28 hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        148,
+                        155,
+                        "crystal",
+                        "evidence"
+                    ],
+                    [
+                        200,
+                        207,
+                        "hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        234,
+                        244,
+                        "\u03b11-\u03b12 loop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 72,
+                "sent": "This conformational change substantially altered the geometry of the substrate binding site, which could potentially change the way in which the substrate accesses the active site of the enzyme.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        69,
+                        91,
+                        "substrate binding site",
+                        "site"
+                    ],
+                    [
+                        168,
+                        179,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 73,
+                "sent": "In hNaa60(1-242), the \u03b27-\u03b28 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        3,
+                        9,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        10,
+                        15,
+                        "1-242",
+                        "residue_range"
+                    ],
+                    [
+                        22,
+                        35,
+                        "\u03b27-\u03b28 hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        47,
+                        58,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        96,
+                        102,
+                        "hNaa50",
+                        "protein"
+                    ],
+                    [
+                        168,
+                        179,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        231,
+                        237,
+                        "tunnel",
+                        "site"
+                    ],
+                    [
+                        244,
+                        250,
+                        "Ac-CoA",
+                        "chemical"
+                    ],
+                    [
+                        251,
+                        254,
+                        "CoA",
+                        "chemical"
+                    ],
+                    [
+                        306,
+                        309,
+                        "NAT",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 74,
+                "sent": "KAT activity of hNaa60 toward histone H4 has been noted in previous study, and our enzyme kinetic data also indicated that hNaa60 can acetylate H3-H4 tetramer in vitro (Figure S3).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "KAT",
+                        "protein_type"
+                    ],
+                    [
+                        16,
+                        22,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        30,
+                        37,
+                        "histone",
+                        "protein_type"
+                    ],
+                    [
+                        38,
+                        40,
+                        "H4",
+                        "protein_type"
+                    ],
+                    [
+                        83,
+                        102,
+                        "enzyme kinetic data",
+                        "evidence"
+                    ],
+                    [
+                        123,
+                        129,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        144,
+                        149,
+                        "H3-H4",
+                        "complex_assembly"
+                    ],
+                    [
+                        150,
+                        158,
+                        "tetramer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 75,
+                "sent": "Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        40,
+                        "acetylation",
+                        "ptm"
+                    ],
+                    [
+                        51,
+                        58,
+                        "histone",
+                        "protein_type"
+                    ],
+                    [
+                        59,
+                        64,
+                        "H3-H4",
+                        "complex_assembly"
+                    ],
+                    [
+                        65,
+                        73,
+                        "tetramer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        80,
+                        97,
+                        "mass spectrometry",
+                        "experimental_method"
+                    ],
+                    [
+                        125,
+                        131,
+                        "lysine",
+                        "residue_name"
+                    ],
+                    [
+                        187,
+                        198,
+                        "acetylation",
+                        "ptm"
+                    ],
+                    [
+                        217,
+                        228,
+                        "acetylation",
+                        "ptm"
+                    ],
+                    [
+                        257,
+                        270,
+                        "hNaa60(1-199)",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 76,
+                "sent": "We also conducted liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis on a synthetic peptide (NH2-MKGKEEKEGGAR-COOH) after treatment with hNaa60(1-199), and the data confirmed that both the N-terminal \u03b1-amine and lysine side-chain \u03b5-amine were robustly acetylated after the treatment (Table S1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        64,
+                        "liquid chromatography-tandem mass spectrometry",
+                        "experimental_method"
+                    ],
+                    [
+                        66,
+                        74,
+                        "LC/MS/MS",
+                        "experimental_method"
+                    ],
+                    [
+                        100,
+                        107,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        109,
+                        130,
+                        "NH2-MKGKEEKEGGAR-COOH",
+                        "chemical"
+                    ],
+                    [
+                        153,
+                        166,
+                        "hNaa60(1-199)",
+                        "mutant"
+                    ],
+                    [
+                        228,
+                        234,
+                        "lysine",
+                        "residue_name"
+                    ],
+                    [
+                        268,
+                        278,
+                        "acetylated",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 77,
+                "sent": "Recent structural investigation of other NATs proposed that the \u03b26-\u03b27 loop, corresponding to the \u03b27-\u03b28 hairpin in hNaa60, and the \u03b11-\u03b12 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        7,
+                        31,
+                        "structural investigation",
+                        "experimental_method"
+                    ],
+                    [
+                        41,
+                        45,
+                        "NATs",
+                        "protein_type"
+                    ],
+                    [
+                        64,
+                        74,
+                        "\u03b26-\u03b27 loop",
+                        "structure_element"
+                    ],
+                    [
+                        97,
+                        110,
+                        "\u03b27-\u03b28 hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        114,
+                        120,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        130,
+                        140,
+                        "\u03b11-\u03b12 loop",
+                        "structure_element"
+                    ],
+                    [
+                        154,
+                        176,
+                        "substrate-binding site",
+                        "site"
+                    ],
+                    [
+                        180,
+                        184,
+                        "NATs",
+                        "protein_type"
+                    ],
+                    [
+                        198,
+                        204,
+                        "lysine",
+                        "residue_name"
+                    ],
+                    [
+                        223,
+                        226,
+                        "KAT",
+                        "protein_type"
+                    ],
+                    [
+                        262,
+                        273,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 78,
+                "sent": "Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the \u03b27-\u03b28 hairpin of hNaa60(1-242) (Fig. 2D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        21,
+                        "superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        25,
+                        31,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        32,
+                        37,
+                        "1-242",
+                        "residue_range"
+                    ],
+                    [
+                        39,
+                        48,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        60,
+                        65,
+                        "Hat1p",
+                        "protein"
+                    ],
+                    [
+                        77,
+                        80,
+                        "KAT",
+                        "protein_type"
+                    ],
+                    [
+                        82,
+                        97,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        100,
+                        107,
+                        "histone",
+                        "protein_type"
+                    ],
+                    [
+                        108,
+                        110,
+                        "H4",
+                        "protein_type"
+                    ],
+                    [
+                        111,
+                        118,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        164,
+                        166,
+                        "H4",
+                        "protein_type"
+                    ],
+                    [
+                        167,
+                        174,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        178,
+                        181,
+                        "KAT",
+                        "protein_type"
+                    ],
+                    [
+                        202,
+                        215,
+                        "\u03b27-\u03b28 hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        219,
+                        225,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        226,
+                        231,
+                        "1-242",
+                        "residue_range"
+                    ]
+                ]
+            },
+            {
+                "sid": 79,
+                "sent": "Interestingly, in the hNaa60(1-199) crystal structure, the displaced \u03b27-\u03b28 hairpin opened a second way for the substrate to access the active center that would readily accommodate the binding of the H4 peptide (Fig. 2E), thus implied a potential explanation for KAT activity of this enzyme from a structural biological view.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        22,
+                        35,
+                        "hNaa60(1-199)",
+                        "mutant"
+                    ],
+                    [
+                        36,
+                        53,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        69,
+                        82,
+                        "\u03b27-\u03b28 hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        135,
+                        148,
+                        "active center",
+                        "site"
+                    ],
+                    [
+                        199,
+                        201,
+                        "H4",
+                        "protein_type"
+                    ],
+                    [
+                        202,
+                        209,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        262,
+                        265,
+                        "KAT",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 80,
+                "sent": "However, since hNaa60(1-242) and hNaa60(1-199) were crystallized in different crystal forms, the observed conformational change of the \u03b27-\u03b28 hairpin may simply be an artifact related to the different crystal packing.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        15,
+                        21,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        22,
+                        27,
+                        "1-242",
+                        "residue_range"
+                    ],
+                    [
+                        33,
+                        39,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        52,
+                        64,
+                        "crystallized",
+                        "experimental_method"
+                    ],
+                    [
+                        78,
+                        91,
+                        "crystal forms",
+                        "evidence"
+                    ],
+                    [
+                        135,
+                        148,
+                        "\u03b27-\u03b28 hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        200,
+                        215,
+                        "crystal packing",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 81,
+                "sent": "Whether the KAT substrates bind to the \u03b27-\u03b28 hairpin displaced conformation of the enzyme needs to be verified by further structural and functional studies.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        12,
+                        15,
+                        "KAT",
+                        "protein_type"
+                    ],
+                    [
+                        39,
+                        52,
+                        "\u03b27-\u03b28 hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        122,
+                        155,
+                        "structural and functional studies",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 82,
+                "sent": "Phe 34 facilitates proper positioning of the cofactor for acetyl-transfer",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        6,
+                        "Phe 34",
+                        "residue_name_number"
+                    ],
+                    [
+                        58,
+                        64,
+                        "acetyl",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 83,
+                "sent": "The electron density of Phe 34 side-chain is well defined in the hNaa60(1-242)/Ac-CoA structure, but becomes invisible in the hNaa60(1-199)/CoA structure, indicating displacement of the Phe 34 side-chain in the latter (Fig. 3A,B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        20,
+                        "electron density",
+                        "evidence"
+                    ],
+                    [
+                        24,
+                        30,
+                        "Phe 34",
+                        "residue_name_number"
+                    ],
+                    [
+                        65,
+                        85,
+                        "hNaa60(1-242)/Ac-CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        86,
+                        95,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        126,
+                        143,
+                        "hNaa60(1-199)/CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        144,
+                        153,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        186,
+                        192,
+                        "Phe 34",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 84,
+                "sent": "A solvent-derived malonate molecule is found beside Phe 34 and the ethanethioate moiety of Ac-CoA in the high-resolution hNaa60(1-242)/Ac-CoA structure (Fig. 3A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        26,
+                        "malonate",
+                        "chemical"
+                    ],
+                    [
+                        52,
+                        58,
+                        "Phe 34",
+                        "residue_name_number"
+                    ],
+                    [
+                        67,
+                        80,
+                        "ethanethioate",
+                        "chemical"
+                    ],
+                    [
+                        91,
+                        97,
+                        "Ac-CoA",
+                        "chemical"
+                    ],
+                    [
+                        121,
+                        141,
+                        "hNaa60(1-242)/Ac-CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        142,
+                        151,
+                        "structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 85,
+                "sent": "Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        13,
+                        "Superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        22,
+                        31,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        43,
+                        62,
+                        "hNaa50p/CoA/peptide",
+                        "complex_assembly"
+                    ],
+                    [
+                        78,
+                        86,
+                        "malonate",
+                        "chemical"
+                    ],
+                    [
+                        128,
+                        138,
+                        "methionine",
+                        "residue_name"
+                    ],
+                    [
+                        156,
+                        163,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        176,
+                        182,
+                        "Phe 34",
+                        "residue_name_number"
+                    ],
+                    [
+                        186,
+                        192,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        210,
+                        216,
+                        "Phe 27",
+                        "residue_name_number"
+                    ],
+                    [
+                        220,
+                        226,
+                        "hNaa50",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 86,
+                "sent": "Interestingly, in the structure of hNaa60(1-199)/CoA, the terminal thiol of CoA adopts alternative conformations.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        22,
+                        31,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        35,
+                        52,
+                        "hNaa60(1-199)/CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        76,
+                        79,
+                        "CoA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 87,
+                "sent": "One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the \u03b11-\u03b12 loop and away from the substrate amine (Fig. 3B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        33,
+                        38,
+                        "amine",
+                        "chemical"
+                    ],
+                    [
+                        60,
+                        72,
+                        "superimposed",
+                        "experimental_method"
+                    ],
+                    [
+                        73,
+                        91,
+                        "hNaa50/CoA/peptide",
+                        "complex_assembly"
+                    ],
+                    [
+                        92,
+                        101,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        128,
+                        141,
+                        "ethanethioate",
+                        "chemical"
+                    ],
+                    [
+                        145,
+                        151,
+                        "Ac-CoA",
+                        "chemical"
+                    ],
+                    [
+                        159,
+                        168,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        172,
+                        192,
+                        "hNaa60(1-242)/Ac-CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        223,
+                        233,
+                        "\u03b11-\u03b12 loop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 88,
+                "sent": "To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        37,
+                        53,
+                        "electron density",
+                        "evidence"
+                    ],
+                    [
+                        130,
+                        146,
+                        "electron density",
+                        "evidence"
+                    ],
+                    [
+                        178,
+                        184,
+                        "Phe 34",
+                        "residue_name_number"
+                    ],
+                    [
+                        189,
+                        195,
+                        "solved",
+                        "experimental_method"
+                    ],
+                    [
+                        200,
+                        217,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        221,
+                        243,
+                        "hNaa60(1-199) F34A/CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        249,
+                        258,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        267,
+                        273,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        295,
+                        312,
+                        "hNaa60(1-199)/CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        347,
+                        363,
+                        "electron density",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 89,
+                "sent": "Phe 27 in hNaa50p (equivalent to Phe 34 in hNaa60) has been implicated to facilitate the binding of N-terminal methionine of the substrate peptide through hydrophobic interaction.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        6,
+                        "Phe 27",
+                        "residue_name_number"
+                    ],
+                    [
+                        10,
+                        17,
+                        "hNaa50p",
+                        "protein"
+                    ],
+                    [
+                        33,
+                        39,
+                        "Phe 34",
+                        "residue_name_number"
+                    ],
+                    [
+                        43,
+                        49,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        111,
+                        121,
+                        "methionine",
+                        "residue_name"
+                    ],
+                    [
+                        139,
+                        146,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        155,
+                        178,
+                        "hydrophobic interaction",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 90,
+                "sent": "However, in the hNaa60/Ac-CoA structure, a hydrophilic malonate molecule is found at the same location where the N-terminal methionine should bind as is indicated by the superposition (Fig. 3A), suggesting that Phe 34 may accommodate binding of hydrophilic substrate, too.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        16,
+                        29,
+                        "hNaa60/Ac-CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        30,
+                        39,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        55,
+                        63,
+                        "malonate",
+                        "chemical"
+                    ],
+                    [
+                        124,
+                        134,
+                        "methionine",
+                        "residue_name"
+                    ],
+                    [
+                        170,
+                        183,
+                        "superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        211,
+                        217,
+                        "Phe 34",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 91,
+                "sent": "Moreover, orientation of Phe 34 side-chain seems to be co-related to positioning of the terminus of the co-enzyme and important for placing it at a location in close proximity to the substrate amine.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        31,
+                        "Phe 34",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 92,
+                "sent": "We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        23,
+                        29,
+                        "Phe 34",
+                        "residue_name_number"
+                    ],
+                    [
+                        97,
+                        100,
+                        "Met",
+                        "residue_name"
+                    ],
+                    [
+                        113,
+                        119,
+                        "mutate",
+                        "experimental_method"
+                    ],
+                    [
+                        128,
+                        131,
+                        "Phe",
+                        "residue_name"
+                    ],
+                    [
+                        135,
+                        138,
+                        "Ala",
+                        "residue_name"
+                    ],
+                    [
+                        205,
+                        211,
+                        "Phe 34",
+                        "residue_name_number"
+                    ],
+                    [
+                        257,
+                        270,
+                        "ethanethioate",
+                        "chemical"
+                    ],
+                    [
+                        281,
+                        287,
+                        "Ac-CoA",
+                        "chemical"
+                    ],
+                    [
+                        293,
+                        301,
+                        "mutation",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 93,
+                "sent": "Indeed, our enzyme kinetic data showed that hNaa60(1-199) F34A mutant showed no detectable activity (Fig. 5A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        12,
+                        31,
+                        "enzyme kinetic data",
+                        "evidence"
+                    ],
+                    [
+                        44,
+                        57,
+                        "hNaa60(1-199)",
+                        "mutant"
+                    ],
+                    [
+                        58,
+                        62,
+                        "F34A",
+                        "mutant"
+                    ],
+                    [
+                        63,
+                        69,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 94,
+                "sent": "In order to rule out the possibility that the observed loss of activity may be related to bad folding of the mutant protein, we studied the circular dichroism (CD) spectrum of the protein (Fig. 5B) and determined its crystal structure (Fig. 3C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        109,
+                        115,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        140,
+                        158,
+                        "circular dichroism",
+                        "experimental_method"
+                    ],
+                    [
+                        160,
+                        162,
+                        "CD",
+                        "experimental_method"
+                    ],
+                    [
+                        164,
+                        172,
+                        "spectrum",
+                        "evidence"
+                    ],
+                    [
+                        217,
+                        234,
+                        "crystal structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 95,
+                "sent": "Both studies proved that the F34A mutant protein is well-folded.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        33,
+                        "F34A",
+                        "mutant"
+                    ],
+                    [
+                        34,
+                        40,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        52,
+                        63,
+                        "well-folded",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 96,
+                "sent": "Many studies have addressed the crucial effect of \u03b11-\u03b12 loop on catalysis, showing that some residues located in this area are involved in the binding of substrates.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        50,
+                        60,
+                        "\u03b11-\u03b12 loop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 97,
+                "sent": "We propose that Phe 34 may play a dual role both in interacting with the peptide substrate (recognition) and in positioning of the ethanethioate moiety of Ac-CoA to the right location to facilitate acetyl-transfer.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        16,
+                        22,
+                        "Phe 34",
+                        "residue_name_number"
+                    ],
+                    [
+                        73,
+                        80,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        131,
+                        144,
+                        "ethanethioate",
+                        "chemical"
+                    ],
+                    [
+                        155,
+                        161,
+                        "Ac-CoA",
+                        "chemical"
+                    ],
+                    [
+                        198,
+                        204,
+                        "acetyl",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 98,
+                "sent": "Structural basis for hNaa60 substrate binding",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        21,
+                        27,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 99,
+                "sent": "Several studies have demonstrated that the substrate specificities of hNaa60 and hNaa50 are highly overlapped.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        70,
+                        76,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        81,
+                        87,
+                        "hNaa50",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 100,
+                "sent": "The structure of hNaa50p/CoA/peptide provides detailed information about the position of substrate N-terminal residues in the active site of hNaa50.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        17,
+                        36,
+                        "hNaa50p/CoA/peptide",
+                        "complex_assembly"
+                    ],
+                    [
+                        126,
+                        137,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        141,
+                        147,
+                        "hNaa50",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 101,
+                "sent": "Comparing the active site of hNaa60(1-242)/Ac-CoA with hNaa50p/CoA/peptide revealed that key catalytic and substrate binding residues are highly conserved in both proteins (Fig. 4A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        25,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        29,
+                        49,
+                        "hNaa60(1-242)/Ac-CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        55,
+                        74,
+                        "hNaa50p/CoA/peptide",
+                        "complex_assembly"
+                    ],
+                    [
+                        93,
+                        133,
+                        "catalytic and substrate binding residues",
+                        "site"
+                    ],
+                    [
+                        138,
+                        154,
+                        "highly conserved",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 102,
+                "sent": "With respect to catalysis, hNaa50p has been shown to employ residues Tyr 73 and His 112 to abstract proton from the \u03b1-amino group from the substrate\u2019s first residue through a well-ordered water.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        27,
+                        34,
+                        "hNaa50p",
+                        "protein"
+                    ],
+                    [
+                        69,
+                        75,
+                        "Tyr 73",
+                        "residue_name_number"
+                    ],
+                    [
+                        80,
+                        87,
+                        "His 112",
+                        "residue_name_number"
+                    ],
+                    [
+                        175,
+                        187,
+                        "well-ordered",
+                        "protein_state"
+                    ],
+                    [
+                        188,
+                        193,
+                        "water",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 103,
+                "sent": "A well-ordered water was also found between Tyr 97 and His 138 in hNaa60 (1-199)/CoA and hNaa60 (1-242)/Ac-CoA (Fig. 4B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        2,
+                        14,
+                        "well-ordered",
+                        "protein_state"
+                    ],
+                    [
+                        15,
+                        20,
+                        "water",
+                        "chemical"
+                    ],
+                    [
+                        44,
+                        50,
+                        "Tyr 97",
+                        "residue_name_number"
+                    ],
+                    [
+                        55,
+                        62,
+                        "His 138",
+                        "residue_name_number"
+                    ],
+                    [
+                        66,
+                        84,
+                        "hNaa60 (1-199)/CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        89,
+                        110,
+                        "hNaa60 (1-242)/Ac-CoA",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 104,
+                "sent": "To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        35,
+                        "Tyr 97",
+                        "residue_name_number"
+                    ],
+                    [
+                        40,
+                        47,
+                        "His 138",
+                        "residue_name_number"
+                    ],
+                    [
+                        51,
+                        57,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        72,
+                        79,
+                        "mutated",
+                        "experimental_method"
+                    ],
+                    [
+                        98,
+                        105,
+                        "alanine",
+                        "residue_name"
+                    ],
+                    [
+                        110,
+                        123,
+                        "phenylalanine",
+                        "residue_name"
+                    ],
+                    [
+                        168,
+                        175,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        188,
+                        202,
+                        "kinetic assays",
+                        "experimental_method"
+                    ],
+                    [
+                        207,
+                        218,
+                        "well-folded",
+                        "protein_state"
+                    ],
+                    [
+                        222,
+                        224,
+                        "CD",
+                        "experimental_method"
+                    ],
+                    [
+                        225,
+                        232,
+                        "spectra",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 105,
+                "sent": "Purity of all proteins were also analyzed by SDS-PAGE (Figure S5).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        45,
+                        53,
+                        "SDS-PAGE",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 106,
+                "sent": "As show in Fig. 5A, the mutants Y97A, Y97F, H138A and H138F abolished the activity of hNaa60.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        24,
+                        31,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        32,
+                        36,
+                        "Y97A",
+                        "mutant"
+                    ],
+                    [
+                        38,
+                        42,
+                        "Y97F",
+                        "mutant"
+                    ],
+                    [
+                        44,
+                        49,
+                        "H138A",
+                        "mutant"
+                    ],
+                    [
+                        54,
+                        59,
+                        "H138F",
+                        "mutant"
+                    ],
+                    [
+                        60,
+                        82,
+                        "abolished the activity",
+                        "protein_state"
+                    ],
+                    [
+                        86,
+                        92,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 107,
+                "sent": "In contrast, to mutate the nearby solvent exposed residue Glu 37 to Ala (E37A) has little impact on the activity of hNaa60 (Figs 4B and 5A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        16,
+                        22,
+                        "mutate",
+                        "experimental_method"
+                    ],
+                    [
+                        34,
+                        49,
+                        "solvent exposed",
+                        "protein_state"
+                    ],
+                    [
+                        58,
+                        64,
+                        "Glu 37",
+                        "residue_name_number"
+                    ],
+                    [
+                        68,
+                        71,
+                        "Ala",
+                        "residue_name"
+                    ],
+                    [
+                        73,
+                        77,
+                        "E37A",
+                        "mutant"
+                    ],
+                    [
+                        116,
+                        122,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 108,
+                "sent": "In conclusion, the structural and functional studies indicate that hNaa60 applies the same two base mechanism through Tyr 97, His 138 and a well-ordered water as was described for hNaa50.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        52,
+                        "structural and functional studies",
+                        "experimental_method"
+                    ],
+                    [
+                        67,
+                        73,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        118,
+                        124,
+                        "Tyr 97",
+                        "residue_name_number"
+                    ],
+                    [
+                        126,
+                        133,
+                        "His 138",
+                        "residue_name_number"
+                    ],
+                    [
+                        140,
+                        152,
+                        "well-ordered",
+                        "protein_state"
+                    ],
+                    [
+                        153,
+                        158,
+                        "water",
+                        "chemical"
+                    ],
+                    [
+                        180,
+                        186,
+                        "hNaa50",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 109,
+                "sent": "The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        12,
+                        "malonate",
+                        "chemical"
+                    ],
+                    [
+                        38,
+                        58,
+                        "hNaa60(1-242)/Ac-CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        59,
+                        76,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        132,
+                        138,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        166,
+                        177,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        206,
+                        209,
+                        "Met",
+                        "residue_name"
+                    ],
+                    [
+                        227,
+                        234,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        242,
+                        255,
+                        "superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        265,
+                        284,
+                        "hNaa50p/CoA/peptide",
+                        "complex_assembly"
+                    ],
+                    [
+                        285,
+                        294,
+                        "structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 110,
+                "sent": "Residues Tyr 38, Asn 143 and Tyr 165 are located around the malonate and interact with it through direct hydrogen bonds or water bridge (Fig. 4C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        15,
+                        "Tyr 38",
+                        "residue_name_number"
+                    ],
+                    [
+                        17,
+                        24,
+                        "Asn 143",
+                        "residue_name_number"
+                    ],
+                    [
+                        29,
+                        36,
+                        "Tyr 165",
+                        "residue_name_number"
+                    ],
+                    [
+                        60,
+                        68,
+                        "malonate",
+                        "chemical"
+                    ],
+                    [
+                        105,
+                        119,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        123,
+                        135,
+                        "water bridge",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 111,
+                "sent": "Although malonate is negatively charged, which is different from that of lysine \u03b5-amine or peptide N-terminal amine, similar hydrophilic interactions may take place when substrate amine presents in the same position, since Tyr 38, Asn 143 and Tyr 165 are not positively or negatively charged.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        17,
+                        "malonate",
+                        "chemical"
+                    ],
+                    [
+                        73,
+                        79,
+                        "lysine",
+                        "residue_name"
+                    ],
+                    [
+                        91,
+                        98,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        125,
+                        149,
+                        "hydrophilic interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        223,
+                        229,
+                        "Tyr 38",
+                        "residue_name_number"
+                    ],
+                    [
+                        231,
+                        238,
+                        "Asn 143",
+                        "residue_name_number"
+                    ],
+                    [
+                        243,
+                        250,
+                        "Tyr 165",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 112,
+                "sent": "In agreement with this hypothesis, it was found that the Y38A, N143A and Y165A mutants all showed remarkably reduced activities as compared to WT, implying that these residues may be critical for substrate binding (Figs 4C and 5A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        57,
+                        61,
+                        "Y38A",
+                        "mutant"
+                    ],
+                    [
+                        63,
+                        68,
+                        "N143A",
+                        "mutant"
+                    ],
+                    [
+                        73,
+                        78,
+                        "Y165A",
+                        "mutant"
+                    ],
+                    [
+                        79,
+                        86,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        143,
+                        145,
+                        "WT",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 113,
+                "sent": "The \u03b23-\u03b24 loop participates in the regulation of hNaa60-activity",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        14,
+                        "\u03b23-\u03b24 loop",
+                        "structure_element"
+                    ],
+                    [
+                        49,
+                        55,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 114,
+                "sent": "Residues between \u03b23 and \u03b24 of hNaa60 form a unique 20-residue long loop (residues 73\u201392) that is a short turn in many other NAT members (Fig. 1D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        17,
+                        19,
+                        "\u03b23",
+                        "structure_element"
+                    ],
+                    [
+                        24,
+                        26,
+                        "\u03b24",
+                        "structure_element"
+                    ],
+                    [
+                        30,
+                        36,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        51,
+                        71,
+                        "20-residue long loop",
+                        "structure_element"
+                    ],
+                    [
+                        82,
+                        87,
+                        "73\u201392",
+                        "residue_range"
+                    ],
+                    [
+                        99,
+                        109,
+                        "short turn",
+                        "structure_element"
+                    ],
+                    [
+                        124,
+                        127,
+                        "NAT",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 115,
+                "sent": "Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        30,
+                        46,
+                        "auto-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        50,
+                        56,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        56,
+                        59,
+                        "K79",
+                        "residue_name_number"
+                    ],
+                    [
+                        92,
+                        98,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        142,
+                        148,
+                        "Lys 79",
+                        "residue_name_number"
+                    ],
+                    [
+                        152,
+                        162,
+                        "acetylated",
+                        "protein_state"
+                    ],
+                    [
+                        170,
+                        188,
+                        "crystal structures",
+                        "evidence"
+                    ],
+                    [
+                        216,
+                        232,
+                        "electron density",
+                        "evidence"
+                    ],
+                    [
+                        236,
+                        242,
+                        "Lys 79",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 116,
+                "sent": "We therefore used mass spectrometry to analyze if Lys 79 was acetylated in our bacterially purified proteins, and observed no modification on this residue (Figure S6).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        35,
+                        "mass spectrometry",
+                        "experimental_method"
+                    ],
+                    [
+                        50,
+                        56,
+                        "Lys 79",
+                        "residue_name_number"
+                    ],
+                    [
+                        61,
+                        71,
+                        "acetylated",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 117,
+                "sent": "To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        24,
+                        30,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        30,
+                        33,
+                        "K79",
+                        "residue_name_number"
+                    ],
+                    [
+                        34,
+                        50,
+                        "auto-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        79,
+                        83,
+                        "K79R",
+                        "mutant"
+                    ],
+                    [
+                        88,
+                        92,
+                        "K79Q",
+                        "mutant"
+                    ],
+                    [
+                        93,
+                        100,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        117,
+                        130,
+                        "un-acetylated",
+                        "protein_state"
+                    ],
+                    [
+                        135,
+                        145,
+                        "acetylated",
+                        "protein_state"
+                    ],
+                    [
+                        154,
+                        160,
+                        "Lys 79",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 118,
+                "sent": "Interestingly, both K79R and K79Q mutants led to an increase in the catalytic activity of hNaa60, while K79A mutant led to modest decrease of the activity (Fig. 5A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        20,
+                        24,
+                        "K79R",
+                        "mutant"
+                    ],
+                    [
+                        29,
+                        33,
+                        "K79Q",
+                        "mutant"
+                    ],
+                    [
+                        34,
+                        41,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        90,
+                        96,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        104,
+                        108,
+                        "K79A",
+                        "mutant"
+                    ],
+                    [
+                        109,
+                        115,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 119,
+                "sent": "These data indicate that the acetylation of Lys 79 is not required for optimal catalytic activity of hNaa60 in vitro.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        40,
+                        "acetylation",
+                        "ptm"
+                    ],
+                    [
+                        44,
+                        50,
+                        "Lys 79",
+                        "residue_name_number"
+                    ],
+                    [
+                        101,
+                        107,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 120,
+                "sent": "It is noted that the \u03b23-\u03b24 loop of hNaa60 acts like a door leaf to partly cover the substrate-binding pathway.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        21,
+                        31,
+                        "\u03b23-\u03b24 loop",
+                        "structure_element"
+                    ],
+                    [
+                        35,
+                        41,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        84,
+                        109,
+                        "substrate-binding pathway",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 121,
+                "sent": "We hence hypothesize that the \u03b23-\u03b24 loop may interfere with the access of the peptide substrates and that the solvent-exposing Lys 79 may play a potential role to remove the door leaf when it hovers in solvent (Fig. 4D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        30,
+                        40,
+                        "\u03b23-\u03b24 loop",
+                        "structure_element"
+                    ],
+                    [
+                        78,
+                        85,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        110,
+                        126,
+                        "solvent-exposing",
+                        "protein_state"
+                    ],
+                    [
+                        127,
+                        133,
+                        "Lys 79",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 122,
+                "sent": "Acidic residues Glu 80, Asp 81 and Asp 83 interact with His 138, His 159 and His 158 to maintain the conformation of the \u03b23-\u03b24 loop, thus contribute to control the substrate binding (Fig. 4D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        16,
+                        22,
+                        "Glu 80",
+                        "residue_name_number"
+                    ],
+                    [
+                        24,
+                        30,
+                        "Asp 81",
+                        "residue_name_number"
+                    ],
+                    [
+                        35,
+                        41,
+                        "Asp 83",
+                        "residue_name_number"
+                    ],
+                    [
+                        56,
+                        63,
+                        "His 138",
+                        "residue_name_number"
+                    ],
+                    [
+                        65,
+                        72,
+                        "His 159",
+                        "residue_name_number"
+                    ],
+                    [
+                        77,
+                        84,
+                        "His 158",
+                        "residue_name_number"
+                    ],
+                    [
+                        121,
+                        131,
+                        "\u03b23-\u03b24 loop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 123,
+                "sent": "To verify this hypothesis, we mutated Glu 80, Asp 81 and Asp 83 to Ala respectively.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        30,
+                        37,
+                        "mutated",
+                        "experimental_method"
+                    ],
+                    [
+                        38,
+                        44,
+                        "Glu 80",
+                        "residue_name_number"
+                    ],
+                    [
+                        46,
+                        52,
+                        "Asp 81",
+                        "residue_name_number"
+                    ],
+                    [
+                        57,
+                        63,
+                        "Asp 83",
+                        "residue_name_number"
+                    ],
+                    [
+                        67,
+                        70,
+                        "Ala",
+                        "residue_name"
+                    ]
+                ]
+            },
+            {
+                "sid": 124,
+                "sent": "In line with our hypothesis, E80A, D81A and D83A mutants exhibit at least 2-fold increase in hNaa60-activity (Fig. 5A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        33,
+                        "E80A",
+                        "mutant"
+                    ],
+                    [
+                        35,
+                        39,
+                        "D81A",
+                        "mutant"
+                    ],
+                    [
+                        44,
+                        48,
+                        "D83A",
+                        "mutant"
+                    ],
+                    [
+                        49,
+                        56,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        93,
+                        99,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 125,
+                "sent": "Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between \u03b23 and \u03b24, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        28,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        45,
+                        48,
+                        "NAT",
+                        "protein_type"
+                    ],
+                    [
+                        54,
+                        69,
+                        "S. solfataricus",
+                        "species"
+                    ],
+                    [
+                        86,
+                        111,
+                        "10-residue long extension",
+                        "structure_element"
+                    ],
+                    [
+                        120,
+                        122,
+                        "\u03b23",
+                        "structure_element"
+                    ],
+                    [
+                        127,
+                        129,
+                        "\u03b24",
+                        "structure_element"
+                    ],
+                    [
+                        139,
+                        172,
+                        "structure and biochemical studies",
+                        "experimental_method"
+                    ],
+                    [
+                        189,
+                        198,
+                        "extension",
+                        "structure_element"
+                    ],
+                    [
+                        202,
+                        207,
+                        "SsNat",
+                        "protein"
+                    ],
+                    [
+                        254,
+                        265,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        281,
+                        286,
+                        "SsNat",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 126,
+                "sent": "Nt-acetylation, which is carried out by the NAT family acetyltransferases, is an ancient and essential modification of proteins.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        14,
+                        "Nt-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        44,
+                        73,
+                        "NAT family acetyltransferases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 127,
+                "sent": "Although many NATs are highly conserved from lower to higher eukaryotes and the substrate bias of them appears to be partially overlapped, there is a significant increase in the overall level of N-terminal acetylation from lower to higher eukaryotes.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        14,
+                        18,
+                        "NATs",
+                        "protein_type"
+                    ],
+                    [
+                        23,
+                        39,
+                        "highly conserved",
+                        "protein_state"
+                    ],
+                    [
+                        45,
+                        50,
+                        "lower",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        54,
+                        71,
+                        "higher eukaryotes",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        195,
+                        217,
+                        "N-terminal acetylation",
+                        "ptm"
+                    ],
+                    [
+                        223,
+                        228,
+                        "lower",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        232,
+                        249,
+                        "higher eukaryotes",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 128,
+                "sent": "In this study we provide structural insights into Naa60 found only in multicellular eukaryotes.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        50,
+                        55,
+                        "Naa60",
+                        "protein"
+                    ],
+                    [
+                        70,
+                        94,
+                        "multicellular eukaryotes",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 129,
+                "sent": "The N-terminus of hNaa60 harbors three hydrophobic residues (VVP) that makes it very difficult to express and purify the protein.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        18,
+                        24,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        61,
+                        64,
+                        "VVP",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 130,
+                "sent": "This problem was solved by replacing residues 4\u20136 from VVP to EER that are found in Naa60 from Xenopus Laevis.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        27,
+                        36,
+                        "replacing",
+                        "experimental_method"
+                    ],
+                    [
+                        46,
+                        49,
+                        "4\u20136",
+                        "residue_range"
+                    ],
+                    [
+                        55,
+                        58,
+                        "VVP",
+                        "structure_element"
+                    ],
+                    [
+                        62,
+                        65,
+                        "EER",
+                        "structure_element"
+                    ],
+                    [
+                        84,
+                        89,
+                        "Naa60",
+                        "protein"
+                    ],
+                    [
+                        95,
+                        109,
+                        "Xenopus Laevis",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 131,
+                "sent": "Since Naa60 from human and from Xenopus Laevis are highly homologous (Fig. 1A), we speculate that these two proteins should have the same biological function.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        6,
+                        11,
+                        "Naa60",
+                        "protein"
+                    ],
+                    [
+                        17,
+                        22,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        32,
+                        46,
+                        "Xenopus Laevis",
+                        "species"
+                    ],
+                    [
+                        51,
+                        68,
+                        "highly homologous",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 132,
+                "sent": "Therefore it is deduced that the VVP to EER replacement on the N-terminus of hNaa60 may not interfere with its function.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        33,
+                        43,
+                        "VVP to EER",
+                        "mutant"
+                    ],
+                    [
+                        44,
+                        55,
+                        "replacement",
+                        "experimental_method"
+                    ],
+                    [
+                        77,
+                        83,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 133,
+                "sent": "However, in the hNaa60(1-242) structure the N-terminus adopts an \u03b1-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        16,
+                        22,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        23,
+                        28,
+                        "1-242",
+                        "residue_range"
+                    ],
+                    [
+                        30,
+                        39,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        65,
+                        84,
+                        "\u03b1-helical structure",
+                        "structure_element"
+                    ],
+                    [
+                        126,
+                        127,
+                        "6",
+                        "residue_number"
+                    ],
+                    [
+                        131,
+                        138,
+                        "proline",
+                        "residue_name"
+                    ],
+                    [
+                        161,
+                        174,
+                        "hNaa60(1-199)",
+                        "mutant"
+                    ],
+                    [
+                        175,
+                        184,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        219,
+                        241,
+                        "semi-helical structure",
+                        "structure_element"
+                    ],
+                    [
+                        276,
+                        291,
+                        "crystal packing",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 134,
+                "sent": "Hence it is not clear if the N-terminal end of wild-type hNaa60 is an \u03b1-helix, and what roles the hydrophobic residues 4\u20136 play in structure and function of wild-type hNaa60.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        47,
+                        56,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        57,
+                        63,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        70,
+                        77,
+                        "\u03b1-helix",
+                        "structure_element"
+                    ],
+                    [
+                        119,
+                        122,
+                        "4\u20136",
+                        "residue_range"
+                    ],
+                    [
+                        157,
+                        166,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        167,
+                        173,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 135,
+                "sent": "In addition to the three-residue mutation (VVP to EER), we also tried many other hNaa60 constructs, but only the full-length protein and the truncated variant 1-199 behaved well.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        33,
+                        41,
+                        "mutation",
+                        "experimental_method"
+                    ],
+                    [
+                        43,
+                        46,
+                        "VVP",
+                        "structure_element"
+                    ],
+                    [
+                        50,
+                        53,
+                        "EER",
+                        "structure_element"
+                    ],
+                    [
+                        81,
+                        87,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        113,
+                        124,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        141,
+                        150,
+                        "truncated",
+                        "protein_state"
+                    ],
+                    [
+                        159,
+                        164,
+                        "1-199",
+                        "residue_range"
+                    ]
+                ]
+            },
+            {
+                "sid": 136,
+                "sent": "The finding that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) is intriguing.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        43,
+                        49,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        50,
+                        55,
+                        "1-242",
+                        "residue_range"
+                    ],
+                    [
+                        84,
+                        97,
+                        "hNaa60(1-199)",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 137,
+                "sent": "We speculate that low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies or there remains some undiscovered auto-inhibitory regulation in the full-length protein.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        38,
+                        49,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        50,
+                        56,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        211,
+                        222,
+                        "full-length",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 138,
+                "sent": "The hNaa60 protein was proven to be localized on Golgi apparatus.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        10,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 139,
+                "sent": "Aksnes and colleagues predicted putative transmembrane domains and two putative sites of S-palmitoylation, by bioinformatics means, to account for Golgi localization of the protein.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        41,
+                        62,
+                        "transmembrane domains",
+                        "structure_element"
+                    ],
+                    [
+                        89,
+                        105,
+                        "S-palmitoylation",
+                        "ptm"
+                    ]
+                ]
+            },
+            {
+                "sid": 140,
+                "sent": "They then mutated all five cysteine residues of hNaa60\u2019s to serine, including the two putative S-palmitoylation sites.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        10,
+                        17,
+                        "mutated",
+                        "experimental_method"
+                    ],
+                    [
+                        27,
+                        35,
+                        "cysteine",
+                        "residue_name"
+                    ],
+                    [
+                        48,
+                        54,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        60,
+                        66,
+                        "serine",
+                        "residue_name"
+                    ],
+                    [
+                        95,
+                        117,
+                        "S-palmitoylation sites",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 141,
+                "sent": "However, these mutations did not abolish Naa60 membrane localization, indicating that S-palmitoylation is unlikely to (solely) account for targeting hNaa60 on Golgi.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        15,
+                        24,
+                        "mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        41,
+                        46,
+                        "Naa60",
+                        "protein"
+                    ],
+                    [
+                        86,
+                        102,
+                        "S-palmitoylation",
+                        "ptm"
+                    ],
+                    [
+                        149,
+                        155,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 142,
+                "sent": "Furthermore, adding residues 217\u2013242 of hNaa60 (containing residues 217\u2013236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182\u2013216 are important for Golgi localization of hNaa60.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        13,
+                        19,
+                        "adding",
+                        "experimental_method"
+                    ],
+                    [
+                        29,
+                        36,
+                        "217\u2013242",
+                        "residue_range"
+                    ],
+                    [
+                        40,
+                        46,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        68,
+                        75,
+                        "217\u2013236",
+                        "residue_range"
+                    ],
+                    [
+                        97,
+                        118,
+                        "transmembrane domains",
+                        "structure_element"
+                    ],
+                    [
+                        141,
+                        145,
+                        "eGFP",
+                        "experimental_method"
+                    ],
+                    [
+                        216,
+                        220,
+                        "eGFP",
+                        "experimental_method"
+                    ],
+                    [
+                        221,
+                        234,
+                        "hNaa60182-242",
+                        "mutant"
+                    ],
+                    [
+                        279,
+                        286,
+                        "182\u2013216",
+                        "residue_range"
+                    ],
+                    [
+                        327,
+                        333,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 143,
+                "sent": "We found that residues 190\u2013202 formed an amphipathic helix with an array of hydrophobic residues located on one side.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        23,
+                        30,
+                        "190\u2013202",
+                        "residue_range"
+                    ],
+                    [
+                        41,
+                        58,
+                        "amphipathic helix",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 144,
+                "sent": "This observation is reminiscent of the protein/membrane interaction through amphipathic helices in the cases of KalSec14, Atg3, PB1-F2 etc.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        76,
+                        95,
+                        "amphipathic helices",
+                        "structure_element"
+                    ],
+                    [
+                        112,
+                        120,
+                        "KalSec14",
+                        "protein"
+                    ],
+                    [
+                        122,
+                        126,
+                        "Atg3",
+                        "protein"
+                    ],
+                    [
+                        128,
+                        134,
+                        "PB1-F2",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 145,
+                "sent": "In this model an amphipathic helix can immerse its hydrophobic side into the lipid bilayer through hydrophobic interactions.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        17,
+                        34,
+                        "amphipathic helix",
+                        "structure_element"
+                    ],
+                    [
+                        99,
+                        123,
+                        "hydrophobic interactions",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 146,
+                "sent": "Therefore we propose that the amphipathic helix \u03b15 may contribute to Golgi localization of hNaa60.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        30,
+                        47,
+                        "amphipathic helix",
+                        "structure_element"
+                    ],
+                    [
+                        48,
+                        50,
+                        "\u03b15",
+                        "structure_element"
+                    ],
+                    [
+                        91,
+                        97,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 147,
+                "sent": "Previous studies indicated that members of NAT family are bi-functional NAT and KAT enzymes.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        43,
+                        46,
+                        "NAT",
+                        "protein_type"
+                    ],
+                    [
+                        72,
+                        75,
+                        "NAT",
+                        "protein_type"
+                    ],
+                    [
+                        80,
+                        83,
+                        "KAT",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 148,
+                "sent": "However, known structures of NATs do not well support this hypothesis, since the \u03b26-\u03b27 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the \u03b11-\u03b12 loop, which would be good for the NAT but not KAT activity of the enzyme.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        15,
+                        25,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        29,
+                        33,
+                        "NATs",
+                        "protein_type"
+                    ],
+                    [
+                        81,
+                        94,
+                        "\u03b26-\u03b27 hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        95,
+                        99,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        111,
+                        115,
+                        "NATs",
+                        "protein_type"
+                    ],
+                    [
+                        150,
+                        184,
+                        "tunnel-like substrate-binding site",
+                        "site"
+                    ],
+                    [
+                        194,
+                        204,
+                        "\u03b11-\u03b12 loop",
+                        "structure_element"
+                    ],
+                    [
+                        234,
+                        237,
+                        "NAT",
+                        "protein_type"
+                    ],
+                    [
+                        246,
+                        249,
+                        "KAT",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 149,
+                "sent": "Kinetic studies have been conducted to compare the NAT and KAT activity of hNaa50 in vitro, and indicate that the NAT activity of Naa50 is much higher than KAT activity.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        15,
+                        "Kinetic studies",
+                        "experimental_method"
+                    ],
+                    [
+                        51,
+                        54,
+                        "NAT",
+                        "protein_type"
+                    ],
+                    [
+                        59,
+                        62,
+                        "KAT",
+                        "protein_type"
+                    ],
+                    [
+                        75,
+                        81,
+                        "hNaa50",
+                        "protein"
+                    ],
+                    [
+                        114,
+                        117,
+                        "NAT",
+                        "protein_type"
+                    ],
+                    [
+                        130,
+                        135,
+                        "Naa50",
+                        "protein"
+                    ],
+                    [
+                        156,
+                        159,
+                        "KAT",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 150,
+                "sent": "However, the substrate used in this study for assessing KAT activity was a small peptide which could not really mimic the 3D structure of a folded protein substrate in vivo.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        56,
+                        59,
+                        "KAT",
+                        "protein_type"
+                    ],
+                    [
+                        81,
+                        88,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        122,
+                        134,
+                        "3D structure",
+                        "evidence"
+                    ],
+                    [
+                        140,
+                        146,
+                        "folded",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 151,
+                "sent": "Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        21,
+                        "mass spectrometry",
+                        "experimental_method"
+                    ],
+                    [
+                        22,
+                        26,
+                        "data",
+                        "evidence"
+                    ],
+                    [
+                        60,
+                        71,
+                        "acetylation",
+                        "ptm"
+                    ],
+                    [
+                        75,
+                        82,
+                        "histone",
+                        "protein_type"
+                    ],
+                    [
+                        83,
+                        88,
+                        "H3-H4",
+                        "complex_assembly"
+                    ],
+                    [
+                        89,
+                        97,
+                        "tetramer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        98,
+                        105,
+                        "lysines",
+                        "residue_name"
+                    ],
+                    [
+                        115,
+                        137,
+                        "N-terminal acetylation",
+                        "ptm"
+                    ],
+                    [
+                        142,
+                        160,
+                        "lysine acetylation",
+                        "ptm"
+                    ],
+                    [
+                        168,
+                        175,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        188,
+                        202,
+                        "activity assay",
+                        "experimental_method"
+                    ],
+                    [
+                        223,
+                        226,
+                        "KAT",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 152,
+                "sent": "Conformational change of the \u03b27-\u03b28 hairpin (corresponding to the \u03b26-\u03b27 loop of other NATs) is noted in our structures (Figs 1D and 2C), which might provide an explanation to the NAT/KAT dual-activity in a structural biological view, but we were unable to rule out the possibility that the observed conformational change of this hairpin might be an artifact related to crystal packing or truncation of the C-terminal end of the protein.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        29,
+                        42,
+                        "\u03b27-\u03b28 hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        65,
+                        75,
+                        "\u03b26-\u03b27 loop",
+                        "structure_element"
+                    ],
+                    [
+                        85,
+                        89,
+                        "NATs",
+                        "protein_type"
+                    ],
+                    [
+                        107,
+                        117,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        178,
+                        181,
+                        "NAT",
+                        "protein_type"
+                    ],
+                    [
+                        182,
+                        185,
+                        "KAT",
+                        "protein_type"
+                    ],
+                    [
+                        328,
+                        335,
+                        "hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        368,
+                        383,
+                        "crystal packing",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 153,
+                "sent": "Further studies are therefore needed to reveal the mechanism for the KAT activity of this enzyme.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        69,
+                        72,
+                        "KAT",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 154,
+                "sent": "In early years, researchers found adjustment of GCN5 histone acetyltransferase structure when it binds CoA molecule.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        48,
+                        78,
+                        "GCN5 histone acetyltransferase",
+                        "protein_type"
+                    ],
+                    [
+                        79,
+                        88,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        103,
+                        106,
+                        "CoA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 155,
+                "sent": "The complexed form of NatA is more suitable for catalytic activation, since the \u03b11-\u03b12 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "complexed",
+                        "protein_state"
+                    ],
+                    [
+                        22,
+                        26,
+                        "NatA",
+                        "complex_assembly"
+                    ],
+                    [
+                        80,
+                        90,
+                        "\u03b11-\u03b12 loop",
+                        "structure_element"
+                    ],
+                    [
+                        158,
+                        180,
+                        "substrate-binding site",
+                        "site"
+                    ],
+                    [
+                        208,
+                        213,
+                        "Naa15",
+                        "protein"
+                    ],
+                    [
+                        229,
+                        234,
+                        "Naa10",
+                        "protein"
+                    ],
+                    [
+                        240,
+                        249,
+                        "catalytic",
+                        "protein_state"
+                    ],
+                    [
+                        250,
+                        257,
+                        "subunit",
+                        "structure_element"
+                    ],
+                    [
+                        261,
+                        265,
+                        "NatA",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 156,
+                "sent": "In the structure of hNaa50/CoA/peptide, Phe 27 in the \u03b11-\u03b12 loop appears to make hydrophobic interaction with the N-terminal Met of substrate peptide.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        7,
+                        16,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        20,
+                        38,
+                        "hNaa50/CoA/peptide",
+                        "complex_assembly"
+                    ],
+                    [
+                        40,
+                        46,
+                        "Phe 27",
+                        "residue_name_number"
+                    ],
+                    [
+                        54,
+                        64,
+                        "\u03b11-\u03b12 loop",
+                        "structure_element"
+                    ],
+                    [
+                        81,
+                        104,
+                        "hydrophobic interaction",
+                        "bond_interaction"
+                    ],
+                    [
+                        125,
+                        128,
+                        "Met",
+                        "residue_name"
+                    ],
+                    [
+                        142,
+                        149,
+                        "peptide",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 157,
+                "sent": "However, the hNaa60(1-242)/Ac-CoA crystal structure indicated that its counterpart in hNaa60, Phe 34, could also accommodate the binding of a hydrophilic malonate that occupied the substrate binding site although it maintained the same conformation as that observed in hNaa50.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        13,
+                        33,
+                        "hNaa60(1-242)/Ac-CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        34,
+                        51,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        86,
+                        92,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        94,
+                        100,
+                        "Phe 34",
+                        "residue_name_number"
+                    ],
+                    [
+                        154,
+                        162,
+                        "malonate",
+                        "chemical"
+                    ],
+                    [
+                        181,
+                        203,
+                        "substrate binding site",
+                        "site"
+                    ],
+                    [
+                        269,
+                        275,
+                        "hNaa50",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 158,
+                "sent": "Interestingly, the terminal thiol of CoA adopted alternative conformations in the structure of hNaa60(1-199)/CoA. One was to approach the substrate amine; the other was to approach the \u03b11-\u03b12 loop and away from the substrate amine.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        28,
+                        33,
+                        "thiol",
+                        "chemical"
+                    ],
+                    [
+                        37,
+                        40,
+                        "CoA",
+                        "chemical"
+                    ],
+                    [
+                        82,
+                        91,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        95,
+                        112,
+                        "hNaa60(1-199)/CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        185,
+                        195,
+                        "\u03b11-\u03b12 loop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 159,
+                "sent": "Same alternative conformations of CoA were observed in the hNaa60(1-199)(F34A) crystal structure, and our kinetic data showed that the F34A mutation abolished the activity of the enzyme.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        34,
+                        37,
+                        "CoA",
+                        "chemical"
+                    ],
+                    [
+                        59,
+                        78,
+                        "hNaa60(1-199)(F34A)",
+                        "mutant"
+                    ],
+                    [
+                        79,
+                        96,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        106,
+                        118,
+                        "kinetic data",
+                        "evidence"
+                    ],
+                    [
+                        135,
+                        139,
+                        "F34A",
+                        "mutant"
+                    ],
+                    [
+                        140,
+                        148,
+                        "mutation",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 160,
+                "sent": "Taken together, our data indicated that Phe 34 in hNaa60 may play a role in placing co-enzyme at the right location to facilitate the acetyl-transfer.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        40,
+                        46,
+                        "Phe 34",
+                        "residue_name_number"
+                    ],
+                    [
+                        50,
+                        56,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        134,
+                        140,
+                        "acetyl",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 161,
+                "sent": "However, these data did not rule out that possibility that Phe 34 may coordinate the binding of the N-terminal Met through hydrophobic interaction as was proposed by previous studies.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        59,
+                        65,
+                        "Phe 34",
+                        "residue_name_number"
+                    ],
+                    [
+                        111,
+                        114,
+                        "Met",
+                        "residue_name"
+                    ],
+                    [
+                        123,
+                        146,
+                        "hydrophobic interaction",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 162,
+                "sent": "Furthermore, we showed that hNaa60 adopts the classical two base mechanism to catalyze acetyl-transfer.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        28,
+                        34,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        87,
+                        93,
+                        "acetyl",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 163,
+                "sent": "Although sequence identity between hNaa60 and hNaa50 is low, key residues in the active site of both enzymes are highly conserved.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        35,
+                        41,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        46,
+                        52,
+                        "hNaa50",
+                        "protein"
+                    ],
+                    [
+                        81,
+                        92,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        113,
+                        129,
+                        "highly conserved",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 164,
+                "sent": "This can reasonably explain the high overlapping substrates specificities between hNaa60 and hNaa50.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        82,
+                        88,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        93,
+                        99,
+                        "hNaa50",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 165,
+                "sent": "Another structural feature of hNaa60 that distinguishes it from other NATs is the \u03b23-\u03b24 long loop which appears to inhibit the catalytic activity of hNaa60.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        30,
+                        36,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        70,
+                        74,
+                        "NATs",
+                        "protein_type"
+                    ],
+                    [
+                        82,
+                        97,
+                        "\u03b23-\u03b24 long loop",
+                        "structure_element"
+                    ],
+                    [
+                        149,
+                        155,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 166,
+                "sent": "However, this loop also seems to stabilize the whole hNaa60 structure, because deletion mutations of this region led to protein precipitation and aggregation (Figure S7).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        14,
+                        18,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        53,
+                        59,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        60,
+                        69,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        79,
+                        97,
+                        "deletion mutations",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 167,
+                "sent": "A previous study suggested that the auto-acetylation of Lys 79 was important for hNaa60-activity, whereas the point mutation K79R did not decrease the activity of hNaa60 in our study.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        36,
+                        52,
+                        "auto-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        56,
+                        62,
+                        "Lys 79",
+                        "residue_name_number"
+                    ],
+                    [
+                        81,
+                        87,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        110,
+                        124,
+                        "point mutation",
+                        "experimental_method"
+                    ],
+                    [
+                        125,
+                        129,
+                        "K79R",
+                        "mutant"
+                    ],
+                    [
+                        163,
+                        169,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 168,
+                "sent": "Meanwhile, no electron density of acetyl group was found on Lys 79 in our structures and mass spectrometry analysis.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        14,
+                        30,
+                        "electron density",
+                        "evidence"
+                    ],
+                    [
+                        34,
+                        40,
+                        "acetyl",
+                        "chemical"
+                    ],
+                    [
+                        60,
+                        66,
+                        "Lys 79",
+                        "residue_name_number"
+                    ],
+                    [
+                        74,
+                        84,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        89,
+                        106,
+                        "mass spectrometry",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 169,
+                "sent": "Hence, it appears that the auto-acetylation of hNaa60 is not an essential modification for its activity for the protein we used here.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        27,
+                        43,
+                        "auto-acetylation",
+                        "ptm"
+                    ],
+                    [
+                        47,
+                        53,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 170,
+                "sent": "As for the reason why K79R in Yang\u2019s previous studies reduced the activity of the enzyme, but in our studies it didn\u2019t, we suspect that the stability of this mutant may play some role.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        22,
+                        26,
+                        "K79R",
+                        "mutant"
+                    ],
+                    [
+                        158,
+                        164,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 171,
+                "sent": "K79R is less stable than the wild-type enzyme as was judged by its poorer gel-filtration behavior and tendency to precipitate.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "K79R",
+                        "mutant"
+                    ],
+                    [
+                        13,
+                        19,
+                        "stable",
+                        "protein_state"
+                    ],
+                    [
+                        29,
+                        38,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        74,
+                        88,
+                        "gel-filtration",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 172,
+                "sent": "In our studies we have paid special attention and carefully handled this protein to ensure that we did get enough of the protein in good condition for kinetic assays.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        151,
+                        165,
+                        "kinetic assays",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 173,
+                "sent": "The intracellular environment is more complicated than our in vitro assay and the substrate specificity of hNaa60 most focuses on transmembrane proteins.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        107,
+                        113,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 174,
+                "sent": "The interaction between hNaa60 and its substrates may involve the protein-membrane interaction which would further increase the complexity.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        24,
+                        30,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 175,
+                "sent": "It is not clear if the structure of hNaa60 is different in vivo or if other potential partner proteins may help to regulate its activity.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        23,
+                        32,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        36,
+                        42,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 176,
+                "sent": "Nevertheless, our study may be an inspiration for further studies on the functions and regulation of this youngest member of the NAT family.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        129,
+                        132,
+                        "NAT",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 177,
+                "sent": "Overall structure of Naa60.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        8,
+                        17,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        26,
+                        "Naa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 178,
+                "sent": "(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        22,
+                        "Sequence alignment",
+                        "experimental_method"
+                    ],
+                    [
+                        26,
+                        31,
+                        "Naa60",
+                        "protein"
+                    ],
+                    [
+                        33,
+                        37,
+                        "NatF",
+                        "complex_assembly"
+                    ],
+                    [
+                        39,
+                        43,
+                        "HAT4",
+                        "protein"
+                    ],
+                    [
+                        78,
+                        90,
+                        "Homo sapiens",
+                        "species"
+                    ],
+                    [
+                        92,
+                        96,
+                        "Homo",
+                        "species"
+                    ],
+                    [
+                        99,
+                        108,
+                        "Bos mutus",
+                        "species"
+                    ],
+                    [
+                        110,
+                        113,
+                        "Bos",
+                        "species"
+                    ],
+                    [
+                        116,
+                        127,
+                        "Salmo salar",
+                        "species"
+                    ],
+                    [
+                        129,
+                        134,
+                        "Salmo",
+                        "species"
+                    ],
+                    [
+                        140,
+                        147,
+                        "Xenopus",
+                        "species"
+                    ],
+                    [
+                        149,
+                        157,
+                        "Silurana",
+                        "species"
+                    ],
+                    [
+                        159,
+                        169,
+                        "tropicalis",
+                        "species"
+                    ],
+                    [
+                        171,
+                        178,
+                        "Xenopus",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 179,
+                "sent": "Alignment was generated using NPS@ and ESPript.3.0 (http://espript.ibcp.fr/ESPript/ESPript/).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Alignment",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 180,
+                "sent": "Residues 4\u20136 are highlighted in red box.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        9,
+                        12,
+                        "4\u20136",
+                        "residue_range"
+                    ]
+                ]
+            },
+            {
+                "sid": 181,
+                "sent": "(B) The structure of hNaa60(1-199)/CoA complex is shown as a yellow cartoon model.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        8,
+                        17,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        38,
+                        "hNaa60(1-199)/CoA",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 182,
+                "sent": "The CoA molecule is shown as sticks. (C) The structure of hNaa60(1-242)/Ac-CoA complex is presented as a cartoon model in cyan.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "CoA",
+                        "chemical"
+                    ],
+                    [
+                        45,
+                        54,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        58,
+                        78,
+                        "hNaa60(1-242)/Ac-CoA",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 183,
+                "sent": "The Ac-CoA and malonate molecules are shown as cyan and purple sticks, respectively.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        10,
+                        "Ac-CoA",
+                        "chemical"
+                    ],
+                    [
+                        15,
+                        23,
+                        "malonate",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 184,
+                "sent": "The secondary structures are labeled starting with \u03b10. (D) Superposition of hNaa60(1-242) (cyan), hNaa60(1-199) (yellow) and hNaa50 (pink, PDB 3TFY).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        51,
+                        53,
+                        "\u03b10",
+                        "structure_element"
+                    ],
+                    [
+                        59,
+                        72,
+                        "Superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        76,
+                        82,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        83,
+                        88,
+                        "1-242",
+                        "residue_range"
+                    ],
+                    [
+                        98,
+                        111,
+                        "hNaa60(1-199)",
+                        "mutant"
+                    ],
+                    [
+                        125,
+                        131,
+                        "hNaa50",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 185,
+                "sent": "The Ac-CoA of hNaa60(1-242)/Ac-CoA complex is represented as cyan sticks.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        10,
+                        "Ac-CoA",
+                        "chemical"
+                    ],
+                    [
+                        14,
+                        34,
+                        "hNaa60(1-242)/Ac-CoA",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 186,
+                "sent": "Amphipathicity of the \u03b15 helix and alternative conformations of the \u03b27-\u03b28 hairpin.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        14,
+                        "Amphipathicity",
+                        "protein_state"
+                    ],
+                    [
+                        22,
+                        30,
+                        "\u03b15 helix",
+                        "structure_element"
+                    ],
+                    [
+                        68,
+                        81,
+                        "\u03b27-\u03b28 hairpin",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 187,
+                "sent": "(A) The \u03b15 helix of hNaa60(1-242) in one asymmetric unit (slate) interacts with another hNaa60 molecule in a neighboring asymmetric unit (cyan).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        8,
+                        16,
+                        "\u03b15 helix",
+                        "structure_element"
+                    ],
+                    [
+                        20,
+                        26,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        27,
+                        32,
+                        "1-242",
+                        "residue_range"
+                    ],
+                    [
+                        88,
+                        94,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 188,
+                "sent": "Side-chains of hydrophobic residues on \u03b15 helix and the neighboring molecule participating in the interaction are shown as yellow and green sticks, respectively. (B) The \u03b15 helix of hNaa60(1-199) in one asymmetric unit (yellow) interacts with another hNaa60 molecule in the neighboring asymmetric units (green).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        39,
+                        47,
+                        "\u03b15 helix",
+                        "structure_element"
+                    ],
+                    [
+                        170,
+                        178,
+                        "\u03b15 helix",
+                        "structure_element"
+                    ],
+                    [
+                        182,
+                        195,
+                        "hNaa60(1-199)",
+                        "mutant"
+                    ],
+                    [
+                        251,
+                        257,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 189,
+                "sent": "Side-chains of hydrophobic residues on \u03b15 helix and the neighboring molecule (green) participating in the interaction are shown as yellow and green sticks, respectively.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        39,
+                        47,
+                        "\u03b15 helix",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 190,
+                "sent": "The third molecule (pink) does not directly interact with the \u03b15 helix.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        62,
+                        70,
+                        "\u03b15 helix",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 191,
+                "sent": "(C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the \u03b27-\u03b28 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        17,
+                        "Superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        21,
+                        34,
+                        "hNaa60(1-199)",
+                        "mutant"
+                    ],
+                    [
+                        48,
+                        54,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        55,
+                        60,
+                        "1-242",
+                        "residue_range"
+                    ],
+                    [
+                        106,
+                        119,
+                        "\u03b27-\u03b28 hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        133,
+                        143,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        151,
+                        164,
+                        "Superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        168,
+                        173,
+                        "Hat1p",
+                        "protein"
+                    ],
+                    [
+                        174,
+                        176,
+                        "H4",
+                        "protein_type"
+                    ],
+                    [
+                        210,
+                        216,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        217,
+                        222,
+                        "1-242",
+                        "residue_range"
+                    ],
+                    [
+                        237,
+                        250,
+                        "hNaa60(1-199)",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 192,
+                "sent": "The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "histone",
+                        "protein_type"
+                    ],
+                    [
+                        12,
+                        14,
+                        "H4",
+                        "protein_type"
+                    ],
+                    [
+                        15,
+                        22,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        26,
+                        29,
+                        "KAT",
+                        "protein_type"
+                    ],
+                    [
+                        41,
+                        49,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        50,
+                        55,
+                        "Hat1p",
+                        "protein"
+                    ],
+                    [
+                        92,
+                        99,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        100,
+                        108,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        109,
+                        115,
+                        "hNaa50",
+                        "protein"
+                    ],
+                    [
+                        119,
+                        122,
+                        "NAT",
+                        "protein_type"
+                    ],
+                    [
+                        175,
+                        185,
+                        "Nt-peptide",
+                        "chemical"
+                    ],
+                    [
+                        193,
+                        206,
+                        "superimposing",
+                        "experimental_method"
+                    ],
+                    [
+                        207,
+                        213,
+                        "hNaa50",
+                        "protein"
+                    ],
+                    [
+                        239,
+                        245,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 193,
+                "sent": "The \u03b1-amine of the NAT substrate and \u03b5-amine of the KAT substrate (along with the lysine side-chain) subject to acetylation are shown as sticks.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        19,
+                        22,
+                        "NAT",
+                        "protein_type"
+                    ],
+                    [
+                        52,
+                        55,
+                        "KAT",
+                        "protein_type"
+                    ],
+                    [
+                        82,
+                        88,
+                        "lysine",
+                        "residue_name"
+                    ],
+                    [
+                        112,
+                        123,
+                        "acetylation",
+                        "ptm"
+                    ]
+                ]
+            },
+            {
+                "sid": 194,
+                "sent": "Electron density map of the active site.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        20,
+                        "Electron density map",
+                        "evidence"
+                    ],
+                    [
+                        28,
+                        39,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 195,
+                "sent": "The 2Fo-Fc maps contoured at 1.0\u03c3 are shown for hNaa60(1-242)/Ac-CoA (A), hNaa60(1-199)/CoA (B) and hNaa60(1-199) F34A/CoA (C).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        15,
+                        "2Fo-Fc maps",
+                        "evidence"
+                    ],
+                    [
+                        48,
+                        68,
+                        "hNaa60(1-242)/Ac-CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        74,
+                        91,
+                        "hNaa60(1-199)/CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        100,
+                        122,
+                        "hNaa60(1-199) F34A/CoA",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 196,
+                "sent": "The putative substrate peptide binding site is indicated by the peptide (shown as pink sticks) from the hNaa50/CoA/peptide complex structure after superimposing hNaa50 on the hNaa60 structures determined in this study.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        13,
+                        43,
+                        "substrate peptide binding site",
+                        "site"
+                    ],
+                    [
+                        64,
+                        71,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        104,
+                        122,
+                        "hNaa50/CoA/peptide",
+                        "complex_assembly"
+                    ],
+                    [
+                        131,
+                        140,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        147,
+                        160,
+                        "superimposing",
+                        "experimental_method"
+                    ],
+                    [
+                        161,
+                        167,
+                        "hNaa50",
+                        "protein"
+                    ],
+                    [
+                        175,
+                        181,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        182,
+                        192,
+                        "structures",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 197,
+                "sent": "The black arrow indicates the \u03b1-amine of the first Met (M1) (all panels).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        45,
+                        54,
+                        "first Met",
+                        "residue_name_number"
+                    ],
+                    [
+                        56,
+                        58,
+                        "M1",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 198,
+                "sent": "The purple arrow indicates the acetyl moiety of Ac-CoA (A).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        31,
+                        37,
+                        "acetyl",
+                        "chemical"
+                    ],
+                    [
+                        48,
+                        54,
+                        "Ac-CoA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 199,
+                "sent": "The red arrow indicates the alternative conformation of the thiol moiety of the co-enzyme when Phe 34 side-chain is displaced (B) or mutated to Ala (C).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        95,
+                        101,
+                        "Phe 34",
+                        "residue_name_number"
+                    ],
+                    [
+                        133,
+                        140,
+                        "mutated",
+                        "experimental_method"
+                    ],
+                    [
+                        144,
+                        147,
+                        "Ala",
+                        "residue_name"
+                    ]
+                ]
+            },
+            {
+                "sid": 200,
+                "sent": "Structural basis for hNaa60 catalytic activity.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        21,
+                        27,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 201,
+                "sent": "(A) Superposition of hNaa60 active site (cyan) on that of hNaa50 (pink, PDB 3TFY).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        17,
+                        "Superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        21,
+                        27,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        28,
+                        39,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        58,
+                        64,
+                        "hNaa50",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 202,
+                "sent": "Side-chains of key catalytic and substrate-binding residues are highlighted as sticks.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        19,
+                        59,
+                        "catalytic and substrate-binding residues",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 203,
+                "sent": "The malonate molecule in the hNaa60(1-242)/Ac-CoA structure and the peptide in the hNaa50/CoA/peptide structure are shown as purple and yellow sticks respectively. (B) A close view of the active site of hNaa60.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        12,
+                        "malonate",
+                        "chemical"
+                    ],
+                    [
+                        29,
+                        49,
+                        "hNaa60(1-242)/Ac-CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        50,
+                        59,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        68,
+                        75,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        83,
+                        101,
+                        "hNaa50/CoA/peptide",
+                        "complex_assembly"
+                    ],
+                    [
+                        102,
+                        111,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        188,
+                        199,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        203,
+                        209,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 204,
+                "sent": "Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        9,
+                        15,
+                        "Glu 37",
+                        "residue_name_number"
+                    ],
+                    [
+                        17,
+                        23,
+                        "Tyr 97",
+                        "residue_name_number"
+                    ],
+                    [
+                        28,
+                        35,
+                        "His 138",
+                        "residue_name_number"
+                    ],
+                    [
+                        39,
+                        45,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        81,
+                        87,
+                        "Tyr 73",
+                        "residue_name_number"
+                    ],
+                    [
+                        92,
+                        99,
+                        "His 112",
+                        "residue_name_number"
+                    ],
+                    [
+                        104,
+                        110,
+                        "hNaa50",
+                        "protein"
+                    ],
+                    [
+                        171,
+                        177,
+                        "Glu 24",
+                        "residue_name_number"
+                    ],
+                    [
+                        179,
+                        185,
+                        "His 72",
+                        "residue_name_number"
+                    ],
+                    [
+                        190,
+                        197,
+                        "His 111",
+                        "residue_name_number"
+                    ],
+                    [
+                        202,
+                        211,
+                        "complexed",
+                        "protein_state"
+                    ],
+                    [
+                        219,
+                        226,
+                        "hNaa10p",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 205,
+                "sent": "The water molecules participating in catalysis in the hNaa60 and hNaa50 structures are showed as green and red spheres, separately. (C) The interaction between the malonate molecule and surrounding residues observed in the hNaa60(1-242)/Ac-CoA structure.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        9,
+                        "water",
+                        "chemical"
+                    ],
+                    [
+                        54,
+                        60,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        65,
+                        71,
+                        "hNaa50",
+                        "protein"
+                    ],
+                    [
+                        72,
+                        82,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        164,
+                        172,
+                        "malonate",
+                        "chemical"
+                    ],
+                    [
+                        223,
+                        243,
+                        "hNaa60(1-242)/Ac-CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        244,
+                        253,
+                        "structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 206,
+                "sent": "The yellow dotted lines indicate the hydrogen bonds. (D) A zoomed view of \u03b23-\u03b24 loop of hNaa60.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        37,
+                        51,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        74,
+                        84,
+                        "\u03b23-\u03b24 loop",
+                        "structure_element"
+                    ],
+                    [
+                        88,
+                        94,
+                        "hNaa60",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 207,
+                "sent": "Key residues discussed in the text (cyan), the malonate (purple) and Ac-CoA (gray) are shown as sticks.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        47,
+                        55,
+                        "malonate",
+                        "chemical"
+                    ],
+                    [
+                        69,
+                        75,
+                        "Ac-CoA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 208,
+                "sent": "The yellow dotted lines indicate the salt bridges.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        37,
+                        49,
+                        "salt bridges",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 209,
+                "sent": "Catalytic activity of hNaa60 and mutant proteins.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        22,
+                        28,
+                        "hNaa60",
+                        "protein"
+                    ],
+                    [
+                        33,
+                        39,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 210,
+                "sent": "(A) Catalytic efficiency (shown as kcat/Km values) of hNaa60 (1-199) WT and mutants.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        24,
+                        "Catalytic efficiency",
+                        "evidence"
+                    ],
+                    [
+                        35,
+                        39,
+                        "kcat",
+                        "evidence"
+                    ],
+                    [
+                        40,
+                        42,
+                        "Km",
+                        "evidence"
+                    ],
+                    [
+                        54,
+                        68,
+                        "hNaa60 (1-199)",
+                        "mutant"
+                    ],
+                    [
+                        69,
+                        71,
+                        "WT",
+                        "protein_state"
+                    ],
+                    [
+                        76,
+                        83,
+                        "mutants",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 211,
+                "sent": "(B) CD spectra of wild-type and mutant proteins from 250\u2009nm to 190\u2009nm.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        6,
+                        "CD",
+                        "experimental_method"
+                    ],
+                    [
+                        7,
+                        14,
+                        "spectra",
+                        "evidence"
+                    ],
+                    [
+                        18,
+                        27,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        32,
+                        38,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 212,
+                "sent": "The sample concentration was 4.5\u2009\u03bcM in 20\u2009mM Tris, pH 8.0, 150\u2009mM NaCl, 1% glycerol and 1\u2009mM TCEP at room temperature.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        93,
+                        97,
+                        "TCEP",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 213,
+                "sent": "Data collection and refinement statistics.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        41,
+                        "Data collection and refinement statistics",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 214,
+                "sent": "Structure and PDB ID\thNaa60(1-242)/Ac-CoA 5HGZ\thNaa60(1-199)/CoA 5HH0\thNaa60(1-199)F34A/CoA 5HH1\t \tData collection*\t \t\u2003Space group\tP212121\tP21212\tP21212\t \tCell dimensions\t \t\u2003a, b, c (\u00c5)\t53.3, 57.4, 68.8\t67.8, 73.8, 43.2\t66.7, 74.0, 43.5\t \t\u2003\u03b1,\u03b2,\u03b3 (\u00b0)\t90.0, 90.0, 90.0\t90.0, 90.0, 90.0\t90.0, 90.0, 90.0\t \tResolution (\u00c5)\t50\u20131.38 (1.42\u20131.38)\t50\u20131.60 (1.66\u20131.60)\t50\u20131.80 (1.86\u20131.80)\t \tRp.i.m.(%)**\t3.0 (34.4)\t2.1 (32.5)\t2.6 (47.8)\t \tI/\u03c3\t21.5 (2.0)\t31.8 (2.0)\t28.0 (2.4)\t \tCompleteness (%)\t99.8 (99.1)\t99.6 (98.5)\t99.9 (99.7)\t \tRedundancy\t6.9 (5.0)\t6.9 (6.2)\t6.3 (5.9)\t \tRefinement\t \t\u2003Resolution (\u00c5)\t25.81\u20131.38\t33.55\u20131.60\t43.52\u20131.80\t \t\u2003No. reflections\t43660\t28588\t20490\t \t\u2003Rwork/Rfree\t0.182/0.192\t0.181/0.184\t0.189/0.209\t \tNo. atoms\t \t\u2003Protein\t1717\t1576\t1566\t \t\u2003Ligand/ion\t116\t96\t96\t \t\u2003Water\t289\t258\t168\t \tB-factors\t \t\u2003Protein\t23.8\t32.0\t37.4\t \t\u2003Ligand/ion\t22.2\t34.6\t43.7\t \t\u2003Water\t35.1\t46.4\t49.1\t \tR.m.s.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        21,
+                        41,
+                        "hNaa60(1-242)/Ac-CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        47,
+                        64,
+                        "hNaa60(1-199)/CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        70,
+                        91,
+                        "hNaa60(1-199)F34A/CoA",
+                        "complex_assembly"
+                    ],
+                    [
+                        780,
+                        785,
+                        "Water",
+                        "chemical"
+                    ],
+                    [
+                        868,
+                        873,
+                        "Water",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 215,
+                "sent": "One crystal was used for each data set.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "crystal",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 216,
+                "sent": "**Rp.i.m., a redundancy-independent R factor was used to evaluate the diffraction data quality as was proposed by Evans.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        36,
+                        44,
+                        "R factor",
+                        "evidence"
+                    ],
+                    [
+                        70,
+                        86,
+                        "diffraction data",
+                        "evidence"
+                    ]
+                ]
+            }
+        ]
+    },
+    "PMC5014086": {
+        "annotations": [
+            {
+                "sid": 0,
+                "sent": "Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf",
+                "section": "TITLE",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Structure",
+                        "evidence"
+                    ],
+                    [
+                        27,
+                        30,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        41,
+                        48,
+                        "Kremen1",
+                        "protein"
+                    ],
+                    [
+                        97,
+                        101,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        106,
+                        114,
+                        "Dickkopf",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 1,
+                "sent": "Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        14,
+                        "Kremen 1 and 2",
+                        "protein_type"
+                    ],
+                    [
+                        39,
+                        51,
+                        "co-receptors",
+                        "protein_type"
+                    ],
+                    [
+                        56,
+                        64,
+                        "Dickkopf",
+                        "protein_type"
+                    ],
+                    [
+                        66,
+                        69,
+                        "Dkk",
+                        "protein_type"
+                    ],
+                    [
+                        124,
+                        127,
+                        "Wnt",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 2,
+                "sent": "We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2\u00a0\u00c5. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        22,
+                        40,
+                        "crystal structures",
+                        "evidence"
+                    ],
+                    [
+                        48,
+                        58,
+                        "ectodomain",
+                        "structure_element"
+                    ],
+                    [
+                        62,
+                        67,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        68,
+                        75,
+                        "Kremen1",
+                        "protein"
+                    ],
+                    [
+                        77,
+                        81,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        81,
+                        84,
+                        "ECD",
+                        "structure_element"
+                    ],
+                    [
+                        124,
+                        128,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        128,
+                        131,
+                        "ECD",
+                        "structure_element"
+                    ],
+                    [
+                        198,
+                        220,
+                        "triangular arrangement",
+                        "protein_state"
+                    ],
+                    [
+                        228,
+                        235,
+                        "Kringle",
+                        "structure_element"
+                    ],
+                    [
+                        237,
+                        240,
+                        "WSC",
+                        "structure_element"
+                    ],
+                    [
+                        246,
+                        249,
+                        "CUB",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 3,
+                "sent": "The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        4,
+                        14,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        53,
+                        56,
+                        "WSC",
+                        "structure_element"
+                    ],
+                    [
+                        67,
+                        91,
+                        "hepatocyte growth factor",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 4,
+                "sent": "We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between \u03b2-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        80,
+                        83,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        84,
+                        95,
+                        "co-receptor",
+                        "protein_type"
+                    ],
+                    [
+                        96,
+                        102,
+                        "Lrp5/6",
+                        "protein_type"
+                    ],
+                    [
+                        104,
+                        107,
+                        "Dkk",
+                        "protein_type"
+                    ],
+                    [
+                        113,
+                        116,
+                        "Krm",
+                        "protein_type"
+                    ],
+                    [
+                        159,
+                        176,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        185,
+                        221,
+                        "\u03b2-propeller/EGF repeats (PE) 3 and 4",
+                        "structure_element"
+                    ],
+                    [
+                        229,
+                        232,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        233,
+                        244,
+                        "co-receptor",
+                        "protein_type"
+                    ],
+                    [
+                        245,
+                        249,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        251,
+                        255,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        255,
+                        261,
+                        "PE3PE4",
+                        "structure_element"
+                    ],
+                    [
+                        268,
+                        290,
+                        "cysteine-rich domain 2",
+                        "structure_element"
+                    ],
+                    [
+                        292,
+                        296,
+                        "CRD2",
+                        "structure_element"
+                    ],
+                    [
+                        301,
+                        305,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        311,
+                        315,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        315,
+                        318,
+                        "ECD",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 5,
+                "sent": "DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        4,
+                        8,
+                        "CRD2",
+                        "structure_element"
+                    ],
+                    [
+                        31,
+                        35,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        35,
+                        38,
+                        "PE3",
+                        "structure_element"
+                    ],
+                    [
+                        43,
+                        47,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        47,
+                        58,
+                        "Kringle-WSC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 6,
+                "sent": "Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "Modeling",
+                        "experimental_method"
+                    ],
+                    [
+                        30,
+                        55,
+                        "surface plasmon resonance",
+                        "experimental_method"
+                    ],
+                    [
+                        73,
+                        89,
+                        "interaction site",
+                        "site"
+                    ],
+                    [
+                        98,
+                        102,
+                        "Krm1",
+                        "protein"
+                    ],
+                    [
+                        102,
+                        105,
+                        "CUB",
+                        "structure_element"
+                    ],
+                    [
+                        110,
+                        114,
+                        "Lrp6",
+                        "protein"
+                    ],
+                    [
+                        114,
+                        117,
+                        "PE2",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 7,
+                "sent": "The structure of the KREMEN 1 ectodomain is solved from three crystal forms",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        29,
+                        "KREMEN 1",
+                        "protein"
+                    ],
+                    [
+                        30,
+                        40,
+                        "ectodomain",
+                        "structure_element"
+                    ],
+                    [
+                        44,
+                        50,
+                        "solved",
+                        "experimental_method"
+                    ],
+                    [
+                        62,
+                        75,
+                        "crystal forms",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 8,
+                "sent": "Kringle, WSC, and CUB subdomains interact tightly to form a single structural unit",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "Kringle",
+                        "structure_element"
+                    ],
+                    [
+                        9,
+                        12,
+                        "WSC",
+                        "structure_element"
+                    ],
+                    [
+                        18,
+                        21,
+                        "CUB",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 9,
+                "sent": "The interface to DKKs is formed from the Kringle and WSC domains",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "interface",
+                        "site"
+                    ],
+                    [
+                        17,
+                        21,
+                        "DKKs",
+                        "protein_type"
+                    ],
+                    [
+                        41,
+                        48,
+                        "Kringle",
+                        "structure_element"
+                    ],
+                    [
+                        53,
+                        56,
+                        "WSC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 10,
+                "sent": "The CUB domain is found to interact directly with LRP6PE1PE2",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "CUB",
+                        "structure_element"
+                    ],
+                    [
+                        50,
+                        54,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        54,
+                        60,
+                        "PE1PE2",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 11,
+                "sent": "Zebisch et\u00a0al. describe the ectodomain structure of KREMEN 1, a receptor for Wnt antagonists of the DKK family.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        28,
+                        38,
+                        "ectodomain",
+                        "structure_element"
+                    ],
+                    [
+                        39,
+                        48,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        52,
+                        60,
+                        "KREMEN 1",
+                        "protein"
+                    ],
+                    [
+                        64,
+                        72,
+                        "receptor",
+                        "protein_type"
+                    ],
+                    [
+                        77,
+                        80,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        100,
+                        103,
+                        "DKK",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 12,
+                "sent": "Apo structures and a complex with functional fragments of DKK1 and LRP6 shed light on the function of this dual-mode regulator of Wnt signaling.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "Apo",
+                        "protein_state"
+                    ],
+                    [
+                        4,
+                        14,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        33,
+                        "complex with",
+                        "protein_state"
+                    ],
+                    [
+                        34,
+                        54,
+                        "functional fragments",
+                        "protein_state"
+                    ],
+                    [
+                        58,
+                        62,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        67,
+                        71,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        130,
+                        133,
+                        "Wnt",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 13,
+                "sent": "Signaling by Wnt morphogens is renowned for its fundamental roles in embryonic development, tissue homeostasis, and stem cell maintenance.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        13,
+                        16,
+                        "Wnt",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 14,
+                "sent": "Due to these functions, generation, delivery, and interpretation of Wnt signals are all heavily regulated in the animal body.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        68,
+                        71,
+                        "Wnt",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 15,
+                "sent": "Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        10,
+                        "Vertebrate",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        11,
+                        19,
+                        "Dickkopf",
+                        "protein_type"
+                    ],
+                    [
+                        30,
+                        34,
+                        "Dkk1",
+                        "protein_type"
+                    ],
+                    [
+                        36,
+                        37,
+                        "2",
+                        "protein_type"
+                    ],
+                    [
+                        43,
+                        44,
+                        "4",
+                        "protein_type"
+                    ],
+                    [
+                        86,
+                        89,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        129,
+                        132,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        133,
+                        144,
+                        "co-receptor",
+                        "protein_type"
+                    ],
+                    [
+                        145,
+                        151,
+                        "LRP5/6",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 16,
+                "sent": "Kremen proteins (Krm1 and Krm2) have been identified as additional high-affinity transmembrane receptors for Dkk.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        6,
+                        "Kremen",
+                        "protein_type"
+                    ],
+                    [
+                        17,
+                        21,
+                        "Krm1",
+                        "protein_type"
+                    ],
+                    [
+                        26,
+                        30,
+                        "Krm2",
+                        "protein_type"
+                    ],
+                    [
+                        81,
+                        104,
+                        "transmembrane receptors",
+                        "protein_type"
+                    ],
+                    [
+                        109,
+                        112,
+                        "Dkk",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 17,
+                "sent": "Krm and Dkk synergize in Wnt inhibition during Xenopus embryogenesis to regulate anterior-posterior patterning.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "Krm",
+                        "protein_type"
+                    ],
+                    [
+                        8,
+                        11,
+                        "Dkk",
+                        "protein_type"
+                    ],
+                    [
+                        25,
+                        28,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        47,
+                        54,
+                        "Xenopus",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 18,
+                "sent": "Mechanistically it is thought that, in the presence of Dkk, Krm forms a ternary complex with Lrp6, which is then rapidly endocytosed.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        43,
+                        54,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        55,
+                        58,
+                        "Dkk",
+                        "protein_type"
+                    ],
+                    [
+                        60,
+                        63,
+                        "Krm",
+                        "protein_type"
+                    ],
+                    [
+                        80,
+                        92,
+                        "complex with",
+                        "protein_state"
+                    ],
+                    [
+                        93,
+                        97,
+                        "Lrp6",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 19,
+                "sent": "This amplifies the intrinsic Wnt antagonistic activity of Dkk by efficiently depleting the cell surface of the Wnt co-receptor.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        29,
+                        32,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        58,
+                        61,
+                        "Dkk",
+                        "protein_type"
+                    ],
+                    [
+                        111,
+                        114,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        115,
+                        126,
+                        "co-receptor",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 20,
+                "sent": "In accordance with this, Krm1\u2212/\u2212 and Krm2\u2212/\u2212 double knockout mice show a high bone mass phenotype typical of increased Wnt signaling, as well as growth of ectopic forelimb digits.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        25,
+                        29,
+                        "Krm1",
+                        "protein_type"
+                    ],
+                    [
+                        37,
+                        41,
+                        "Krm2",
+                        "protein_type"
+                    ],
+                    [
+                        45,
+                        60,
+                        "double knockout",
+                        "experimental_method"
+                    ],
+                    [
+                        61,
+                        65,
+                        "mice",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        119,
+                        122,
+                        "Wnt",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 21,
+                "sent": "Growth of ectopic digits is further enhanced upon additional loss of dkk expression.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        69,
+                        72,
+                        "dkk",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 22,
+                "sent": "The Wnt antagonistic activity of Krm1 is also linked to its importance for correct thymus epithelium formation in mice.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        33,
+                        37,
+                        "Krm1",
+                        "protein_type"
+                    ],
+                    [
+                        114,
+                        118,
+                        "mice",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 23,
+                "sent": "The importance of intact KRM1 for normal human development and health is highlighted by the recent finding that a homozygous mutation in the ectodomain of KRM1 leads to severe ectodermal dysplasia including oligodontia.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        18,
+                        24,
+                        "intact",
+                        "protein_state"
+                    ],
+                    [
+                        25,
+                        29,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        41,
+                        46,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        141,
+                        151,
+                        "ectodomain",
+                        "structure_element"
+                    ],
+                    [
+                        155,
+                        159,
+                        "KRM1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 24,
+                "sent": "Interestingly, the Wnt antagonistic activity of Krm is context dependent, and Krm proteins are actually dual-mode Wnt regulators.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        19,
+                        22,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        48,
+                        51,
+                        "Krm",
+                        "protein_type"
+                    ],
+                    [
+                        78,
+                        81,
+                        "Krm",
+                        "protein_type"
+                    ],
+                    [
+                        114,
+                        117,
+                        "Wnt",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 25,
+                "sent": "In the absence of Dkk, Krm1 and 2 change their function from inhibition to enhancement of Lrp6-mediated signaling.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        7,
+                        17,
+                        "absence of",
+                        "protein_state"
+                    ],
+                    [
+                        18,
+                        21,
+                        "Dkk",
+                        "protein_type"
+                    ],
+                    [
+                        23,
+                        27,
+                        "Krm1",
+                        "protein_type"
+                    ],
+                    [
+                        32,
+                        33,
+                        "2",
+                        "protein_type"
+                    ],
+                    [
+                        90,
+                        94,
+                        "Lrp6",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 26,
+                "sent": "By direct binding to Lrp6 via the ectodomains, Krm proteins promote Lrp6 cell-surface localization and hence increase receptor availability.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        21,
+                        25,
+                        "Lrp6",
+                        "protein_type"
+                    ],
+                    [
+                        34,
+                        45,
+                        "ectodomains",
+                        "structure_element"
+                    ],
+                    [
+                        47,
+                        50,
+                        "Krm",
+                        "protein_type"
+                    ],
+                    [
+                        68,
+                        72,
+                        "Lrp6",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 27,
+                "sent": "Further increasing the complexity of Krm functionality, it was recently found that Krm1 (but not Krm2) can also act independently of LRP5/6 and Wnt as a dependence receptor, triggering apoptosis unless bound to Dkk.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        37,
+                        40,
+                        "Krm",
+                        "protein_type"
+                    ],
+                    [
+                        83,
+                        87,
+                        "Krm1",
+                        "protein_type"
+                    ],
+                    [
+                        97,
+                        101,
+                        "Krm2",
+                        "protein_type"
+                    ],
+                    [
+                        133,
+                        139,
+                        "LRP5/6",
+                        "protein"
+                    ],
+                    [
+                        144,
+                        147,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        202,
+                        210,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        211,
+                        214,
+                        "Dkk",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 28,
+                "sent": "Structurally, Krm1 and 2 are type I transmembrane proteins with a 40\u00a0kDa ectodomain and a flexible cytoplasmic tail consisting of 60\u201375 residues.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        14,
+                        18,
+                        "Krm1",
+                        "protein_type"
+                    ],
+                    [
+                        23,
+                        24,
+                        "2",
+                        "protein_type"
+                    ],
+                    [
+                        29,
+                        58,
+                        "type I transmembrane proteins",
+                        "protein_type"
+                    ],
+                    [
+                        73,
+                        83,
+                        "ectodomain",
+                        "structure_element"
+                    ],
+                    [
+                        90,
+                        98,
+                        "flexible",
+                        "protein_state"
+                    ],
+                    [
+                        99,
+                        115,
+                        "cytoplasmic tail",
+                        "structure_element"
+                    ],
+                    [
+                        130,
+                        132,
+                        "60",
+                        "residue_range"
+                    ],
+                    [
+                        133,
+                        135,
+                        "75",
+                        "residue_range"
+                    ]
+                ]
+            },
+            {
+                "sid": 29,
+                "sent": "The ectodomain consists of three similarly sized structural domains of around 10\u00a0kDa each: the N-terminal Kringle domain (KR) is followed by a WSC domain of unknown fold.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        14,
+                        "ectodomain",
+                        "structure_element"
+                    ],
+                    [
+                        106,
+                        113,
+                        "Kringle",
+                        "structure_element"
+                    ],
+                    [
+                        122,
+                        124,
+                        "KR",
+                        "structure_element"
+                    ],
+                    [
+                        143,
+                        146,
+                        "WSC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 30,
+                "sent": "The third structural domain is a CUB domain.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        33,
+                        36,
+                        "CUB",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 31,
+                "sent": "An approximately 70-residue linker connects the CUB domain to the transmembrane span.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        3,
+                        27,
+                        "approximately 70-residue",
+                        "residue_range"
+                    ],
+                    [
+                        28,
+                        34,
+                        "linker",
+                        "structure_element"
+                    ],
+                    [
+                        48,
+                        51,
+                        "CUB",
+                        "structure_element"
+                    ],
+                    [
+                        66,
+                        84,
+                        "transmembrane span",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 32,
+                "sent": "An intact KR-WSC-CUB domain triplet and membrane attachment is required for Wnt antagonism.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        3,
+                        9,
+                        "intact",
+                        "protein_state"
+                    ],
+                    [
+                        10,
+                        20,
+                        "KR-WSC-CUB",
+                        "structure_element"
+                    ],
+                    [
+                        76,
+                        79,
+                        "Wnt",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 33,
+                "sent": "The transmembrane span and cytoplasmic tail can be replaced with a GPI linker without impact on Wnt antagonism.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        22,
+                        "transmembrane span",
+                        "structure_element"
+                    ],
+                    [
+                        27,
+                        43,
+                        "cytoplasmic tail",
+                        "structure_element"
+                    ],
+                    [
+                        67,
+                        70,
+                        "GPI",
+                        "structure_element"
+                    ],
+                    [
+                        71,
+                        77,
+                        "linker",
+                        "structure_element"
+                    ],
+                    [
+                        96,
+                        99,
+                        "Wnt",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 34,
+                "sent": "The structures presented here reveal the unknown fold of the WSC domain and the tight interactions of all three domains.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        14,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        61,
+                        64,
+                        "WSC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 35,
+                "sent": "We further succeeded in determination of a low-resolution LRP6PE3PE4-DKK1CRD2-KRM1ECD complex, defining the architecture of the Wnt inhibitory complex that leads to Lrp6 cell-surface depletion.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        58,
+                        85,
+                        "LRP6PE3PE4-DKK1CRD2-KRM1ECD",
+                        "complex_assembly"
+                    ],
+                    [
+                        128,
+                        131,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        132,
+                        150,
+                        "inhibitory complex",
+                        "complex_assembly"
+                    ],
+                    [
+                        165,
+                        169,
+                        "Lrp6",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 36,
+                "sent": "The recombinant production of the extracellular domain of Krm for structural studies proved challenging (see Experimental Procedures).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        54,
+                        "extracellular domain",
+                        "structure_element"
+                    ],
+                    [
+                        58,
+                        61,
+                        "Krm",
+                        "protein_type"
+                    ],
+                    [
+                        66,
+                        84,
+                        "structural studies",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 37,
+                "sent": "We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        26,
+                        30,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        30,
+                        33,
+                        "ECD",
+                        "structure_element"
+                    ],
+                    [
+                        34,
+                        48,
+                        "complexes with",
+                        "protein_state"
+                    ],
+                    [
+                        49,
+                        55,
+                        "DKK1fl",
+                        "protein"
+                    ],
+                    [
+                        57,
+                        61,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        61,
+                        72,
+                        "Linker-CRD2",
+                        "structure_element"
+                    ],
+                    [
+                        78,
+                        82,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        82,
+                        86,
+                        "CRD2",
+                        "structure_element"
+                    ],
+                    [
+                        124,
+                        138,
+                        "gel filtration",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 38,
+                "sent": "Several crystal forms were obtained from these complexes, however, crystals always contained only KRM1 protein.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        21,
+                        "crystal forms",
+                        "evidence"
+                    ],
+                    [
+                        67,
+                        75,
+                        "crystals",
+                        "evidence"
+                    ],
+                    [
+                        98,
+                        102,
+                        "KRM1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 39,
+                "sent": "We solved the structure of KRM1ECD in three crystal forms at 1.9, 2.8, and 3.2\u00a0\u00c5 resolution (Table 1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        3,
+                        9,
+                        "solved",
+                        "experimental_method"
+                    ],
+                    [
+                        14,
+                        23,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        27,
+                        31,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        31,
+                        34,
+                        "ECD",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 40,
+                "sent": "The high-resolution structure is a near full-length model (Figure\u00a01).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        20,
+                        29,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        40,
+                        51,
+                        "full-length",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 41,
+                "sent": "The small, flexible, and charged 98AEHED102 loop could only be modeled in a slightly lower resolution structure and in crystal form III.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        9,
+                        "small",
+                        "protein_state"
+                    ],
+                    [
+                        11,
+                        19,
+                        "flexible",
+                        "protein_state"
+                    ],
+                    [
+                        25,
+                        32,
+                        "charged",
+                        "protein_state"
+                    ],
+                    [
+                        33,
+                        48,
+                        "98AEHED102 loop",
+                        "structure_element"
+                    ],
+                    [
+                        102,
+                        111,
+                        "structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 42,
+                "sent": "The KR, WSC, and CUB are arranged in a roughly triangular fashion with tight interactions between all three domains.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        6,
+                        "KR",
+                        "structure_element"
+                    ],
+                    [
+                        8,
+                        11,
+                        "WSC",
+                        "structure_element"
+                    ],
+                    [
+                        17,
+                        20,
+                        "CUB",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 43,
+                "sent": "The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        6,
+                        "KR",
+                        "structure_element"
+                    ],
+                    [
+                        43,
+                        62,
+                        "glycosylation sites",
+                        "site"
+                    ],
+                    [
+                        93,
+                        110,
+                        "disulfide bridges",
+                        "ptm"
+                    ],
+                    [
+                        112,
+                        115,
+                        "C32",
+                        "residue_name_number"
+                    ],
+                    [
+                        116,
+                        120,
+                        "C114",
+                        "residue_name_number"
+                    ],
+                    [
+                        122,
+                        125,
+                        "C55",
+                        "residue_name_number"
+                    ],
+                    [
+                        126,
+                        129,
+                        "C95",
+                        "residue_name_number"
+                    ],
+                    [
+                        131,
+                        134,
+                        "C84",
+                        "residue_name_number"
+                    ],
+                    [
+                        135,
+                        139,
+                        "C109",
+                        "residue_name_number"
+                    ],
+                    [
+                        157,
+                        164,
+                        "Kringle",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 44,
+                "sent": "The structurally most similar Kringle domain is that of human plasminogen (PDB: 1PKR) with an root-mean-square deviation (RMSD) of 1.7\u00a0\u00c5 for 73 aligned C\u03b1 (Figure\u00a01B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        30,
+                        37,
+                        "Kringle",
+                        "structure_element"
+                    ],
+                    [
+                        56,
+                        61,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        62,
+                        73,
+                        "plasminogen",
+                        "protein"
+                    ],
+                    [
+                        94,
+                        120,
+                        "root-mean-square deviation",
+                        "evidence"
+                    ],
+                    [
+                        122,
+                        126,
+                        "RMSD",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 45,
+                "sent": "The KRM1 structure reveals the fold of the WSC domain for the first time.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        9,
+                        18,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        43,
+                        46,
+                        "WSC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 46,
+                "sent": "The structure is best described as a sandwich of a \u03b21-\u03b25-\u03b23-\u03b24-\u03b22 antiparallel \u03b2 sheet and a single \u03b1 helix.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        37,
+                        45,
+                        "sandwich",
+                        "structure_element"
+                    ],
+                    [
+                        51,
+                        86,
+                        "\u03b21-\u03b25-\u03b23-\u03b24-\u03b22 antiparallel \u03b2 sheet",
+                        "structure_element"
+                    ],
+                    [
+                        100,
+                        107,
+                        "\u03b1 helix",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 47,
+                "sent": "The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        30,
+                        35,
+                        "loops",
+                        "structure_element"
+                    ],
+                    [
+                        62,
+                        79,
+                        "disulfide bridges",
+                        "ptm"
+                    ],
+                    [
+                        81,
+                        85,
+                        "C122",
+                        "residue_name_number"
+                    ],
+                    [
+                        86,
+                        90,
+                        "C186",
+                        "residue_name_number"
+                    ],
+                    [
+                        92,
+                        96,
+                        "C147",
+                        "residue_name_number"
+                    ],
+                    [
+                        97,
+                        101,
+                        "C167",
+                        "residue_name_number"
+                    ],
+                    [
+                        103,
+                        107,
+                        "C151",
+                        "residue_name_number"
+                    ],
+                    [
+                        108,
+                        112,
+                        "C169",
+                        "residue_name_number"
+                    ],
+                    [
+                        114,
+                        118,
+                        "C190",
+                        "residue_name_number"
+                    ],
+                    [
+                        119,
+                        123,
+                        "C198",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 48,
+                "sent": "Using the PDBeFold server, we detected a surprising yet significant homology to PAN module domains.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        10,
+                        25,
+                        "PDBeFold server",
+                        "experimental_method"
+                    ],
+                    [
+                        80,
+                        98,
+                        "PAN module domains",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 49,
+                "sent": "The closest structural relative is hepatocyte growth factor (HGF, PDB: 1GP9), which superposes with an RMSD of 2.3\u00a0\u00c5 for 58 aligned C\u03b1 (Figure\u00a01B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        35,
+                        59,
+                        "hepatocyte growth factor",
+                        "protein_type"
+                    ],
+                    [
+                        61,
+                        64,
+                        "HGF",
+                        "protein_type"
+                    ],
+                    [
+                        84,
+                        94,
+                        "superposes",
+                        "experimental_method"
+                    ],
+                    [
+                        103,
+                        107,
+                        "RMSD",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 50,
+                "sent": "The CUB domain bears two glycosylation sites.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "CUB",
+                        "structure_element"
+                    ],
+                    [
+                        25,
+                        44,
+                        "glycosylation sites",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 51,
+                "sent": "Although present, the quality of the electron density around N217 did not allow modeling of the sugar moiety.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        37,
+                        53,
+                        "electron density",
+                        "evidence"
+                    ],
+                    [
+                        61,
+                        65,
+                        "N217",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 52,
+                "sent": "In crystal form I, a calcium ion is present at the canonical position coordinated by the carboxylates of D263, D266 (bidentate), and D306, as well as the carbonyl of N309 and a water molecule.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        3,
+                        17,
+                        "crystal form I",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        28,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        70,
+                        84,
+                        "coordinated by",
+                        "bond_interaction"
+                    ],
+                    [
+                        105,
+                        109,
+                        "D263",
+                        "residue_name_number"
+                    ],
+                    [
+                        111,
+                        115,
+                        "D266",
+                        "residue_name_number"
+                    ],
+                    [
+                        133,
+                        137,
+                        "D306",
+                        "residue_name_number"
+                    ],
+                    [
+                        166,
+                        170,
+                        "N309",
+                        "residue_name_number"
+                    ],
+                    [
+                        177,
+                        182,
+                        "water",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 53,
+                "sent": "The coordination sphere deviates significantly from perfectly octahedral (not shown).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        23,
+                        "coordination sphere",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 54,
+                "sent": "This might result in the site having a low affinity and may explain why calcium is not present in the two low-resolution crystal forms.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        72,
+                        79,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        121,
+                        134,
+                        "crystal forms",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 55,
+                "sent": "Loss of calcium has led to loop rearrangements and partial disorder in these crystal forms.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "Loss of",
+                        "protein_state"
+                    ],
+                    [
+                        8,
+                        15,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        27,
+                        31,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        77,
+                        90,
+                        "crystal forms",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 56,
+                "sent": "The closest structural relative is the CUB_C domain of Tsg-6 (PDB: 2WNO), which superposes with KRMCUB with an RMSD of 1.6\u00a0\u00c5 for 104 C\u03b1 (Figure\u00a01B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        39,
+                        44,
+                        "CUB_C",
+                        "structure_element"
+                    ],
+                    [
+                        55,
+                        60,
+                        "Tsg-6",
+                        "protein"
+                    ],
+                    [
+                        80,
+                        90,
+                        "superposes",
+                        "experimental_method"
+                    ],
+                    [
+                        96,
+                        99,
+                        "KRM",
+                        "protein"
+                    ],
+                    [
+                        99,
+                        102,
+                        "CUB",
+                        "structure_element"
+                    ],
+                    [
+                        111,
+                        115,
+                        "RMSD",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 57,
+                "sent": "A superposition of the three KRM1 structures reveals no major structural differences (Figure\u00a01C) as anticipated from the plethora of interactions between the three domains.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        2,
+                        15,
+                        "superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        29,
+                        33,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        34,
+                        44,
+                        "structures",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 58,
+                "sent": "Minor differences are caused by the collapse of the Ca2+ binding site in crystal forms II and III and loop flexibility in the KR domain.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        52,
+                        69,
+                        "Ca2+ binding site",
+                        "site"
+                    ],
+                    [
+                        73,
+                        97,
+                        "crystal forms II and III",
+                        "evidence"
+                    ],
+                    [
+                        102,
+                        106,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        126,
+                        128,
+                        "KR",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 59,
+                "sent": "The F207S mutation recently found to cause ectodermal dysplasia in Palestinian families maps to the hydrophobic core of the protein at the interface of the three subdomains (Figure\u00a01A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        9,
+                        "F207S",
+                        "mutant"
+                    ],
+                    [
+                        100,
+                        116,
+                        "hydrophobic core",
+                        "site"
+                    ],
+                    [
+                        139,
+                        148,
+                        "interface",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 60,
+                "sent": "Such a mutation is bound to severely destabilize the protein structure of KRM1, leading to disturbance of its Wnt antagonistic, Wnt stimulatory, and Wnt independent activity.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        27,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        74,
+                        78,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        110,
+                        113,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        128,
+                        131,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        149,
+                        152,
+                        "Wnt",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 61,
+                "sent": "Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        18,
+                        "Co-crystallization",
+                        "experimental_method"
+                    ],
+                    [
+                        22,
+                        26,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        26,
+                        32,
+                        "PE3PE4",
+                        "structure_element"
+                    ],
+                    [
+                        38,
+                        42,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        42,
+                        46,
+                        "CRD2",
+                        "structure_element"
+                    ],
+                    [
+                        52,
+                        56,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        56,
+                        59,
+                        "PE1",
+                        "structure_element"
+                    ],
+                    [
+                        90,
+                        94,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        148,
+                        151,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        166,
+                        169,
+                        "Dkk",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 62,
+                "sent": "One face of the rather flat DKK1CRD2 fragment binds to the third \u03b2 propeller of LRP6.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        23,
+                        27,
+                        "flat",
+                        "protein_state"
+                    ],
+                    [
+                        28,
+                        32,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        32,
+                        36,
+                        "CRD2",
+                        "structure_element"
+                    ],
+                    [
+                        46,
+                        54,
+                        "binds to",
+                        "protein_state"
+                    ],
+                    [
+                        59,
+                        76,
+                        "third \u03b2 propeller",
+                        "structure_element"
+                    ],
+                    [
+                        80,
+                        84,
+                        "LRP6",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 63,
+                "sent": "Mutational analyses further implied that the LRP6PE3-averted face of DKK1CRD2 bears the Krm binding site, hence suggesting how Dkk can recruit both receptors into a ternary complex.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        19,
+                        "Mutational analyses",
+                        "experimental_method"
+                    ],
+                    [
+                        45,
+                        49,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        49,
+                        52,
+                        "PE3",
+                        "structure_element"
+                    ],
+                    [
+                        69,
+                        73,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        73,
+                        77,
+                        "CRD2",
+                        "structure_element"
+                    ],
+                    [
+                        88,
+                        104,
+                        "Krm binding site",
+                        "site"
+                    ],
+                    [
+                        127,
+                        130,
+                        "Dkk",
+                        "protein_type"
+                    ],
+                    [
+                        148,
+                        157,
+                        "receptors",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 64,
+                "sent": "To obtain direct insight into ternary complex formation by Lrp5/6, Dkk, and Krm, we subjected an LRP6PE3PE4-DKK1fl-KRM1ECD complex to crystallization trials.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        59,
+                        65,
+                        "Lrp5/6",
+                        "protein_type"
+                    ],
+                    [
+                        67,
+                        70,
+                        "Dkk",
+                        "protein_type"
+                    ],
+                    [
+                        76,
+                        79,
+                        "Krm",
+                        "protein_type"
+                    ],
+                    [
+                        97,
+                        122,
+                        "LRP6PE3PE4-DKK1fl-KRM1ECD",
+                        "complex_assembly"
+                    ],
+                    [
+                        134,
+                        156,
+                        "crystallization trials",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 65,
+                "sent": "Diffraction data collected from the resulting crystals were highly anisotropic with diffraction extending in the best directions to 3.5\u00a0\u00c5 and 3.7\u00a0\u00c5 but only to 6.4\u00a0\u00c5 in the third direction.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        16,
+                        "Diffraction data",
+                        "evidence"
+                    ],
+                    [
+                        46,
+                        54,
+                        "crystals",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 66,
+                "sent": "Despite the lack of high-resolution diffraction, the general architecture of the ternary complex is revealed (Figure\u00a02A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        36,
+                        47,
+                        "diffraction",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 67,
+                "sent": "DKK1CRD2 binds to the top face of the LRP6 PE3 \u03b2 propeller as described earlier for the binary complex.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        4,
+                        8,
+                        "CRD2",
+                        "structure_element"
+                    ],
+                    [
+                        9,
+                        17,
+                        "binds to",
+                        "protein_state"
+                    ],
+                    [
+                        38,
+                        42,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        43,
+                        46,
+                        "PE3",
+                        "structure_element"
+                    ],
+                    [
+                        47,
+                        58,
+                        "\u03b2 propeller",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 68,
+                "sent": "KRM1ECD does indeed bind on the opposite side of DKK1CRD2 with only its KR and WSC domains engaged in binding (Figure\u00a02A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        4,
+                        7,
+                        "ECD",
+                        "structure_element"
+                    ],
+                    [
+                        20,
+                        27,
+                        "bind on",
+                        "protein_state"
+                    ],
+                    [
+                        49,
+                        53,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        53,
+                        57,
+                        "CRD2",
+                        "structure_element"
+                    ],
+                    [
+                        72,
+                        74,
+                        "KR",
+                        "structure_element"
+                    ],
+                    [
+                        79,
+                        82,
+                        "WSC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 69,
+                "sent": "Although present in the complex subjected to crystallization, we observe no density that could correspond to CRD1 or the domain linker\u00a0(L).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        45,
+                        60,
+                        "crystallization",
+                        "experimental_method"
+                    ],
+                    [
+                        76,
+                        83,
+                        "density",
+                        "evidence"
+                    ],
+                    [
+                        109,
+                        113,
+                        "CRD1",
+                        "structure_element"
+                    ],
+                    [
+                        121,
+                        134,
+                        "domain linker",
+                        "structure_element"
+                    ],
+                    [
+                        136,
+                        137,
+                        "L",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 70,
+                "sent": "We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure\u00a02B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        20,
+                        24,
+                        "CRD2",
+                        "structure_element"
+                    ],
+                    [
+                        28,
+                        32,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        75,
+                        79,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        84,
+                        109,
+                        "surface plasmon resonance",
+                        "experimental_method"
+                    ],
+                    [
+                        111,
+                        114,
+                        "SPR",
+                        "experimental_method"
+                    ],
+                    [
+                        144,
+                        152,
+                        "affinity",
+                        "evidence"
+                    ],
+                    [
+                        161,
+                        172,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        173,
+                        177,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        194,
+                        198,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        198,
+                        201,
+                        "ECD",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 71,
+                "sent": "A SUMO fusion of DKK1L-CRD2 displayed a similar (slightly higher) affinity.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        2,
+                        13,
+                        "SUMO fusion",
+                        "experimental_method"
+                    ],
+                    [
+                        17,
+                        27,
+                        "DKK1L-CRD2",
+                        "structure_element"
+                    ],
+                    [
+                        66,
+                        74,
+                        "affinity",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 72,
+                "sent": "In contrast, a SUMO fusion of DKK1CRD1-L did not display binding for concentrations tested up to 325\u00a0\u03bcM (Figure\u00a02B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        15,
+                        26,
+                        "SUMO fusion",
+                        "experimental_method"
+                    ],
+                    [
+                        30,
+                        40,
+                        "DKK1CRD1-L",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 73,
+                "sent": "Overall, the DKK1-KRM1 interface is characterized by a large number of polar interactions but only few hydrophobic contacts (Figure\u00a02C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        32,
+                        "DKK1-KRM1 interface",
+                        "site"
+                    ],
+                    [
+                        71,
+                        89,
+                        "polar interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        103,
+                        123,
+                        "hydrophobic contacts",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 74,
+                "sent": "The crystal structure gives an explanation for DKK1 loss-of-binding mutations identified previously: R191 of DKK1 forms a double salt bridge to D125 and E162 of KRM1 (Figure\u00a02C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        21,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        47,
+                        51,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        101,
+                        105,
+                        "R191",
+                        "residue_name_number"
+                    ],
+                    [
+                        109,
+                        113,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        129,
+                        140,
+                        "salt bridge",
+                        "bond_interaction"
+                    ],
+                    [
+                        144,
+                        148,
+                        "D125",
+                        "residue_name_number"
+                    ],
+                    [
+                        153,
+                        157,
+                        "E162",
+                        "residue_name_number"
+                    ],
+                    [
+                        161,
+                        165,
+                        "KRM1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 75,
+                "sent": "A charge reversal as in the mouse Dkk1 (mDkk1) R197E variant would severely disrupt the binding.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        2,
+                        17,
+                        "charge reversal",
+                        "experimental_method"
+                    ],
+                    [
+                        28,
+                        33,
+                        "mouse",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        34,
+                        38,
+                        "Dkk1",
+                        "protein"
+                    ],
+                    [
+                        40,
+                        45,
+                        "mDkk1",
+                        "protein"
+                    ],
+                    [
+                        47,
+                        52,
+                        "R197E",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 76,
+                "sent": "Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        15,
+                        19,
+                        "K226",
+                        "residue_name_number"
+                    ],
+                    [
+                        34,
+                        38,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        64,
+                        82,
+                        "hydrophobic pocket",
+                        "site"
+                    ],
+                    [
+                        101,
+                        105,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        116,
+                        120,
+                        "Y108",
+                        "residue_name_number"
+                    ],
+                    [
+                        122,
+                        125,
+                        "W94",
+                        "residue_name_number"
+                    ],
+                    [
+                        131,
+                        135,
+                        "W106",
+                        "residue_name_number"
+                    ],
+                    [
+                        143,
+                        155,
+                        "salt bridges",
+                        "bond_interaction"
+                    ],
+                    [
+                        180,
+                        184,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        185,
+                        188,
+                        "D88",
+                        "residue_name_number"
+                    ],
+                    [
+                        193,
+                        196,
+                        "D90",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 77,
+                "sent": "Again, a charge reversal as shown before for mDkk1 K232E would be incompatible with binding.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        24,
+                        "charge reversal",
+                        "experimental_method"
+                    ],
+                    [
+                        45,
+                        50,
+                        "mDkk1",
+                        "protein"
+                    ],
+                    [
+                        51,
+                        56,
+                        "K232E",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 78,
+                "sent": "The side chain of DKK1 S192 was also predicted to be involved in Krm binding.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        22,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        23,
+                        27,
+                        "S192",
+                        "residue_name_number"
+                    ],
+                    [
+                        65,
+                        68,
+                        "Krm",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 79,
+                "sent": "Indeed, we found (Figure\u00a02C) that the side chain of D201 of KRM1 forms two hydrogen bonds to the side-chain hydroxyl and the backbone amide of S192 (mouse, S198).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        52,
+                        56,
+                        "D201",
+                        "residue_name_number"
+                    ],
+                    [
+                        60,
+                        64,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        75,
+                        89,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        143,
+                        147,
+                        "S192",
+                        "residue_name_number"
+                    ],
+                    [
+                        149,
+                        154,
+                        "mouse",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        156,
+                        160,
+                        "S198",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 80,
+                "sent": "Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        29,
+                        "polar interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        53,
+                        57,
+                        "N140",
+                        "residue_name_number"
+                    ],
+                    [
+                        59,
+                        63,
+                        "S163",
+                        "residue_name_number"
+                    ],
+                    [
+                        69,
+                        73,
+                        "Y165",
+                        "residue_name_number"
+                    ],
+                    [
+                        89,
+                        93,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        98,
+                        102,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        125,
+                        129,
+                        "W206",
+                        "residue_name_number"
+                    ],
+                    [
+                        131,
+                        135,
+                        "L190",
+                        "residue_name_number"
+                    ],
+                    [
+                        141,
+                        145,
+                        "C189",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 81,
+                "sent": "The carbonyl of DKK1 R224 is hydrogen bonded to Y105 and W106 of KRM1.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        16,
+                        20,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        21,
+                        25,
+                        "R224",
+                        "residue_name_number"
+                    ],
+                    [
+                        29,
+                        44,
+                        "hydrogen bonded",
+                        "bond_interaction"
+                    ],
+                    [
+                        48,
+                        52,
+                        "Y105",
+                        "residue_name_number"
+                    ],
+                    [
+                        57,
+                        61,
+                        "W106",
+                        "residue_name_number"
+                    ],
+                    [
+                        65,
+                        69,
+                        "KRM1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 82,
+                "sent": "We suspect that the Dkk charge reversal mutations performed in the murine background and shown to\u00a0diminish Krm binding K211E and R203E (mouse K217E and\u00a0R209E) do so likely indirectly by disruption\u00a0of the Dkk fold.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        20,
+                        23,
+                        "Dkk",
+                        "protein_type"
+                    ],
+                    [
+                        24,
+                        49,
+                        "charge reversal mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        67,
+                        73,
+                        "murine",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        107,
+                        110,
+                        "Krm",
+                        "protein_type"
+                    ],
+                    [
+                        119,
+                        124,
+                        "K211E",
+                        "mutant"
+                    ],
+                    [
+                        129,
+                        134,
+                        "R203E",
+                        "mutant"
+                    ],
+                    [
+                        136,
+                        141,
+                        "mouse",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        142,
+                        147,
+                        "K217E",
+                        "mutant"
+                    ],
+                    [
+                        152,
+                        157,
+                        "R209E",
+                        "mutant"
+                    ],
+                    [
+                        204,
+                        207,
+                        "Dkk",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 83,
+                "sent": "We further validated the DKK1 binding site\u00a0on\u00a0KRM1 by introducing glycosylation sites at the KR (90DVS92\u2192NVS) and WSC (189VCF191\u2192NCS) domains pointing toward DKK (Figures 2A and 2D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        42,
+                        "DKK1 binding site",
+                        "site"
+                    ],
+                    [
+                        46,
+                        50,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        54,
+                        65,
+                        "introducing",
+                        "experimental_method"
+                    ],
+                    [
+                        66,
+                        85,
+                        "glycosylation sites",
+                        "site"
+                    ],
+                    [
+                        93,
+                        95,
+                        "KR",
+                        "structure_element"
+                    ],
+                    [
+                        97,
+                        108,
+                        "90DVS92\u2192NVS",
+                        "mutant"
+                    ],
+                    [
+                        114,
+                        117,
+                        "WSC",
+                        "structure_element"
+                    ],
+                    [
+                        119,
+                        132,
+                        "189VCF191\u2192NCS",
+                        "mutant"
+                    ],
+                    [
+                        158,
+                        161,
+                        "DKK",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 84,
+                "sent": "Introduction of N-linked glycans in protein-protein-binding sites is an established way of disrupting protein-binding interfaces.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        16,
+                        32,
+                        "N-linked glycans",
+                        "ptm"
+                    ],
+                    [
+                        36,
+                        65,
+                        "protein-protein-binding sites",
+                        "site"
+                    ],
+                    [
+                        102,
+                        128,
+                        "protein-binding interfaces",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 85,
+                "sent": "Both ectodomain mutants were secreted comparably with the wild-type, indicating correct folding, but failed to achieve any detectable binding in SPR using full-length DKK1 as analyte.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        15,
+                        "ectodomain",
+                        "structure_element"
+                    ],
+                    [
+                        16,
+                        23,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        58,
+                        67,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        145,
+                        148,
+                        "SPR",
+                        "experimental_method"
+                    ],
+                    [
+                        155,
+                        166,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        167,
+                        171,
+                        "DKK1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 86,
+                "sent": "In contrast, a mutant carrying an additional N-glycan outside the interface at the CUB domain (309NQA311\u2192NQS), was wild-type-like in DKK1 binding (Figure\u00a02D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        15,
+                        21,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        45,
+                        53,
+                        "N-glycan",
+                        "ptm"
+                    ],
+                    [
+                        66,
+                        75,
+                        "interface",
+                        "site"
+                    ],
+                    [
+                        83,
+                        86,
+                        "CUB",
+                        "structure_element"
+                    ],
+                    [
+                        95,
+                        108,
+                        "309NQA311\u2192NQS",
+                        "mutant"
+                    ],
+                    [
+                        115,
+                        124,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        133,
+                        137,
+                        "DKK1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 87,
+                "sent": "Identification of a Direct LRP6-KRM1 Binding Site",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        27,
+                        49,
+                        "LRP6-KRM1 Binding Site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 88,
+                "sent": "The LRP6PE3PE4-DKK1CRD2-KRM1ECD complex structure reveals no direct interactions between KRM1 and LRP6.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        31,
+                        "LRP6PE3PE4-DKK1CRD2-KRM1ECD",
+                        "complex_assembly"
+                    ],
+                    [
+                        40,
+                        49,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        89,
+                        93,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        98,
+                        102,
+                        "LRP6",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 89,
+                "sent": "We constructed in\u00a0silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        35,
+                        47,
+                        "complex with",
+                        "protein_state"
+                    ],
+                    [
+                        59,
+                        70,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        71,
+                        75,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        76,
+                        86,
+                        "ectodomain",
+                        "structure_element"
+                    ],
+                    [
+                        88,
+                        100,
+                        "PE1PE2PE3PE4",
+                        "structure_element"
+                    ],
+                    [
+                        101,
+                        111,
+                        "horse shoe",
+                        "structure_element"
+                    ],
+                    [
+                        155,
+                        174,
+                        "electron microscopy",
+                        "experimental_method"
+                    ],
+                    [
+                        176,
+                        178,
+                        "EM",
+                        "experimental_method"
+                    ],
+                    [
+                        183,
+                        211,
+                        "small-angle X-ray scattering",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 90,
+                "sent": "An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure\u00a03A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        19,
+                        "PE3PE4",
+                        "structure_element"
+                    ],
+                    [
+                        33,
+                        45,
+                        "superimposed",
+                        "experimental_method"
+                    ],
+                    [
+                        50,
+                        53,
+                        "PE4",
+                        "structure_element"
+                    ],
+                    [
+                        59,
+                        62,
+                        "PE3",
+                        "structure_element"
+                    ],
+                    [
+                        70,
+                        87,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        97,
+                        101,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        101,
+                        107,
+                        "PE1PE2",
+                        "structure_element"
+                    ],
+                    [
+                        108,
+                        117,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        122,
+                        134,
+                        "superimposed",
+                        "experimental_method"
+                    ],
+                    [
+                        139,
+                        142,
+                        "PE2",
+                        "structure_element"
+                    ],
+                    [
+                        148,
+                        151,
+                        "PE3",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 91,
+                "sent": "For this crude approximation of a true ternary complex, we noted very close proximity between the Ca2+-binding region of KRM1 and the top face of the PE2 \u03b2 propeller of LRP6.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        98,
+                        117,
+                        "Ca2+-binding region",
+                        "site"
+                    ],
+                    [
+                        121,
+                        125,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        150,
+                        153,
+                        "PE2",
+                        "structure_element"
+                    ],
+                    [
+                        154,
+                        165,
+                        "\u03b2 propeller",
+                        "structure_element"
+                    ],
+                    [
+                        169,
+                        173,
+                        "LRP6",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 92,
+                "sent": "The solvent-exposed residues R307, I308, and N309 of the central Ca2+-binding \u03b2 connection loop of KRM1 would be almost ideally positioned for binding to this face, which is commonly used as a binding site on \u03b2 propellers.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        19,
+                        "solvent-exposed",
+                        "protein_state"
+                    ],
+                    [
+                        29,
+                        33,
+                        "R307",
+                        "residue_name_number"
+                    ],
+                    [
+                        35,
+                        39,
+                        "I308",
+                        "residue_name_number"
+                    ],
+                    [
+                        45,
+                        49,
+                        "N309",
+                        "residue_name_number"
+                    ],
+                    [
+                        65,
+                        95,
+                        "Ca2+-binding \u03b2 connection loop",
+                        "structure_element"
+                    ],
+                    [
+                        99,
+                        103,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        193,
+                        205,
+                        "binding site",
+                        "site"
+                    ],
+                    [
+                        209,
+                        221,
+                        "\u03b2 propellers",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 93,
+                "sent": "Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure\u00a03B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        20,
+                        28,
+                        "arginine",
+                        "residue_name"
+                    ],
+                    [
+                        29,
+                        35,
+                        "lysine",
+                        "residue_name"
+                    ],
+                    [
+                        37,
+                        47,
+                        "isoleucine",
+                        "residue_name"
+                    ],
+                    [
+                        53,
+                        63,
+                        "asparagine",
+                        "residue_name"
+                    ],
+                    [
+                        84,
+                        97,
+                        "N-X-I-(G)-R/K",
+                        "structure_element"
+                    ],
+                    [
+                        120,
+                        124,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        129,
+                        133,
+                        "SOST",
+                        "protein"
+                    ],
+                    [
+                        145,
+                        149,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        161,
+                        172,
+                        "propeller 1",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 94,
+                "sent": "To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        31,
+                        35,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        35,
+                        38,
+                        "CUB",
+                        "structure_element"
+                    ],
+                    [
+                        39,
+                        47,
+                        "binds to",
+                        "protein_state"
+                    ],
+                    [
+                        48,
+                        52,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        52,
+                        55,
+                        "PE2",
+                        "structure_element"
+                    ],
+                    [
+                        65,
+                        68,
+                        "SPR",
+                        "experimental_method"
+                    ],
+                    [
+                        97,
+                        106,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        115,
+                        130,
+                        "GlycoCUB mutant",
+                        "protein_state"
+                    ],
+                    [
+                        134,
+                        138,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        138,
+                        141,
+                        "ECD",
+                        "structure_element"
+                    ],
+                    [
+                        154,
+                        174,
+                        "N-glycosylation site",
+                        "site"
+                    ],
+                    [
+                        178,
+                        182,
+                        "N309",
+                        "residue_name_number"
+                    ],
+                    [
+                        200,
+                        204,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        204,
+                        210,
+                        "PE1PE2",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 95,
+                "sent": "Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2 (Figure\u00a03C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        39,
+                        "absence of",
+                        "protein_state"
+                    ],
+                    [
+                        40,
+                        43,
+                        "Dkk",
+                        "protein_type"
+                    ],
+                    [
+                        45,
+                        49,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        49,
+                        52,
+                        "ECD",
+                        "structure_element"
+                    ],
+                    [
+                        53,
+                        58,
+                        "bound",
+                        "protein_state"
+                    ],
+                    [
+                        86,
+                        88,
+                        "to",
+                        "protein_state"
+                    ],
+                    [
+                        89,
+                        93,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        93,
+                        99,
+                        "PE1PE2",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 96,
+                "sent": "In contrast, no saturable binding was observed between KRM1 and LRP6PE3PE4.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        55,
+                        59,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        64,
+                        68,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        68,
+                        74,
+                        "PE3PE4",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 97,
+                "sent": "Introduction of an N-glycosylation site at N309 in KRM1ECD abolished LRP6PE1PE2 binding (Figure\u00a03C), while binding to DKK1 was unaffected (Figure\u00a02D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        15,
+                        "Introduction of",
+                        "experimental_method"
+                    ],
+                    [
+                        19,
+                        39,
+                        "N-glycosylation site",
+                        "site"
+                    ],
+                    [
+                        43,
+                        47,
+                        "N309",
+                        "residue_name_number"
+                    ],
+                    [
+                        51,
+                        55,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        55,
+                        58,
+                        "ECD",
+                        "structure_element"
+                    ],
+                    [
+                        69,
+                        73,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        73,
+                        79,
+                        "PE1PE2",
+                        "structure_element"
+                    ],
+                    [
+                        118,
+                        122,
+                        "DKK1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 98,
+                "sent": "We conclude that the predicted binding site between KRM1CUB and LRP6PE2 is a strong candidate for mediating the direct Lrp6-Krm interaction, which is thought to increase Wnt responsiveness by stabilizing Lrp6 at the cell surface.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        31,
+                        43,
+                        "binding site",
+                        "site"
+                    ],
+                    [
+                        52,
+                        56,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        56,
+                        59,
+                        "CUB",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        68,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        68,
+                        71,
+                        "PE2",
+                        "structure_element"
+                    ],
+                    [
+                        119,
+                        127,
+                        "Lrp6-Krm",
+                        "complex_assembly"
+                    ],
+                    [
+                        170,
+                        173,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        204,
+                        208,
+                        "Lrp6",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 99,
+                "sent": "Further experiments are required to pinpoint the exact binding site.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        55,
+                        67,
+                        "binding site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 100,
+                "sent": "Although LRP6PE1 appears somewhat out of reach in the modeled ternary complex, it cannot be excluded as the Krm binding site in the ternary complex and LRP6-Krm binary complex.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        13,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        13,
+                        16,
+                        "PE1",
+                        "structure_element"
+                    ],
+                    [
+                        108,
+                        124,
+                        "Krm binding site",
+                        "site"
+                    ],
+                    [
+                        152,
+                        160,
+                        "LRP6-Krm",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 101,
+                "sent": "The presence of DKK may govern which propeller (PE1 versus PE2) of LRP6 is available for Krm binding.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        15,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        16,
+                        19,
+                        "DKK",
+                        "protein"
+                    ],
+                    [
+                        37,
+                        46,
+                        "propeller",
+                        "structure_element"
+                    ],
+                    [
+                        48,
+                        51,
+                        "PE1",
+                        "structure_element"
+                    ],
+                    [
+                        59,
+                        62,
+                        "PE2",
+                        "structure_element"
+                    ],
+                    [
+                        67,
+                        71,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        89,
+                        92,
+                        "Krm",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 102,
+                "sent": "Apparent binding across the proposed KRM1CUB-LRP6PE2 interface is expected to be higher once Krm is also cross-linked to LRP6PE3 via DKK1CRD2 (Figure\u00a03D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        37,
+                        62,
+                        "KRM1CUB-LRP6PE2 interface",
+                        "site"
+                    ],
+                    [
+                        93,
+                        96,
+                        "Krm",
+                        "protein_type"
+                    ],
+                    [
+                        121,
+                        125,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        125,
+                        128,
+                        "PE3",
+                        "structure_element"
+                    ],
+                    [
+                        133,
+                        137,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        137,
+                        141,
+                        "CRD2",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 103,
+                "sent": "Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        15,
+                        32,
+                        "negative-stain EM",
+                        "experimental_method"
+                    ],
+                    [
+                        37,
+                        65,
+                        "small-angle X-ray scattering",
+                        "experimental_method"
+                    ],
+                    [
+                        77,
+                        81,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        81,
+                        93,
+                        "PE1PE2PE3PE4",
+                        "structure_element"
+                    ],
+                    [
+                        95,
+                        107,
+                        "in isolation",
+                        "protein_state"
+                    ],
+                    [
+                        112,
+                        127,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        128,
+                        132,
+                        "Dkk1",
+                        "protein_type"
+                    ],
+                    [
+                        139,
+                        156,
+                        "negative-stain EM",
+                        "experimental_method"
+                    ],
+                    [
+                        160,
+                        171,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        172,
+                        176,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        177,
+                        187,
+                        "ectodomain",
+                        "structure_element"
+                    ],
+                    [
+                        204,
+                        210,
+                        "curved",
+                        "protein_state"
+                    ],
+                    [
+                        212,
+                        225,
+                        "platform-like",
+                        "protein_state"
+                    ],
+                    [
+                        279,
+                        282,
+                        "PE2",
+                        "structure_element"
+                    ],
+                    [
+                        287,
+                        290,
+                        "PE3",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 104,
+                "sent": "It is therefore possible that the interplay of Krm and Dkk binding can promote changes in LRP6 ectodomain conformation with functional consequences; however, such ideas await investigation.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        47,
+                        50,
+                        "Krm",
+                        "protein_type"
+                    ],
+                    [
+                        55,
+                        58,
+                        "Dkk",
+                        "protein_type"
+                    ],
+                    [
+                        90,
+                        94,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        95,
+                        105,
+                        "ectodomain",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 105,
+                "sent": "Taken together, the structural and biophysical studies we report here extend our mechanistic understanding of Wnt signal regulation.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        20,
+                        54,
+                        "structural and biophysical studies",
+                        "experimental_method"
+                    ],
+                    [
+                        110,
+                        113,
+                        "Wnt",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 106,
+                "sent": "We describe the ectodomain structure of the dual Wnt regulator Krm1, providing an explanation for the detrimental effect on health and development of a homozygous KRM1 mutation.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        16,
+                        26,
+                        "ectodomain",
+                        "structure_element"
+                    ],
+                    [
+                        27,
+                        36,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        49,
+                        52,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        63,
+                        67,
+                        "Krm1",
+                        "protein_type"
+                    ],
+                    [
+                        163,
+                        167,
+                        "KRM1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 107,
+                "sent": "We also reveal the interaction mode of Krm-Dkk and the architecture of the ternary complex formed by Lrp5/6, Dkk, and Krm.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        39,
+                        46,
+                        "Krm-Dkk",
+                        "complex_assembly"
+                    ],
+                    [
+                        101,
+                        107,
+                        "Lrp5/6",
+                        "protein_type"
+                    ],
+                    [
+                        109,
+                        112,
+                        "Dkk",
+                        "protein_type"
+                    ],
+                    [
+                        118,
+                        121,
+                        "Krm",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 108,
+                "sent": "Furthermore, the ternary crystal structure has guided in\u00a0silico and biophysical analyses to suggest a direct LRP6-KRM1 interaction site.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        42,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        54,
+                        88,
+                        "in\u00a0silico and biophysical analyses",
+                        "experimental_method"
+                    ],
+                    [
+                        109,
+                        135,
+                        "LRP6-KRM1 interaction site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 109,
+                "sent": "Our findings provide a solid foundation for additional studies to probe how ternary complex formation triggers internalization, whereas Krm binding in the absence of Dkk stabilizes the Wnt co-receptor at the cell surface.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        136,
+                        139,
+                        "Krm",
+                        "protein_type"
+                    ],
+                    [
+                        155,
+                        165,
+                        "absence of",
+                        "protein_state"
+                    ],
+                    [
+                        166,
+                        169,
+                        "Dkk",
+                        "protein_type"
+                    ],
+                    [
+                        185,
+                        188,
+                        "Wnt",
+                        "protein_type"
+                    ],
+                    [
+                        189,
+                        200,
+                        "co-receptor",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 110,
+                "sent": "Structure of Unliganded KRM1ECD",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Structure",
+                        "evidence"
+                    ],
+                    [
+                        13,
+                        23,
+                        "Unliganded",
+                        "protein_state"
+                    ],
+                    [
+                        24,
+                        28,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        28,
+                        31,
+                        "ECD",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 111,
+                "sent": "(A) The KRM1ECD fold (crystal form I) colored blue to red from the N to C terminus.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        8,
+                        12,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        12,
+                        15,
+                        "ECD",
+                        "structure_element"
+                    ],
+                    [
+                        22,
+                        36,
+                        "crystal form I",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 112,
+                "sent": "Cysteines as ball and sticks, glycosylation sites as sticks.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Cysteines",
+                        "residue_name"
+                    ],
+                    [
+                        30,
+                        49,
+                        "glycosylation sites",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 113,
+                "sent": "The bound calcium is shown as a gray sphere.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        10,
+                        17,
+                        "calcium",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 114,
+                "sent": "The site of the\u00a0F207S mutation associated with ectodermal dysplasia in humans is shown as mesh.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        16,
+                        21,
+                        "F207S",
+                        "mutant"
+                    ],
+                    [
+                        71,
+                        77,
+                        "humans",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 115,
+                "sent": "(B) Superposition of the three KRM1ECD subdomains (solid) with their next structurally characterized homologs (half transparent).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        17,
+                        "Superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        31,
+                        35,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        35,
+                        38,
+                        "ECD",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 116,
+                "sent": "(C) Superposition of KRM1ECD from the three crystal forms.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        17,
+                        "Superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        21,
+                        25,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        25,
+                        28,
+                        "ECD",
+                        "structure_element"
+                    ],
+                    [
+                        44,
+                        57,
+                        "crystal forms",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 117,
+                "sent": "Alignment scores for each pairing are indicated on the dashed triangle.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        16,
+                        "Alignment scores",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 118,
+                "sent": "(A) The structure of the ternary LRP6PE3PE4-DKK1CRD2-KRM1ECD complex.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        8,
+                        17,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        33,
+                        60,
+                        "LRP6PE3PE4-DKK1CRD2-KRM1ECD",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 119,
+                "sent": "DKK1 (orange) is sandwiched between the PE3 module of LRP6 (blue) and the KR-WSC domain pair of KRM1 (green).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        40,
+                        43,
+                        "PE3",
+                        "structure_element"
+                    ],
+                    [
+                        54,
+                        58,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        74,
+                        80,
+                        "KR-WSC",
+                        "structure_element"
+                    ],
+                    [
+                        96,
+                        100,
+                        "KRM1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 120,
+                "sent": "Colored symbols indicate introduced N-glycan attachment sites (see D).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        36,
+                        61,
+                        "N-glycan attachment sites",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 121,
+                "sent": "(B) SPR data comparing binding of full-length DKK1 and SUMO fusions of DKK1 truncations for binding to immobilized wild-type KRM1ECD.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "SPR",
+                        "experimental_method"
+                    ],
+                    [
+                        34,
+                        45,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        46,
+                        50,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        55,
+                        67,
+                        "SUMO fusions",
+                        "experimental_method"
+                    ],
+                    [
+                        71,
+                        75,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        115,
+                        124,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        125,
+                        129,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        129,
+                        132,
+                        "ECD",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 122,
+                "sent": "(C) Close-up view of the DKK1CRD2-KRM1ECD interface.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        25,
+                        51,
+                        "DKK1CRD2-KRM1ECD interface",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 123,
+                "sent": "Residues involved in interface formation are shown as sticks; those mentioned in the text are labeled.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        21,
+                        30,
+                        "interface",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 124,
+                "sent": "Salt bridges are in pink and hydrogen bonds in black.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        12,
+                        "Salt bridges",
+                        "bond_interaction"
+                    ],
+                    [
+                        29,
+                        43,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 125,
+                "sent": "(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "SPR",
+                        "experimental_method"
+                    ],
+                    [
+                        8,
+                        20,
+                        "binding data",
+                        "evidence"
+                    ],
+                    [
+                        31,
+                        35,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        57,
+                        66,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        67,
+                        71,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        71,
+                        74,
+                        "ECD",
+                        "structure_element"
+                    ],
+                    [
+                        102,
+                        112,
+                        "engineered",
+                        "protein_state"
+                    ],
+                    [
+                        113,
+                        132,
+                        "glycosylation sites",
+                        "site"
+                    ],
+                    [
+                        140,
+                        142,
+                        "KR",
+                        "structure_element"
+                    ],
+                    [
+                        147,
+                        150,
+                        "WSC",
+                        "structure_element"
+                    ],
+                    [
+                        187,
+                        191,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        204,
+                        207,
+                        "CUB",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 126,
+                "sent": "LRP6-KRM1 Direct Interaction and Summary",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "LRP6-KRM1",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 127,
+                "sent": "(A) In a construction of a ternary complex with all four \u03b2 propellers of LRP6 intact, the CUB domain points via its Ca2+-binding region toward the top face of the second \u03b2 propeller.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        35,
+                        47,
+                        "complex with",
+                        "protein_state"
+                    ],
+                    [
+                        57,
+                        69,
+                        "\u03b2 propellers",
+                        "structure_element"
+                    ],
+                    [
+                        73,
+                        77,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        78,
+                        84,
+                        "intact",
+                        "protein_state"
+                    ],
+                    [
+                        90,
+                        93,
+                        "CUB",
+                        "structure_element"
+                    ],
+                    [
+                        116,
+                        135,
+                        "Ca2+-binding region",
+                        "site"
+                    ],
+                    [
+                        163,
+                        181,
+                        "second \u03b2 propeller",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 128,
+                "sent": "(B) Close-up view of the potential interaction site.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        35,
+                        51,
+                        "interaction site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 129,
+                "sent": "In addition, LRP6PE2 has been superimposed with DKK1 (yellow) and SOST (pink) peptide complexes of LRP6PE1.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        13,
+                        17,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        17,
+                        20,
+                        "PE2",
+                        "structure_element"
+                    ],
+                    [
+                        30,
+                        42,
+                        "superimposed",
+                        "experimental_method"
+                    ],
+                    [
+                        48,
+                        52,
+                        "DKK1",
+                        "protein"
+                    ],
+                    [
+                        66,
+                        70,
+                        "SOST",
+                        "protein"
+                    ],
+                    [
+                        99,
+                        103,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        103,
+                        106,
+                        "PE1",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 130,
+                "sent": "(C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        20,
+                        "SPR measurements",
+                        "experimental_method"
+                    ],
+                    [
+                        31,
+                        35,
+                        "LRP6",
+                        "protein"
+                    ],
+                    [
+                        35,
+                        41,
+                        "PE1PE2",
+                        "structure_element"
+                    ],
+                    [
+                        55,
+                        64,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        65,
+                        69,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        69,
+                        72,
+                        "ECD",
+                        "structure_element"
+                    ],
+                    [
+                        81,
+                        96,
+                        "GlycoCUB mutant",
+                        "protein_state"
+                    ],
+                    [
+                        108,
+                        116,
+                        "N-glycan",
+                        "ptm"
+                    ],
+                    [
+                        120,
+                        124,
+                        "N309",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 131,
+                "sent": "(D) Schematic representation of structural and biophysical findings and their implications for Wnt-dependent (left, middle) and independent (right) signaling.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        95,
+                        98,
+                        "Wnt",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 132,
+                "sent": "Conformational differences in the depictions of LRP6 are included purely for ease of representation.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        48,
+                        52,
+                        "LRP6",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 133,
+                "sent": "Diffraction and Refinement Statistics",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        37,
+                        "Diffraction and Refinement Statistics",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 134,
+                "sent": "\tKRM1ECD\tKRM1ECD\tKRM1ECD\tKRM1ECD\tLRP6PE3PE4-DKKCRD2-KRM1ECD\t \tCrystal form\tI\tI\tII\tIII\tI\t \tX-ray source\tDiamond i04\tDiamond i03\tDiamond i03\tDiamond i04\tDiamond i04\t \tWavelength (\u00c5)\t0.9793\t0.9700\t0.9700\t0.9795\t0.9795\t \tSpace group\tP3121\tP3121\tP43\tP41212\tC2221\t \tUnit cell a/\u03b1 (\u00c5/\u00b0)\t50.9/90\t50.5/90\t65.8/90\t67.8/90\t86.9/90\t \tb/\u03b2 (\u00c5/\u00b0)\t50.9/90\t50.5/90\t65.8/90\t67.8/90\t100.1/90\t \tc/\u03b3 (\u00c5/\u00b0)\t188.4/120\t187.4/120\t75.0/90\t198.2/90\t270.7/90\t \tWilson B factor (\u00c52)\t31\t41\t76\t77\tNA\t \tResolution range (\u00c5)\t47.10\u20131.90 (1.95\u20131.90)\t62.47\u20132.10 (2.16\u20132.10)\t75.00\u20132.80 (2.99\u20132.80)\t67.80\u20133.20 (3.42\u20133.20)\t67.68\u20133.50 (7.16\u20136.40, 3.92\u20133.50)\t \tUnique reflections\t23,300 (1,524)\t17,089 (1,428)\t7,964 (1,448)\t8,171 (1,343)\t8,070 (723, 645)\t \tAverage multiplicity\t9.1 (9.2)\t5.2 (5.3)\t3.7 (3.7)\t22.7 (12.6)\t3.8 (3.5, 4.4)\t \tCompleteness (%)\t99.8 (98.5)\t100 (100)\t99.8 (100)\t98.8 (93.4)\t51.6 (98.5, 14.1)\t \t<I/\u03c3I>\t11.4 (1.7)\t12.0 (1.7)\t14.9 (1.5)\t13.1 (1.9)\t4.6 (4.1, 2.2)\t \tRmerge (%)\t14.8 (158.3)\t9.3 (98.0)\t6.2 (98.9)\t29.8 (142.2)\t44.9 (40.5, 114.2)\t \tRpim (%)\t15.7 (55.3)\t10.3 (109.0)\t3.7 (53.8)\t6.3 (40.0)\t24.7 (23.9, 59.9)\t \t\t \tRefinement\t \t\t \tRwork (%)\t17.9\t18.4\t21.6\t20.2\t32.1\t \tRfree (%)\t22.7\t23.2\t30.7\t27.1\t35.5\t \t\t \tNo. of Non-Hydrogen Atoms\t \t\t \tProtein\t2,260\t2,301\t2,102\t2,305\t7,730\t \tN-glycans\t42\t42\t28\t28\t0\t \tWater\t79\t54\t0\t2\t0\t \tLigands\t6\t6\t2\t5\t0\t \t\t \tAverage B factor (\u00c52)\t \t\t \tProtein\t63\t65\t108\t84\t\u2013\t \tN-glycans\t35\t46\t102\t18\t\u2013\t \tWater\t68\t85\t\u2013\t75\t\u2013\t \tLigands\t36\t47\t91\t75\t66\t \t\t \tRMSD from Ideality\t \t\t \tBond lengths (\u00c5)\t0.020\t0.016\t0.019\t0.016\t0.004\t \tBond angles (\u00b0)\t2.050\t1.748\t1.952\t1.796\t0.770\t \t\t \tRamachandran Plot\t \t\t \tFavored (%)\t96.8\t95.5\t96.9\t94.9\t92.3\t \tAllowed (%)\t99.7\t100.0\t100.0\t99.7\t99.8\t \tNumber of outliers\t1\t0\t0\t1\t2\t \tPDB code\t5FWS\t5FWT\t5FWU\t5FWV\t5FWW\t \t",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        1,
+                        5,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        5,
+                        8,
+                        "ECD",
+                        "structure_element"
+                    ],
+                    [
+                        9,
+                        13,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        13,
+                        16,
+                        "ECD",
+                        "structure_element"
+                    ],
+                    [
+                        17,
+                        21,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        21,
+                        24,
+                        "ECD",
+                        "structure_element"
+                    ],
+                    [
+                        25,
+                        29,
+                        "KRM1",
+                        "protein"
+                    ],
+                    [
+                        29,
+                        32,
+                        "ECD",
+                        "structure_element"
+                    ],
+                    [
+                        33,
+                        59,
+                        "LRP6PE3PE4-DKKCRD2-KRM1ECD",
+                        "complex_assembly"
+                    ],
+                    [
+                        1295,
+                        1300,
+                        "Water",
+                        "chemical"
+                    ],
+                    [
+                        1417,
+                        1422,
+                        "Water",
+                        "chemical"
+                    ],
+                    [
+                        1466,
+                        1470,
+                        "RMSD",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 135,
+                "sent": "An additional shell given for the ternary complex corresponds to the last shell with near-complete diffraction data.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        99,
+                        115,
+                        "diffraction data",
+                        "evidence"
+                    ]
+                ]
+            }
+        ]
+    },
+    "PMC5063996": {
+        "annotations": [
+            {
+                "sid": 0,
+                "sent": "The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans*",
+                "section": "TITLE",
+                "ner": [
+                    [
+                        23,
+                        39,
+                        "Arabinoxylanases",
+                        "protein_type"
+                    ],
+                    [
+                        54,
+                        70,
+                        "Highly Decorated",
+                        "protein_state"
+                    ],
+                    [
+                        71,
+                        77,
+                        "Xylans",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 1,
+                "sent": "The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        29,
+                        34,
+                        "plant",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 2,
+                "sent": "Xylan, a major component of the plant cell wall, consists of a backbone of \u03b2-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "Xylan",
+                        "chemical"
+                    ],
+                    [
+                        32,
+                        37,
+                        "plant",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        75,
+                        87,
+                        "\u03b2-1,4-xylose",
+                        "chemical"
+                    ],
+                    [
+                        89,
+                        93,
+                        "Xylp",
+                        "chemical"
+                    ],
+                    [
+                        131,
+                        146,
+                        "arabinofuranose",
+                        "chemical"
+                    ],
+                    [
+                        148,
+                        152,
+                        "Araf",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 3,
+                "sent": "A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        8,
+                        28,
+                        "penta-modular enzyme",
+                        "protein_type"
+                    ],
+                    [
+                        30,
+                        37,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        83,
+                        96,
+                        "arabinoxylans",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 4,
+                "sent": "Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        19,
+                        36,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        44,
+                        59,
+                        "arabinoxylanase",
+                        "protein_type"
+                    ],
+                    [
+                        75,
+                        90,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        91,
+                        98,
+                        "ligands",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 5,
+                "sent": "The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        95,
+                        111,
+                        "catalytic domain",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 6,
+                "sent": "The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        15,
+                        56,
+                        "non-catalytic carbohydrate binding module",
+                        "structure_element"
+                    ],
+                    [
+                        86,
+                        103,
+                        "crystal structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 7,
+                "sent": "The structure of the enzyme in complex with Xylp-\u03b2-1,4-Xylp-\u03b2-1,4-Xylp-[\u03b1-1,3-Araf]-\u03b2-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (\u22122* subsite) that abuts onto the catalytic center.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        28,
+                        43,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        44,
+                        94,
+                        "Xylp-\u03b2-1,4-Xylp-\u03b2-1,4-Xylp-[\u03b1-1,3-Araf]-\u03b2-1,4-Xylp",
+                        "chemical"
+                    ],
+                    [
+                        111,
+                        115,
+                        "Araf",
+                        "chemical"
+                    ],
+                    [
+                        144,
+                        150,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        158,
+                        169,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        188,
+                        194,
+                        "pocket",
+                        "site"
+                    ],
+                    [
+                        196,
+                        207,
+                        "\u22122* subsite",
+                        "site"
+                    ],
+                    [
+                        229,
+                        245,
+                        "catalytic center",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 8,
+                "sent": "The \u22122* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        4,
+                        15,
+                        "\u22122* subsite",
+                        "site"
+                    ],
+                    [
+                        33,
+                        37,
+                        "Xylp",
+                        "chemical"
+                    ],
+                    [
+                        42,
+                        46,
+                        "Arap",
+                        "chemical"
+                    ],
+                    [
+                        86,
+                        92,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        97,
+                        106,
+                        "arabinose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 9,
+                "sent": "Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the \u22122* subsite, abrogates catalytic activity.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        20,
+                        "Alanine substitution",
+                        "experimental_method"
+                    ],
+                    [
+                        24,
+                        29,
+                        "Glu68",
+                        "residue_name_number"
+                    ],
+                    [
+                        31,
+                        36,
+                        "Tyr92",
+                        "residue_name_number"
+                    ],
+                    [
+                        41,
+                        47,
+                        "Asn139",
+                        "residue_name_number"
+                    ],
+                    [
+                        69,
+                        78,
+                        "arabinose",
+                        "chemical"
+                    ],
+                    [
+                        83,
+                        89,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        109,
+                        120,
+                        "\u22122* subsite",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 10,
+                "sent": "Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        14,
+                        25,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        31,
+                        36,
+                        "xylan",
+                        "chemical"
+                    ],
+                    [
+                        115,
+                        130,
+                        "solvent-exposed",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 11,
+                "sent": "This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        18,
+                        25,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        52,
+                        58,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        127,
+                        140,
+                        "endo-xylanase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 12,
+                "sent": "The plant cell wall is an important biological substrate.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        9,
+                        "plant",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 13,
+                "sent": "This complex composite structure is depolymerized by microorganisms that occupy important highly competitive ecological niches, whereas the process makes an important contribution to the carbon cycle.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        53,
+                        67,
+                        "microorganisms",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 14,
+                "sent": "Given that the plant cell wall is the most abundant source of renewable organic carbon on the planet, this macromolecular substrate has substantial industrial potential.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        15,
+                        20,
+                        "plant",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 15,
+                "sent": "An example of the chemical complexity of the plant cell wall is provided by xylan, which is the major hemicellulosic component.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        45,
+                        50,
+                        "plant",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        76,
+                        81,
+                        "xylan",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 16,
+                "sent": "This polysaccharide comprises a backbone of \u03b2-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with \u03b1-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        5,
+                        19,
+                        "polysaccharide",
+                        "chemical"
+                    ],
+                    [
+                        44,
+                        58,
+                        "\u03b2-1,4-d-xylose",
+                        "chemical"
+                    ],
+                    [
+                        77,
+                        85,
+                        "pyranose",
+                        "chemical"
+                    ],
+                    [
+                        101,
+                        105,
+                        "Xylp",
+                        "chemical"
+                    ],
+                    [
+                        137,
+                        165,
+                        "4-O-methyl-d-glucuronic acid",
+                        "chemical"
+                    ],
+                    [
+                        167,
+                        171,
+                        "GlcA",
+                        "chemical"
+                    ],
+                    [
+                        198,
+                        217,
+                        "\u03b1-l-arabinofuranose",
+                        "chemical"
+                    ],
+                    [
+                        219,
+                        223,
+                        "Araf",
+                        "chemical"
+                    ],
+                    [
+                        247,
+                        261,
+                        "polysaccharide",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 17,
+                "sent": "In addition, the Araf side chain decorations can also be esterified to ferulic acid that, in some species, provide a chemical link between hemicellulose and lignin.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        17,
+                        21,
+                        "Araf",
+                        "chemical"
+                    ],
+                    [
+                        71,
+                        83,
+                        "ferulic acid",
+                        "chemical"
+                    ],
+                    [
+                        139,
+                        152,
+                        "hemicellulose",
+                        "chemical"
+                    ],
+                    [
+                        157,
+                        163,
+                        "lignin",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 18,
+                "sent": "The precise structure of xylans varies between plant species, in particular in different tissues and during cellular differentiation.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        25,
+                        31,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        47,
+                        52,
+                        "plant",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 19,
+                "sent": "In specialized plant tissues, such as the outer layer of cereal grains, xylans are extremely complex, and side chains may comprise a range of other sugars including l- and d-galactose and \u03b2- and \u03b1-Xylp units.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        15,
+                        20,
+                        "plant",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        57,
+                        63,
+                        "cereal",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        72,
+                        78,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        148,
+                        154,
+                        "sugars",
+                        "chemical"
+                    ],
+                    [
+                        165,
+                        183,
+                        "l- and d-galactose",
+                        "chemical"
+                    ],
+                    [
+                        188,
+                        201,
+                        "\u03b2- and \u03b1-Xylp",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 20,
+                "sent": "Indeed, in these cereal brans, xylans have very few backbone Xylp units that are undecorated, and the side chains can contain up to six sugars.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        17,
+                        23,
+                        "cereal",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        31,
+                        37,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        61,
+                        65,
+                        "Xylp",
+                        "chemical"
+                    ],
+                    [
+                        136,
+                        142,
+                        "sugars",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 21,
+                "sent": "Reflecting the chemical and physical complexity of the plant cell wall, microorganisms that utilize these composite structures express a large number of polysaccharide-degrading enzymes, primarily glycoside hydrolases, but also polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        55,
+                        60,
+                        "plant",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        72,
+                        86,
+                        "microorganisms",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        153,
+                        185,
+                        "polysaccharide-degrading enzymes",
+                        "protein_type"
+                    ],
+                    [
+                        197,
+                        217,
+                        "glycoside hydrolases",
+                        "protein_type"
+                    ],
+                    [
+                        228,
+                        249,
+                        "polysaccharide lyases",
+                        "protein_type"
+                    ],
+                    [
+                        251,
+                        273,
+                        "carbohydrate esterases",
+                        "protein_type"
+                    ],
+                    [
+                        279,
+                        314,
+                        "lytic polysaccharide monooxygenases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 22,
+                "sent": "These carbohydrate active enzymes are grouped into sequence-based families in the CAZy database.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        6,
+                        33,
+                        "carbohydrate active enzymes",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 23,
+                "sent": "With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        16,
+                        21,
+                        "xylan",
+                        "chemical"
+                    ],
+                    [
+                        58,
+                        64,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        82,
+                        103,
+                        "endo-acting xylanases",
+                        "protein_type"
+                    ],
+                    [
+                        142,
+                        161,
+                        "glycoside hydrolase",
+                        "protein_type"
+                    ],
+                    [
+                        163,
+                        165,
+                        "GH",
+                        "protein_type"
+                    ],
+                    [
+                        166,
+                        167,
+                        "5",
+                        "protein_type"
+                    ],
+                    [
+                        177,
+                        181,
+                        "GH10",
+                        "protein_type"
+                    ],
+                    [
+                        186,
+                        190,
+                        "GH11",
+                        "protein_type"
+                    ],
+                    [
+                        226,
+                        229,
+                        "GH8",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 24,
+                "sent": "The extensive decoration of the xylan backbone generally restricts the capacity of these enzymes to attack the polysaccharide prior to removal of the side chains by a range of \u03b1-glucuronidases, \u03b1-arabinofuranosidases, and esterases.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        32,
+                        37,
+                        "xylan",
+                        "chemical"
+                    ],
+                    [
+                        111,
+                        125,
+                        "polysaccharide",
+                        "chemical"
+                    ],
+                    [
+                        176,
+                        192,
+                        "\u03b1-glucuronidases",
+                        "protein_type"
+                    ],
+                    [
+                        194,
+                        216,
+                        "\u03b1-arabinofuranosidases",
+                        "protein_type"
+                    ],
+                    [
+                        222,
+                        231,
+                        "esterases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 25,
+                "sent": "Two xylanases, however, utilize the side chains as essential specificity determinants and thus target decorated forms of the hemicellulose.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "xylanases",
+                        "protein_type"
+                    ],
+                    [
+                        125,
+                        138,
+                        "hemicellulose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 26,
+                "sent": "The GH30 glucuronoxylanases require the Xylp bound at the \u22122 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites \u22121 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "GH30",
+                        "protein_type"
+                    ],
+                    [
+                        9,
+                        27,
+                        "glucuronoxylanases",
+                        "protein_type"
+                    ],
+                    [
+                        40,
+                        44,
+                        "Xylp",
+                        "chemical"
+                    ],
+                    [
+                        45,
+                        53,
+                        "bound at",
+                        "protein_state"
+                    ],
+                    [
+                        58,
+                        60,
+                        "\u22122",
+                        "site"
+                    ],
+                    [
+                        74,
+                        78,
+                        "GlcA",
+                        "chemical"
+                    ],
+                    [
+                        121,
+                        141,
+                        "glycoside hydrolases",
+                        "protein_type"
+                    ],
+                    [
+                        153,
+                        171,
+                        "subsites \u22121 and +1",
+                        "site"
+                    ],
+                    [
+                        177,
+                        185,
+                        "subsites",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 27,
+                "sent": "The GH5 arabinoxylanase (CtXyl5A) derived from Clostridium thermocellum displays an absolute requirement for xylans that contain Araf side chains.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        8,
+                        23,
+                        "arabinoxylanase",
+                        "protein_type"
+                    ],
+                    [
+                        25,
+                        32,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        47,
+                        71,
+                        "Clostridium thermocellum",
+                        "species"
+                    ],
+                    [
+                        109,
+                        115,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        129,
+                        133,
+                        "Araf",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 28,
+                "sent": "In this enzyme, the key specificity determinant is the Araf appended to O3 of the Xylp bound in the active site (\u22121 subsite).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        55,
+                        59,
+                        "Araf",
+                        "chemical"
+                    ],
+                    [
+                        82,
+                        86,
+                        "Xylp",
+                        "chemical"
+                    ],
+                    [
+                        87,
+                        95,
+                        "bound in",
+                        "protein_state"
+                    ],
+                    [
+                        100,
+                        111,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        113,
+                        123,
+                        "\u22121 subsite",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 29,
+                "sent": "The reaction products generated from arabinoxylans, however, suggest that Araf can be accommodated at subsites distal to the active site.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        37,
+                        50,
+                        "arabinoxylans",
+                        "chemical"
+                    ],
+                    [
+                        74,
+                        78,
+                        "Araf",
+                        "chemical"
+                    ],
+                    [
+                        102,
+                        110,
+                        "subsites",
+                        "site"
+                    ],
+                    [
+                        125,
+                        136,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 30,
+                "sent": "CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        64,
+                        67,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        68,
+                        84,
+                        "catalytic module",
+                        "structure_element"
+                    ],
+                    [
+                        86,
+                        91,
+                        "CtGH5",
+                        "structure_element"
+                    ],
+                    [
+                        100,
+                        142,
+                        "non-catalytic carbohydrate binding modules",
+                        "structure_element"
+                    ],
+                    [
+                        144,
+                        148,
+                        "CBMs",
+                        "structure_element"
+                    ],
+                    [
+                        172,
+                        173,
+                        "6",
+                        "protein_type"
+                    ],
+                    [
+                        175,
+                        181,
+                        "CtCBM6",
+                        "structure_element"
+                    ],
+                    [
+                        184,
+                        186,
+                        "13",
+                        "protein_type"
+                    ],
+                    [
+                        188,
+                        195,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        202,
+                        204,
+                        "62",
+                        "protein_type"
+                    ],
+                    [
+                        206,
+                        213,
+                        "CtCBM62",
+                        "structure_element"
+                    ],
+                    [
+                        216,
+                        234,
+                        "fibronectin type 3",
+                        "protein_type"
+                    ],
+                    [
+                        236,
+                        239,
+                        "Fn3",
+                        "structure_element"
+                    ],
+                    [
+                        266,
+                        274,
+                        "dockerin",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 31,
+                "sent": "Previous studies of Fn3 domains have indicated that they might function as ligand-binding modules, as a compact form of peptide linkers or spacers between other domains, as cellulose-disrupting modules, or as proteins that help large enzyme complexes remain soluble.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        20,
+                        23,
+                        "Fn3",
+                        "structure_element"
+                    ],
+                    [
+                        75,
+                        97,
+                        "ligand-binding modules",
+                        "structure_element"
+                    ],
+                    [
+                        173,
+                        201,
+                        "cellulose-disrupting modules",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 32,
+                "sent": "The dockerin domain recruits the enzyme into the cellulosome, a multienzyme plant cell wall degrading complex presented on the surface of C. thermocellum.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        12,
+                        "dockerin",
+                        "structure_element"
+                    ],
+                    [
+                        49,
+                        60,
+                        "cellulosome",
+                        "complex_assembly"
+                    ],
+                    [
+                        76,
+                        81,
+                        "plant",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        138,
+                        153,
+                        "C. thermocellum",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 33,
+                "sent": "CtCBM6 stabilizes CtGH5, and CtCBM62 binds to d-galactopyranose and l-arabinopyranose.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        6,
+                        "CtCBM6",
+                        "structure_element"
+                    ],
+                    [
+                        18,
+                        23,
+                        "CtGH5",
+                        "structure_element"
+                    ],
+                    [
+                        29,
+                        36,
+                        "CtCBM62",
+                        "structure_element"
+                    ],
+                    [
+                        46,
+                        63,
+                        "d-galactopyranose",
+                        "chemical"
+                    ],
+                    [
+                        68,
+                        85,
+                        "l-arabinopyranose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 34,
+                "sent": "The function of the CtCBM13 and Fn3 modules remains unclear.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        20,
+                        27,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        32,
+                        35,
+                        "Fn3",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 35,
+                "sent": "This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        25,
+                        42,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        46,
+                        52,
+                        "mature",
+                        "protein_state"
+                    ],
+                    [
+                        53,
+                        60,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        61,
+                        68,
+                        "lacking",
+                        "protein_state"
+                    ],
+                    [
+                        84,
+                        92,
+                        "dockerin",
+                        "structure_element"
+                    ],
+                    [
+                        101,
+                        112,
+                        "CtXyl5A-Doc",
+                        "mutant"
+                    ],
+                    [
+                        130,
+                        145,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        146,
+                        153,
+                        "ligands",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 36,
+                "sent": "The data show that the plasticity in substrate recognition enables the enzyme to hydrolyze highly complex xylans that are not accessible to classical GH10 and GH11 endo-xylanases.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        106,
+                        112,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        150,
+                        154,
+                        "GH10",
+                        "protein_type"
+                    ],
+                    [
+                        159,
+                        163,
+                        "GH11",
+                        "protein_type"
+                    ],
+                    [
+                        164,
+                        178,
+                        "endo-xylanases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 37,
+                "sent": "Molecular architecture of GH5_34 enzymes.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        26,
+                        32,
+                        "GH5_34",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 38,
+                "sent": "Modules prefaced by GH, CBM, or CE are modules in the indicated glycoside hydrolase, carbohydrate binding module, or carbohydrate esterase families, respectively.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        20,
+                        22,
+                        "GH",
+                        "structure_element"
+                    ],
+                    [
+                        24,
+                        27,
+                        "CBM",
+                        "structure_element"
+                    ],
+                    [
+                        32,
+                        34,
+                        "CE",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        83,
+                        "glycoside hydrolase",
+                        "protein_type"
+                    ],
+                    [
+                        85,
+                        112,
+                        "carbohydrate binding module",
+                        "structure_element"
+                    ],
+                    [
+                        117,
+                        138,
+                        "carbohydrate esterase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 39,
+                "sent": "Laminin_3_G domain belongs to the concanavalin A lectin superfamily, and FN3 denotes a fibronectin type 3 domain.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Laminin_3_G",
+                        "structure_element"
+                    ],
+                    [
+                        34,
+                        67,
+                        "concanavalin A lectin superfamily",
+                        "protein_type"
+                    ],
+                    [
+                        73,
+                        76,
+                        "FN3",
+                        "structure_element"
+                    ],
+                    [
+                        87,
+                        112,
+                        "fibronectin type 3 domain",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 40,
+                "sent": "Segments labeled D are dockerin domains.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        23,
+                        31,
+                        "dockerin",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 41,
+                "sent": "Substrate Specificity of CtXyl5A",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        32,
+                        "CtXyl5A",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 42,
+                "sent": "Previous studies showed that CtXyl5A is an arabinoxylan-specific xylanase that generates xylooligosaccharides with an arabinose linked O3 to the reducing end xylose.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        36,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        43,
+                        73,
+                        "arabinoxylan-specific xylanase",
+                        "protein_type"
+                    ],
+                    [
+                        89,
+                        109,
+                        "xylooligosaccharides",
+                        "chemical"
+                    ],
+                    [
+                        118,
+                        127,
+                        "arabinose",
+                        "chemical"
+                    ],
+                    [
+                        158,
+                        164,
+                        "xylose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 43,
+                "sent": "The enzyme is active against both wheat and rye arabinoxylans (abbreviated as WAX and RAX, respectively).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        39,
+                        "wheat",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        44,
+                        47,
+                        "rye",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        48,
+                        61,
+                        "arabinoxylans",
+                        "chemical"
+                    ],
+                    [
+                        78,
+                        81,
+                        "WAX",
+                        "chemical"
+                    ],
+                    [
+                        86,
+                        89,
+                        "RAX",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 44,
+                "sent": "It was proposed that arabinose decorations make productive interactions with a pocket (\u22122*) that is abutted onto the active site or \u22121 subsite.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        21,
+                        30,
+                        "arabinose",
+                        "chemical"
+                    ],
+                    [
+                        79,
+                        85,
+                        "pocket",
+                        "site"
+                    ],
+                    [
+                        87,
+                        90,
+                        "\u22122*",
+                        "site"
+                    ],
+                    [
+                        117,
+                        128,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        132,
+                        142,
+                        "\u22121 subsite",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 45,
+                "sent": "Arabinose side chains of the other backbone xylose units in the oligosaccharides generated by CtXyl5A were essentially random.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Arabinose",
+                        "chemical"
+                    ],
+                    [
+                        44,
+                        50,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        64,
+                        80,
+                        "oligosaccharides",
+                        "chemical"
+                    ],
+                    [
+                        94,
+                        101,
+                        "CtXyl5A",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 46,
+                "sent": "These data suggest that O3, and possibly O2, on the xylose residues at subsites distal to the active site and \u22122* pocket are solvent-exposed, implying that the enzyme can access highly decorated xylans.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        52,
+                        58,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        71,
+                        79,
+                        "subsites",
+                        "site"
+                    ],
+                    [
+                        94,
+                        105,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        110,
+                        120,
+                        "\u22122* pocket",
+                        "site"
+                    ],
+                    [
+                        125,
+                        140,
+                        "solvent-exposed",
+                        "protein_state"
+                    ],
+                    [
+                        195,
+                        201,
+                        "xylans",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 47,
+                "sent": "To test this hypothesis, the activity of CtXyl5A against xylans from cereal brans was assessed.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        41,
+                        48,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        57,
+                        63,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        69,
+                        75,
+                        "cereal",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 48,
+                "sent": "CtXyl5a was incubated with a range of xylans for 16 h at 60 \u00b0C, and the limit products were visualized by TLC.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "CtXyl5a",
+                        "protein"
+                    ],
+                    [
+                        12,
+                        21,
+                        "incubated",
+                        "experimental_method"
+                    ],
+                    [
+                        38,
+                        44,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        106,
+                        109,
+                        "TLC",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 49,
+                "sent": "These xylans are highly decorated not only with Araf and GlcA units but also with l-Gal, d-Gal, and d-Xyl.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        6,
+                        12,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        48,
+                        52,
+                        "Araf",
+                        "chemical"
+                    ],
+                    [
+                        57,
+                        61,
+                        "GlcA",
+                        "chemical"
+                    ],
+                    [
+                        82,
+                        87,
+                        "l-Gal",
+                        "chemical"
+                    ],
+                    [
+                        89,
+                        94,
+                        "d-Gal",
+                        "chemical"
+                    ],
+                    [
+                        100,
+                        105,
+                        "d-Xyl",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 50,
+                "sent": "Indeed, very few xylose units in the backbone of bran xylans lack side chains.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        17,
+                        23,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        54,
+                        60,
+                        "xylans",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 51,
+                "sent": "The data presented in Table 1 showed that CtXyl5A was active against corn bran xylan (CX).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        42,
+                        49,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        69,
+                        73,
+                        "corn",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        79,
+                        84,
+                        "xylan",
+                        "chemical"
+                    ],
+                    [
+                        86,
+                        88,
+                        "CX",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 52,
+                "sent": "In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        20,
+                        34,
+                        "endo-xylanases",
+                        "protein_type"
+                    ],
+                    [
+                        40,
+                        44,
+                        "GH10",
+                        "protein_type"
+                    ],
+                    [
+                        49,
+                        53,
+                        "GH11",
+                        "protein_type"
+                    ],
+                    [
+                        76,
+                        78,
+                        "CX",
+                        "chemical"
+                    ],
+                    [
+                        95,
+                        102,
+                        "lack of",
+                        "protein_state"
+                    ],
+                    [
+                        115,
+                        121,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        149,
+                        160,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        187,
+                        194,
+                        "bind to",
+                        "protein_state"
+                    ],
+                    [
+                        211,
+                        217,
+                        "xylose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 53,
+                "sent": "The limit products generated by CtXyl5A from CX consisted of an extensive range of oligosaccharides.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        32,
+                        39,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        45,
+                        47,
+                        "CX",
+                        "chemical"
+                    ],
+                    [
+                        83,
+                        99,
+                        "oligosaccharides",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 54,
+                "sent": "These data support the view that in subsites out with the active site the O2 and O3 groups of the bound xylose units are solvent-exposed and will thus tolerate decoration.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        36,
+                        44,
+                        "subsites",
+                        "site"
+                    ],
+                    [
+                        58,
+                        69,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        104,
+                        110,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        121,
+                        136,
+                        "solvent-exposed",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 55,
+                "sent": "Kinetics of GH5_34 arabinoxylanases",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "Kinetics",
+                        "evidence"
+                    ],
+                    [
+                        12,
+                        18,
+                        "GH5_34",
+                        "protein_type"
+                    ],
+                    [
+                        19,
+                        35,
+                        "arabinoxylanases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 56,
+                "sent": "Enzyme\tVariant\tkcat/Km\t \tWAX\tRAX\tCX\t \t\t\tmin\u22121mg\u22121ml\t \tCtXyl5A\tCtGH5-CBM6-CBM13-Fn3-CBM62\t800\tND\t460\t \tCtXyl5A\tCtGH5-CBM6-CBM13-Fn3\t1,232\tND\t659\t \tCtXyl5A\tCtGH5-CBM6-CBM13\t1,307\tND\t620\t \tCtXyl5A\tCtGH5-CBM6\t488\tND\t102\t \tCtXyl5A\tCtGH5-CBM6: E68A\tNA\tNA\tNA\t \tCtXyl5A\tCtGH5-CBM6: Y92A\tNA\tNA\tNA\t \tCtXyl5A\tCtGH5-CBM6: N135A\t260\tND\tND\t \tCtXyl5A\tCtGH5-CBM6: N139A\tNA\tNA\tNA\t \tAcGH5\tWild type\t628\t1,641\t289\t \tGpGH5\tWild type\t2,600\t9,986\t314\t \tVbGH5\tWild type\tND\tND\tND\t \tVbGH5\tD45A\t102\t203\t23\t \t",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        15,
+                        19,
+                        "kcat",
+                        "evidence"
+                    ],
+                    [
+                        20,
+                        22,
+                        "Km",
+                        "evidence"
+                    ],
+                    [
+                        25,
+                        28,
+                        "WAX",
+                        "chemical"
+                    ],
+                    [
+                        29,
+                        32,
+                        "RAX",
+                        "chemical"
+                    ],
+                    [
+                        33,
+                        35,
+                        "CX",
+                        "chemical"
+                    ],
+                    [
+                        54,
+                        61,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        62,
+                        88,
+                        "CtGH5-CBM6-CBM13-Fn3-CBM62",
+                        "structure_element"
+                    ],
+                    [
+                        102,
+                        109,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        110,
+                        130,
+                        "CtGH5-CBM6-CBM13-Fn3",
+                        "structure_element"
+                    ],
+                    [
+                        146,
+                        153,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        154,
+                        170,
+                        "CtGH5-CBM6-CBM13",
+                        "structure_element"
+                    ],
+                    [
+                        186,
+                        193,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        194,
+                        204,
+                        "CtGH5-CBM6",
+                        "structure_element"
+                    ],
+                    [
+                        218,
+                        225,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        226,
+                        236,
+                        "CtGH5-CBM6",
+                        "structure_element"
+                    ],
+                    [
+                        238,
+                        242,
+                        "E68A",
+                        "mutant"
+                    ],
+                    [
+                        254,
+                        261,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        262,
+                        272,
+                        "CtGH5-CBM6",
+                        "structure_element"
+                    ],
+                    [
+                        274,
+                        278,
+                        "Y92A",
+                        "mutant"
+                    ],
+                    [
+                        290,
+                        297,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        298,
+                        308,
+                        "CtGH5-CBM6",
+                        "structure_element"
+                    ],
+                    [
+                        310,
+                        315,
+                        "N135A",
+                        "mutant"
+                    ],
+                    [
+                        328,
+                        335,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        336,
+                        346,
+                        "CtGH5-CBM6",
+                        "structure_element"
+                    ],
+                    [
+                        348,
+                        353,
+                        "N139A",
+                        "mutant"
+                    ],
+                    [
+                        365,
+                        370,
+                        "AcGH5",
+                        "protein"
+                    ],
+                    [
+                        371,
+                        380,
+                        "Wild type",
+                        "protein_state"
+                    ],
+                    [
+                        397,
+                        402,
+                        "GpGH5",
+                        "protein"
+                    ],
+                    [
+                        403,
+                        412,
+                        "Wild type",
+                        "protein_state"
+                    ],
+                    [
+                        431,
+                        436,
+                        "VbGH5",
+                        "protein"
+                    ],
+                    [
+                        437,
+                        446,
+                        "Wild type",
+                        "protein_state"
+                    ],
+                    [
+                        458,
+                        463,
+                        "VbGH5",
+                        "protein"
+                    ],
+                    [
+                        464,
+                        468,
+                        "D45A",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 57,
+                "sent": "To explore whether substrate bound only at \u22122* and \u22121 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        42,
+                        "bound only at",
+                        "protein_state"
+                    ],
+                    [
+                        43,
+                        46,
+                        "\u22122*",
+                        "site"
+                    ],
+                    [
+                        51,
+                        53,
+                        "\u22121",
+                        "site"
+                    ],
+                    [
+                        61,
+                        78,
+                        "negative subsites",
+                        "site"
+                    ],
+                    [
+                        97,
+                        104,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        128,
+                        130,
+                        "CX",
+                        "chemical"
+                    ],
+                    [
+                        147,
+                        162,
+                        "arabinoxylanase",
+                        "protein_type"
+                    ],
+                    [
+                        181,
+                        210,
+                        "size exclusion chromatography",
+                        "experimental_method"
+                    ],
+                    [
+                        249,
+                        265,
+                        "oligosaccharides",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 58,
+                "sent": "HPAEC analysis of the smallest oligosaccharide fraction (pool 4) contained two species with retention times of 14.0 min (oligosaccharide 1) and 20.8 min (oligosaccharide 2) (Fig. 2).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "HPAEC",
+                        "experimental_method"
+                    ],
+                    [
+                        31,
+                        46,
+                        "oligosaccharide",
+                        "chemical"
+                    ],
+                    [
+                        121,
+                        136,
+                        "oligosaccharide",
+                        "chemical"
+                    ],
+                    [
+                        154,
+                        169,
+                        "oligosaccharide",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 59,
+                "sent": "Positive mode electrospray mass spectrometry showed that pool 4 contained exclusively molecular ions with a m/z = 305 [M + Na]+, which corresponds to a pentose-pentose disaccharide (molecular mass = 282 Da) as a sodium ion adduct, whereas a dimer of the disaccharide with a sodium adduct (m/z = 587 [2M+Na]+) was also evident.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        44,
+                        "Positive mode electrospray mass spectrometry",
+                        "experimental_method"
+                    ],
+                    [
+                        152,
+                        159,
+                        "pentose",
+                        "chemical"
+                    ],
+                    [
+                        160,
+                        167,
+                        "pentose",
+                        "chemical"
+                    ],
+                    [
+                        168,
+                        180,
+                        "disaccharide",
+                        "chemical"
+                    ],
+                    [
+                        254,
+                        266,
+                        "disaccharide",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 60,
+                "sent": "The monosaccharide composition of pool 4 determined by TFA hydrolysis contained xylose and arabinose in a 3:1 ratio.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        55,
+                        69,
+                        "TFA hydrolysis",
+                        "experimental_method"
+                    ],
+                    [
+                        80,
+                        86,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        91,
+                        100,
+                        "arabinose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 61,
+                "sent": "This suggests that the two oligosaccharides consist of two disaccharides: one consisting of two xylose residues and the other consisting of an arabinose linked to a xylose.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        27,
+                        43,
+                        "oligosaccharides",
+                        "chemical"
+                    ],
+                    [
+                        59,
+                        72,
+                        "disaccharides",
+                        "chemical"
+                    ],
+                    [
+                        96,
+                        102,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        143,
+                        152,
+                        "arabinose",
+                        "chemical"
+                    ],
+                    [
+                        165,
+                        171,
+                        "xylose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 62,
+                "sent": "Treatment of pool 4 with the nonspecific arabinofuranosidase, CjAbf51A, resulted in the loss of oligosaccharide 2 and the production of both xylose and arabinose, indicative of a disaccharide of xylose and arabinose.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        60,
+                        "nonspecific arabinofuranosidase",
+                        "protein_type"
+                    ],
+                    [
+                        62,
+                        70,
+                        "CjAbf51A",
+                        "protein"
+                    ],
+                    [
+                        96,
+                        111,
+                        "oligosaccharide",
+                        "chemical"
+                    ],
+                    [
+                        141,
+                        147,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        152,
+                        161,
+                        "arabinose",
+                        "chemical"
+                    ],
+                    [
+                        179,
+                        191,
+                        "disaccharide",
+                        "chemical"
+                    ],
+                    [
+                        195,
+                        201,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        206,
+                        215,
+                        "arabinose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 63,
+                "sent": "Incubation of pool 4 with a \u03b2-1,3-xylosidase (XynB) converted oligosaccharide 1 into xylose, demonstrating that this molecule is the disaccharide \u03b2-1,3-xylobiose.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        28,
+                        44,
+                        "\u03b2-1,3-xylosidase",
+                        "protein_type"
+                    ],
+                    [
+                        46,
+                        50,
+                        "XynB",
+                        "protein"
+                    ],
+                    [
+                        62,
+                        77,
+                        "oligosaccharide",
+                        "chemical"
+                    ],
+                    [
+                        85,
+                        91,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        133,
+                        145,
+                        "disaccharide",
+                        "chemical"
+                    ],
+                    [
+                        146,
+                        161,
+                        "\u03b2-1,3-xylobiose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 64,
+                "sent": "This view is supported by the inability of a \u03b2-1,4-specific xylosidase to hydrolyze oligosaccharide 1 or oligosaccharide 2 (data not shown).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        45,
+                        70,
+                        "\u03b2-1,4-specific xylosidase",
+                        "protein_type"
+                    ],
+                    [
+                        84,
+                        99,
+                        "oligosaccharide",
+                        "chemical"
+                    ],
+                    [
+                        105,
+                        120,
+                        "oligosaccharide",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 65,
+                "sent": "The crucial importance of occupancy of the \u22122* pocket for catalytic competence is illustrated by the inability of the enzyme to hydrolyze linear \u03b2-1,4-xylooligosaccharides.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        43,
+                        53,
+                        "\u22122* pocket",
+                        "site"
+                    ],
+                    [
+                        145,
+                        171,
+                        "\u03b2-1,4-xylooligosaccharides",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 66,
+                "sent": "The generation of Araf-Xylp and Xyl-\u03b2-1,3-Xyl as reaction products demonstrates that occupancy of the \u22122 subsite is not essential for catalytic activity, which is in contrast to all endo-acting xylanases where this subsite plays a critical role in enzyme activity.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        27,
+                        "Araf-Xylp",
+                        "chemical"
+                    ],
+                    [
+                        32,
+                        45,
+                        "Xyl-\u03b2-1,3-Xyl",
+                        "chemical"
+                    ],
+                    [
+                        102,
+                        112,
+                        "\u22122 subsite",
+                        "site"
+                    ],
+                    [
+                        182,
+                        203,
+                        "endo-acting xylanases",
+                        "protein_type"
+                    ],
+                    [
+                        215,
+                        222,
+                        "subsite",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 67,
+                "sent": "Indeed, the data demonstrate that \u22122* plays a more important role in productive substrate binding than the \u22122 subsite.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        37,
+                        "\u22122*",
+                        "site"
+                    ],
+                    [
+                        107,
+                        117,
+                        "\u22122 subsite",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 68,
+                "sent": "Unfortunately, the inability to generate highly purified (Xyl-\u03b2-1,4)n-[\u03b2-1,3-Xyl/Ara]-Xyl oligosaccharides from arabinoxylans prevented the precise binding energies at the negative subsites to be determined.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        57,
+                        89,
+                        "(Xyl-\u03b2-1,4)n-[\u03b2-1,3-Xyl/Ara]-Xyl",
+                        "chemical"
+                    ],
+                    [
+                        90,
+                        106,
+                        "oligosaccharides",
+                        "chemical"
+                    ],
+                    [
+                        112,
+                        125,
+                        "arabinoxylans",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 69,
+                "sent": "Identification of the disaccharide reaction products generated from CX.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        22,
+                        34,
+                        "disaccharide",
+                        "chemical"
+                    ],
+                    [
+                        68,
+                        70,
+                        "CX",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 70,
+                "sent": "The smallest reaction products were purified by size exclusion chromatography and analyzed by HPAEC (A) and positive mode ESI-MS (B), respectively.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        48,
+                        77,
+                        "size exclusion chromatography",
+                        "experimental_method"
+                    ],
+                    [
+                        94,
+                        99,
+                        "HPAEC",
+                        "experimental_method"
+                    ],
+                    [
+                        122,
+                        128,
+                        "ESI-MS",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 71,
+                "sent": "The samples were treated with a nonspecific arabinofuranosidase (CjAbf51A) and a GH3 xylosidase (XynB) that targeted \u03b2-1,3-xylosidic bonds.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        32,
+                        63,
+                        "nonspecific arabinofuranosidase",
+                        "protein_type"
+                    ],
+                    [
+                        65,
+                        73,
+                        "CjAbf51A",
+                        "protein"
+                    ],
+                    [
+                        81,
+                        95,
+                        "GH3 xylosidase",
+                        "protein_type"
+                    ],
+                    [
+                        97,
+                        101,
+                        "XynB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 72,
+                "sent": "X, xylose; A, arabinose.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        3,
+                        9,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        14,
+                        23,
+                        "arabinose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 73,
+                "sent": "The m/z = 305 species denotes a pentose disaccharide as a sodium adduct [M + Na]+, whereas the m/z = 587 signal corresponds to an ESI-MS dimer of the pentose disaccharide also as a sodium adduct [2M + Na]+.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        32,
+                        39,
+                        "pentose",
+                        "chemical"
+                    ],
+                    [
+                        40,
+                        52,
+                        "disaccharide",
+                        "chemical"
+                    ],
+                    [
+                        130,
+                        136,
+                        "ESI-MS",
+                        "experimental_method"
+                    ],
+                    [
+                        150,
+                        157,
+                        "pentose",
+                        "chemical"
+                    ],
+                    [
+                        158,
+                        170,
+                        "disaccharide",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 74,
+                "sent": "Crystal Structure of the Catalytic Module of CtXyl5A in Complex with Ligands",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        17,
+                        "Crystal Structure",
+                        "evidence"
+                    ],
+                    [
+                        25,
+                        41,
+                        "Catalytic Module",
+                        "structure_element"
+                    ],
+                    [
+                        45,
+                        52,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        53,
+                        68,
+                        "in Complex with",
+                        "protein_state"
+                    ],
+                    [
+                        69,
+                        76,
+                        "Ligands",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 75,
+                "sent": "To understand the structural basis for the biochemical properties of CtXyl5A, the crystal structure of the enzyme with ligands that occupy the substrate binding cleft and the critical \u22122* subsite were sought.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        69,
+                        76,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        82,
+                        99,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        143,
+                        166,
+                        "substrate binding cleft",
+                        "site"
+                    ],
+                    [
+                        184,
+                        195,
+                        "\u22122* subsite",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 76,
+                "sent": "The data presented in Fig. 3A show the structure of the CtXyl5A derivative CtGH5-CtCBM6 in complex with arabinose bound in the \u22122* pocket.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        39,
+                        48,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        56,
+                        63,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        75,
+                        87,
+                        "CtGH5-CtCBM6",
+                        "structure_element"
+                    ],
+                    [
+                        88,
+                        103,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        104,
+                        113,
+                        "arabinose",
+                        "chemical"
+                    ],
+                    [
+                        114,
+                        122,
+                        "bound in",
+                        "protein_state"
+                    ],
+                    [
+                        127,
+                        137,
+                        "\u22122* pocket",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 77,
+                "sent": "Interestingly, the bound arabinose was in the pyranose conformation rather than in its furanose form found in arabinoxylans.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        24,
+                        "bound",
+                        "protein_state"
+                    ],
+                    [
+                        25,
+                        34,
+                        "arabinose",
+                        "chemical"
+                    ],
+                    [
+                        46,
+                        54,
+                        "pyranose",
+                        "chemical"
+                    ],
+                    [
+                        87,
+                        95,
+                        "furanose",
+                        "chemical"
+                    ],
+                    [
+                        110,
+                        123,
+                        "arabinoxylans",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 78,
+                "sent": "O1 was facing toward the active site \u22121 subsite, indicative of the bound arabinose being in the right orientation to be linked to the xylan backbone via an \u03b1-1,3 linkage.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        36,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        37,
+                        47,
+                        "\u22121 subsite",
+                        "site"
+                    ],
+                    [
+                        67,
+                        72,
+                        "bound",
+                        "protein_state"
+                    ],
+                    [
+                        73,
+                        82,
+                        "arabinose",
+                        "chemical"
+                    ],
+                    [
+                        134,
+                        139,
+                        "xylan",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 79,
+                "sent": "As discussed on below, the axial O4 of the Arap did not interact with the \u22122* subsite, suggesting that the pocket might be capable of binding a xylose molecule.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        43,
+                        47,
+                        "Arap",
+                        "chemical"
+                    ],
+                    [
+                        74,
+                        85,
+                        "\u22122* subsite",
+                        "site"
+                    ],
+                    [
+                        107,
+                        113,
+                        "pocket",
+                        "site"
+                    ],
+                    [
+                        144,
+                        150,
+                        "xylose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 80,
+                "sent": "Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the \u22122* subsite in its pyranose conformation (Fig. 3B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        15,
+                        "soaking",
+                        "experimental_method"
+                    ],
+                    [
+                        16,
+                        19,
+                        "apo",
+                        "protein_state"
+                    ],
+                    [
+                        20,
+                        28,
+                        "crystals",
+                        "evidence"
+                    ],
+                    [
+                        34,
+                        40,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        57,
+                        64,
+                        "pentose",
+                        "chemical"
+                    ],
+                    [
+                        65,
+                        70,
+                        "sugar",
+                        "chemical"
+                    ],
+                    [
+                        76,
+                        84,
+                        "bound in",
+                        "protein_state"
+                    ],
+                    [
+                        89,
+                        100,
+                        "\u22122* subsite",
+                        "site"
+                    ],
+                    [
+                        108,
+                        116,
+                        "pyranose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 81,
+                "sent": "These crystal structures support the biochemical data presented above showing that the enzyme generated \u03b2-1,3-xylobiose from CX, which would require the disaccharide to bind at the \u22121 and \u22122* subsites.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        6,
+                        24,
+                        "crystal structures",
+                        "evidence"
+                    ],
+                    [
+                        104,
+                        119,
+                        "\u03b2-1,3-xylobiose",
+                        "chemical"
+                    ],
+                    [
+                        125,
+                        127,
+                        "CX",
+                        "chemical"
+                    ],
+                    [
+                        153,
+                        165,
+                        "disaccharide",
+                        "chemical"
+                    ],
+                    [
+                        181,
+                        200,
+                        "\u22121 and \u22122* subsites",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 82,
+                "sent": "A third product complex was generated by co-crystallizing the nucleophile inactive mutant CtGH5E279S-CtCBM6 with a WAX-derived oligosaccharide (Fig. 3C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        41,
+                        57,
+                        "co-crystallizing",
+                        "experimental_method"
+                    ],
+                    [
+                        62,
+                        82,
+                        "nucleophile inactive",
+                        "protein_state"
+                    ],
+                    [
+                        83,
+                        89,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        90,
+                        100,
+                        "CtGH5E279S",
+                        "mutant"
+                    ],
+                    [
+                        101,
+                        107,
+                        "CtCBM6",
+                        "structure_element"
+                    ],
+                    [
+                        115,
+                        118,
+                        "WAX",
+                        "chemical"
+                    ],
+                    [
+                        127,
+                        142,
+                        "oligosaccharide",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 83,
+                "sent": "The data revealed a pentasaccharide bound to the enzyme, comprising \u03b2-1,4-xylotetraose with an Araf linked \u03b1-1,3 to the reducing end xylose.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        20,
+                        35,
+                        "pentasaccharide",
+                        "chemical"
+                    ],
+                    [
+                        36,
+                        44,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        68,
+                        86,
+                        "\u03b2-1,4-xylotetraose",
+                        "chemical"
+                    ],
+                    [
+                        95,
+                        99,
+                        "Araf",
+                        "chemical"
+                    ],
+                    [
+                        133,
+                        139,
+                        "xylose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 84,
+                "sent": "The xylotetraose was positioned in subsites \u22121 to \u22124 and the Araf in the \u22122* pocket.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        16,
+                        "xylotetraose",
+                        "chemical"
+                    ],
+                    [
+                        35,
+                        52,
+                        "subsites \u22121 to \u22124",
+                        "site"
+                    ],
+                    [
+                        61,
+                        65,
+                        "Araf",
+                        "chemical"
+                    ],
+                    [
+                        73,
+                        83,
+                        "\u22122* pocket",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 85,
+                "sent": "Analysis of the three structures showed that O1, O2, O3, and the endocyclic oxygen occupied identical positions in the Arap, Araf, and Xylp ligands bound in the \u22122* subsite and thus made identical interactions with the pocket.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        22,
+                        32,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        119,
+                        123,
+                        "Arap",
+                        "chemical"
+                    ],
+                    [
+                        125,
+                        129,
+                        "Araf",
+                        "chemical"
+                    ],
+                    [
+                        135,
+                        139,
+                        "Xylp",
+                        "chemical"
+                    ],
+                    [
+                        148,
+                        156,
+                        "bound in",
+                        "protein_state"
+                    ],
+                    [
+                        161,
+                        172,
+                        "\u22122* subsite",
+                        "site"
+                    ],
+                    [
+                        219,
+                        225,
+                        "pocket",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 86,
+                "sent": "O1 makes a polar contact with N\u03b42 of Asn139, O2 is within hydrogen bonding distance with O\u03b41 of Asn139 and the backbone N of Asn135, and O3 interacts with the N of Gly136 and O\u03f52 of Glu68.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        24,
+                        "polar contact",
+                        "bond_interaction"
+                    ],
+                    [
+                        37,
+                        43,
+                        "Asn139",
+                        "residue_name_number"
+                    ],
+                    [
+                        58,
+                        74,
+                        "hydrogen bonding",
+                        "bond_interaction"
+                    ],
+                    [
+                        96,
+                        102,
+                        "Asn139",
+                        "residue_name_number"
+                    ],
+                    [
+                        125,
+                        131,
+                        "Asn135",
+                        "residue_name_number"
+                    ],
+                    [
+                        164,
+                        170,
+                        "Gly136",
+                        "residue_name_number"
+                    ],
+                    [
+                        182,
+                        187,
+                        "Glu68",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 87,
+                "sent": "Although O4 of Arap does not make a direct interaction with the enzyme, O4 and O5 of Xylp and Araf, respectively, form hydrogen bonds with O\u03f51 of Glu68.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        15,
+                        19,
+                        "Arap",
+                        "chemical"
+                    ],
+                    [
+                        85,
+                        89,
+                        "Xylp",
+                        "chemical"
+                    ],
+                    [
+                        94,
+                        98,
+                        "Araf",
+                        "chemical"
+                    ],
+                    [
+                        119,
+                        133,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        146,
+                        151,
+                        "Glu68",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 88,
+                "sent": "Finally Tyr92 makes apolar parallel interactions with the pyranose or furanose rings of the three sugars.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        13,
+                        "Tyr92",
+                        "residue_name_number"
+                    ],
+                    [
+                        27,
+                        48,
+                        "parallel interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        58,
+                        66,
+                        "pyranose",
+                        "chemical"
+                    ],
+                    [
+                        70,
+                        78,
+                        "furanose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 89,
+                "sent": "Representation of the residues involved in the ligands recognition at the \u22122* subsite.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        74,
+                        85,
+                        "\u22122* subsite",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 90,
+                "sent": "Interacting residues are represented as stick in blue, and the catalytic residues and the mutated glutamate (into a serine) are in magenta.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        63,
+                        81,
+                        "catalytic residues",
+                        "site"
+                    ],
+                    [
+                        90,
+                        97,
+                        "mutated",
+                        "experimental_method"
+                    ],
+                    [
+                        98,
+                        107,
+                        "glutamate",
+                        "residue_name"
+                    ],
+                    [
+                        116,
+                        122,
+                        "serine",
+                        "residue_name"
+                    ]
+                ]
+            },
+            {
+                "sid": 91,
+                "sent": "A, CtGH5-CBM6 in complex with an arabinopyranose.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        3,
+                        13,
+                        "CtGH5-CBM6",
+                        "structure_element"
+                    ],
+                    [
+                        14,
+                        29,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        33,
+                        48,
+                        "arabinopyranose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 92,
+                "sent": "B, CtGH5-CBM6 in complex with a xylopyranose.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        3,
+                        13,
+                        "CtGH5-CBM6",
+                        "structure_element"
+                    ],
+                    [
+                        14,
+                        29,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        32,
+                        44,
+                        "xylopyranose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 93,
+                "sent": "C, CtGH5E279S-CBM6 in complex with a pentasaccharide (\u03b21,4-xylotetraose with an l-Araf linked \u03b11,3 to the reducing end xylose).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        3,
+                        13,
+                        "CtGH5E279S",
+                        "mutant"
+                    ],
+                    [
+                        14,
+                        18,
+                        "CBM6",
+                        "structure_element"
+                    ],
+                    [
+                        19,
+                        34,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        37,
+                        52,
+                        "pentasaccharide",
+                        "chemical"
+                    ],
+                    [
+                        54,
+                        71,
+                        "\u03b21,4-xylotetraose",
+                        "chemical"
+                    ],
+                    [
+                        80,
+                        86,
+                        "l-Araf",
+                        "chemical"
+                    ],
+                    [
+                        119,
+                        125,
+                        "xylose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 94,
+                "sent": "The xylan backbone is shown transparently for more clarity.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        9,
+                        "xylan",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 95,
+                "sent": "Densities shown in blue are RefMac maximum-likelihood \u03c3A-weighted 2Fo \u2212 Fc at 1.5 \u03c3.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Densities",
+                        "evidence"
+                    ],
+                    [
+                        35,
+                        83,
+                        "maximum-likelihood \u03c3A-weighted 2Fo \u2212 Fc at 1.5 \u03c3",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 96,
+                "sent": "The importance of the interactions between the ligands and the side chains of the residues in the \u22122* pocket were evaluated by alanine substitution of these amino acids.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        98,
+                        108,
+                        "\u22122* pocket",
+                        "site"
+                    ],
+                    [
+                        127,
+                        147,
+                        "alanine substitution",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 97,
+                "sent": "The mutants E68A, Y92A, and N139A were all inactive (Table 1), demonstrating the importance of the interactions of these residues with the substrate and reinforcing the critical role the \u22122* subsite plays in the activity of the enzyme.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        12,
+                        16,
+                        "E68A",
+                        "mutant"
+                    ],
+                    [
+                        18,
+                        22,
+                        "Y92A",
+                        "mutant"
+                    ],
+                    [
+                        28,
+                        33,
+                        "N139A",
+                        "mutant"
+                    ],
+                    [
+                        43,
+                        51,
+                        "inactive",
+                        "protein_state"
+                    ],
+                    [
+                        187,
+                        198,
+                        "\u22122* subsite",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 98,
+                "sent": "N135A retained wild type activity because the O2 of the sugars interacts with the backbone N of Asn135 and not with the side chain.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "N135A",
+                        "mutant"
+                    ],
+                    [
+                        15,
+                        24,
+                        "wild type",
+                        "protein_state"
+                    ],
+                    [
+                        96,
+                        102,
+                        "Asn135",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 99,
+                "sent": "Because the hydroxyls of Xylp or Araf in the \u22122* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        29,
+                        "Xylp",
+                        "chemical"
+                    ],
+                    [
+                        33,
+                        37,
+                        "Araf",
+                        "chemical"
+                    ],
+                    [
+                        45,
+                        55,
+                        "\u22122* pocket",
+                        "site"
+                    ],
+                    [
+                        64,
+                        79,
+                        "solvent-exposed",
+                        "protein_state"
+                    ],
+                    [
+                        85,
+                        96,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        104,
+                        119,
+                        "arabinoxylanase",
+                        "protein_type"
+                    ],
+                    [
+                        137,
+                        143,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        175,
+                        181,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        185,
+                        194,
+                        "arabinose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 100,
+                "sent": "This may explain why the kcat/Km for CtXyl5A against WAX was 2-fold higher than against CX (Table 1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        29,
+                        "kcat",
+                        "evidence"
+                    ],
+                    [
+                        30,
+                        32,
+                        "Km",
+                        "evidence"
+                    ],
+                    [
+                        37,
+                        44,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        53,
+                        56,
+                        "WAX",
+                        "chemical"
+                    ],
+                    [
+                        88,
+                        90,
+                        "CX",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 101,
+                "sent": "WAX is likely to have a higher concentration of single Araf decorations compared with CX and thus contain more substrate available to the arabinoxylanase.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "WAX",
+                        "chemical"
+                    ],
+                    [
+                        55,
+                        59,
+                        "Araf",
+                        "chemical"
+                    ],
+                    [
+                        86,
+                        88,
+                        "CX",
+                        "chemical"
+                    ],
+                    [
+                        138,
+                        153,
+                        "arabinoxylanase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 102,
+                "sent": "In the active site of CtXyl5A the \u03b1-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A\u2013C): O1 hydrogen bonds with the N\u03b41 of His253 and O\u03f52 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and O\u03b3 of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with N\u03b42 of Asn170 and OH of Tyr92.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        7,
+                        18,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        22,
+                        29,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        34,
+                        42,
+                        "\u03b1-d-Xylp",
+                        "chemical"
+                    ],
+                    [
+                        153,
+                        167,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        184,
+                        190,
+                        "His253",
+                        "residue_name_number"
+                    ],
+                    [
+                        202,
+                        208,
+                        "Glu171",
+                        "residue_name_number"
+                    ],
+                    [
+                        257,
+                        270,
+                        "polar contact",
+                        "bond_interaction"
+                    ],
+                    [
+                        286,
+                        292,
+                        "Tyr255",
+                        "residue_name_number"
+                    ],
+                    [
+                        303,
+                        309,
+                        "Ser279",
+                        "residue_name_number"
+                    ],
+                    [
+                        354,
+                        368,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        381,
+                        387,
+                        "Asn170",
+                        "residue_name_number"
+                    ],
+                    [
+                        398,
+                        403,
+                        "Tyr92",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 103,
+                "sent": "O3 (O1 of the Araf at the \u22122* subsite) makes a polar contact with N\u03b42 of Asn139; the endocyclic oxygen hydrogens bonds with the OH of Tyr255.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        18,
+                        "Araf",
+                        "chemical"
+                    ],
+                    [
+                        26,
+                        37,
+                        "\u22122* subsite",
+                        "site"
+                    ],
+                    [
+                        47,
+                        60,
+                        "polar contact",
+                        "bond_interaction"
+                    ],
+                    [
+                        73,
+                        79,
+                        "Asn139",
+                        "residue_name_number"
+                    ],
+                    [
+                        103,
+                        118,
+                        "hydrogens bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        134,
+                        140,
+                        "Tyr255",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 104,
+                "sent": "The Xylp in the active site makes strong parallel apolar interactions with Phe310.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "Xylp",
+                        "chemical"
+                    ],
+                    [
+                        16,
+                        27,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        41,
+                        69,
+                        "parallel apolar interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        75,
+                        81,
+                        "Phe310",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 105,
+                "sent": "Substrate recognition in the active site is conserved between CtXyl5A and the closest GH5 structural homolog, the endoglucanase BaCel5A (PDB code 1qi2) as noted previously.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        40,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        44,
+                        53,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        62,
+                        69,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        86,
+                        89,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        114,
+                        127,
+                        "endoglucanase",
+                        "protein_type"
+                    ],
+                    [
+                        128,
+                        135,
+                        "BaCel5A",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 106,
+                "sent": "Comparison of the ligand recognition at the distal negative subsites between CtGH5E279S-CBM6, the cellulase BaCel5A, and the xylanase GH10.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        51,
+                        68,
+                        "negative subsites",
+                        "site"
+                    ],
+                    [
+                        77,
+                        87,
+                        "CtGH5E279S",
+                        "mutant"
+                    ],
+                    [
+                        88,
+                        92,
+                        "CBM6",
+                        "structure_element"
+                    ],
+                    [
+                        98,
+                        107,
+                        "cellulase",
+                        "protein_type"
+                    ],
+                    [
+                        108,
+                        115,
+                        "BaCel5A",
+                        "protein"
+                    ],
+                    [
+                        125,
+                        133,
+                        "xylanase",
+                        "protein_type"
+                    ],
+                    [
+                        134,
+                        138,
+                        "GH10",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 107,
+                "sent": " A\u2013C show CtGH5E279S-CBM6 is in complex with a pentasaccharide (\u03b21,4-xylotetraose with an l-Araf linked \u03b11,3 to the reducing end xylose).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        10,
+                        20,
+                        "CtGH5E279S",
+                        "mutant"
+                    ],
+                    [
+                        29,
+                        44,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        47,
+                        62,
+                        "pentasaccharide",
+                        "chemical"
+                    ],
+                    [
+                        64,
+                        81,
+                        "\u03b21,4-xylotetraose",
+                        "chemical"
+                    ],
+                    [
+                        90,
+                        96,
+                        "l-Araf",
+                        "chemical"
+                    ],
+                    [
+                        129,
+                        135,
+                        "xylose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 108,
+                "sent": "A, Poseview representation highlighting the hydrogen bonding and the hydrophobic interactions that occur in the negative subsites.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        44,
+                        60,
+                        "hydrogen bonding",
+                        "bond_interaction"
+                    ],
+                    [
+                        69,
+                        93,
+                        "hydrophobic interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        112,
+                        129,
+                        "negative subsites",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 109,
+                "sent": "C, density of the ligand shown in blue is RefMac maximum-likelihood \u03c3A-weighted 2Fo \u2212 Fc at 1.5 \u03c3.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        3,
+                        10,
+                        "density",
+                        "evidence"
+                    ],
+                    [
+                        49,
+                        97,
+                        "maximum-likelihood \u03c3A-weighted 2Fo \u2212 Fc at 1.5 \u03c3",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 110,
+                "sent": "D and E display BaCel5A in complex with deoxy-2-fluoro-\u03b2-d-cellotrioside (PDB code 1qi2), and F and G show CmXyn10B in complex with a xylotriose (PDB code 1uqy).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        16,
+                        23,
+                        "BaCel5A",
+                        "protein"
+                    ],
+                    [
+                        24,
+                        39,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        40,
+                        72,
+                        "deoxy-2-fluoro-\u03b2-d-cellotrioside",
+                        "chemical"
+                    ],
+                    [
+                        107,
+                        115,
+                        "CmXyn10B",
+                        "protein"
+                    ],
+                    [
+                        116,
+                        131,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        134,
+                        144,
+                        "xylotriose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 111,
+                "sent": "B, D, and F are surface representations (CtGH5E279S-CBM6 in gray, BaCel5A in cyan, and the xylanase GH10 in light brown).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        41,
+                        51,
+                        "CtGH5E279S",
+                        "mutant"
+                    ],
+                    [
+                        66,
+                        73,
+                        "BaCel5A",
+                        "protein"
+                    ],
+                    [
+                        91,
+                        99,
+                        "xylanase",
+                        "protein_type"
+                    ],
+                    [
+                        100,
+                        104,
+                        "GH10",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 112,
+                "sent": "The black dashes represent the hydrogen bonds.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        31,
+                        45,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        31,
+                        45,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 113,
+                "sent": "The capacity of CtXyl5A to act on the highly decorated xylan CX indicates that O3 and possibly O2 of the backbone Xylp units are solvent-exposed.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        16,
+                        23,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        55,
+                        60,
+                        "xylan",
+                        "chemical"
+                    ],
+                    [
+                        61,
+                        63,
+                        "CX",
+                        "chemical"
+                    ],
+                    [
+                        114,
+                        118,
+                        "Xylp",
+                        "chemical"
+                    ],
+                    [
+                        129,
+                        144,
+                        "solvent-exposed",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 114,
+                "sent": "This is consistent with the interaction of the xylotetraose backbone with the enzyme distal to the active site.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        47,
+                        59,
+                        "xylotetraose",
+                        "chemical"
+                    ],
+                    [
+                        99,
+                        110,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 115,
+                "sent": "A surface representation of the enzyme (Fig. 4B) shows that O3 and O2 of xylose units at subsites \u22122 to \u22124 are solvent-exposed and are thus available for decoration.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        73,
+                        79,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        89,
+                        106,
+                        "subsites \u22122 to \u22124",
+                        "site"
+                    ],
+                    [
+                        111,
+                        126,
+                        "solvent-exposed",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 116,
+                "sent": "Indeed, these pyranose sugars make very weak apolar interactions with the arabinoxylanase.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        22,
+                        "pyranose",
+                        "chemical"
+                    ],
+                    [
+                        23,
+                        29,
+                        "sugars",
+                        "chemical"
+                    ],
+                    [
+                        45,
+                        64,
+                        "apolar interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        74,
+                        89,
+                        "arabinoxylanase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 117,
+                "sent": "At \u22122, Xylp makes planar apolar interactions with the Araf bound to the \u22122* subsite (Fig. 4C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        3,
+                        5,
+                        "\u22122",
+                        "site"
+                    ],
+                    [
+                        7,
+                        11,
+                        "Xylp",
+                        "chemical"
+                    ],
+                    [
+                        18,
+                        44,
+                        "planar apolar interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        54,
+                        58,
+                        "Araf",
+                        "chemical"
+                    ],
+                    [
+                        59,
+                        67,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        72,
+                        83,
+                        "\u22122* subsite",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 118,
+                "sent": "Xylp at subsites \u22122 and \u22123, respectively, make weak hydrophobic contact with Val318, the \u22123 Xylp makes planar apolar interactions with Ala137, whereas the xylose at \u22124 forms parallel apolar contacts with Trp69.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "Xylp",
+                        "chemical"
+                    ],
+                    [
+                        8,
+                        26,
+                        "subsites \u22122 and \u22123",
+                        "site"
+                    ],
+                    [
+                        52,
+                        71,
+                        "hydrophobic contact",
+                        "bond_interaction"
+                    ],
+                    [
+                        77,
+                        83,
+                        "Val318",
+                        "residue_name_number"
+                    ],
+                    [
+                        89,
+                        91,
+                        "\u22123",
+                        "site"
+                    ],
+                    [
+                        92,
+                        96,
+                        "Xylp",
+                        "chemical"
+                    ],
+                    [
+                        103,
+                        129,
+                        "planar apolar interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        135,
+                        141,
+                        "Ala137",
+                        "residue_name_number"
+                    ],
+                    [
+                        155,
+                        161,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        165,
+                        167,
+                        "\u22124",
+                        "site"
+                    ],
+                    [
+                        174,
+                        198,
+                        "parallel apolar contacts",
+                        "bond_interaction"
+                    ],
+                    [
+                        204,
+                        209,
+                        "Trp69",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 119,
+                "sent": "Comparison of the distal negative subsites of CtXyl5A with BaCel5A and a typical GH10 xylanase (CmXyn10B, PDB code 1uqy) highlights the paucity of interactions between the arabinoxylanase and its substrate out with the active site (Fig. 4).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        42,
+                        "negative subsites",
+                        "site"
+                    ],
+                    [
+                        46,
+                        53,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        59,
+                        66,
+                        "BaCel5A",
+                        "protein"
+                    ],
+                    [
+                        81,
+                        85,
+                        "GH10",
+                        "protein_type"
+                    ],
+                    [
+                        86,
+                        94,
+                        "xylanase",
+                        "protein_type"
+                    ],
+                    [
+                        96,
+                        104,
+                        "CmXyn10B",
+                        "protein"
+                    ],
+                    [
+                        172,
+                        187,
+                        "arabinoxylanase",
+                        "protein_type"
+                    ],
+                    [
+                        219,
+                        230,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 120,
+                "sent": "Thus, the cellulase contains three negative subsites and the sugars bound in the \u22122 and \u22123 subsites make a total of 9 polar interactions with the enzyme (Fig. 4, D and E).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        10,
+                        19,
+                        "cellulase",
+                        "protein_type"
+                    ],
+                    [
+                        35,
+                        52,
+                        "negative subsites",
+                        "site"
+                    ],
+                    [
+                        61,
+                        67,
+                        "sugars",
+                        "chemical"
+                    ],
+                    [
+                        68,
+                        76,
+                        "bound in",
+                        "protein_state"
+                    ],
+                    [
+                        81,
+                        99,
+                        "\u22122 and \u22123 subsites",
+                        "site"
+                    ],
+                    [
+                        118,
+                        136,
+                        "polar interactions",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 121,
+                "sent": "The GH10 xylanase also contains a \u22122 subsite that, similar to the cellulase, makes numerous interactions with the substrate (Fig. 4, F and G).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "GH10",
+                        "protein_type"
+                    ],
+                    [
+                        9,
+                        17,
+                        "xylanase",
+                        "protein_type"
+                    ],
+                    [
+                        34,
+                        44,
+                        "\u22122 subsite",
+                        "site"
+                    ],
+                    [
+                        66,
+                        75,
+                        "cellulase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 122,
+                "sent": "The Influence of the Modular Architecture of CtXyl5A on Catalytic Activity",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        45,
+                        52,
+                        "CtXyl5A",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 123,
+                "sent": "CtXyl5A, in addition to its catalytic module, contains three CBMs (CtCBM6, CtCBM13, and CtCBM62) and a fibronectin domain (CtFn3).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        28,
+                        44,
+                        "catalytic module",
+                        "structure_element"
+                    ],
+                    [
+                        61,
+                        65,
+                        "CBMs",
+                        "structure_element"
+                    ],
+                    [
+                        67,
+                        73,
+                        "CtCBM6",
+                        "structure_element"
+                    ],
+                    [
+                        75,
+                        82,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        88,
+                        95,
+                        "CtCBM62",
+                        "structure_element"
+                    ],
+                    [
+                        103,
+                        121,
+                        "fibronectin domain",
+                        "structure_element"
+                    ],
+                    [
+                        123,
+                        128,
+                        "CtFn3",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 124,
+                "sent": "A previous study showed that although the CBM6 bound in an exo-mode to xylo- and cellulooligosaccharides, the primary role of this module was to stabilize the structure of the GH5 catalytic module.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        42,
+                        46,
+                        "CBM6",
+                        "structure_element"
+                    ],
+                    [
+                        47,
+                        55,
+                        "bound in",
+                        "protein_state"
+                    ],
+                    [
+                        59,
+                        67,
+                        "exo-mode",
+                        "protein_state"
+                    ],
+                    [
+                        71,
+                        104,
+                        "xylo- and cellulooligosaccharides",
+                        "chemical"
+                    ],
+                    [
+                        176,
+                        179,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        180,
+                        196,
+                        "catalytic module",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 125,
+                "sent": "To explore the contribution of the other non-catalytic modules to CtXyl5A function, the activity of a series of truncated derivatives of the arabinoxylanase were assessed.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        41,
+                        62,
+                        "non-catalytic modules",
+                        "structure_element"
+                    ],
+                    [
+                        66,
+                        73,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        112,
+                        121,
+                        "truncated",
+                        "protein_state"
+                    ],
+                    [
+                        141,
+                        156,
+                        "arabinoxylanase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 126,
+                "sent": "The data in Table 1 show that removal of CtCBM62 caused a modest increase in activity against both WAX and CX, whereas deletion of the Fn3 domain had no further impact on catalytic performance.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        30,
+                        40,
+                        "removal of",
+                        "experimental_method"
+                    ],
+                    [
+                        41,
+                        48,
+                        "CtCBM62",
+                        "structure_element"
+                    ],
+                    [
+                        99,
+                        102,
+                        "WAX",
+                        "chemical"
+                    ],
+                    [
+                        107,
+                        109,
+                        "CX",
+                        "chemical"
+                    ],
+                    [
+                        119,
+                        130,
+                        "deletion of",
+                        "experimental_method"
+                    ],
+                    [
+                        135,
+                        138,
+                        "Fn3",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 127,
+                "sent": "Truncation of CtCBM13, however, caused a 4\u20135-fold reduction in activity against both substrates.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        10,
+                        "Truncation",
+                        "experimental_method"
+                    ],
+                    [
+                        14,
+                        21,
+                        "CtCBM13",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 128,
+                "sent": "Members of CBM13 have been shown to bind to xylans, mannose, and galactose residues in complex glycans, hinting that the function of CtCBM13 is to increase the proximity of substrate to the catalytic module of CtXyl5A.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        16,
+                        "CBM13",
+                        "structure_element"
+                    ],
+                    [
+                        44,
+                        50,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        52,
+                        59,
+                        "mannose",
+                        "chemical"
+                    ],
+                    [
+                        65,
+                        74,
+                        "galactose",
+                        "chemical"
+                    ],
+                    [
+                        87,
+                        102,
+                        "complex glycans",
+                        "chemical"
+                    ],
+                    [
+                        133,
+                        140,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        190,
+                        206,
+                        "catalytic module",
+                        "structure_element"
+                    ],
+                    [
+                        210,
+                        217,
+                        "CtXyl5A",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 129,
+                "sent": "Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        15,
+                        "Binding studies",
+                        "experimental_method"
+                    ],
+                    [
+                        38,
+                        45,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        92,
+                        99,
+                        "glycans",
+                        "chemical"
+                    ],
+                    [
+                        110,
+                        113,
+                        "WAX",
+                        "chemical"
+                    ],
+                    [
+                        115,
+                        117,
+                        "CX",
+                        "chemical"
+                    ],
+                    [
+                        119,
+                        125,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        127,
+                        134,
+                        "mannose",
+                        "chemical"
+                    ],
+                    [
+                        136,
+                        145,
+                        "galactose",
+                        "chemical"
+                    ],
+                    [
+                        151,
+                        166,
+                        "birchwood xylan",
+                        "chemical"
+                    ],
+                    [
+                        168,
+                        170,
+                        "BX",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 130,
+                "sent": "It would appear, therefore, that CtCBM13 makes a structural contribution to the function of CtXyl5A.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        33,
+                        40,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        92,
+                        99,
+                        "CtXyl5A",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 131,
+                "sent": "Crystal Structure of CtXyl5A-D",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        17,
+                        "Crystal Structure",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        30,
+                        "CtXyl5A-D",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 132,
+                "sent": "To explore further the role of the non-catalytic modules in CtXyl5A the crystal structure of CtXyl5A extending from CtGH5 to CtCBM62 was sought.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        35,
+                        56,
+                        "non-catalytic modules",
+                        "structure_element"
+                    ],
+                    [
+                        60,
+                        67,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        72,
+                        89,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        93,
+                        100,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        116,
+                        121,
+                        "CtGH5",
+                        "structure_element"
+                    ],
+                    [
+                        125,
+                        132,
+                        "CtCBM62",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 133,
+                "sent": "To obtain a construct that could potentially be crystallized, the protein was generated without the C-terminal dockerin domain because it is known to be unstable and prone to cleavage.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        48,
+                        60,
+                        "crystallized",
+                        "experimental_method"
+                    ],
+                    [
+                        88,
+                        95,
+                        "without",
+                        "protein_state"
+                    ],
+                    [
+                        111,
+                        119,
+                        "dockerin",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 134,
+                "sent": "Using this construct (CtXyl5A-D) the crystal structure of the arabinoxylanase was determined by molecular replacement to a resolution of 2.64 \u212b with Rwork and Rfree at 23.7% and 27.8%, respectively.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        22,
+                        31,
+                        "CtXyl5A-D",
+                        "mutant"
+                    ],
+                    [
+                        37,
+                        54,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        62,
+                        77,
+                        "arabinoxylanase",
+                        "protein_type"
+                    ],
+                    [
+                        96,
+                        117,
+                        "molecular replacement",
+                        "experimental_method"
+                    ],
+                    [
+                        149,
+                        154,
+                        "Rwork",
+                        "evidence"
+                    ],
+                    [
+                        159,
+                        164,
+                        "Rfree",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 135,
+                "sent": "The structure comprises a continuous polypeptide extending from Ala36 to Trp742 displaying four modules GH5-CBM6-CBM13-Fn3.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        64,
+                        79,
+                        "Ala36 to Trp742",
+                        "residue_range"
+                    ],
+                    [
+                        104,
+                        122,
+                        "GH5-CBM6-CBM13-Fn3",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 136,
+                "sent": "Although there was some electron density for CtCBM62, it was not sufficient to confidently build the module (Fig. 5).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        24,
+                        40,
+                        "electron density",
+                        "evidence"
+                    ],
+                    [
+                        45,
+                        52,
+                        "CtCBM62",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 137,
+                "sent": "Further investigation of the crystal packing revealed a large solvent channel adjacent to the area the CBM62 occupies.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        44,
+                        "crystal packing",
+                        "evidence"
+                    ],
+                    [
+                        62,
+                        77,
+                        "solvent channel",
+                        "site"
+                    ],
+                    [
+                        103,
+                        108,
+                        "CBM62",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 138,
+                "sent": "We postulate that the reason for the poor electron density is due to the CtCBM62 being mobile compared with the rest of the protein.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        42,
+                        58,
+                        "electron density",
+                        "evidence"
+                    ],
+                    [
+                        73,
+                        80,
+                        "CtCBM62",
+                        "structure_element"
+                    ],
+                    [
+                        87,
+                        93,
+                        "mobile",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 139,
+                "sent": "The structures of CtGH5 and CtCBM6 have been described previously.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        14,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        18,
+                        23,
+                        "CtGH5",
+                        "structure_element"
+                    ],
+                    [
+                        28,
+                        34,
+                        "CtCBM6",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 140,
+                "sent": "Surface representation of the tetra-modular arabinoxylanase and zoom view on the CtGH5 loop.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        44,
+                        59,
+                        "arabinoxylanase",
+                        "protein_type"
+                    ],
+                    [
+                        81,
+                        86,
+                        "CtGH5",
+                        "structure_element"
+                    ],
+                    [
+                        87,
+                        91,
+                        "loop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 141,
+                "sent": "The blue module is the CtGH5 catalytic domain, the green module corresponds to the CtCBM6, the yellow module is the CtCBM13, and the salmon module is the fibronectin domain.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        23,
+                        28,
+                        "CtGH5",
+                        "structure_element"
+                    ],
+                    [
+                        29,
+                        45,
+                        "catalytic domain",
+                        "structure_element"
+                    ],
+                    [
+                        83,
+                        89,
+                        "CtCBM6",
+                        "structure_element"
+                    ],
+                    [
+                        116,
+                        123,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        154,
+                        172,
+                        "fibronectin domain",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 142,
+                "sent": "The CtGH5 loop is stabilized between the CtCBM6 and the CtCBM13 modules.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        9,
+                        "CtGH5",
+                        "structure_element"
+                    ],
+                    [
+                        10,
+                        14,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        41,
+                        47,
+                        "CtCBM6",
+                        "structure_element"
+                    ],
+                    [
+                        56,
+                        63,
+                        "CtCBM13",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 143,
+                "sent": "CtCBM13 extends from Gly567 to Pro648.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        21,
+                        37,
+                        "Gly567 to Pro648",
+                        "residue_range"
+                    ]
+                ]
+            },
+            {
+                "sid": 144,
+                "sent": "Typical of CBM13 proteins CtCBM13 displays a \u03b2-trefoil fold comprising the canonical pseudo 3-fold symmetry with a 3-fold repeating unit of 40\u201350 amino acid residues characteristic of the Ricin superfamily.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        16,
+                        "CBM13",
+                        "protein_type"
+                    ],
+                    [
+                        26,
+                        33,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        45,
+                        59,
+                        "\u03b2-trefoil fold",
+                        "structure_element"
+                    ],
+                    [
+                        115,
+                        136,
+                        "3-fold repeating unit",
+                        "structure_element"
+                    ],
+                    [
+                        140,
+                        156,
+                        "40\u201350 amino acid",
+                        "residue_range"
+                    ],
+                    [
+                        188,
+                        205,
+                        "Ricin superfamily",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 145,
+                "sent": "Each repeat contains two pairs of antiparallel \u03b2-strands.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        11,
+                        "repeat",
+                        "structure_element"
+                    ],
+                    [
+                        34,
+                        56,
+                        "antiparallel \u03b2-strands",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 146,
+                "sent": "A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 \u212b and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-\u03b2-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis \u03b2-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        2,
+                        13,
+                        "Dali search",
+                        "experimental_method"
+                    ],
+                    [
+                        52,
+                        57,
+                        "CBM13",
+                        "protein_type"
+                    ],
+                    [
+                        73,
+                        99,
+                        "root mean square deviation",
+                        "evidence"
+                    ],
+                    [
+                        205,
+                        220,
+                        "C. thermocellum",
+                        "species"
+                    ],
+                    [
+                        221,
+                        242,
+                        "exo-\u03b2-1,3-galactanase",
+                        "protein_type"
+                    ],
+                    [
+                        260,
+                        284,
+                        "Streptomyces avermitilis",
+                        "species"
+                    ],
+                    [
+                        285,
+                        308,
+                        "\u03b2-l-arabinopyranosidase",
+                        "protein_type"
+                    ],
+                    [
+                        326,
+                        347,
+                        "Streptomyces lividans",
+                        "species"
+                    ],
+                    [
+                        348,
+                        360,
+                        "xylanase 10A",
+                        "protein"
+                    ],
+                    [
+                        383,
+                        416,
+                        "Streptomyces olivaceoviridis E-86",
+                        "species"
+                    ],
+                    [
+                        417,
+                        429,
+                        "xylanase 10A",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 147,
+                "sent": "The Fn3 module displays a typical \u03b2-sandwich fold with the two sheets comprising, primarily, three antiparallel strands in the order \u03b21-\u03b22-\u03b25 in \u03b2-sheet 1 and \u03b24-\u03b23-\u03b26 in \u03b2-sheet 2.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "Fn3",
+                        "structure_element"
+                    ],
+                    [
+                        34,
+                        49,
+                        "\u03b2-sandwich fold",
+                        "structure_element"
+                    ],
+                    [
+                        63,
+                        69,
+                        "sheets",
+                        "structure_element"
+                    ],
+                    [
+                        99,
+                        119,
+                        "antiparallel strands",
+                        "structure_element"
+                    ],
+                    [
+                        133,
+                        141,
+                        "\u03b21-\u03b22-\u03b25",
+                        "structure_element"
+                    ],
+                    [
+                        145,
+                        154,
+                        "\u03b2-sheet 1",
+                        "structure_element"
+                    ],
+                    [
+                        159,
+                        167,
+                        "\u03b24-\u03b23-\u03b26",
+                        "structure_element"
+                    ],
+                    [
+                        171,
+                        180,
+                        "\u03b2-sheet 2",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 148,
+                "sent": "Although \u03b2-sheet 2 presents a cleft-like topology, typical of endo-binding CBMs, the surface lacks aromatic residues that play a key role in ligand recognition, and in the context of the full-length enzyme, the cleft abuts into CtCBM13 and thus would not be able to accommodate an extended polysaccharide chain (see below).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        18,
+                        "\u03b2-sheet 2",
+                        "structure_element"
+                    ],
+                    [
+                        30,
+                        35,
+                        "cleft",
+                        "site"
+                    ],
+                    [
+                        62,
+                        79,
+                        "endo-binding CBMs",
+                        "protein_type"
+                    ],
+                    [
+                        187,
+                        198,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        199,
+                        205,
+                        "enzyme",
+                        "protein"
+                    ],
+                    [
+                        211,
+                        216,
+                        "cleft",
+                        "site"
+                    ],
+                    [
+                        228,
+                        235,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        290,
+                        304,
+                        "polysaccharide",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 149,
+                "sent": "In the structure of CtXyl5A-D, the four modules form a three-leaf clover-like structure (Fig. 5).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        7,
+                        16,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        20,
+                        29,
+                        "CtXyl5A-D",
+                        "mutant"
+                    ],
+                    [
+                        40,
+                        47,
+                        "modules",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 150,
+                "sent": "Between the interfaces of CtGH5-CBM6-CBM13 there are a number of interactions that maintain the modules in a fixed position relative to each other.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        12,
+                        22,
+                        "interfaces",
+                        "site"
+                    ],
+                    [
+                        26,
+                        42,
+                        "CtGH5-CBM6-CBM13",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 151,
+                "sent": "The interaction of CtGH5 and CtCBM6, which buries a substantial apolar solvent-exposed surface of the two modules, has been described previously.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        24,
+                        "CtGH5",
+                        "structure_element"
+                    ],
+                    [
+                        29,
+                        35,
+                        "CtCBM6",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        94,
+                        "apolar solvent-exposed surface",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 152,
+                "sent": "The polar interactions between these two modules comprise 14 hydrogen bonds and 5 salt bridges.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        22,
+                        "polar interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        61,
+                        75,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        82,
+                        94,
+                        "salt bridges",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 153,
+                "sent": "The apolar and polar interactions between these two modules likely explaining why they do not fold independently compared with other glycoside hydrolases that contain CBMs.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        33,
+                        "apolar and polar interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        133,
+                        153,
+                        "glycoside hydrolases",
+                        "protein_type"
+                    ],
+                    [
+                        167,
+                        171,
+                        "CBMs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 154,
+                "sent": "CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of \u223c450, 350, and 500 \u212b2, respectively, to form a compact heterotetramer.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        20,
+                        34,
+                        "central domain",
+                        "structure_element"
+                    ],
+                    [
+                        42,
+                        56,
+                        "interacts with",
+                        "protein_state"
+                    ],
+                    [
+                        57,
+                        62,
+                        "CtGH5",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        70,
+                        "CtCBM6",
+                        "structure_element"
+                    ],
+                    [
+                        76,
+                        81,
+                        "CtFn3",
+                        "structure_element"
+                    ],
+                    [
+                        98,
+                        112,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        201,
+                        208,
+                        "compact",
+                        "protein_state"
+                    ],
+                    [
+                        209,
+                        223,
+                        "heterotetramer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 155,
+                "sent": "With respect to the CtCBM6-CBM13 interface, the linker (SPISTGTIP) between the two modules, extending from Ser514 to Pro522, adopts a fixed conformation.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        20,
+                        42,
+                        "CtCBM6-CBM13 interface",
+                        "site"
+                    ],
+                    [
+                        48,
+                        54,
+                        "linker",
+                        "structure_element"
+                    ],
+                    [
+                        56,
+                        65,
+                        "SPISTGTIP",
+                        "structure_element"
+                    ],
+                    [
+                        83,
+                        90,
+                        "modules",
+                        "structure_element"
+                    ],
+                    [
+                        107,
+                        113,
+                        "Ser514",
+                        "residue_name_number"
+                    ],
+                    [
+                        117,
+                        123,
+                        "Pro522",
+                        "residue_name_number"
+                    ],
+                    [
+                        134,
+                        152,
+                        "fixed conformation",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 156,
+                "sent": "Such sequences are normally extremely flexible; however, the two Ile residues make extensive apolar contacts within the linker and with the two CBMs, leading to conformational stabilization.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        65,
+                        68,
+                        "Ile",
+                        "residue_name"
+                    ],
+                    [
+                        93,
+                        108,
+                        "apolar contacts",
+                        "bond_interaction"
+                    ],
+                    [
+                        120,
+                        126,
+                        "linker",
+                        "structure_element"
+                    ],
+                    [
+                        144,
+                        148,
+                        "CBMs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 157,
+                "sent": "The interactions between CtGH5 and the two CBMs, which are mediated by the tip of the loop between \u03b2-7 and \u03b1-7 (loop 7) of CtGH5, not only stabilize the trimodular clover-like structure but also make a contribution to catalytic function.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        30,
+                        "CtGH5",
+                        "structure_element"
+                    ],
+                    [
+                        43,
+                        47,
+                        "CBMs",
+                        "structure_element"
+                    ],
+                    [
+                        86,
+                        90,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        99,
+                        102,
+                        "\u03b2-7",
+                        "structure_element"
+                    ],
+                    [
+                        107,
+                        110,
+                        "\u03b1-7",
+                        "structure_element"
+                    ],
+                    [
+                        112,
+                        118,
+                        "loop 7",
+                        "structure_element"
+                    ],
+                    [
+                        123,
+                        128,
+                        "CtGH5",
+                        "structure_element"
+                    ],
+                    [
+                        153,
+                        170,
+                        "trimodular clover",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 158,
+                "sent": "Central to the interactions between the three modules is Trp285, which is intercalated between the two CBMs.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        46,
+                        53,
+                        "modules",
+                        "structure_element"
+                    ],
+                    [
+                        57,
+                        63,
+                        "Trp285",
+                        "residue_name_number"
+                    ],
+                    [
+                        74,
+                        94,
+                        "intercalated between",
+                        "bond_interaction"
+                    ],
+                    [
+                        103,
+                        107,
+                        "CBMs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 159,
+                "sent": "The N\u03f5 of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        38,
+                        52,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        83,
+                        89,
+                        "Val615",
+                        "residue_name_number"
+                    ],
+                    [
+                        94,
+                        100,
+                        "Gly616",
+                        "residue_name_number"
+                    ],
+                    [
+                        104,
+                        111,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        147,
+                        162,
+                        "apolar contacts",
+                        "bond_interaction"
+                    ],
+                    [
+                        168,
+                        174,
+                        "CtCBM6",
+                        "structure_element"
+                    ],
+                    [
+                        176,
+                        182,
+                        "Pro440",
+                        "residue_name_number"
+                    ],
+                    [
+                        184,
+                        190,
+                        "Phe489",
+                        "residue_name_number"
+                    ],
+                    [
+                        192,
+                        198,
+                        "Gly491",
+                        "residue_name_number"
+                    ],
+                    [
+                        204,
+                        210,
+                        "Ala492",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 160,
+                "sent": "Indeed, loop 7 is completely disordered in the truncated derivative of CtXyl5A comprising CtGH5 and CtCBM6, demonstrating that the interactions with CtCBM13 stabilize the conformation of this loop.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        14,
+                        "loop 7",
+                        "structure_element"
+                    ],
+                    [
+                        18,
+                        39,
+                        "completely disordered",
+                        "protein_state"
+                    ],
+                    [
+                        47,
+                        56,
+                        "truncated",
+                        "protein_state"
+                    ],
+                    [
+                        71,
+                        78,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        90,
+                        95,
+                        "CtGH5",
+                        "structure_element"
+                    ],
+                    [
+                        100,
+                        106,
+                        "CtCBM6",
+                        "structure_element"
+                    ],
+                    [
+                        149,
+                        156,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        192,
+                        196,
+                        "loop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 161,
+                "sent": "Although the tip of loop 7 does not directly contribute to the topology of the active site, it is only \u223c12 \u212b from the catalytic nucleophile Glu279.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        20,
+                        26,
+                        "loop 7",
+                        "structure_element"
+                    ],
+                    [
+                        79,
+                        90,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        140,
+                        146,
+                        "Glu279",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 162,
+                "sent": "Thus, any perturbation of the loop (through the removal of CtCBM13) is likely to influence the electrostatic and apolar environment of the catalytic apparatus, which could explain the reduction in activity associated with the deletion of CtCBM13.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        30,
+                        34,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        48,
+                        55,
+                        "removal",
+                        "experimental_method"
+                    ],
+                    [
+                        59,
+                        66,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        226,
+                        234,
+                        "deletion",
+                        "experimental_method"
+                    ],
+                    [
+                        238,
+                        245,
+                        "CtCBM13",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 163,
+                "sent": "Similar to the interactions between CtCBM6 and CtCBM13, there are extensive hydrophobic interactions between CtCBM13 and CtFn3, resulting in very little flexibility between these modules.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        36,
+                        42,
+                        "CtCBM6",
+                        "structure_element"
+                    ],
+                    [
+                        47,
+                        54,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        76,
+                        100,
+                        "hydrophobic interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        109,
+                        116,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        121,
+                        126,
+                        "CtFn3",
+                        "structure_element"
+                    ],
+                    [
+                        179,
+                        186,
+                        "modules",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 164,
+                "sent": "As stated above, the absence of CtCBM62 in the structure suggests that the module can adopt multiple positions with respect to the rest of the protein.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        21,
+                        31,
+                        "absence of",
+                        "protein_state"
+                    ],
+                    [
+                        32,
+                        39,
+                        "CtCBM62",
+                        "structure_element"
+                    ],
+                    [
+                        47,
+                        56,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        75,
+                        81,
+                        "module",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 165,
+                "sent": "The CtCBM62, by binding to its ligands (d-Galp and l-Arap) in plant cell walls, may be able to recruit the enzyme onto its target substrate.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "CtCBM62",
+                        "structure_element"
+                    ],
+                    [
+                        16,
+                        26,
+                        "binding to",
+                        "protein_state"
+                    ],
+                    [
+                        40,
+                        46,
+                        "d-Galp",
+                        "chemical"
+                    ],
+                    [
+                        51,
+                        57,
+                        "l-Arap",
+                        "chemical"
+                    ],
+                    [
+                        62,
+                        67,
+                        "plant",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 166,
+                "sent": "Xylans are not generally thought to contain such sugars.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        6,
+                        "Xylans",
+                        "chemical"
+                    ],
+                    [
+                        49,
+                        55,
+                        "sugars",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 167,
+                "sent": "d-Galp, however, has been detected in xylans in the outer layer of cereal grains and in eucalyptus trees, which are substrates used by CtXyl5A.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        6,
+                        "d-Galp",
+                        "chemical"
+                    ],
+                    [
+                        38,
+                        44,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        67,
+                        73,
+                        "cereal",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        88,
+                        104,
+                        "eucalyptus trees",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        135,
+                        142,
+                        "CtXyl5A",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 168,
+                "sent": "Thus, CtCBM62 may direct the enzyme to particularly complex xylans containing d-Galp at the non-reducing termini of the side chains, consistent with the open substrate binding cleft of the arabinoxylanase that is optimized to bind highly decorated forms of the hemicellulose.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        6,
+                        13,
+                        "CtCBM62",
+                        "structure_element"
+                    ],
+                    [
+                        60,
+                        66,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        78,
+                        84,
+                        "d-Galp",
+                        "chemical"
+                    ],
+                    [
+                        153,
+                        157,
+                        "open",
+                        "protein_state"
+                    ],
+                    [
+                        158,
+                        181,
+                        "substrate binding cleft",
+                        "site"
+                    ],
+                    [
+                        189,
+                        204,
+                        "arabinoxylanase",
+                        "protein_type"
+                    ],
+                    [
+                        261,
+                        274,
+                        "hemicellulose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 169,
+                "sent": "In general CBMs have little influence on enzyme activity against soluble substrates but have a significant impact on glycans within plant cell walls.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        15,
+                        "CBMs",
+                        "structure_element"
+                    ],
+                    [
+                        117,
+                        124,
+                        "glycans",
+                        "chemical"
+                    ],
+                    [
+                        132,
+                        137,
+                        "plant",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 170,
+                "sent": "Thus, the role of CBM62 will likely only be evident against insoluble composite substrates.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        23,
+                        "CBM62",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 171,
+                "sent": "Exploring GH5 Subfamily 34",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        10,
+                        26,
+                        "GH5 Subfamily 34",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 172,
+                "sent": "CtXyl5A is a member of a seven-protein subfamily of GH5, GH5_34.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        52,
+                        55,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        57,
+                        63,
+                        "GH5_34",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 173,
+                "sent": "Four of these proteins are distinct, whereas the other three members are essentially identical (derived from different strains of C. thermocellum).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        130,
+                        145,
+                        "C. thermocellum",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 174,
+                "sent": "To investigate further the substrate specificity within this subfamily, recombinant forms of three members of GH5_34 that were distinct from CtXyl5A were generated.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        110,
+                        116,
+                        "GH5_34",
+                        "protein_type"
+                    ],
+                    [
+                        141,
+                        148,
+                        "CtXyl5A",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 175,
+                "sent": "AcGH5 has a similar molecular architecture to CtXyl5A with the exception of an additional carbohydrate esterase family 6 module at the C terminus (Fig. 1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "AcGH5",
+                        "protein"
+                    ],
+                    [
+                        46,
+                        53,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        90,
+                        127,
+                        "carbohydrate esterase family 6 module",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 176,
+                "sent": "The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        10,
+                        "GH5_34",
+                        "protein_type"
+                    ],
+                    [
+                        16,
+                        32,
+                        "Verrucomicrobiae",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        33,
+                        42,
+                        "bacterium",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        44,
+                        49,
+                        "VbGH5",
+                        "protein"
+                    ],
+                    [
+                        64,
+                        78,
+                        "GH5-CBM6-CBM13",
+                        "structure_element"
+                    ],
+                    [
+                        114,
+                        132,
+                        "Fn3-CBM62-dockerin",
+                        "structure_element"
+                    ],
+                    [
+                        153,
+                        160,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        182,
+                        200,
+                        "Laminin_3_G domain",
+                        "structure_element"
+                    ],
+                    [
+                        283,
+                        296,
+                        "carbohydrates",
+                        "chemical"
+                    ],
+                    [
+                        312,
+                        318,
+                        "glycan",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 177,
+                "sent": "The Verrucomicobiae enzyme also has an N-terminal GH43 subfamily 10 (GH43_10) catalytic module.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        19,
+                        "Verrucomicobiae",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        50,
+                        67,
+                        "GH43 subfamily 10",
+                        "protein_type"
+                    ],
+                    [
+                        69,
+                        76,
+                        "GH43_10",
+                        "protein_type"
+                    ],
+                    [
+                        78,
+                        94,
+                        "catalytic module",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 178,
+                "sent": "The fungal GH5_34, GpGH5, unlike the two bacterial homologs, comprises a single GH5 catalytic module lacking all of the other accessory modules (Fig. 1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        10,
+                        "fungal",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        11,
+                        17,
+                        "GH5_34",
+                        "protein_type"
+                    ],
+                    [
+                        19,
+                        24,
+                        "GpGH5",
+                        "protein"
+                    ],
+                    [
+                        41,
+                        50,
+                        "bacterial",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        80,
+                        83,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        84,
+                        100,
+                        "catalytic module",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 179,
+                "sent": "GpGh5 is particularly interesting as Gonapodya prolifera is the only fungus of the several hundred fungal genomes that encodes a GH5_34 enzyme.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "GpGh5",
+                        "protein"
+                    ],
+                    [
+                        37,
+                        56,
+                        "Gonapodya prolifera",
+                        "species"
+                    ],
+                    [
+                        69,
+                        75,
+                        "fungus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        99,
+                        105,
+                        "fungal",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        129,
+                        135,
+                        "GH5_34",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 180,
+                "sent": "In fact there are four potential GH5_34 sequences in the G. prolifera genome, all of which show high sequence homology to Clostridium GH5_34 sequences.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        33,
+                        39,
+                        "GH5_34",
+                        "protein_type"
+                    ],
+                    [
+                        57,
+                        69,
+                        "G. prolifera",
+                        "species"
+                    ],
+                    [
+                        122,
+                        133,
+                        "Clostridium",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        134,
+                        140,
+                        "GH5_34",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 181,
+                "sent": "G. prolifera and Clostridium occupy similar environments, suggesting that the GpGH5_34 gene was acquired from a Clostridium species, which was followed by duplication of the gene in the fungal genome.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        12,
+                        "G. prolifera",
+                        "species"
+                    ],
+                    [
+                        17,
+                        28,
+                        "Clostridium",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        78,
+                        86,
+                        "GpGH5_34",
+                        "protein"
+                    ],
+                    [
+                        112,
+                        123,
+                        "Clostridium",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        186,
+                        192,
+                        "fungal",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 182,
+                "sent": "The sequence identity of the GH5_34 catalytic modules with CtXyl5A ranged from 55 to 80% (supplemental Fig. S1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        35,
+                        "GH5_34",
+                        "protein_type"
+                    ],
+                    [
+                        36,
+                        53,
+                        "catalytic modules",
+                        "structure_element"
+                    ],
+                    [
+                        59,
+                        66,
+                        "CtXyl5A",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 183,
+                "sent": "All the GH5_34 enzymes were active on the arabinoxylans RAX, WAX, and CX but displayed no activity on BX (Table 1 and Fig. 6) and are thus defined as arabinoxylanases.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        14,
+                        "GH5_34",
+                        "protein_type"
+                    ],
+                    [
+                        42,
+                        55,
+                        "arabinoxylans",
+                        "chemical"
+                    ],
+                    [
+                        56,
+                        59,
+                        "RAX",
+                        "chemical"
+                    ],
+                    [
+                        61,
+                        64,
+                        "WAX",
+                        "chemical"
+                    ],
+                    [
+                        70,
+                        72,
+                        "CX",
+                        "chemical"
+                    ],
+                    [
+                        102,
+                        104,
+                        "BX",
+                        "chemical"
+                    ],
+                    [
+                        150,
+                        166,
+                        "arabinoxylanases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 184,
+                "sent": "The limit products generated by CtXyl5A, AcGH5, and GpGH5 comprised a range of oligosaccharides with some high molecular weight material.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        32,
+                        39,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        41,
+                        46,
+                        "AcGH5",
+                        "protein"
+                    ],
+                    [
+                        52,
+                        57,
+                        "GpGH5",
+                        "protein"
+                    ],
+                    [
+                        79,
+                        95,
+                        "oligosaccharides",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 185,
+                "sent": "The oligosaccharides with low degrees of polymerization were absent in the VbGH5 reaction products.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        20,
+                        "oligosaccharides",
+                        "chemical"
+                    ],
+                    [
+                        75,
+                        80,
+                        "VbGH5",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 186,
+                "sent": "However, the enzyme generated a large amount of arabinose, which was not produced by the other arabinoxylanases.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        48,
+                        57,
+                        "arabinose",
+                        "chemical"
+                    ],
+                    [
+                        95,
+                        111,
+                        "arabinoxylanases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 187,
+                "sent": "Given that GH43_10 is predominantly an arabinofuranosidase subfamily of GH43, the arabinose generated by VbGH5 is likely mediated by the N-terminal catalytic module (see below).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        18,
+                        "GH43_10",
+                        "protein_type"
+                    ],
+                    [
+                        39,
+                        58,
+                        "arabinofuranosidase",
+                        "protein_type"
+                    ],
+                    [
+                        72,
+                        76,
+                        "GH43",
+                        "protein_type"
+                    ],
+                    [
+                        82,
+                        91,
+                        "arabinose",
+                        "chemical"
+                    ],
+                    [
+                        105,
+                        110,
+                        "VbGH5",
+                        "protein"
+                    ],
+                    [
+                        148,
+                        164,
+                        "catalytic module",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 188,
+                "sent": "Kinetic analysis showed that AcGH5 displayed similar activity to CtXyl5A against both WAX and RAX and was 2-fold less active against CX.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        34,
+                        "AcGH5",
+                        "protein"
+                    ],
+                    [
+                        65,
+                        72,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        86,
+                        89,
+                        "WAX",
+                        "chemical"
+                    ],
+                    [
+                        94,
+                        97,
+                        "RAX",
+                        "chemical"
+                    ],
+                    [
+                        133,
+                        135,
+                        "CX",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 189,
+                "sent": "When initially measuring the activity of wild type VbGH5 against the different substrates, no clear data could be obtained, regardless of the concentration of enzyme used the reaction appeared to cease after a few minutes.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        41,
+                        50,
+                        "wild type",
+                        "protein_state"
+                    ],
+                    [
+                        51,
+                        56,
+                        "VbGH5",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 190,
+                "sent": "We hypothesized that the N-terminal GH43_10 rapidly removed single arabinose decorations from the arabinoxylans depleting the substrate available to the arabinoxylanase, explaining why this activity was short lived.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        36,
+                        43,
+                        "GH43_10",
+                        "protein_type"
+                    ],
+                    [
+                        67,
+                        76,
+                        "arabinose",
+                        "chemical"
+                    ],
+                    [
+                        98,
+                        111,
+                        "arabinoxylans",
+                        "chemical"
+                    ],
+                    [
+                        153,
+                        168,
+                        "arabinoxylanase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 191,
+                "sent": "To test this hypothesis, the conserved catalytic base (Asp45) of the GH43_10 module of VbGH5 was substituted with alanine, which is predicted to inactivate this catalytic module.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        38,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        55,
+                        60,
+                        "Asp45",
+                        "residue_name_number"
+                    ],
+                    [
+                        69,
+                        76,
+                        "GH43_10",
+                        "structure_element"
+                    ],
+                    [
+                        87,
+                        92,
+                        "VbGH5",
+                        "protein"
+                    ],
+                    [
+                        97,
+                        113,
+                        "substituted with",
+                        "experimental_method"
+                    ],
+                    [
+                        114,
+                        121,
+                        "alanine",
+                        "residue_name"
+                    ],
+                    [
+                        161,
+                        177,
+                        "catalytic module",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 192,
+                "sent": "The D45A mutant did not produce arabinose consistent with the arabinofuranosidase activity displayed by the GH43_10 module in the wild type enzyme (Fig. 6).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "D45A",
+                        "mutant"
+                    ],
+                    [
+                        9,
+                        15,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        32,
+                        41,
+                        "arabinose",
+                        "chemical"
+                    ],
+                    [
+                        62,
+                        81,
+                        "arabinofuranosidase",
+                        "protein_type"
+                    ],
+                    [
+                        108,
+                        115,
+                        "GH43_10",
+                        "structure_element"
+                    ],
+                    [
+                        130,
+                        139,
+                        "wild type",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 193,
+                "sent": "The kinetics of the GH5_34 arabinoxylanase catalytic module was now measurable, and activities were determined to be between \u223c6- and 10-fold lower than that of CtXyl5A.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        12,
+                        "kinetics",
+                        "evidence"
+                    ],
+                    [
+                        20,
+                        26,
+                        "GH5_34",
+                        "protein_type"
+                    ],
+                    [
+                        27,
+                        42,
+                        "arabinoxylanase",
+                        "protein_type"
+                    ],
+                    [
+                        43,
+                        59,
+                        "catalytic module",
+                        "structure_element"
+                    ],
+                    [
+                        160,
+                        167,
+                        "CtXyl5A",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 194,
+                "sent": "Interestingly, the fungal arabinoxylanase displays the highest activities against WAX and RAX, \u223c4- and 6-fold higher, respectively, than CtXyl5A; however, there is very little difference in the activity between the eukaryotic and prokaryotic enzymes against CX.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        25,
+                        "fungal",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        26,
+                        41,
+                        "arabinoxylanase",
+                        "protein_type"
+                    ],
+                    [
+                        82,
+                        85,
+                        "WAX",
+                        "chemical"
+                    ],
+                    [
+                        90,
+                        93,
+                        "RAX",
+                        "chemical"
+                    ],
+                    [
+                        137,
+                        144,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        215,
+                        225,
+                        "eukaryotic",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        230,
+                        241,
+                        "prokaryotic",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        258,
+                        260,
+                        "CX",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 195,
+                "sent": "Attempts to express individual modules of a variety of truncations of AcGH5 and VbGH5 were unsuccessful.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        70,
+                        75,
+                        "AcGH5",
+                        "protein"
+                    ],
+                    [
+                        80,
+                        85,
+                        "VbGH5",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 196,
+                "sent": "This may indicate that the individual modules can only fold correctly when incorporated into the full-length enzyme, demonstrating the importance of intermodule interactions to maintain the structural integrity of these enzymes.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        97,
+                        108,
+                        "full-length",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 197,
+                "sent": "Products profile generated of GH5_34 enzymes.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        30,
+                        36,
+                        "GH5_34",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 198,
+                "sent": "The enzymes at 1 \u03bcm were incubated with the four different xylans at 1% in 50 mm sodium phosphate buffer for 16 h at 37 \u00b0C (GpGH5, VbGH5, and AcGH5) or 60 \u00b0C.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        25,
+                        34,
+                        "incubated",
+                        "experimental_method"
+                    ],
+                    [
+                        59,
+                        65,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        124,
+                        129,
+                        "GpGH5",
+                        "protein"
+                    ],
+                    [
+                        131,
+                        136,
+                        "VbGH5",
+                        "protein"
+                    ],
+                    [
+                        142,
+                        147,
+                        "AcGH5",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 199,
+                "sent": "The limit products were separated by TLC.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        37,
+                        40,
+                        "TLC",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 200,
+                "sent": "The xylooligosaccharide standards (X) are indicated by their degrees of polymerization.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        23,
+                        "xylooligosaccharide",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 201,
+                "sent": "A characteristic feature of enzymes that attack the plant cell wall is their complex molecular architecture.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        52,
+                        57,
+                        "plant",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 202,
+                "sent": "The CBMs in these enzymes generally play a role in substrate targeting and are appended to the catalytic modules through flexible linker sequences.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "CBMs",
+                        "structure_element"
+                    ],
+                    [
+                        95,
+                        112,
+                        "catalytic modules",
+                        "structure_element"
+                    ],
+                    [
+                        121,
+                        146,
+                        "flexible linker sequences",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 203,
+                "sent": "CtXyl5A provides a rare visualization of the structure of multiple modules within a single enzyme.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        45,
+                        54,
+                        "structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 204,
+                "sent": "The central feature of these data is the structural role played by two of the CBMs, CtCBM6 and CtCBM13, in maintaining the active conformation of the catalytic module, CtGH5.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        78,
+                        82,
+                        "CBMs",
+                        "structure_element"
+                    ],
+                    [
+                        84,
+                        90,
+                        "CtCBM6",
+                        "structure_element"
+                    ],
+                    [
+                        95,
+                        102,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        123,
+                        129,
+                        "active",
+                        "protein_state"
+                    ],
+                    [
+                        150,
+                        166,
+                        "catalytic module",
+                        "structure_element"
+                    ],
+                    [
+                        168,
+                        173,
+                        "CtGH5",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 205,
+                "sent": "The crystallographic data described here are supported by biochemical data showing either that these two modules do not bind to glycans (CtCBM13) or that the recognition of the non-reducing end of xylan or cellulose chains (CtCBM6) is unlikely to be biologically significant.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        25,
+                        "crystallographic data",
+                        "evidence"
+                    ],
+                    [
+                        128,
+                        135,
+                        "glycans",
+                        "chemical"
+                    ],
+                    [
+                        137,
+                        144,
+                        "CtCBM13",
+                        "structure_element"
+                    ],
+                    [
+                        197,
+                        202,
+                        "xylan",
+                        "chemical"
+                    ],
+                    [
+                        206,
+                        215,
+                        "cellulose",
+                        "chemical"
+                    ],
+                    [
+                        224,
+                        230,
+                        "CtCBM6",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 206,
+                "sent": "It should be emphasized, however, that glycan binding and substrate targeting may only be evident in the full-length enzyme acting on highly complex structures such as the plant cell wall, as observed recently by a CBM46 module in the Bacillus xyloglucanase/mixed linked glucanase BhCel5B.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        39,
+                        45,
+                        "glycan",
+                        "chemical"
+                    ],
+                    [
+                        105,
+                        116,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        172,
+                        177,
+                        "plant",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        215,
+                        220,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        235,
+                        243,
+                        "Bacillus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        244,
+                        257,
+                        "xyloglucanase",
+                        "protein_type"
+                    ],
+                    [
+                        258,
+                        280,
+                        "mixed linked glucanase",
+                        "protein_type"
+                    ],
+                    [
+                        281,
+                        288,
+                        "BhCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 207,
+                "sent": "CtXyl5A is a member of GH5 that contains 6644 members.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        23,
+                        26,
+                        "GH5",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 208,
+                "sent": "CtXyl5A is a member of subfamily GH5_34.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        33,
+                        39,
+                        "GH5_34",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 209,
+                "sent": "Despite differences in sequence identity all of the homologs were shown to be arabinoxylanases.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        78,
+                        94,
+                        "arabinoxylanases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 210,
+                "sent": "Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the \u22122* subsite, which are 1,3-linked to the xylose positioned in the active site.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        68,
+                        74,
+                        "GH5_34",
+                        "protein_type"
+                    ],
+                    [
+                        89,
+                        113,
+                        "specificity determinants",
+                        "site"
+                    ],
+                    [
+                        114,
+                        119,
+                        "Glu68",
+                        "residue_name_number"
+                    ],
+                    [
+                        121,
+                        126,
+                        "Tyr92",
+                        "residue_name_number"
+                    ],
+                    [
+                        132,
+                        138,
+                        "Asn139",
+                        "residue_name_number"
+                    ],
+                    [
+                        182,
+                        188,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        192,
+                        201,
+                        "arabinose",
+                        "chemical"
+                    ],
+                    [
+                        209,
+                        220,
+                        "\u22122* subsite",
+                        "site"
+                    ],
+                    [
+                        250,
+                        256,
+                        "xylose",
+                        "chemical"
+                    ],
+                    [
+                        275,
+                        286,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 211,
+                "sent": "The presence of a CBM62 in CtXyl5A and AcGH5 suggests that these enzymes target highly complex xylans that contain d-galactose in their side chains.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        18,
+                        23,
+                        "CBM62",
+                        "structure_element"
+                    ],
+                    [
+                        27,
+                        34,
+                        "CtXyl5A",
+                        "protein"
+                    ],
+                    [
+                        39,
+                        44,
+                        "AcGH5",
+                        "protein"
+                    ],
+                    [
+                        95,
+                        101,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        115,
+                        126,
+                        "d-galactose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 212,
+                "sent": "The absence of a \u201cnon-structural\u201d CBM in GpGH5 may indicate that this arabinoxylanase is designed to target simpler arabinoxylans present in the endosperm of cereals.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        14,
+                        "absence of",
+                        "protein_state"
+                    ],
+                    [
+                        34,
+                        37,
+                        "CBM",
+                        "structure_element"
+                    ],
+                    [
+                        41,
+                        46,
+                        "GpGH5",
+                        "protein"
+                    ],
+                    [
+                        70,
+                        85,
+                        "arabinoxylanase",
+                        "protein_type"
+                    ],
+                    [
+                        116,
+                        129,
+                        "arabinoxylans",
+                        "chemical"
+                    ],
+                    [
+                        158,
+                        165,
+                        "cereals",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 213,
+                "sent": "Although the characterization of all members of GH5_34 suggests that this subfamily is monospecific, differences in specificity are observed in other subfamilies of GHs including GH43 and GH5.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        48,
+                        54,
+                        "GH5_34",
+                        "protein_type"
+                    ],
+                    [
+                        165,
+                        168,
+                        "GHs",
+                        "protein_type"
+                    ],
+                    [
+                        179,
+                        183,
+                        "GH43",
+                        "protein_type"
+                    ],
+                    [
+                        188,
+                        191,
+                        "GH5",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 214,
+                "sent": "Thus, as new members of GH5_34 are identified from genomic sequence data and subsequently characterized, the specificity of this family may require reinterpretation.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        24,
+                        30,
+                        "GH5_34",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 215,
+                "sent": "An intriguing feature of VbGH5 is that the limited products generated by this enzymes are much larger than those produced by the other arabinoxylanases.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        25,
+                        30,
+                        "VbGH5",
+                        "protein"
+                    ],
+                    [
+                        135,
+                        151,
+                        "arabinoxylanases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 216,
+                "sent": "This suggests that although arabinose decorations contribute to enzyme specificity (VbGH5 is not active on xylans lacking arabinose side chains), the enzyme requires other specificity determinants that occur less frequently in arabinoxylans.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        28,
+                        37,
+                        "arabinose",
+                        "chemical"
+                    ],
+                    [
+                        84,
+                        89,
+                        "VbGH5",
+                        "protein"
+                    ],
+                    [
+                        107,
+                        113,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        122,
+                        131,
+                        "arabinose",
+                        "chemical"
+                    ],
+                    [
+                        227,
+                        240,
+                        "arabinoxylans",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 217,
+                "sent": "This has some resonance with a recently described GH98 xylanase that also exploits specificity determinants that occur infrequently and are only evident in highly complex xylans (e.g. CX).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        50,
+                        54,
+                        "GH98",
+                        "protein_type"
+                    ],
+                    [
+                        55,
+                        63,
+                        "xylanase",
+                        "protein_type"
+                    ],
+                    [
+                        171,
+                        177,
+                        "xylans",
+                        "chemical"
+                    ],
+                    [
+                        184,
+                        186,
+                        "CX",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 218,
+                "sent": "To conclude, this study provides the molecular basis for the specificity displayed by arabinoxylanases.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        86,
+                        102,
+                        "arabinoxylanases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 219,
+                "sent": "Substrate specificity is dominated by the pocket that binds single arabinose or xylose side chains.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        42,
+                        48,
+                        "pocket",
+                        "site"
+                    ],
+                    [
+                        67,
+                        76,
+                        "arabinose",
+                        "chemical"
+                    ],
+                    [
+                        80,
+                        86,
+                        "xylose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 220,
+                "sent": "The open xylan binding cleft explains why the enzyme is able to attack highly decorated forms of the hemicellulose.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "open",
+                        "protein_state"
+                    ],
+                    [
+                        9,
+                        28,
+                        "xylan binding cleft",
+                        "site"
+                    ],
+                    [
+                        101,
+                        114,
+                        "hemicellulose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 221,
+                "sent": "It is also evident that appending additional catalytic modules and CBMs onto the core components of these enzymes generates bespoke arabinoxylanases with activities optimized for specific functions.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        45,
+                        62,
+                        "catalytic modules",
+                        "structure_element"
+                    ],
+                    [
+                        67,
+                        71,
+                        "CBMs",
+                        "structure_element"
+                    ],
+                    [
+                        132,
+                        148,
+                        "arabinoxylanases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 222,
+                "sent": "The specificities of the arabinoxylanases described here are distinct from the classical endo-xylanases and thus have the potential to contribute to the toolbox of biocatalysts required by industries that exploit the plant cell wall as a sustainable substrate.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        25,
+                        41,
+                        "arabinoxylanases",
+                        "protein_type"
+                    ],
+                    [
+                        89,
+                        103,
+                        "endo-xylanases",
+                        "protein_type"
+                    ],
+                    [
+                        217,
+                        222,
+                        "plant",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 223,
+                "sent": "Data collection and refinement statistics",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        41,
+                        "Data collection and refinement statistics",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 224,
+                "sent": "\tCtXyl5A-D\tGH5-CBM6-Arap\tGH5-CBM6-Xylp\tGH5-CBM6- (Araf-Xylp4)\t \tData collection\t\t\t\t\t \t\u2003\u2003\u2003\u2003Source\tESRF-ID14-1\tDiamond I04\u20131\tDiamond I24\tDiamond I02\t \t\u2003\u2003\u2003\u2003Wavelength (\u00c5)\t0.9334\t0.9173\t0.9772\t0.9791\t \t\u2003\u2003\u2003\u2003Space group\tP21212\tP212121\tP212121\tP212121\t \t\u2003\u2003\u2003\u2003Cell dimensions\t\t\t\t\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003a, b, c (\u00c5)\t147.4, 191.7, 50.7\t67.1, 72.4, 109.1\t67.9, 72.5, 109.5\t76.3, 123.2, 125.4\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u03b1, \u03b2, \u03b3 (\u00b0)\t90, 90, 90\t90, 90, 90\t90, 90, 90\t90, 90, 90\t \t\u2003\u2003\u2003\u2003No. of measured reflections\t244,475 (29,324)\t224,842 (11,281)\t152,004 (4,996)\t463,237 (23,068)\t \t\u2003\u2003\u2003\u2003No. of independent reflections\t42246 (5,920)\t63,523 (3,175)\t42,716 (2,334)\t140,288 (6,879)\t \t\u2003\u2003\u2003\u2003Resolution (\u00c5)\t50.70\u20132.64 (2.78\u20132.64)\t44.85\u20131.65 (1.68\u20131.65)\t45.16\u20131.90 (1.94\u20131.90)\t48.43\u20131.65 (1.68\u20131.65)\t \t\u2003\u2003\u2003\u2003Rmerge (%)\t16.5 (69.5)\t6.7 (65.1)\t2.8 (8.4)\t5.7 (74.9)\t \t\u2003\u2003\u2003\u2003CC1/2\t0.985 (0.478)\t0.998 (0.594)\t0.999 (0.982)\t0.998 (0.484)\t \t\u2003\u2003\u2003\u2003I/\u03c3I\t8.0 (2.0)\t13 (1.6)\t26.6 (8.0)\t11.2 (1.6)\t \t\u2003\u2003\u2003\u2003Completeness (%)\t98.5 (96.4)\t98.5 (99.4)\t98.6 (85.0)\t98.8 (99.4)\t \t\u2003\u2003\u2003\u2003Redundancy\t5.8 (5.0)\t3.5 (3.6)\t3.6 (2.1)\t3.3 (3.4)\t \t\t \tRefinement\t\t\t\t\t \t\u2003\u2003\u2003\u2003Rwork/Rfree\t23.7/27.8\t12.2/17.0\t12.9/16.1\t14.5/19.9\t \t\u2003\u2003\u2003\u2003No. atoms\t\t\t\t\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Protein\t5446\t3790\t3729\t7333\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Ligand\t19\t20\t20\t92\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Water\t227\t579\t601\t923\t \t\u2003\u2003\u2003\u2003B-factors\t\t\t\t\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Protein\t41.6\t17.8\t15.8\t21.0\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Ligand\t65.0\t19.4\t24.2\t39.5\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Water\t35.4\t38.5\t32.2\t37.6\t \t\u2003\u2003\u2003\u2003R.m.s deviations\t\t\t\t\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Bond lengths (\u00c5)\t0.008\t0.015\t0.012\t0.012\t \t\u2003\u2003\u2003\u2003\u2003\u2003\u2003\u2003Bond angles (\u00b0)\t1.233\t1.502\t1.624\t1.554\t \t\u2003\u2003\u2003\u2003Protein Data Bank code\t5G56\t5LA0\t5LA1\t2LA2\t \t",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        1,
+                        10,
+                        "CtXyl5A-D",
+                        "mutant"
+                    ],
+                    [
+                        11,
+                        24,
+                        "GH5-CBM6-Arap",
+                        "complex_assembly"
+                    ],
+                    [
+                        25,
+                        38,
+                        "GH5-CBM6-Xylp",
+                        "complex_assembly"
+                    ],
+                    [
+                        39,
+                        61,
+                        "GH5-CBM6- (Araf-Xylp4)",
+                        "complex_assembly"
+                    ],
+                    [
+                        1079,
+                        1084,
+                        "Rwork",
+                        "evidence"
+                    ],
+                    [
+                        1085,
+                        1090,
+                        "Rfree",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 225,
+                "sent": "GH",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        2,
+                        "GH",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 226,
+                "sent": "glycoside hydrolase",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        19,
+                        "glycoside hydrolase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 227,
+                "sent": "CtXyl5A",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "CtXyl5A",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 228,
+                "sent": "C. thermocellum arabinoxylanase",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        15,
+                        "C. thermocellum",
+                        "species"
+                    ],
+                    [
+                        16,
+                        31,
+                        "arabinoxylanase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 229,
+                "sent": "CBM",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "CBM",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 230,
+                "sent": "non-catalytic carbohydrate binding module",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        41,
+                        "non-catalytic carbohydrate binding module",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 231,
+                "sent": "Fn",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        2,
+                        "Fn",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 232,
+                "sent": "fibronectin",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "fibronectin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 233,
+                "sent": "WAX",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "WAX",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 234,
+                "sent": "wheat arabinoxylan",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "wheat",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        6,
+                        18,
+                        "arabinoxylan",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 235,
+                "sent": "RAX",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "RAX",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 236,
+                "sent": "rye arabinoxylan",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "rye",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        4,
+                        16,
+                        "arabinoxylan",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 237,
+                "sent": "CX",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        2,
+                        "CX",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 238,
+                "sent": "corn bran xylan",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "corn",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        10,
+                        15,
+                        "xylan",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 239,
+                "sent": "HPAEC",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "HPAEC",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 240,
+                "sent": "high performance anion exchange chromatography",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        46,
+                        "high performance anion exchange chromatography",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 241,
+                "sent": "birchwood xylan",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "birchwood",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        10,
+                        15,
+                        "xylan",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 242,
+                "sent": "electrospray ionization.",
+                "section": "SUPPL",
+                "ner": [
+                    [
+                        0,
+                        23,
+                        "electrospray ionization",
+                        "experimental_method"
+                    ]
+                ]
+            }
+        ]
+    },
+    "PMC4980666": {
+        "annotations": [
+            {
+                "sid": 0,
+                "sent": "N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes",
+                "section": "TITLE",
+                "ner": [
+                    [
+                        0,
+                        15,
+                        "N-acylhydrazone",
+                        "chemical"
+                    ],
+                    [
+                        30,
+                        39,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        40,
+                        45,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        46,
+                        48,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        49,
+                        61,
+                        "endonuclease",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 1,
+                "sent": "Influenza virus PA endonuclease has recently emerged as an attractive target for the development of novel antiviral therapeutics.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        10,
+                        15,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        16,
+                        18,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        19,
+                        31,
+                        "endonuclease",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 2,
+                "sent": "This is an enzyme with divalent metal ion(s) (Mg2+ or Mn2+) in its catalytic site: chelation of these metal cofactors is an attractive strategy to inhibit enzymatic activity.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        46,
+                        50,
+                        "Mg2+",
+                        "chemical"
+                    ],
+                    [
+                        54,
+                        58,
+                        "Mn2+",
+                        "chemical"
+                    ],
+                    [
+                        67,
+                        81,
+                        "catalytic site",
+                        "site"
+                    ],
+                    [
+                        83,
+                        92,
+                        "chelation",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 3,
+                "sent": "Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        43,
+                        59,
+                        "N-acylhydrazones",
+                        "chemical"
+                    ],
+                    [
+                        66,
+                        81,
+                        "enzymatic assay",
+                        "experimental_method"
+                    ],
+                    [
+                        87,
+                        89,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        90,
+                        94,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        95,
+                        107,
+                        "endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        123,
+                        163,
+                        "cell-based influenza vRNP reconstitution",
+                        "experimental_method"
+                    ],
+                    [
+                        168,
+                        186,
+                        "virus yield assays",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 4,
+                "sent": "Several N-acylhydrazones were found to have promising anti-influenza activity in the low micromolar concentration range and good selectivity.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        8,
+                        24,
+                        "N-acylhydrazones",
+                        "chemical"
+                    ],
+                    [
+                        59,
+                        68,
+                        "influenza",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 5,
+                "sent": "Computational docking studies are carried on to investigate the key features that determine inhibition of the endonuclease enzyme by N-acylhydrazones.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        29,
+                        "Computational docking studies",
+                        "experimental_method"
+                    ],
+                    [
+                        110,
+                        122,
+                        "endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        133,
+                        149,
+                        "N-acylhydrazones",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 6,
+                "sent": "Moreover, we here describe the crystal structure of PA-Nter in complex with one of the most active inhibitors, revealing its interactions within the protein\u2019s active site.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        31,
+                        48,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        52,
+                        54,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        55,
+                        59,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        60,
+                        75,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        159,
+                        170,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 7,
+                "sent": "Influenza virus is an enveloped virus with a segmented negative-oriented single-stranded RNA genome, belonging to the Orthomyxoviridae.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        10,
+                        15,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        22,
+                        37,
+                        "enveloped virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        55,
+                        92,
+                        "negative-oriented single-stranded RNA",
+                        "chemical"
+                    ],
+                    [
+                        118,
+                        134,
+                        "Orthomyxoviridae",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 8,
+                "sent": "Seasonal influenza A and B viruses affect each year approximately 5\u201310% of the adult and 20\u201330% of the paediatric population, and there is a permanent risk of sudden influenza pandemics, such as the notorious \u2018Spanish flu\u2019 in 1918 and the swine-origin H1N1 pandemic in 2009.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        9,
+                        20,
+                        "influenza A",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        25,
+                        26,
+                        "B",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        27,
+                        34,
+                        "viruses",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        166,
+                        175,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        252,
+                        256,
+                        "H1N1",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 9,
+                "sent": "Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        20,
+                        29,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        30,
+                        35,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        71,
+                        76,
+                        "viral",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        77,
+                        91,
+                        "M2 ion-channel",
+                        "protein_type"
+                    ],
+                    [
+                        93,
+                        103,
+                        "amantadine",
+                        "chemical"
+                    ],
+                    [
+                        108,
+                        119,
+                        "rimantadine",
+                        "chemical"
+                    ],
+                    [
+                        131,
+                        136,
+                        "viral",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        137,
+                        150,
+                        "neuraminidase",
+                        "protein_type"
+                    ],
+                    [
+                        152,
+                        161,
+                        "zanamivir",
+                        "chemical"
+                    ],
+                    [
+                        166,
+                        177,
+                        "oseltamivir",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 10,
+                "sent": "The M2 inhibitors have limited clinical utility due to their central nervous system side effects and widespread resistance, as in the case of the 2009 pandemic H1N1 virus; resistance is also a growing concern for oseltamivir.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        6,
+                        "M2",
+                        "protein_type"
+                    ],
+                    [
+                        160,
+                        164,
+                        "H1N1",
+                        "species"
+                    ],
+                    [
+                        165,
+                        170,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        213,
+                        224,
+                        "oseltamivir",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 11,
+                "sent": "The influenza virus polymerase complex is composed of three subunits: PB1, PB2 and PA.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        14,
+                        19,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        20,
+                        30,
+                        "polymerase",
+                        "protein_type"
+                    ],
+                    [
+                        70,
+                        73,
+                        "PB1",
+                        "protein"
+                    ],
+                    [
+                        75,
+                        78,
+                        "PB2",
+                        "protein"
+                    ],
+                    [
+                        83,
+                        85,
+                        "PA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 12,
+                "sent": "The PA subunit performs the \u2018cap-snatching\u2019 endonuclease reaction, the PB2 subunit is responsible for initial binding of the capped RNAs, while the actual RNA synthesis is performed by the PB1 protein.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        6,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        7,
+                        14,
+                        "subunit",
+                        "structure_element"
+                    ],
+                    [
+                        44,
+                        56,
+                        "endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        71,
+                        74,
+                        "PB2",
+                        "protein"
+                    ],
+                    [
+                        75,
+                        82,
+                        "subunit",
+                        "structure_element"
+                    ],
+                    [
+                        125,
+                        136,
+                        "capped RNAs",
+                        "chemical"
+                    ],
+                    [
+                        155,
+                        158,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        189,
+                        192,
+                        "PB1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 13,
+                "sent": "Given its crucial role in the viral life cycle, the influenza virus polymerase is widely recognized as a superior target for antiviral drug development and, in particular, inhibition of the PA endonuclease has deserved much attention in recent years.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        30,
+                        35,
+                        "viral",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        52,
+                        61,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        62,
+                        67,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        68,
+                        78,
+                        "polymerase",
+                        "protein_type"
+                    ],
+                    [
+                        190,
+                        192,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        193,
+                        205,
+                        "endonuclease",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 14,
+                "sent": "The endonuclease catalytic site resides in the N-terminal domain of PA (PA-Nter; residues 1~195).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        16,
+                        "endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        17,
+                        31,
+                        "catalytic site",
+                        "site"
+                    ],
+                    [
+                        47,
+                        64,
+                        "N-terminal domain",
+                        "structure_element"
+                    ],
+                    [
+                        68,
+                        70,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        72,
+                        74,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        75,
+                        79,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        90,
+                        95,
+                        "1~195",
+                        "residue_range"
+                    ]
+                ]
+            },
+            {
+                "sid": 15,
+                "sent": "It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        15,
+                        24,
+                        "histidine",
+                        "residue_name"
+                    ],
+                    [
+                        26,
+                        31,
+                        "His41",
+                        "residue_name_number"
+                    ],
+                    [
+                        56,
+                        74,
+                        "strictly conserved",
+                        "protein_state"
+                    ],
+                    [
+                        75,
+                        81,
+                        "acidic",
+                        "protein_state"
+                    ],
+                    [
+                        92,
+                        97,
+                        "Glu80",
+                        "residue_name_number"
+                    ],
+                    [
+                        99,
+                        105,
+                        "Asp108",
+                        "residue_name_number"
+                    ],
+                    [
+                        107,
+                        113,
+                        "Glu119",
+                        "residue_name_number"
+                    ],
+                    [
+                        122,
+                        132,
+                        "coordinate",
+                        "bond_interaction"
+                    ],
+                    [
+                        148,
+                        154,
+                        "Ile120",
+                        "residue_name_number"
+                    ],
+                    [
+                        175,
+                        184,
+                        "manganese",
+                        "chemical"
+                    ],
+                    [
+                        188,
+                        197,
+                        "magnesium",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 16,
+                "sent": "Since the intracellular concentration of Mg2+ is at least 1000-fold higher than that of Mn2+, magnesium may be more biologically relevant.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        41,
+                        45,
+                        "Mg2+",
+                        "chemical"
+                    ],
+                    [
+                        88,
+                        93,
+                        "Mn2+,",
+                        "chemical"
+                    ],
+                    [
+                        94,
+                        103,
+                        "magnesium",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 17,
+                "sent": "A controversy about number and type of metal ions exists also for the active site of HIV-1 integrase.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        70,
+                        81,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        85,
+                        90,
+                        "HIV-1",
+                        "species"
+                    ],
+                    [
+                        91,
+                        100,
+                        "integrase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 18,
+                "sent": "HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "HIV-1",
+                        "species"
+                    ],
+                    [
+                        6,
+                        15,
+                        "integrase",
+                        "protein_type"
+                    ],
+                    [
+                        113,
+                        118,
+                        "metal",
+                        "chemical"
+                    ],
+                    [
+                        134,
+                        139,
+                        "viral",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        168,
+                        170,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        242,
+                        251,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        252,
+                        264,
+                        "endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        296,
+                        318,
+                        "2,4-dioxobutanoic acid",
+                        "chemical"
+                    ],
+                    [
+                        332,
+                        341,
+                        "flutimide",
+                        "chemical"
+                    ],
+                    [
+                        363,
+                        384,
+                        "2-hydroxyphenyl amide",
+                        "chemical"
+                    ],
+                    [
+                        409,
+                        423,
+                        "tetramic acids",
+                        "chemical"
+                    ],
+                    [
+                        425,
+                        449,
+                        "5-hydroxypyrimidin-4-one",
+                        "chemical"
+                    ],
+                    [
+                        463,
+                        474,
+                        "marchantins",
+                        "chemical"
+                    ],
+                    [
+                        479,
+                        488,
+                        "green tea",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        489,
+                        498,
+                        "catechins",
+                        "chemical"
+                    ],
+                    [
+                        505,
+                        531,
+                        "epigallocatechin-3-gallate",
+                        "chemical"
+                    ],
+                    [
+                        533,
+                        537,
+                        "EGCG",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 19,
+                "sent": "In recent years, we focused our research on chemical scaffolds that are able to chelate metal ions of PA-Nter, resulting in inhibition of influenza virus replication.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        102,
+                        104,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        105,
+                        109,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        138,
+                        147,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        148,
+                        153,
+                        "virus",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 20,
+                "sent": "N-acylhydrazones represent an appealing class of chelating ligands with a broad spectrum of biological activities, such as activity against HIV, hepatitis A, vaccinia and influenza virus.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        16,
+                        "N-acylhydrazones",
+                        "chemical"
+                    ],
+                    [
+                        80,
+                        88,
+                        "spectrum",
+                        "evidence"
+                    ],
+                    [
+                        140,
+                        143,
+                        "HIV",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        145,
+                        156,
+                        "hepatitis A",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        158,
+                        166,
+                        "vaccinia",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        171,
+                        180,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        181,
+                        186,
+                        "virus",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 21,
+                "sent": "In the present work, we report the biological activity of a series of N-acylhydrazones (Fig. 2), as determined in an enzymatic assay with PA-Nter endonuclease as well as in cell-based influenza viral ribonucleoprotein (vRNP) reconstitution and virus yield assays.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        70,
+                        86,
+                        "N-acylhydrazones",
+                        "chemical"
+                    ],
+                    [
+                        117,
+                        132,
+                        "enzymatic assay",
+                        "experimental_method"
+                    ],
+                    [
+                        138,
+                        140,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        141,
+                        145,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        146,
+                        158,
+                        "endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        173,
+                        239,
+                        "cell-based influenza viral ribonucleoprotein (vRNP) reconstitution",
+                        "experimental_method"
+                    ],
+                    [
+                        244,
+                        262,
+                        "virus yield assays",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 22,
+                "sent": "Several N-acylhydrazones were found to have promising anti-influenza activity with 50% effective concentration values (EC50) in the range of 3\u201320\u2009\u03bcM and good selectivity (Table 1 and Fig. 3).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        8,
+                        24,
+                        "N-acylhydrazones",
+                        "chemical"
+                    ],
+                    [
+                        59,
+                        68,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        83,
+                        110,
+                        "50% effective concentration",
+                        "evidence"
+                    ],
+                    [
+                        119,
+                        123,
+                        "EC50",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 23,
+                "sent": "Computational docking studies of two candidate ligands in the PA-Nter active site gave information about the features that could determine inhibition of endonuclease activity.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        29,
+                        "Computational docking studies",
+                        "experimental_method"
+                    ],
+                    [
+                        62,
+                        64,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        65,
+                        69,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        70,
+                        81,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        153,
+                        165,
+                        "endonuclease",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 24,
+                "sent": "Moreover, we describe the X-ray crystal structure of PA-Nter in complex with one of the most active inhibitors.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        26,
+                        49,
+                        "X-ray crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        53,
+                        55,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        56,
+                        60,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        61,
+                        76,
+                        "in complex with",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 25,
+                "sent": "N-acylhydrazones 1\u201327 (Fig. 2) were prepared in high yields by following literature methods (Fig. 2A); they were characterized by spectroscopic tools, mass spectrometry and elemental analysis.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        16,
+                        "N-acylhydrazones",
+                        "chemical"
+                    ],
+                    [
+                        17,
+                        21,
+                        "1\u201327",
+                        "chemical"
+                    ],
+                    [
+                        151,
+                        168,
+                        "mass spectrometry",
+                        "experimental_method"
+                    ],
+                    [
+                        173,
+                        191,
+                        "elemental analysis",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 26,
+                "sent": "Even if isomerism around the C\u2009=\u2009N bond is possible, 1\u201327 are present in the E form in solution, as evidenced by the chemical shift values of the HC\u2009=\u2009N and NH protons in the 1H-NMR spectrum.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        53,
+                        57,
+                        "1\u201327",
+                        "chemical"
+                    ],
+                    [
+                        175,
+                        181,
+                        "1H-NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        182,
+                        190,
+                        "spectrum",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 27,
+                "sent": "Exceptions are represented by the alkyl-derivatives 3 and 4 (2:1 and 5:3 E:Z ratio, respectively).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        52,
+                        53,
+                        "3",
+                        "chemical"
+                    ],
+                    [
+                        58,
+                        59,
+                        "4",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 28,
+                "sent": "If R\u2019 (Fig. 2A) is a 2-hydroxy substituted phenyl ring, the corresponding acylhydrazones can coordinate one or, depending on denticity, two metal centers (modes A and B in Fig. 4).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        74,
+                        88,
+                        "acylhydrazones",
+                        "chemical"
+                    ],
+                    [
+                        93,
+                        103,
+                        "coordinate",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 29,
+                "sent": "Starting from N\u2019-(2,3-dihydroxybenzylidene)-semicarbazide (1) and its methoxy-analogue (2), we modified the acylhydrazonic substituent R\u201d (3\u20138, 18, 19, Fig. 2A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        57,
+                        "N\u2019-(2,3-dihydroxybenzylidene)-semicarbazide",
+                        "chemical"
+                    ],
+                    [
+                        59,
+                        60,
+                        "1",
+                        "chemical"
+                    ],
+                    [
+                        88,
+                        89,
+                        "2",
+                        "chemical"
+                    ],
+                    [
+                        139,
+                        142,
+                        "3\u20138",
+                        "chemical"
+                    ],
+                    [
+                        144,
+                        146,
+                        "18",
+                        "chemical"
+                    ],
+                    [
+                        148,
+                        150,
+                        "19",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 30,
+                "sent": "In 18 and 19, also the gallic moiety can be involved in the chelation of the metal cofactors (mode C, Fig. 4).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        3,
+                        5,
+                        "18",
+                        "chemical"
+                    ],
+                    [
+                        10,
+                        12,
+                        "19",
+                        "chemical"
+                    ],
+                    [
+                        23,
+                        29,
+                        "gallic",
+                        "chemical"
+                    ],
+                    [
+                        60,
+                        69,
+                        "chelation",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 31,
+                "sent": "In order to investigate the role of hydroxyl substituents 9\u201311, 13\u201317, 20\u201323 and 27 were also synthesized.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        58,
+                        62,
+                        "9\u201311",
+                        "chemical"
+                    ],
+                    [
+                        64,
+                        69,
+                        "13\u201317",
+                        "chemical"
+                    ],
+                    [
+                        71,
+                        76,
+                        "20\u201323",
+                        "chemical"
+                    ],
+                    [
+                        81,
+                        83,
+                        "27",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 32,
+                "sent": "Compound 12 was synthesized in order to confirm the crucial influence of the gallic moiety.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        11,
+                        "12",
+                        "chemical"
+                    ],
+                    [
+                        77,
+                        83,
+                        "gallic",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 33,
+                "sent": "Finally, 26 was here considered, because it is an inhibitor of HIV RNase H, another enzyme with two magnesium ions in its active site.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        11,
+                        "26",
+                        "chemical"
+                    ],
+                    [
+                        63,
+                        66,
+                        "HIV",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        67,
+                        74,
+                        "RNase H",
+                        "protein"
+                    ],
+                    [
+                        100,
+                        109,
+                        "magnesium",
+                        "chemical"
+                    ],
+                    [
+                        122,
+                        133,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 34,
+                "sent": "Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        37,
+                        53,
+                        "N-acylhydrazones",
+                        "chemical"
+                    ],
+                    [
+                        74,
+                        83,
+                        "chelation",
+                        "bond_interaction"
+                    ],
+                    [
+                        100,
+                        105,
+                        "metal",
+                        "chemical"
+                    ],
+                    [
+                        125,
+                        134,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        135,
+                        137,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        138,
+                        142,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        143,
+                        154,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        226,
+                        228,
+                        "19",
+                        "chemical"
+                    ],
+                    [
+                        230,
+                        233,
+                        "H2L",
+                        "chemical"
+                    ],
+                    [
+                        243,
+                        247,
+                        "Mg2+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 35,
+                "sent": "Different reaction conditions were used (1:1 and 1:2 metal to ligand ratio, up to 4 equivalents of triethylamine), but in any case the same chemical species Mg(HL)2\u22194H2O was recovered and conveniently characterized.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        99,
+                        112,
+                        "triethylamine",
+                        "chemical"
+                    ],
+                    [
+                        157,
+                        169,
+                        "Mg(HL)2\u22194H2O",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 36,
+                "sent": "The use of a coordinating solvent as d6-DMSO causes partial decoordination of the ligand, but the 1H-NMR spectrum in MeOD, instead, shows only the signals attributable to the complex.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        37,
+                        44,
+                        "d6-DMSO",
+                        "chemical"
+                    ],
+                    [
+                        98,
+                        104,
+                        "1H-NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        105,
+                        113,
+                        "spectrum",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 37,
+                "sent": "In the 13C-NMR spectrum, the signal of the C\u2009=\u2009O quaternary carbon is practically unaffected by complexation, suggesting that the C\u2009=\u2009O group is weakly involved in the coordination to the metal ion.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        7,
+                        14,
+                        "13C-NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        15,
+                        23,
+                        "spectrum",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 38,
+                "sent": "This is confirmed, in the IR spectrum, by the shift of about 20\u2009cm\u22121 of the C\u2009=\u2009O absorption, while a shift of 30\u201350\u2009cm\u22121 is expected when the carbonylic oxygen is tightly bound to the metal ion.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        26,
+                        28,
+                        "IR",
+                        "experimental_method"
+                    ],
+                    [
+                        29,
+                        37,
+                        "spectrum",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 39,
+                "sent": "ESI-mass spectra and elemental analysis confirmed the formula Mg(HL)2\u22194H2O.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "ESI-mass",
+                        "experimental_method"
+                    ],
+                    [
+                        9,
+                        16,
+                        "spectra",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        39,
+                        "elemental analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        62,
+                        74,
+                        "Mg(HL)2\u22194H2O",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 40,
+                "sent": "The interaction between the N-acylhydrazone ligands and the magnesium cation was investigated also by means of UV-visible spectroscopy (UV-visible titrations of 23 and 19 with increasing amount of Mg(CH3COO)2 are shown in Figure S1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        28,
+                        43,
+                        "N-acylhydrazone",
+                        "chemical"
+                    ],
+                    [
+                        60,
+                        69,
+                        "magnesium",
+                        "chemical"
+                    ],
+                    [
+                        111,
+                        134,
+                        "UV-visible spectroscopy",
+                        "experimental_method"
+                    ],
+                    [
+                        136,
+                        157,
+                        "UV-visible titrations",
+                        "experimental_method"
+                    ],
+                    [
+                        161,
+                        163,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        168,
+                        170,
+                        "19",
+                        "chemical"
+                    ],
+                    [
+                        176,
+                        193,
+                        "increasing amount",
+                        "experimental_method"
+                    ],
+                    [
+                        197,
+                        208,
+                        "Mg(CH3COO)2",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 41,
+                "sent": "The spectrum of 19 includes a band at 313\u2009nm assignable to n-\u03c0* transitions of the C\u2009=\u2009N and C\u2009=\u2009O groups.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        12,
+                        "spectrum",
+                        "evidence"
+                    ],
+                    [
+                        16,
+                        18,
+                        "19",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 42,
+                "sent": "By adding increasing equivalents of Mg(CH3COO)2, the absorption around 400\u2009nm increases, and a new band appears with a maximum at 397\u2009nm.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        36,
+                        47,
+                        "Mg(CH3COO)2",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 43,
+                "sent": "When the same experiment was performed with 23, a different behavior was observed.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        44,
+                        46,
+                        "23",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 44,
+                "sent": "Increasing concentration of Mg2+, in fact, caused a diminution in the maximum absorption, an isosbestic point is visible at about 345\u2009nm, but a new band at 400\u2009nm does not appear.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        28,
+                        33,
+                        "Mg2+,",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 45,
+                "sent": "Ligands 19 and 23 coordinate the Mg2+ ions in different ways: 19 chelates the metal ion by using the deprotonated salicyl oxygen and the iminic nitrogen, while for 23, the gallic moiety is supposed to be involved (Fig. 4A,B versus C), leading to different, less extensive, modifications of the UV spectrum.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        10,
+                        "19",
+                        "chemical"
+                    ],
+                    [
+                        15,
+                        17,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        18,
+                        28,
+                        "coordinate",
+                        "bond_interaction"
+                    ],
+                    [
+                        33,
+                        37,
+                        "Mg2+",
+                        "chemical"
+                    ],
+                    [
+                        62,
+                        64,
+                        "19",
+                        "chemical"
+                    ],
+                    [
+                        164,
+                        166,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        294,
+                        296,
+                        "UV",
+                        "experimental_method"
+                    ],
+                    [
+                        297,
+                        305,
+                        "spectrum",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 46,
+                "sent": "Inhibition of the PA-Nter enzyme",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        20,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        21,
+                        25,
+                        "Nter",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 47,
+                "sent": "All the compounds were tested for their ability to inhibit the influenza endonuclease in an enzymatic plasmid-based assay with recombinant PA-Nter, as well as in cell-based influenza methods (i.e. virus yield and vRNP reconstitution assays).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        63,
+                        72,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        73,
+                        85,
+                        "endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        92,
+                        121,
+                        "enzymatic plasmid-based assay",
+                        "experimental_method"
+                    ],
+                    [
+                        139,
+                        141,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        142,
+                        146,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        162,
+                        190,
+                        "cell-based influenza methods",
+                        "experimental_method"
+                    ],
+                    [
+                        197,
+                        239,
+                        "virus yield and vRNP reconstitution assays",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 48,
+                "sent": "The results are shown in Table 1 and summarized in Fig. 3 to visualize the structure-activity relationships; Figure S2 shows the dose-response curves for three representative compounds (i.e. 10, 13 and 23) in either the PA-enzyme or vRNP reconstitution assay.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        129,
+                        149,
+                        "dose-response curves",
+                        "evidence"
+                    ],
+                    [
+                        191,
+                        193,
+                        "10",
+                        "chemical"
+                    ],
+                    [
+                        195,
+                        197,
+                        "13",
+                        "chemical"
+                    ],
+                    [
+                        202,
+                        204,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        220,
+                        258,
+                        "PA-enzyme or vRNP reconstitution assay",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 49,
+                "sent": "The moderate activity (IC50\u2009=\u200924\u2009\u03bcM) of N\u2019-2,3-dihydroxybenzylidene semicarbazide (1) was completely lost when the NH2 moiety was replaced by a hydrophobic heptyl chain (3), but it is less affected when a phenyl or a 2-hydroxyphenyl is present (5 and 7, IC50\u2009=\u200984 and 54\u2009\u03bcM, respectively).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        23,
+                        27,
+                        "IC50",
+                        "evidence"
+                    ],
+                    [
+                        40,
+                        81,
+                        "N\u2019-2,3-dihydroxybenzylidene semicarbazide",
+                        "chemical"
+                    ],
+                    [
+                        83,
+                        84,
+                        "1",
+                        "chemical"
+                    ],
+                    [
+                        170,
+                        171,
+                        "3",
+                        "chemical"
+                    ],
+                    [
+                        245,
+                        246,
+                        "5",
+                        "chemical"
+                    ],
+                    [
+                        251,
+                        252,
+                        "7",
+                        "chemical"
+                    ],
+                    [
+                        254,
+                        258,
+                        "IC50",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 50,
+                "sent": "When the hydroxyl in position 3 on R1 (2,3-dihydroxybenzylidene) was replaced by a methoxy group (2-hydroxy-3-methoxybenzylidene), the activity disappeared (compounds 2, 4, 6 and 8).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        39,
+                        63,
+                        "2,3-dihydroxybenzylidene",
+                        "chemical"
+                    ],
+                    [
+                        98,
+                        128,
+                        "2-hydroxy-3-methoxybenzylidene",
+                        "chemical"
+                    ],
+                    [
+                        167,
+                        168,
+                        "2",
+                        "chemical"
+                    ],
+                    [
+                        170,
+                        171,
+                        "4",
+                        "chemical"
+                    ],
+                    [
+                        173,
+                        174,
+                        "6",
+                        "chemical"
+                    ],
+                    [
+                        179,
+                        180,
+                        "8",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 51,
+                "sent": "The activity is unaffected (IC50 values ranging from 45 to 75\u2009\u03bcM) when going from two hydroxyls in R1 (7) to compounds with three hydroxyls (i.e. 9, 10 and 11).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        28,
+                        32,
+                        "IC50",
+                        "evidence"
+                    ],
+                    [
+                        103,
+                        104,
+                        "7",
+                        "chemical"
+                    ],
+                    [
+                        146,
+                        147,
+                        "9",
+                        "chemical"
+                    ],
+                    [
+                        149,
+                        151,
+                        "10",
+                        "chemical"
+                    ],
+                    [
+                        156,
+                        158,
+                        "11",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 52,
+                "sent": "Similarly, 11 (R1\u2009=\u20093,4,5-trihydroxyphenyl, R2\u2009=\u20092-hydroxyphenyl) had comparable activity as 27 (R1\u2009=\u20093,4,5-trihydroxyphenyl, R2\u2009=\u2009NH2).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        13,
+                        "11",
+                        "chemical"
+                    ],
+                    [
+                        93,
+                        95,
+                        "27",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 53,
+                "sent": "Within the series carrying a 2-hydroxyphenyl R2 group, the activity of 11 is particularly intriguing.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        71,
+                        73,
+                        "11",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 54,
+                "sent": "11 does not have the possibility to chelate in a tridentate ONO fashion (mode A in Fig. 4), but it can coordinate two cations by means of its three OH groups in R1 (mode C, Fig. 4).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        2,
+                        "11",
+                        "chemical"
+                    ],
+                    [
+                        103,
+                        113,
+                        "coordinate",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 55,
+                "sent": "Note that a similar chelating mode was observed in a crystal structure, solved by Cusack and coworkers, of PA-Nter endonuclease in complex with the inhibitor EGCG.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        53,
+                        70,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        107,
+                        109,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        110,
+                        114,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        115,
+                        127,
+                        "endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        128,
+                        143,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        158,
+                        162,
+                        "EGCG",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 56,
+                "sent": "The PA-Nter inhibitory activity strongly depends on the number and position of hydroxyl substituents in R1 and R2: this is clearly highlighted by the data obtained with compounds 13\u201323, in which R2 is a 3,4,5-trihydroxyphenyl (gallic) group, the most active scaffold in our series.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        6,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        7,
+                        11,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        179,
+                        184,
+                        "13\u201323",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 57,
+                "sent": "The analogue carrying an unsubstituted aromatic ring as R1 (compound 13) had moderate activity (IC50\u2009=\u200969\u2009\u03bcM).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        69,
+                        71,
+                        "13",
+                        "chemical"
+                    ],
+                    [
+                        96,
+                        100,
+                        "IC50",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 58,
+                "sent": "When one OH was added at position 2 of the R1 ring (14), the activity was lost.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        52,
+                        54,
+                        "14",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 59,
+                "sent": "Adding a second OH substituent at position 5 resulted in strong activity (compound 15, IC50\u2009=\u20099\u2009\u03bcM); medium activity for a 3-OH (18; IC50\u2009=\u200983\u2009\u03bcM), and marginal activity when the second OH is at position 4 (17, IC50\u2009\u2265\u2009370\u2009\u03bcM).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        83,
+                        85,
+                        "15",
+                        "chemical"
+                    ],
+                    [
+                        87,
+                        91,
+                        "IC50",
+                        "evidence"
+                    ],
+                    [
+                        129,
+                        131,
+                        "18",
+                        "chemical"
+                    ],
+                    [
+                        133,
+                        137,
+                        "IC50",
+                        "evidence"
+                    ],
+                    [
+                        207,
+                        209,
+                        "17",
+                        "chemical"
+                    ],
+                    [
+                        211,
+                        215,
+                        "IC50",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 60,
+                "sent": "The addition of a 3-methoxy group (19) abolished all inhibitory activity.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        35,
+                        37,
+                        "19",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 61,
+                "sent": "This cannot be related to variations in the chelating features displayed by the R1 moiety, since compounds 14\u201319 all have, in theory, the capacity to chelate one metal ion through the ortho-OH and iminic nitrogen (mode A in Fig. 4).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        107,
+                        112,
+                        "14\u201319",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 62,
+                "sent": "Moreover, compound 18 can, in principle, chelate the two M2+ ions in the active site according to mode B (Fig. 4), yet it (IC50\u2009=\u200983\u2009\u03bcM) has nine-fold lower activity than 15, that does not possess this two-metal chelating feature.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        21,
+                        "18",
+                        "chemical"
+                    ],
+                    [
+                        57,
+                        60,
+                        "M2+",
+                        "chemical"
+                    ],
+                    [
+                        73,
+                        84,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        123,
+                        127,
+                        "IC50",
+                        "evidence"
+                    ],
+                    [
+                        171,
+                        173,
+                        "15",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 63,
+                "sent": "Therefore, we hypothesized that the inhibitory activity of the series containing the gallic moiety is determined by: (i) the capacity of the moiety R2 to chelate two metal ions in the active site of the enzyme, according to mode C (Fig. 4); and (ii) the presence and position of one or more hydroxyl substituents in R1, which may possibly result in ligand-protein interactions (e.g. through hydrogen bonds).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        184,
+                        195,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        391,
+                        405,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 64,
+                "sent": "This assumption was supported by molecular docking calculations and X-ray analysis of inhibitor 23 in complex with PA-Nter (vide infra).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        33,
+                        63,
+                        "molecular docking calculations",
+                        "experimental_method"
+                    ],
+                    [
+                        68,
+                        82,
+                        "X-ray analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        96,
+                        98,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        99,
+                        114,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        115,
+                        117,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        118,
+                        122,
+                        "Nter",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 65,
+                "sent": "Substitution of the 5-hydroxyl in 15 by a methoxy group (16) causes a dramatic drop in activity (IC50\u2009=\u20099 and 454\u2009\u03bcM for 15 and 16, respectively).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        36,
+                        "15",
+                        "chemical"
+                    ],
+                    [
+                        57,
+                        59,
+                        "16",
+                        "chemical"
+                    ],
+                    [
+                        97,
+                        101,
+                        "IC50",
+                        "evidence"
+                    ],
+                    [
+                        121,
+                        123,
+                        "15",
+                        "chemical"
+                    ],
+                    [
+                        128,
+                        130,
+                        "16",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 66,
+                "sent": "In particular, all the compounds with a trihydroxylated phenyl group as R1 (i.e. 20, 21, 22 and 23) were able to inhibit PA-Nter quite potently.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        81,
+                        83,
+                        "20",
+                        "chemical"
+                    ],
+                    [
+                        85,
+                        87,
+                        "21",
+                        "chemical"
+                    ],
+                    [
+                        89,
+                        91,
+                        "22",
+                        "chemical"
+                    ],
+                    [
+                        96,
+                        98,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        121,
+                        123,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        124,
+                        128,
+                        "Nter",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 67,
+                "sent": "The lowest IC50 values were obtained for 21 and 23 (IC50\u2009=\u200913 and 7\u2009\u03bcM, respectively), which both have one of their three hydroxyl groups at position 5.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        15,
+                        "IC50",
+                        "evidence"
+                    ],
+                    [
+                        41,
+                        43,
+                        "21",
+                        "chemical"
+                    ],
+                    [
+                        48,
+                        50,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        52,
+                        56,
+                        "IC50",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 68,
+                "sent": "The most active compound in this series was 23, which lacks the hydroxyl group at position 2 of R1, further confirming that this function is undesirable or even detrimental for inhibitory activity against PA-Nter, as already noticed above for 14.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        44,
+                        46,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        205,
+                        207,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        208,
+                        212,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        243,
+                        245,
+                        "14",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 69,
+                "sent": "Consistent with a crucial role of the R2 gallic moiety in metal chelation, the strong activity of 15 was completely lost in its 3,4,5-trimethoxy analogue 12.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        64,
+                        73,
+                        "chelation",
+                        "bond_interaction"
+                    ],
+                    [
+                        98,
+                        100,
+                        "15",
+                        "chemical"
+                    ],
+                    [
+                        154,
+                        156,
+                        "12",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 70,
+                "sent": "On the other hand, the R2 gallic containing compounds displayed moderate activity (IC50 values around 40\u2009\u03bcM) when R1 was absent (i.e. the 3,4,5-trihydroxybenzohydrazide 28, Fig. 2), or composed of an extended ring system (26) or a pyrrole ring (25).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        83,
+                        87,
+                        "IC50",
+                        "evidence"
+                    ],
+                    [
+                        138,
+                        168,
+                        "3,4,5-trihydroxybenzohydrazide",
+                        "chemical"
+                    ],
+                    [
+                        169,
+                        171,
+                        "28",
+                        "chemical"
+                    ],
+                    [
+                        222,
+                        224,
+                        "26",
+                        "chemical"
+                    ],
+                    [
+                        245,
+                        247,
+                        "25",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 71,
+                "sent": "Still lower activity was seen with the pyridine analogue 24.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        57,
+                        59,
+                        "24",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 72,
+                "sent": "Evidently, the 3,4,5-trihydroxybenzyl moiety at R2 is fundamental but not sufficient to ensure potent PA-Nter endonuclease inhibition, since the interactions of R1 with the amino acid side chains of the protein appear crucial in modulating activity.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        102,
+                        104,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        105,
+                        109,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        110,
+                        122,
+                        "endonuclease",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 73,
+                "sent": "Inhibition of vRNP activity or virus replication in cells",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        18,
+                        "vRNP",
+                        "complex_assembly"
+                    ],
+                    [
+                        31,
+                        36,
+                        "virus",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 74,
+                "sent": "To determine the anti-influenza virus activity of compounds 1\u201328 in cell culture, we performed an influenza vRNP reconstitution assay in human embryonic kidney 293\u2009T (HEK293T) cells, then subjected the active compounds (i.e. EC50\u2009<\u2009100\u2009\u03bcM) to a virus yield assay in influenza virus-infected Madin-Darby canine kidney (MDCK) cells (Table 1 and Fig. 3).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        22,
+                        31,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        32,
+                        37,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        60,
+                        64,
+                        "1\u201328",
+                        "chemical"
+                    ],
+                    [
+                        98,
+                        133,
+                        "influenza vRNP reconstitution assay",
+                        "experimental_method"
+                    ],
+                    [
+                        137,
+                        142,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        225,
+                        229,
+                        "EC50",
+                        "evidence"
+                    ],
+                    [
+                        245,
+                        262,
+                        "virus yield assay",
+                        "experimental_method"
+                    ],
+                    [
+                        266,
+                        275,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        276,
+                        281,
+                        "virus",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 75,
+                "sent": "For some N-acylhydrazone compounds, we observed quite potent and selective activity in the vRNP reconstitution assay.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        24,
+                        "N-acylhydrazone",
+                        "chemical"
+                    ],
+                    [
+                        91,
+                        116,
+                        "vRNP reconstitution assay",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 76,
+                "sent": "This indicates that they are able to inhibit viral RNA synthesis and suggests that they could be classified as original PA inhibitors.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        45,
+                        50,
+                        "viral",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        51,
+                        54,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        120,
+                        122,
+                        "PA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 77,
+                "sent": "Values for EC50 (vRNP) or EC90 (virus yield) in the range of 0.4\u201318\u2009\u03bcM were obtained for compounds 15 and 20\u201323, which all carry a 3,4,5-trihydroxyphenyl as R2, and possess either two (15) or three (20\u201323) hydroxyl substituents in the R1 moiety.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        15,
+                        "EC50",
+                        "evidence"
+                    ],
+                    [
+                        17,
+                        21,
+                        "vRNP",
+                        "complex_assembly"
+                    ],
+                    [
+                        26,
+                        30,
+                        "EC90",
+                        "evidence"
+                    ],
+                    [
+                        32,
+                        37,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        99,
+                        101,
+                        "15",
+                        "chemical"
+                    ],
+                    [
+                        106,
+                        111,
+                        "20\u201323",
+                        "chemical"
+                    ],
+                    [
+                        185,
+                        187,
+                        "15",
+                        "chemical"
+                    ],
+                    [
+                        199,
+                        201,
+                        "20",
+                        "chemical"
+                    ],
+                    [
+                        202,
+                        204,
+                        "23",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 78,
+                "sent": "As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        10,
+                        34,
+                        "enzymatic PA-Nter assays",
+                        "experimental_method"
+                    ],
+                    [
+                        88,
+                        90,
+                        "21",
+                        "chemical"
+                    ],
+                    [
+                        92,
+                        94,
+                        "22",
+                        "chemical"
+                    ],
+                    [
+                        99,
+                        101,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        186,
+                        187,
+                        "9",
+                        "chemical"
+                    ],
+                    [
+                        189,
+                        191,
+                        "10",
+                        "chemical"
+                    ],
+                    [
+                        196,
+                        198,
+                        "11",
+                        "chemical"
+                    ],
+                    [
+                        201,
+                        203,
+                        "10",
+                        "chemical"
+                    ],
+                    [
+                        208,
+                        210,
+                        "22",
+                        "chemical"
+                    ],
+                    [
+                        239,
+                        243,
+                        "EC50",
+                        "evidence"
+                    ],
+                    [
+                        251,
+                        276,
+                        "vRNP reconstitution assay",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 79,
+                "sent": "The hydrazide 28 displayed weak (virus yield) to moderate (vRNP reconstitution) activity, albeit less than the most active molecules in the 3,4,5-trihydroxyphenyl series (i.e. 18 and 21\u201323).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "hydrazide",
+                        "chemical"
+                    ],
+                    [
+                        14,
+                        16,
+                        "28",
+                        "chemical"
+                    ],
+                    [
+                        33,
+                        38,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        59,
+                        78,
+                        "vRNP reconstitution",
+                        "experimental_method"
+                    ],
+                    [
+                        176,
+                        178,
+                        "18",
+                        "chemical"
+                    ],
+                    [
+                        183,
+                        188,
+                        "21\u201323",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 80,
+                "sent": "Even if there are no data indicating that the compounds reported in the paper are subject to hydrolysis, the activity of 28 could raise the concern that for some N-acylhydrazones the antiviral activity in cell culture may be related to their intracellular hydrolysis.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        121,
+                        123,
+                        "28",
+                        "chemical"
+                    ],
+                    [
+                        162,
+                        178,
+                        "N-acylhydrazones",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 81,
+                "sent": "However, this is unlikely, since the antiviral potency showed large differences (i.e. EC50 values between 0.42 and 29\u2009\u03bcM) for compounds with the same R2 but different R1 groups, meaning that R1 does play a role in modulating the antiviral effect.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        86,
+                        90,
+                        "EC50",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 82,
+                "sent": "Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50\u2009\u03bcM and a selectivity index (ratio of CC50 to EC50) below 8.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        32,
+                        56,
+                        "2,3-dihydroxybenzylidene",
+                        "chemical"
+                    ],
+                    [
+                        63,
+                        64,
+                        "3",
+                        "chemical"
+                    ],
+                    [
+                        66,
+                        67,
+                        "5",
+                        "chemical"
+                    ],
+                    [
+                        72,
+                        73,
+                        "7",
+                        "chemical"
+                    ],
+                    [
+                        78,
+                        108,
+                        "2-hydroxy-3-methoxybenzylidene",
+                        "chemical"
+                    ],
+                    [
+                        122,
+                        123,
+                        "4",
+                        "chemical"
+                    ],
+                    [
+                        125,
+                        126,
+                        "6",
+                        "chemical"
+                    ],
+                    [
+                        131,
+                        132,
+                        "8",
+                        "chemical"
+                    ],
+                    [
+                        177,
+                        187,
+                        "vRNP assay",
+                        "experimental_method"
+                    ],
+                    [
+                        194,
+                        198,
+                        "CC50",
+                        "evidence"
+                    ],
+                    [
+                        224,
+                        241,
+                        "selectivity index",
+                        "evidence"
+                    ],
+                    [
+                        252,
+                        256,
+                        "CC50",
+                        "evidence"
+                    ],
+                    [
+                        260,
+                        264,
+                        "EC50",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 83,
+                "sent": "Two notable exceptions are 18 and 19 (containing a 2,3-dihydroxybenzylidene or 2-hydroxy-3-methoxybenzylidene R1, respectively) which were not cytotoxic at 200\u2009\u03bcM and displayed favorable antiviral selectivity.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        27,
+                        29,
+                        "18",
+                        "chemical"
+                    ],
+                    [
+                        34,
+                        36,
+                        "19",
+                        "chemical"
+                    ],
+                    [
+                        51,
+                        75,
+                        "2,3-dihydroxybenzylidene",
+                        "chemical"
+                    ],
+                    [
+                        79,
+                        109,
+                        "2-hydroxy-3-methoxybenzylidene",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 84,
+                "sent": "Some N-acylhydrazone compounds were devoid of activity in the enzymatic assay, yet showed good to moderate efficacy in cell culture (e.g. 14 and 19, having EC50 values of 2.2 and 7.1\u2009\u03bcM, respectively).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        20,
+                        "N-acylhydrazone",
+                        "chemical"
+                    ],
+                    [
+                        62,
+                        77,
+                        "enzymatic assay",
+                        "experimental_method"
+                    ],
+                    [
+                        138,
+                        140,
+                        "14",
+                        "chemical"
+                    ],
+                    [
+                        145,
+                        147,
+                        "19",
+                        "chemical"
+                    ],
+                    [
+                        156,
+                        160,
+                        "EC50",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 85,
+                "sent": "For most of the active compounds (i.e. 9, 11, 13, 15\u201321, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        39,
+                        40,
+                        "9",
+                        "chemical"
+                    ],
+                    [
+                        42,
+                        44,
+                        "11",
+                        "chemical"
+                    ],
+                    [
+                        46,
+                        48,
+                        "13",
+                        "chemical"
+                    ],
+                    [
+                        50,
+                        55,
+                        "15\u201321",
+                        "chemical"
+                    ],
+                    [
+                        57,
+                        59,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        61,
+                        63,
+                        "24",
+                        "chemical"
+                    ],
+                    [
+                        68,
+                        70,
+                        "26",
+                        "chemical"
+                    ],
+                    [
+                        112,
+                        129,
+                        "cell-based assays",
+                        "experimental_method"
+                    ],
+                    [
+                        141,
+                        145,
+                        "EC50",
+                        "evidence"
+                    ],
+                    [
+                        169,
+                        179,
+                        "vRNP assay",
+                        "experimental_method"
+                    ],
+                    [
+                        219,
+                        223,
+                        "EC90",
+                        "evidence"
+                    ],
+                    [
+                        238,
+                        255,
+                        "virus yield assay",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 86,
+                "sent": "On the other hand, this difference was 8-fold or more for 7, 10, 14, 22, 25 and 28.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        58,
+                        59,
+                        "7",
+                        "chemical"
+                    ],
+                    [
+                        61,
+                        63,
+                        "10",
+                        "chemical"
+                    ],
+                    [
+                        65,
+                        67,
+                        "14",
+                        "chemical"
+                    ],
+                    [
+                        69,
+                        71,
+                        "22",
+                        "chemical"
+                    ],
+                    [
+                        73,
+                        75,
+                        "25",
+                        "chemical"
+                    ],
+                    [
+                        80,
+                        82,
+                        "28",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 87,
+                "sent": "Some N-acylhydrazone compounds showed good to moderate efficacy in the vRNP assay (e.g. 14 and 19, having EC50 values of 2.3 and 5.7\u2009\u03bcM, respectively), yet were devoid of activity in the enzymatic assay.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        20,
+                        "N-acylhydrazone",
+                        "chemical"
+                    ],
+                    [
+                        71,
+                        81,
+                        "vRNP assay",
+                        "experimental_method"
+                    ],
+                    [
+                        88,
+                        90,
+                        "14",
+                        "chemical"
+                    ],
+                    [
+                        95,
+                        97,
+                        "19",
+                        "chemical"
+                    ],
+                    [
+                        106,
+                        110,
+                        "EC50",
+                        "evidence"
+                    ],
+                    [
+                        187,
+                        202,
+                        "enzymatic assay",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 88,
+                "sent": "This observation suggests that they may inhibit the viral polymerase in an endonuclease-independent manner.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        52,
+                        57,
+                        "viral",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        58,
+                        68,
+                        "polymerase",
+                        "protein_type"
+                    ],
+                    [
+                        75,
+                        87,
+                        "endonuclease",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 89,
+                "sent": "To achieve a clear insight into the antiviral profile of the N-acylhydrazones, specific mechanistic experiments are currently ongoing in our laboratory, in which we are analyzing in full depth their effects on virus entry, polymerase-dependent RNA synthesis or the late stage (maturation and release) of the virus replication cycle.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        61,
+                        77,
+                        "N-acylhydrazones",
+                        "chemical"
+                    ],
+                    [
+                        210,
+                        215,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        223,
+                        233,
+                        "polymerase",
+                        "protein_type"
+                    ],
+                    [
+                        244,
+                        247,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        308,
+                        313,
+                        "virus",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 90,
+                "sent": "Docking studies",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        15,
+                        "Docking studies",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 91,
+                "sent": "In order to explore the possible binding mode of the synthesized compounds, docking simulations by GOLD program were performed by using the structural coordinates (PDB code 4AWM) for the PA-Nter endonuclease in complex with EGCG.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        76,
+                        95,
+                        "docking simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        99,
+                        111,
+                        "GOLD program",
+                        "experimental_method"
+                    ],
+                    [
+                        187,
+                        189,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        190,
+                        194,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        195,
+                        207,
+                        "endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        208,
+                        223,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        224,
+                        228,
+                        "EGCG",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 92,
+                "sent": "Considering that the position of the side-chains of some residues changes depending on which pocket the ligand is occupying, we superimposed some X-ray structures of complexes between PA-Nter endonuclease and known active ligands.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        128,
+                        140,
+                        "superimposed",
+                        "experimental_method"
+                    ],
+                    [
+                        146,
+                        162,
+                        "X-ray structures",
+                        "evidence"
+                    ],
+                    [
+                        184,
+                        186,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        187,
+                        191,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        192,
+                        204,
+                        "endonuclease",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 93,
+                "sent": "It was observed that the side-chain of amino acid Tyr24 shows greater movement than the other residues and for this reason we considered it as a flexible residue during the docking procedure.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        50,
+                        55,
+                        "Tyr24",
+                        "residue_name_number"
+                    ],
+                    [
+                        145,
+                        153,
+                        "flexible",
+                        "protein_state"
+                    ],
+                    [
+                        173,
+                        190,
+                        "docking procedure",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 94,
+                "sent": "First, test docking calculations, using EGCG, L-742,001 and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one (Fig. 1), were carried out to compare experimental and predicted binding modes and validate docking procedure.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        7,
+                        32,
+                        "test docking calculations",
+                        "experimental_method"
+                    ],
+                    [
+                        40,
+                        44,
+                        "EGCG",
+                        "chemical"
+                    ],
+                    [
+                        46,
+                        55,
+                        "L-742,001",
+                        "chemical"
+                    ],
+                    [
+                        60,
+                        119,
+                        "2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one",
+                        "chemical"
+                    ],
+                    [
+                        212,
+                        229,
+                        "docking procedure",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 95,
+                "sent": "Their best docking poses agreed well with the experimental binding modes (rmsd values of 0.8, 1.2 and 0.7, respectively).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        74,
+                        78,
+                        "rmsd",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 96,
+                "sent": "Next, docking of several N-acylhydrazones was performed and this generated a number of possible binding conformations, highlighting that the active site cavity of the PA endonuclease is quite spacious, as already demonstrated by crystallographic studies, and confirming the ability of this scaffold to chelate the two M2+ ions in different ways (Mode A-C in Fig. 4).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        6,
+                        13,
+                        "docking",
+                        "experimental_method"
+                    ],
+                    [
+                        25,
+                        41,
+                        "N-acylhydrazones",
+                        "chemical"
+                    ],
+                    [
+                        141,
+                        159,
+                        "active site cavity",
+                        "site"
+                    ],
+                    [
+                        167,
+                        169,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        170,
+                        182,
+                        "endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        229,
+                        253,
+                        "crystallographic studies",
+                        "experimental_method"
+                    ],
+                    [
+                        318,
+                        321,
+                        "M2+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 97,
+                "sent": "Figure 5 displays the first (panel A) and second (panel B) GOLD cluster docked solutions for compound 23.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        59,
+                        78,
+                        "GOLD cluster docked",
+                        "experimental_method"
+                    ],
+                    [
+                        102,
+                        104,
+                        "23",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 98,
+                "sent": "These two complex structures represent the largest clusters with similar fitness values (59.20 and 58.65, respectively).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        28,
+                        "structures",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 99,
+                "sent": "In both cases, 23 appears able to coordinate the two M2+ ions in the active site through the three contiguous OH groups (Fig. 5).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        15,
+                        17,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        34,
+                        44,
+                        "coordinate",
+                        "bond_interaction"
+                    ],
+                    [
+                        53,
+                        56,
+                        "M2+",
+                        "chemical"
+                    ],
+                    [
+                        69,
+                        80,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 100,
+                "sent": "In addition, 23 was predicted to form two hydrogen bonding interactions, i.e. with the catalytic Lys134 on the one side and Glu26 on the other side.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        15,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        42,
+                        71,
+                        "hydrogen bonding interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        87,
+                        96,
+                        "catalytic",
+                        "protein_state"
+                    ],
+                    [
+                        97,
+                        103,
+                        "Lys134",
+                        "residue_name_number"
+                    ],
+                    [
+                        124,
+                        129,
+                        "Glu26",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 101,
+                "sent": "Furthermore, in these two different binding modes, 23 forms \u03c0\u2013\u03c0 interactions with the aromatic ring of Tyr24, in a fashion similar to that described for other endonuclease inhibitors, i.e. EGCG and L-742,001.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        51,
+                        53,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        60,
+                        76,
+                        "\u03c0\u2013\u03c0 interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        103,
+                        108,
+                        "Tyr24",
+                        "residue_name_number"
+                    ],
+                    [
+                        159,
+                        171,
+                        "endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        189,
+                        193,
+                        "EGCG",
+                        "chemical"
+                    ],
+                    [
+                        198,
+                        207,
+                        "L-742,001",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 102,
+                "sent": "The best docked conformation for compound 15 (Fig. 6, fitness value 68.56), which has an activity slightly lower than 23, reveals a different role for the gallic moiety.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        42,
+                        44,
+                        "15",
+                        "chemical"
+                    ],
+                    [
+                        54,
+                        67,
+                        "fitness value",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 103,
+                "sent": "The ligand seems to form two hydrogen bonding interactions with Tyr130 as well as a cation\u2013\u03c0 interaction with Lys134.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        58,
+                        "hydrogen bonding interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        64,
+                        70,
+                        "Tyr130",
+                        "residue_name_number"
+                    ],
+                    [
+                        84,
+                        104,
+                        "cation\u2013\u03c0 interaction",
+                        "bond_interaction"
+                    ],
+                    [
+                        110,
+                        116,
+                        "Lys134",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 104,
+                "sent": "Tyr130 lies in a pocket that also contains Arg124, a residue that was proposed to have a crucial role in binding of the RNA substrate.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        6,
+                        "Tyr130",
+                        "residue_name_number"
+                    ],
+                    [
+                        17,
+                        23,
+                        "pocket",
+                        "site"
+                    ],
+                    [
+                        43,
+                        49,
+                        "Arg124",
+                        "residue_name_number"
+                    ],
+                    [
+                        120,
+                        123,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 105,
+                "sent": "Compound 15 appears further stabilized by hydrogen bonding interactions between two hydroxyl groups and Arg82 and Asp108.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        11,
+                        "15",
+                        "chemical"
+                    ],
+                    [
+                        42,
+                        71,
+                        "hydrogen bonding interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        104,
+                        109,
+                        "Arg82",
+                        "residue_name_number"
+                    ],
+                    [
+                        114,
+                        120,
+                        "Asp108",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 106,
+                "sent": "In this case, chelation of the two M2+ ions is carried out by involving the imine group (mode A in Fig. 4).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        23,
+                        "chelation",
+                        "bond_interaction"
+                    ],
+                    [
+                        35,
+                        38,
+                        "M2+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 107,
+                "sent": "It is important to highlight that compounds 23 and 15, although in different ways, both are able to chelate the metal cofactors and to establish interactions with highly conserved aminoacids (Tyr24, Glu26, Arg124, Tyr130 and Lys134) that are very important for both endonuclease activity and transcription in vitro.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        44,
+                        46,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        51,
+                        53,
+                        "15",
+                        "chemical"
+                    ],
+                    [
+                        163,
+                        179,
+                        "highly conserved",
+                        "protein_state"
+                    ],
+                    [
+                        192,
+                        197,
+                        "Tyr24",
+                        "residue_name_number"
+                    ],
+                    [
+                        199,
+                        204,
+                        "Glu26",
+                        "residue_name_number"
+                    ],
+                    [
+                        206,
+                        212,
+                        "Arg124",
+                        "residue_name_number"
+                    ],
+                    [
+                        214,
+                        220,
+                        "Tyr130",
+                        "residue_name_number"
+                    ],
+                    [
+                        225,
+                        231,
+                        "Lys134",
+                        "residue_name_number"
+                    ],
+                    [
+                        266,
+                        278,
+                        "endonuclease",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 108,
+                "sent": "The crucial role of such interactions is underlined by the differences in activity between 15 (IC50\u2009=\u20099.0\u2009\u03bcM) and 19 (>500\u2009\u03bcM): their coordinating features are similar, since both coordinate to the divalent metal ion through the phenolic oxygen, the iminic nitrogen and the carbonylic oxygen (mode A in Fig. 4), but the biological activity could be related to their different ability to engage interactions with the protein environment.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        91,
+                        93,
+                        "15",
+                        "chemical"
+                    ],
+                    [
+                        95,
+                        99,
+                        "IC50",
+                        "evidence"
+                    ],
+                    [
+                        114,
+                        116,
+                        "19",
+                        "chemical"
+                    ],
+                    [
+                        180,
+                        190,
+                        "coordinate",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 109,
+                "sent": "Crystallographic Studies",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        24,
+                        "Crystallographic Studies",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 110,
+                "sent": "Attempts were made to co-crystallize PA-Nter with 15, 20, 21 and 23 in one to four molar excess.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        22,
+                        36,
+                        "co-crystallize",
+                        "experimental_method"
+                    ],
+                    [
+                        37,
+                        39,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        40,
+                        44,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        50,
+                        52,
+                        "15",
+                        "chemical"
+                    ],
+                    [
+                        54,
+                        56,
+                        "20",
+                        "chemical"
+                    ],
+                    [
+                        58,
+                        60,
+                        "21",
+                        "chemical"
+                    ],
+                    [
+                        65,
+                        67,
+                        "23",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 111,
+                "sent": "While crystals appeared and diffracted well, upon data processing, no or very little electron density for the inhibitors was observed.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        6,
+                        14,
+                        "crystals",
+                        "evidence"
+                    ],
+                    [
+                        85,
+                        101,
+                        "electron density",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 112,
+                "sent": "Attempts to soak apo crystals in crystallization solution containing 5 mM inhibitor overnight also did not result in substantial electron density for the inhibitor.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        17,
+                        20,
+                        "apo",
+                        "protein_state"
+                    ],
+                    [
+                        21,
+                        29,
+                        "crystals",
+                        "evidence"
+                    ],
+                    [
+                        129,
+                        145,
+                        "electron density",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 113,
+                "sent": "As a last resort, dry powder of the inhibitor was sprinkled over the crystallization drop containing apo crystals and left over night.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        101,
+                        104,
+                        "apo",
+                        "protein_state"
+                    ],
+                    [
+                        105,
+                        113,
+                        "crystals",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 114,
+                "sent": "This experiment was successful for compound 23, the crystals diffracted to 2.15 \u00c5 and diffraction data were collected (PDB ID 5EGA).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        44,
+                        46,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        52,
+                        60,
+                        "crystals",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 115,
+                "sent": "The refined structure shows unambiguous electron density for the inhibitor (Table S1 and Fig. 7).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        12,
+                        21,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        40,
+                        56,
+                        "electron density",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 116,
+                "sent": "The complex structure confirms one of the two binding modes predicted by the docking simulations (Fig. 5, panel B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        21,
+                        "complex structure",
+                        "evidence"
+                    ],
+                    [
+                        77,
+                        96,
+                        "docking simulations",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 117,
+                "sent": "The galloyl moiety chelates the manganese ions, while the trihydroxyphenyl group stacks against the Tyr24 side chain.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        32,
+                        41,
+                        "manganese",
+                        "chemical"
+                    ],
+                    [
+                        100,
+                        105,
+                        "Tyr24",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 118,
+                "sent": "It is interesting to note that two of these hydroxyl groups are in position to form hydrogen bonds with the side chain of Glu26 and Lys34 (Fig. 7).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        84,
+                        98,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        122,
+                        127,
+                        "Glu26",
+                        "residue_name_number"
+                    ],
+                    [
+                        132,
+                        137,
+                        "Lys34",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 119,
+                "sent": "These interactions suggest that other functional groups, e.g. halogens, could be used in place of the hydroxyl groups for better interactions with Glu26 and Lys34 side chains, and the inhibitory potency of these compounds could be further improved.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        147,
+                        152,
+                        "Glu26",
+                        "residue_name_number"
+                    ],
+                    [
+                        157,
+                        162,
+                        "Lys34",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 120,
+                "sent": "The development of new agents for the treatment of influenza infection that exert their action by inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is a strategy that recently is gaining a lot of interest.",
+                "section": "CONCL",
+                "ner": [
+                    [
+                        51,
+                        60,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        116,
+                        128,
+                        "endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        141,
+                        150,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        151,
+                        179,
+                        "RNA-dependent RNA polymerase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 121,
+                "sent": "The results here presented add the N-acylhydrazone scaffold to the library of the chelating molecules with potent antiviral activity (EC90\u2009<\u20095 \u03bcM, virus yield assay in influenza virus-infected MDCK cells).",
+                "section": "CONCL",
+                "ner": [
+                    [
+                        35,
+                        50,
+                        "N-acylhydrazone",
+                        "chemical"
+                    ],
+                    [
+                        134,
+                        138,
+                        "EC90",
+                        "evidence"
+                    ],
+                    [
+                        147,
+                        164,
+                        "virus yield assay",
+                        "experimental_method"
+                    ],
+                    [
+                        168,
+                        177,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        178,
+                        183,
+                        "virus",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 122,
+                "sent": "The structure of the N-acylhydrazone 23 co-crystallized with PA-Nter is important not only because confirms that the polyhydroxypheyl group efficiently coordinates two metal ions in the active site of the enzyme, but also because highlights the importance of the (flexible) inhibitor backbone in order to engage effective interactions with crucial aminoacids of the protein.",
+                "section": "CONCL",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        36,
+                        "N-acylhydrazone",
+                        "chemical"
+                    ],
+                    [
+                        37,
+                        39,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        40,
+                        55,
+                        "co-crystallized",
+                        "experimental_method"
+                    ],
+                    [
+                        61,
+                        63,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        64,
+                        68,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        152,
+                        163,
+                        "coordinates",
+                        "bond_interaction"
+                    ],
+                    [
+                        168,
+                        173,
+                        "metal",
+                        "chemical"
+                    ],
+                    [
+                        186,
+                        197,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 123,
+                "sent": "Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase could represent another example, after carbonic anhydrase, histone deacetylase, and HIV-1 integrase, of metal binding as a successful strategy in drug design.",
+                "section": "CONCL",
+                "ner": [
+                    [
+                        18,
+                        30,
+                        "endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        43,
+                        52,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        53,
+                        81,
+                        "RNA-dependent RNA polymerase",
+                        "protein_type"
+                    ],
+                    [
+                        121,
+                        139,
+                        "carbonic anhydrase",
+                        "protein_type"
+                    ],
+                    [
+                        141,
+                        160,
+                        "histone deacetylase",
+                        "protein_type"
+                    ],
+                    [
+                        166,
+                        171,
+                        "HIV-1",
+                        "species"
+                    ],
+                    [
+                        172,
+                        181,
+                        "integrase",
+                        "protein_type"
+                    ],
+                    [
+                        186,
+                        191,
+                        "metal",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 124,
+                "sent": "The ligand and water molecules were discarded and the hydrogens were added to the protein by Discovery Studio 2.5.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        15,
+                        20,
+                        "water",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 125,
+                "sent": "One microgram of recombinant PA-Nter (residues 1\u2013217 from the PA protein of influenza virus strain A/X-31) was incubated with 1 \u03bcg (16.7 nM) of single-stranded circular DNA plasmid M13mp18 (Bayou Biolabs, Metairie, Louisiana) in the presence of the test compounds and at a final volume of 25 \u03bcL. The assay buffer contained 50 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM \u03b2-mercaptoethanol and 1 mM MnCl2.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        233,
+                        244,
+                        "presence of",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 126,
+                "sent": "After incubation at 37\u2009\u00b0C for 24 h in the presence of serial dilutions of the test compounds, the ONE-Glo luciferase assay system (Promega, Madison, WI) was used to determine luciferase activity.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        42,
+                        53,
+                        "presence of",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 127,
+                "sent": "The compound concentration values causing a 2-log10 (EC99) and a 1-log10 (EC90) reduction in viral RNA (vRNA) copy number at 24 h p.i., as compared to the virus control receiving no compound, were calculated by interpolation from data of at least three experiments.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        53,
+                        57,
+                        "EC99",
+                        "evidence"
+                    ],
+                    [
+                        74,
+                        78,
+                        "EC90",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 128,
+                "sent": "A PAN construct (PAN\u0394Loop) with a loop (residues 51\u201372) deleted and replaced with GGS from A/California/04/2009 H1N1 strain was used for the crystallographic studies.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        17,
+                        25,
+                        "PAN\u0394Loop",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 129,
+                "sent": "The apo structure of PAN\u0394Loop (PDB ID: 5DES) was used as starting model for molecular replacement.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        21,
+                        29,
+                        "PAN\u0394Loop",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 130,
+                "sent": "Chemical structures of some prototype inhibitors of influenza virus endonuclease.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        52,
+                        61,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        62,
+                        67,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        68,
+                        80,
+                        "endonuclease",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 131,
+                "sent": "Inhibitor activity in enzymatic assays (IC50,\u2009\u03bcM) as reported in: aref., bref., cref., dref..",
+                "section": "FIG",
+                "ner": [
+                    [
+                        22,
+                        38,
+                        "enzymatic assays",
+                        "experimental_method"
+                    ],
+                    [
+                        40,
+                        44,
+                        "IC50",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 132,
+                "sent": "General synthesis for N-acylhydrazones 1\u201327 and hydrazides 28 and 29 (A).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        22,
+                        38,
+                        "N-acylhydrazones",
+                        "chemical"
+                    ],
+                    [
+                        39,
+                        43,
+                        "1\u201327",
+                        "chemical"
+                    ],
+                    [
+                        48,
+                        58,
+                        "hydrazides",
+                        "chemical"
+                    ],
+                    [
+                        59,
+                        61,
+                        "28",
+                        "chemical"
+                    ],
+                    [
+                        66,
+                        68,
+                        "29",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 133,
+                "sent": "Chemical structures of compounds 1\u201327 (B).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        33,
+                        37,
+                        "1\u201327",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 134,
+                "sent": "Overview of the structure-activity relationship for compounds 1\u201327.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        62,
+                        66,
+                        "1\u201327",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 135,
+                "sent": "Scheme of possible binding modes of the studied N-acylhydrazones.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        48,
+                        64,
+                        "N-acylhydrazones",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 136,
+                "sent": "First (A) and second (B) GOLD cluster docked solutions of compound 23 (orange and cyan, respectively) in complex with PA endonuclease.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        25,
+                        44,
+                        "GOLD cluster docked",
+                        "experimental_method"
+                    ],
+                    [
+                        67,
+                        69,
+                        "23",
+                        "chemical"
+                    ],
+                    [
+                        102,
+                        117,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        118,
+                        120,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        121,
+                        133,
+                        "endonuclease",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 137,
+                "sent": "Key residues of the pocket are presented using PyMOL [ http://www.pymol.org] and LIGPLUS [Laskowski, R. A.; Swindells, M. B. Journal of chemical information and modeling 2011, 51, 2778].",
+                "section": "FIG",
+                "ner": [
+                    [
+                        20,
+                        26,
+                        "pocket",
+                        "site"
+                    ],
+                    [
+                        81,
+                        88,
+                        "LIGPLUS",
+                        "experimental_method"
+                    ],
+                    [
+                        20,
+                        26,
+                        "pocket",
+                        "site"
+                    ],
+                    [
+                        81,
+                        88,
+                        "LIGPLUS",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 138,
+                "sent": "Hydrogen bonds are illustrated by dotted lines, while the divalent metal ions are shown as purple spheres.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        14,
+                        "Hydrogen bonds",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 139,
+                "sent": "Schematic drawings of the interactions of the first (C) and second (D) GOLD cluster docked solutions generated using LIGPLUS.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        71,
+                        90,
+                        "GOLD cluster docked",
+                        "experimental_method"
+                    ],
+                    [
+                        117,
+                        124,
+                        "LIGPLUS",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 140,
+                "sent": "Dashed lines are hydrogen bonds and \u2018eyelashes\u2019 show residues involved in hydrophobic interactions.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        17,
+                        31,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        74,
+                        98,
+                        "hydrophobic interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        17,
+                        31,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        74,
+                        98,
+                        "hydrophobic interactions",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 141,
+                "sent": "(A) Binding mode of compound 15 (orange) in complex with PA endonuclease.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        29,
+                        31,
+                        "15",
+                        "chemical"
+                    ],
+                    [
+                        41,
+                        56,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        57,
+                        59,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        60,
+                        72,
+                        "endonuclease",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 142,
+                "sent": "Hydrogen bonds are illustrated by dotted lines while the divalent metal ions are shown as purple spheres.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        14,
+                        "Hydrogen bonds",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 143,
+                "sent": "(B) Schematic drawing of the interactions of compound 15 generated using LIGPLUS.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        54,
+                        56,
+                        "15",
+                        "chemical"
+                    ],
+                    [
+                        73,
+                        80,
+                        "LIGPLUS",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 144,
+                "sent": "Crystal structure of PAN\u0394Loop in complex with compound 23.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        17,
+                        "Crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        29,
+                        "PAN\u0394Loop",
+                        "mutant"
+                    ],
+                    [
+                        30,
+                        45,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        55,
+                        57,
+                        "23",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 145,
+                "sent": "Active site residues are shown in sticks with green carbons, manganese atoms are shown as purple spheres and water molecules as red spheres.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Active site",
+                        "site"
+                    ],
+                    [
+                        61,
+                        70,
+                        "manganese",
+                        "chemical"
+                    ],
+                    [
+                        109,
+                        114,
+                        "water",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 146,
+                "sent": "Compound 23 is shown in sticks with yellow carbons.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        9,
+                        11,
+                        "23",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 147,
+                "sent": "2Fo-Fc electron density map contoured at 1\u03c3 is shown as blue mesh.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        27,
+                        "2Fo-Fc electron density map",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 148,
+                "sent": "Hydrogen bonds and metal coordination are shown with dotted lines.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        14,
+                        "Hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        19,
+                        37,
+                        "metal coordination",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 149,
+                "sent": "The H-bond distances from the side chain carboxyl group of Glu26 to p-OH and m-OH of the trihydroxyphenyl group of the inhibitor are 2.7 \u00c5 and 3.0 \u00c5, respectively.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        10,
+                        "H-bond",
+                        "bond_interaction"
+                    ],
+                    [
+                        59,
+                        64,
+                        "Glu26",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 150,
+                "sent": "The H-bond distance from the side chain of Lys34 to p-OH of the trihydroxyphenyl group is 3.6 \u00c5. The H-bond distance to the water molecule from m-OH of the galloyl moiety is 3.0 \u00c5, which in turn is H-bonded to the side chain of Tyr130 with a distance of 2.7 \u00c5. Crystal structure has been deposited in the RCSB Protein Data Bank with PDB ID: 5EGA.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        10,
+                        "H-bond",
+                        "bond_interaction"
+                    ],
+                    [
+                        43,
+                        48,
+                        "Lys34",
+                        "residue_name_number"
+                    ],
+                    [
+                        101,
+                        107,
+                        "H-bond",
+                        "bond_interaction"
+                    ],
+                    [
+                        124,
+                        129,
+                        "water",
+                        "chemical"
+                    ],
+                    [
+                        198,
+                        206,
+                        "H-bonded",
+                        "bond_interaction"
+                    ],
+                    [
+                        228,
+                        234,
+                        "Tyr130",
+                        "residue_name_number"
+                    ],
+                    [
+                        261,
+                        278,
+                        "Crystal structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 151,
+                "sent": "Inhibitory activity of the N-acylhydrazones 1\u201327 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        27,
+                        43,
+                        "N-acylhydrazones",
+                        "chemical"
+                    ],
+                    [
+                        44,
+                        48,
+                        "1\u201327",
+                        "chemical"
+                    ],
+                    [
+                        53,
+                        62,
+                        "hydrazide",
+                        "chemical"
+                    ],
+                    [
+                        63,
+                        65,
+                        "28",
+                        "chemical"
+                    ],
+                    [
+                        73,
+                        88,
+                        "enzymatic assay",
+                        "experimental_method"
+                    ],
+                    [
+                        94,
+                        103,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        104,
+                        109,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        110,
+                        112,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        113,
+                        117,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        118,
+                        130,
+                        "endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        138,
+                        169,
+                        "cellular influenza virus assays",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 152,
+                "sent": "Compound\tEnzyme assay with PA-Ntera\tVirus yield assay in influenza virus-infected MDCK cellsb\tvRNP reconstitution assay in HEK293T cellsc\t \tAntiviral activity\tCytotoxicity\tSId\tActivity\tCytotoxicity\t \tIC50\tEC99\tEC90\tCC50\tEC50\tCC50\t \t(1)\t24\tNDf\tND\tND\t\u00a0\t107\t>200\t \t(2)\t>500\tND\tND\tND\t\u00a0\t>100\t>200\t \t(3)\t>500\tND\tND\t>200\t\u00a0\t5.9\t48\t \t(4)\t>500\tND\tND\t>200\t\u00a0\t6.3\t33\t \t(5)\t67\t>25\t>25\t\u2265146\t\u00a0\t2.6\t10\t \t(6)\t>500\t>50\t>50\t>200\t\u00a0\t15\t14\t \t(7)\t54\t172\t100\t>200\t>2.0\t3.2\t8.9\t \t(8)\t>500\t>12.5\t>12.5\t>200\t\u00a0\t1.9\t15\t \t(9)\t34\t16\t5.3\t>200\t>38\t5.5\t>200\t \t(10)\t68\t14\t8.5\t111\t>13\t0.40\t132\t \t(11)\t45\t30\t12\t>200\t>17\t5.6\t>200\t \t(12)\t>500\t>12.5\t>12.5\t>200\t\u00a0\t20\t39\t \t(13)\t69\t71\t34\t>200\t>5.9\t6.3\t>200\t \t(14)\t>500\t63\t37\t>200\t>5.4\t2.3\t>200\t \t(15)\t8.9\t18\t7.5\t\u2265172\t\u226523\t14\t>200\t \t(16)\t454\t67\t28\t>200\t>7.1\t5.2\t>200\t \t(17)\t482\t21\t8.1\t>200\t>25\t7.1\t>200\t \t(18)\t83\t6.2\t2.2\t>200\t>91\t3.3\t>200\t \t(19)\t>500\t53\t26\t>200\t>7.7\t5.7\t>200\t \t(20)\t18\t35\t11\t>200\t>18\t2.2\t>200\t \t(21)\t13\t8.3\t3.6\t>200\t>56\t2.5\t>200\t \t(22)\t75\t7.4\t3.4\t>200\t>59\t0.42\t>200\t \t(23)\t8.7\t11\t3.5\t>200\t>57\t3.1\t>200\t \t(24)\t131\t58\t26\t>200\t>7.7\t25\t>200\t \t(25)\t40\t132\t70\t>200\t>2.9\t4.1\t>200\t \t(26)\t30\t36\t13\t>200\t>15\t5.5\t>200\t \t(27)\t36\tND\tND\tND\t\u00a0\t21\t>200\t \t(28)\t40\t158\t85\t>200\t>2.4\t7.2\t>200\t \tDPBAe\t5.3\tND\tND\tND\t\u00a0\tND\tND\t \tRibavirin\tND\t13\t8.5\t>200\t>24\t9.4\t>200\t \t",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        21,
+                        "Compound\tEnzyme assay",
+                        "experimental_method"
+                    ],
+                    [
+                        27,
+                        29,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        36,
+                        53,
+                        "Virus yield assay",
+                        "experimental_method"
+                    ],
+                    [
+                        57,
+                        66,
+                        "influenza",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        67,
+                        72,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        94,
+                        119,
+                        "vRNP reconstitution assay",
+                        "experimental_method"
+                    ],
+                    [
+                        200,
+                        204,
+                        "IC50",
+                        "evidence"
+                    ],
+                    [
+                        205,
+                        209,
+                        "EC99",
+                        "evidence"
+                    ],
+                    [
+                        210,
+                        214,
+                        "EC90",
+                        "evidence"
+                    ],
+                    [
+                        215,
+                        219,
+                        "CC50",
+                        "evidence"
+                    ],
+                    [
+                        220,
+                        224,
+                        "EC50",
+                        "evidence"
+                    ],
+                    [
+                        225,
+                        229,
+                        "CC50",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 153,
+                "sent": "aRecombinant PA-Nter was incubated with the ssDNA plasmid substrate, a Mn2+-containing buffer and test compounds.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        13,
+                        15,
+                        "PA",
+                        "protein"
+                    ],
+                    [
+                        16,
+                        20,
+                        "Nter",
+                        "structure_element"
+                    ],
+                    [
+                        25,
+                        34,
+                        "incubated",
+                        "experimental_method"
+                    ],
+                    [
+                        44,
+                        49,
+                        "ssDNA",
+                        "chemical"
+                    ],
+                    [
+                        71,
+                        75,
+                        "Mn2+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 154,
+                "sent": "The IC50 represents the compound concentration (in \u03bcM) required to obtain 50% inhibition of cleavage, calculated by nonlinear least-squares regression analysis (using GraphPad Prism software) of the results from 2\u20134 independent experiments.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "IC50",
+                        "evidence"
+                    ],
+                    [
+                        116,
+                        159,
+                        "nonlinear least-squares regression analysis",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 155,
+                "sent": "bMDCK cells were infected with influenza A virus (strain A/PR/8/34) and incubated with the compounds during 24 h. The virus yield in the supernatant was assessed by real-time qPCR.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        31,
+                        42,
+                        "influenza A",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        43,
+                        48,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        118,
+                        123,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        165,
+                        179,
+                        "real-time qPCR",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 156,
+                "sent": "The EC99 and EC90 values represent the compound concentrations (in \u03bcM) producing a 2-log10 or 1-log10 reduction in virus titer, respectively, determined in 2\u20133 independent experiments.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "EC99",
+                        "evidence"
+                    ],
+                    [
+                        13,
+                        17,
+                        "EC90",
+                        "evidence"
+                    ],
+                    [
+                        115,
+                        120,
+                        "virus",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 157,
+                "sent": "The cytotoxicity, assessed in uninfected MDCK cells, was expressed as the CC50 value (50% cytotoxic concentration, determined with the MTS cell viability assay, in \u03bcM).",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        74,
+                        78,
+                        "CC50",
+                        "evidence"
+                    ],
+                    [
+                        135,
+                        159,
+                        "MTS cell viability assay",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 158,
+                "sent": "cHEK293T cells were co-transfected with the four vRNP-reconstituting plasmids and the luciferase reporter plasmid in the presence of the test compounds.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        20,
+                        34,
+                        "co-transfected",
+                        "experimental_method"
+                    ],
+                    [
+                        49,
+                        53,
+                        "vRNP",
+                        "complex_assembly"
+                    ],
+                    [
+                        121,
+                        132,
+                        "presence of",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 159,
+                "sent": "The EC50 represents the compound concentration (in \u03bcM) producing 50% reduction in vRNP-driven firefly reporter signal, estimated at 24\u2009h after transfection.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "EC50",
+                        "evidence"
+                    ],
+                    [
+                        82,
+                        86,
+                        "vRNP",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 160,
+                "sent": "The EC50 value was derived from data from 2\u20134 independent experiments, by nonlinear least-squares regression analysis (using GraphPad Prism software).",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "EC50",
+                        "evidence"
+                    ],
+                    [
+                        74,
+                        117,
+                        "nonlinear least-squares regression analysis",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 161,
+                "sent": "The CC50 (in \u03bcM), i.e. the 50% cytotoxic concentration, was determined in untransfected HEK293T cells by MTS cell viability assay.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "CC50",
+                        "evidence"
+                    ],
+                    [
+                        105,
+                        129,
+                        "MTS cell viability assay",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 162,
+                "sent": "dSI, selectivity index, defined as the ratio between the CC50 and EC90.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "dSI",
+                        "evidence"
+                    ],
+                    [
+                        5,
+                        22,
+                        "selectivity index",
+                        "evidence"
+                    ],
+                    [
+                        57,
+                        61,
+                        "CC50",
+                        "evidence"
+                    ],
+                    [
+                        66,
+                        70,
+                        "EC90",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 163,
+                "sent": "eDPBA, 2,4-dioxo-4-phenylbutanoic acid.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "eDPBA",
+                        "chemical"
+                    ],
+                    [
+                        7,
+                        38,
+                        "2,4-dioxo-4-phenylbutanoic acid",
+                        "chemical"
+                    ]
+                ]
+            }
+        ]
+    },
+    "PMC4817029": {
+        "annotations": [
+            {
+                "sid": 0,
+                "sent": "Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism",
+                "section": "TITLE",
+                "ner": [
+                    [
+                        32,
+                        60,
+                        "family 5 glycoside hydrolase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 1,
+                "sent": "Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        20,
+                        "Glycoside hydrolases",
+                        "protein_type"
+                    ],
+                    [
+                        22,
+                        25,
+                        "GHs",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 2,
+                "sent": "Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        20,
+                        31,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        32,
+                        41,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        47,
+                        56,
+                        "cellulase",
+                        "protein_type"
+                    ],
+                    [
+                        62,
+                        84,
+                        "Bacillus licheniformis",
+                        "species"
+                    ],
+                    [
+                        86,
+                        93,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        112,
+                        127,
+                        "GH5 subfamily 4",
+                        "protein_type"
+                    ],
+                    [
+                        166,
+                        183,
+                        "ancillary modules",
+                        "structure_element"
+                    ],
+                    [
+                        185,
+                        199,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        204,
+                        209,
+                        "CBM46",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 3,
+                "sent": "Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        6,
+                        27,
+                        "X-ray crystallography",
+                        "experimental_method"
+                    ],
+                    [
+                        29,
+                        57,
+                        "small-angle X-ray scattering",
+                        "experimental_method"
+                    ],
+                    [
+                        62,
+                        92,
+                        "molecular dynamics simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        125,
+                        130,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        158,
+                        174,
+                        "catalytic domain",
+                        "structure_element"
+                    ],
+                    [
+                        176,
+                        178,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        195,
+                        211,
+                        "fully functional",
+                        "protein_state"
+                    ],
+                    [
+                        212,
+                        223,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 4,
+                "sent": "The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        4,
+                        18,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        68,
+                        73,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        86,
+                        88,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        112,
+                        127,
+                        "binding subsite",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 5,
+                "sent": "This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        43,
+                        45,
+                        "GH",
+                        "protein_type"
+                    ],
+                    [
+                        88,
+                        93,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        114,
+                        137,
+                        "catalytically competent",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 6,
+                "sent": "Plant biomass-the most abundant source of carbohydrates on Earth-is primarily composed of cellulose microfibrils surrounded by a hydrated heteropolymeric matrix of hemicellulose and lignin.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "Plant",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        42,
+                        55,
+                        "carbohydrates",
+                        "chemical"
+                    ],
+                    [
+                        90,
+                        99,
+                        "cellulose",
+                        "chemical"
+                    ],
+                    [
+                        164,
+                        177,
+                        "hemicellulose",
+                        "chemical"
+                    ],
+                    [
+                        182,
+                        188,
+                        "lignin",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 7,
+                "sent": "Plant biomass may be subjected to thermo-chemical pretreatments and enzymatic reactions to produce soluble fermentable sugars.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "Plant",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        119,
+                        125,
+                        "sugars",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 8,
+                "sent": "The canonical model of hydrolytic degradation of cellulose requires at least three classes of enzymes.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        49,
+                        58,
+                        "cellulose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 9,
+                "sent": "Cellobiohydrolases (CBHs) processively cleave the glycosidic bonds at the reducing and non-reducing ends of cellulose chains in crystalline regions to produce cellobiose.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        18,
+                        "Cellobiohydrolases",
+                        "protein_type"
+                    ],
+                    [
+                        20,
+                        24,
+                        "CBHs",
+                        "protein_type"
+                    ],
+                    [
+                        108,
+                        117,
+                        "cellulose",
+                        "chemical"
+                    ],
+                    [
+                        159,
+                        169,
+                        "cellobiose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 10,
+                "sent": "Endoglucanases (EGs) introduce random cuts in the amorphous regions of cellulose and create new chain extremities for CBH attack; thus, these enzymes act synergistically.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        14,
+                        "Endoglucanases",
+                        "protein_type"
+                    ],
+                    [
+                        16,
+                        19,
+                        "EGs",
+                        "protein_type"
+                    ],
+                    [
+                        71,
+                        80,
+                        "cellulose",
+                        "chemical"
+                    ],
+                    [
+                        118,
+                        121,
+                        "CBH",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 11,
+                "sent": "The released cellobiose molecules are then enzymatically converted into glucose by \u03b2-glucosidases.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        13,
+                        23,
+                        "cellobiose",
+                        "chemical"
+                    ],
+                    [
+                        72,
+                        79,
+                        "glucose",
+                        "chemical"
+                    ],
+                    [
+                        83,
+                        97,
+                        "\u03b2-glucosidases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 12,
+                "sent": "The molecular architecture of glycoside hydrolases (GHs) frequently consists of a catalytic domain (CD), where hydrolysis occurs, and one or more ancillary modules (AMs), which are usually connected by less structured linkers.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        30,
+                        50,
+                        "glycoside hydrolases",
+                        "protein_type"
+                    ],
+                    [
+                        52,
+                        55,
+                        "GHs",
+                        "protein_type"
+                    ],
+                    [
+                        82,
+                        98,
+                        "catalytic domain",
+                        "structure_element"
+                    ],
+                    [
+                        100,
+                        102,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        146,
+                        163,
+                        "ancillary modules",
+                        "structure_element"
+                    ],
+                    [
+                        165,
+                        168,
+                        "AMs",
+                        "structure_element"
+                    ],
+                    [
+                        202,
+                        217,
+                        "less structured",
+                        "protein_state"
+                    ],
+                    [
+                        218,
+                        225,
+                        "linkers",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 13,
+                "sent": "The most common type of AMs are carbohydrate-binding modules (CBMs), which are able to recognize and bind specific carbohydrate chains.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        24,
+                        27,
+                        "AMs",
+                        "structure_element"
+                    ],
+                    [
+                        32,
+                        60,
+                        "carbohydrate-binding modules",
+                        "structure_element"
+                    ],
+                    [
+                        62,
+                        66,
+                        "CBMs",
+                        "structure_element"
+                    ],
+                    [
+                        115,
+                        127,
+                        "carbohydrate",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 14,
+                "sent": "Generally distinct and independent structural domains, the CBMs facilitate carbohydrate hydrolysis by increasing the local concentration of enzymes at the surface of insoluble substrates, thereby targeting the CD component to its cognate ligands.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        59,
+                        63,
+                        "CBMs",
+                        "structure_element"
+                    ],
+                    [
+                        75,
+                        87,
+                        "carbohydrate",
+                        "chemical"
+                    ],
+                    [
+                        210,
+                        212,
+                        "CD",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 15,
+                "sent": "CBMs might also disrupt the crystalline structure of cellulose microfibrils, although the underlying mechanism remains poorly understood.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "CBMs",
+                        "structure_element"
+                    ],
+                    [
+                        53,
+                        62,
+                        "cellulose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 16,
+                "sent": "Thus, CBMs enhance the accessibility of CDs to carbohydrate chains to improve enzymatic activity, making them important candidates for the development of effective biomass-degrading enzymes in industrial settings.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        6,
+                        10,
+                        "CBMs",
+                        "structure_element"
+                    ],
+                    [
+                        40,
+                        43,
+                        "CDs",
+                        "structure_element"
+                    ],
+                    [
+                        47,
+                        59,
+                        "carbohydrate",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 17,
+                "sent": "Although there are examples of active GHs that lack AMs, the majority of the enzymes depend on AMs for activity.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        31,
+                        37,
+                        "active",
+                        "protein_state"
+                    ],
+                    [
+                        38,
+                        41,
+                        "GHs",
+                        "protein_type"
+                    ],
+                    [
+                        47,
+                        51,
+                        "lack",
+                        "protein_state"
+                    ],
+                    [
+                        52,
+                        55,
+                        "AMs",
+                        "structure_element"
+                    ],
+                    [
+                        95,
+                        98,
+                        "AMs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 18,
+                "sent": "In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        18,
+                        22,
+                        "CBMs",
+                        "structure_element"
+                    ],
+                    [
+                        63,
+                        65,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        66,
+                        88,
+                        "substrate-binding site",
+                        "site"
+                    ],
+                    [
+                        105,
+                        132,
+                        "carbohydrate-active enzymes",
+                        "protein_type"
+                    ],
+                    [
+                        142,
+                        159,
+                        "endo/exocellulase",
+                        "protein_type"
+                    ],
+                    [
+                        160,
+                        162,
+                        "E4",
+                        "protein"
+                    ],
+                    [
+                        168,
+                        186,
+                        "Thermobifida fusca",
+                        "species"
+                    ],
+                    [
+                        188,
+                        199,
+                        "chitinase B",
+                        "protein"
+                    ],
+                    [
+                        205,
+                        224,
+                        "Serratia marcescens",
+                        "species"
+                    ],
+                    [
+                        228,
+                        246,
+                        "starch phosphatase",
+                        "protein_type"
+                    ],
+                    [
+                        252,
+                        272,
+                        "Arabidopsis thaliana",
+                        "species"
+                    ],
+                    [
+                        279,
+                        294,
+                        "GH5 subfamily 4",
+                        "protein_type"
+                    ],
+                    [
+                        296,
+                        301,
+                        "GH5_4",
+                        "protein_type"
+                    ],
+                    [
+                        303,
+                        316,
+                        "endoglucanase",
+                        "protein_type"
+                    ],
+                    [
+                        322,
+                        341,
+                        "Bacillus halodurans",
+                        "species"
+                    ],
+                    [
+                        343,
+                        350,
+                        "BhCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 19,
+                "sent": "A pioneer work of Sakon et al. revealed that rigid structural extension of the GH9 CD by a type C CBM3 imprints a processive mode of action to this endoglucanase.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        79,
+                        82,
+                        "GH9",
+                        "protein_type"
+                    ],
+                    [
+                        83,
+                        85,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        91,
+                        102,
+                        "type C CBM3",
+                        "structure_element"
+                    ],
+                    [
+                        148,
+                        161,
+                        "endoglucanase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 20,
+                "sent": "Further publications showed that CBM-based structural extensions of the active site are important for substrate engagement and recognition.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        33,
+                        36,
+                        "CBM",
+                        "structure_element"
+                    ],
+                    [
+                        72,
+                        83,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 21,
+                "sent": "Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        39,
+                        54,
+                        "X-ray structure",
+                        "evidence"
+                    ],
+                    [
+                        62,
+                        73,
+                        "tri-modular",
+                        "structure_element"
+                    ],
+                    [
+                        74,
+                        79,
+                        "GH5_4",
+                        "protein_type"
+                    ],
+                    [
+                        80,
+                        93,
+                        "endoglucanase",
+                        "protein_type"
+                    ],
+                    [
+                        99,
+                        118,
+                        "Bacillus halodurans",
+                        "species"
+                    ],
+                    [
+                        145,
+                        152,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        164,
+                        169,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        187,
+                        198,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        215,
+                        217,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        225,
+                        256,
+                        "immunoglobulin (Ig)-like module",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 22,
+                "sent": "Removal of the CBM46 caused a ~60-fold reduction of the activity of the enzyme against \u03b2-glucans, but showed little or no effect against xyloglucan hydrolysis.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        10,
+                        "Removal of",
+                        "experimental_method"
+                    ],
+                    [
+                        15,
+                        20,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        87,
+                        96,
+                        "\u03b2-glucans",
+                        "chemical"
+                    ],
+                    [
+                        137,
+                        147,
+                        "xyloglucan",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 23,
+                "sent": "Moreover, the CBM46 mediated a significant increase in the BhCel5B activity in plant cell wall settings.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        14,
+                        19,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        59,
+                        66,
+                        "BhCel5B",
+                        "protein"
+                    ],
+                    [
+                        79,
+                        84,
+                        "plant",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 24,
+                "sent": "Modeling of cellotriose in the negative subsites of the active site of BhCel5B demonstrated the structural conservation of the -1 position, but provided little information about direct interactions between CBM46 and the substrate.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "Modeling",
+                        "experimental_method"
+                    ],
+                    [
+                        12,
+                        23,
+                        "cellotriose",
+                        "chemical"
+                    ],
+                    [
+                        31,
+                        48,
+                        "negative subsites",
+                        "site"
+                    ],
+                    [
+                        56,
+                        67,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        71,
+                        78,
+                        "BhCel5B",
+                        "protein"
+                    ],
+                    [
+                        96,
+                        119,
+                        "structural conservation",
+                        "protein_state"
+                    ],
+                    [
+                        127,
+                        129,
+                        "-1",
+                        "residue_number"
+                    ],
+                    [
+                        206,
+                        211,
+                        "CBM46",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 25,
+                "sent": "It was speculated that \u03b2-1,3 kink of the \u03b2-glucan might allow the ligand to reach for the CBM46, whereas pure \u03b2-1,4 linkages in the backbone of xyloglucan chains would restrict binding to the CD, thus explaining the lack of influence of the CBM46 on the enzymatic activity of BhCel5B against xyloglucans in solution.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        41,
+                        49,
+                        "\u03b2-glucan",
+                        "chemical"
+                    ],
+                    [
+                        90,
+                        95,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        144,
+                        154,
+                        "xyloglucan",
+                        "chemical"
+                    ],
+                    [
+                        192,
+                        194,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        241,
+                        246,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        276,
+                        283,
+                        "BhCel5B",
+                        "protein"
+                    ],
+                    [
+                        292,
+                        303,
+                        "xyloglucans",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 26,
+                "sent": "It was also argued that the CBM46 could potentialize the activity by driving BhCel5B towards xyloglucan-rich regions in the context of the plant cell walls, but no large-scale conformational adjustments of the AMs have been shown to occur or suggested to take part in the enzymatic activity.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        28,
+                        33,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        77,
+                        84,
+                        "BhCel5B",
+                        "protein"
+                    ],
+                    [
+                        93,
+                        116,
+                        "xyloglucan-rich regions",
+                        "structure_element"
+                    ],
+                    [
+                        139,
+                        144,
+                        "plant",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        210,
+                        213,
+                        "AMs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 27,
+                "sent": "Although initially introduced as contradictory theories, these two limiting cases can be unified considering the flux description concept or the extended conformational selection model.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        145,
+                        153,
+                        "extended",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 28,
+                "sent": "While local ligand-induced conformational adjustments have been reported for carbohydrate-active enzymes, cognate ligands recognition and hydrolysis mediated by a large-scale conformational mobility of distinct domains in multidomain settings is uncommon for endoglucanases.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        77,
+                        104,
+                        "carbohydrate-active enzymes",
+                        "protein_type"
+                    ],
+                    [
+                        259,
+                        273,
+                        "endoglucanases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 29,
+                "sent": "Here, we report the crystal structure of a full-length GH5_4 enzyme from Bacillus licheniformis (BlCel5B) that exhibits two AMs (Ig-like module and CBM46) appended to the CD.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        20,
+                        37,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        43,
+                        54,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        55,
+                        60,
+                        "GH5_4",
+                        "protein_type"
+                    ],
+                    [
+                        73,
+                        95,
+                        "Bacillus licheniformis",
+                        "species"
+                    ],
+                    [
+                        97,
+                        104,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        124,
+                        127,
+                        "AMs",
+                        "structure_element"
+                    ],
+                    [
+                        129,
+                        143,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        148,
+                        153,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        171,
+                        173,
+                        "CD",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 30,
+                "sent": "We structurally and functionally characterize the enzyme using a combination of protein crystallography, small-angle X-ray scattering (SAXS), molecular dynamics computer simulations and site-directed mutagenesis, and show that the AMs and their conformational mobility are essential for the enzymatic activity of BlCel5B.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        3,
+                        45,
+                        "structurally and functionally characterize",
+                        "experimental_method"
+                    ],
+                    [
+                        80,
+                        103,
+                        "protein crystallography",
+                        "experimental_method"
+                    ],
+                    [
+                        105,
+                        133,
+                        "small-angle X-ray scattering",
+                        "experimental_method"
+                    ],
+                    [
+                        135,
+                        139,
+                        "SAXS",
+                        "experimental_method"
+                    ],
+                    [
+                        142,
+                        181,
+                        "molecular dynamics computer simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        186,
+                        211,
+                        "site-directed mutagenesis",
+                        "experimental_method"
+                    ],
+                    [
+                        231,
+                        234,
+                        "AMs",
+                        "structure_element"
+                    ],
+                    [
+                        313,
+                        320,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 31,
+                "sent": "We find that the large-scale conformational adjustments of the distal CBM46 mediated by the Ig-like hinge domain are crucial in active-site assembly for optimal substrate binding and hydrolysis.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        70,
+                        75,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        92,
+                        112,
+                        "Ig-like hinge domain",
+                        "structure_element"
+                    ],
+                    [
+                        128,
+                        139,
+                        "active-site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 32,
+                "sent": "We propose that the BlCel5B conformational selection/induced-fit mechanism of hydrolysis represents a novel paradigm that applies to several GH5_4 members and, possibly, to a number of other multidomain GHs.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        20,
+                        27,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        141,
+                        146,
+                        "GH5_4",
+                        "protein_type"
+                    ],
+                    [
+                        203,
+                        206,
+                        "GHs",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 33,
+                "sent": "BlCel5B Crystal Structure",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        8,
+                        25,
+                        "Crystal Structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 34,
+                "sent": "BlCel5B crystals in the substrate-free form and complexed with cellopentaose (C5) were obtained and diffracted to 1.7\u2009\u00c5 and 1.75\u2009\u00c5 resolutions, respectively (Supplementary Table 1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        8,
+                        16,
+                        "crystals",
+                        "evidence"
+                    ],
+                    [
+                        24,
+                        38,
+                        "substrate-free",
+                        "protein_state"
+                    ],
+                    [
+                        48,
+                        62,
+                        "complexed with",
+                        "protein_state"
+                    ],
+                    [
+                        63,
+                        76,
+                        "cellopentaose",
+                        "chemical"
+                    ],
+                    [
+                        78,
+                        80,
+                        "C5",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 35,
+                "sent": "The substrate-free and complexed structures exhibited no substantial conformational differences (with the exception of the substrate).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        18,
+                        "substrate-free",
+                        "protein_state"
+                    ],
+                    [
+                        23,
+                        32,
+                        "complexed",
+                        "protein_state"
+                    ],
+                    [
+                        33,
+                        43,
+                        "structures",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 36,
+                "sent": "Because of minor variations in the loops located distal to the substrate-binding site, a root mean squared deviation (rmsd) of 0.33\u2009\u00c5 between the complexed and substrate-free structures was observed.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        35,
+                        40,
+                        "loops",
+                        "structure_element"
+                    ],
+                    [
+                        63,
+                        85,
+                        "substrate-binding site",
+                        "site"
+                    ],
+                    [
+                        89,
+                        116,
+                        "root mean squared deviation",
+                        "evidence"
+                    ],
+                    [
+                        118,
+                        122,
+                        "rmsd",
+                        "evidence"
+                    ],
+                    [
+                        146,
+                        155,
+                        "complexed",
+                        "protein_state"
+                    ],
+                    [
+                        160,
+                        174,
+                        "substrate-free",
+                        "protein_state"
+                    ],
+                    [
+                        175,
+                        185,
+                        "structures",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 37,
+                "sent": "A single protein chain occupies the asymmetric unit, and most of the residues were built, with the exception of the first 17 residues and those in the loop between L398 and P405 due to weak electron density.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        116,
+                        133,
+                        "first 17 residues",
+                        "residue_range"
+                    ],
+                    [
+                        151,
+                        155,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        164,
+                        168,
+                        "L398",
+                        "residue_name_number"
+                    ],
+                    [
+                        173,
+                        177,
+                        "P405",
+                        "residue_name_number"
+                    ],
+                    [
+                        190,
+                        206,
+                        "electron density",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 38,
+                "sent": "The BlCel5B structure comprises three distinct domains: an N-terminal CD (residues 18 to 330), an Ig-like module (residues 335 to 428) and a family 46 CBM (residues 432 to 533) (Fig. 1A,B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        12,
+                        21,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        70,
+                        72,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        83,
+                        92,
+                        "18 to 330",
+                        "residue_range"
+                    ],
+                    [
+                        98,
+                        112,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        123,
+                        133,
+                        "335 to 428",
+                        "residue_range"
+                    ],
+                    [
+                        141,
+                        154,
+                        "family 46 CBM",
+                        "structure_element"
+                    ],
+                    [
+                        165,
+                        175,
+                        "432 to 533",
+                        "residue_range"
+                    ]
+                ]
+            },
+            {
+                "sid": 39,
+                "sent": "Similarly to other members of the GH5 family, the CD of BlCel5B has a typical TIM barrel fold with eight inner \u03b2-strands and eight outer \u03b1 helices that are interconnected by loops and three short \u03b1 helices.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        37,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        50,
+                        52,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        56,
+                        63,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        78,
+                        93,
+                        "TIM barrel fold",
+                        "structure_element"
+                    ],
+                    [
+                        111,
+                        120,
+                        "\u03b2-strands",
+                        "structure_element"
+                    ],
+                    [
+                        137,
+                        146,
+                        "\u03b1 helices",
+                        "structure_element"
+                    ],
+                    [
+                        174,
+                        179,
+                        "loops",
+                        "structure_element"
+                    ],
+                    [
+                        196,
+                        205,
+                        "\u03b1 helices",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 40,
+                "sent": "Very short linkers, D429-D430-P431 and V331-P332-N333-A334, connect the CBM46 to the Ig-like module and the Ig-like module to the CD, respectively.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        18,
+                        "linkers",
+                        "structure_element"
+                    ],
+                    [
+                        20,
+                        34,
+                        "D429-D430-P431",
+                        "structure_element"
+                    ],
+                    [
+                        39,
+                        58,
+                        "V331-P332-N333-A334",
+                        "structure_element"
+                    ],
+                    [
+                        72,
+                        77,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        85,
+                        99,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        108,
+                        122,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        130,
+                        132,
+                        "CD",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 41,
+                "sent": "Both Ig-like module and CBM46 have a \u03b2-sandwich fold composed of two \u03b2-sheets of four and three antiparallel \u03b2-strands interconnected by loops and a short \u03b1 helix between strands \u03b23 and \u03b24 (Fig. 1C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        19,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        24,
+                        29,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        37,
+                        52,
+                        "\u03b2-sandwich fold",
+                        "structure_element"
+                    ],
+                    [
+                        69,
+                        77,
+                        "\u03b2-sheets",
+                        "structure_element"
+                    ],
+                    [
+                        96,
+                        118,
+                        "antiparallel \u03b2-strands",
+                        "structure_element"
+                    ],
+                    [
+                        137,
+                        142,
+                        "loops",
+                        "structure_element"
+                    ],
+                    [
+                        155,
+                        162,
+                        "\u03b1 helix",
+                        "structure_element"
+                    ],
+                    [
+                        171,
+                        178,
+                        "strands",
+                        "structure_element"
+                    ],
+                    [
+                        179,
+                        181,
+                        "\u03b23",
+                        "structure_element"
+                    ],
+                    [
+                        186,
+                        188,
+                        "\u03b24",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 42,
+                "sent": "A structural comparison between the Ig-like module and the CBM46 using the Dali server yielded an rmsd of 2.3\u2009\u00c5 and a Z-score of 10.2.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        2,
+                        23,
+                        "structural comparison",
+                        "experimental_method"
+                    ],
+                    [
+                        36,
+                        50,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        59,
+                        64,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        75,
+                        86,
+                        "Dali server",
+                        "experimental_method"
+                    ],
+                    [
+                        98,
+                        102,
+                        "rmsd",
+                        "evidence"
+                    ],
+                    [
+                        118,
+                        125,
+                        "Z-score",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 43,
+                "sent": "A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd\u2009=\u20091.3\u2009\u00c5 and Z-score\u2009=\u200915.3.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        2,
+                        24,
+                        "structure-based search",
+                        "experimental_method"
+                    ],
+                    [
+                        73,
+                        87,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        106,
+                        120,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        137,
+                        143,
+                        "solved",
+                        "experimental_method"
+                    ],
+                    [
+                        144,
+                        161,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        167,
+                        178,
+                        "tri-modular",
+                        "structure_element"
+                    ],
+                    [
+                        179,
+                        184,
+                        "GH5_4",
+                        "protein_type"
+                    ],
+                    [
+                        197,
+                        216,
+                        "Bacillus halodurans",
+                        "species"
+                    ],
+                    [
+                        218,
+                        225,
+                        "BhCel5B",
+                        "protein"
+                    ],
+                    [
+                        232,
+                        236,
+                        "rmsd",
+                        "evidence"
+                    ],
+                    [
+                        249,
+                        256,
+                        "Z-score",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 44,
+                "sent": "The CBM46 from BhCel5B is the most structurally similar to BlCel5B CBM46, with rmsd\u2009=\u20091.6\u2009\u00c5 and Z-score\u2009=\u200912.4.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        9,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        15,
+                        22,
+                        "BhCel5B",
+                        "protein"
+                    ],
+                    [
+                        59,
+                        66,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        67,
+                        72,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        79,
+                        83,
+                        "rmsd",
+                        "evidence"
+                    ],
+                    [
+                        96,
+                        103,
+                        "Z-score",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 45,
+                "sent": "The sequence identity relative to BhCel5B, however, is low (28% for Ig-like and 25% for CBM46).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        41,
+                        "BhCel5B",
+                        "protein"
+                    ],
+                    [
+                        68,
+                        75,
+                        "Ig-like",
+                        "structure_element"
+                    ],
+                    [
+                        88,
+                        93,
+                        "CBM46",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 46,
+                "sent": "The Ig-like module, adjacent to the CD, contains only one tyrosine (Y367) exposed to solvent and no tryptophan residues.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        18,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        36,
+                        38,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        58,
+                        66,
+                        "tyrosine",
+                        "residue_name"
+                    ],
+                    [
+                        68,
+                        72,
+                        "Y367",
+                        "residue_name_number"
+                    ],
+                    [
+                        100,
+                        110,
+                        "tryptophan",
+                        "residue_name"
+                    ]
+                ]
+            },
+            {
+                "sid": 47,
+                "sent": "Because aromatic residues play a major role in glucose recognition, this observation suggests that substrate binding may not be the primary function of Ig-like module.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        47,
+                        54,
+                        "glucose",
+                        "chemical"
+                    ],
+                    [
+                        152,
+                        166,
+                        "Ig-like module",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 48,
+                "sent": "In contrast, the CBM46 has three tryptophan residues, two of which face the CD substrate binding site (Fig. 1A), indicating that it may be actively engaged in the carbohydrate binding.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        17,
+                        22,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        33,
+                        43,
+                        "tryptophan",
+                        "residue_name"
+                    ],
+                    [
+                        76,
+                        78,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        79,
+                        101,
+                        "substrate binding site",
+                        "site"
+                    ],
+                    [
+                        163,
+                        175,
+                        "carbohydrate",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 49,
+                "sent": "Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        21,
+                        "Electron density maps",
+                        "evidence"
+                    ],
+                    [
+                        41,
+                        52,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        55,
+                        68,
+                        "cellotetraose",
+                        "chemical"
+                    ],
+                    [
+                        70,
+                        72,
+                        "C4",
+                        "chemical"
+                    ],
+                    [
+                        91,
+                        104,
+                        "cellopentaose",
+                        "chemical"
+                    ],
+                    [
+                        106,
+                        108,
+                        "C5",
+                        "chemical"
+                    ],
+                    [
+                        117,
+                        119,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        120,
+                        155,
+                        "negative substrate-binding subsites",
+                        "site"
+                    ],
+                    [
+                        183,
+                        190,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        194,
+                        214,
+                        "catalytically active",
+                        "protein_state"
+                    ],
+                    [
+                        257,
+                        259,
+                        "C5",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 50,
+                "sent": "The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        28,
+                        "lack of electron density",
+                        "evidence"
+                    ],
+                    [
+                        42,
+                        52,
+                        "absence of",
+                        "protein_state"
+                    ],
+                    [
+                        57,
+                        62,
+                        "fifth",
+                        "residue_number"
+                    ],
+                    [
+                        63,
+                        70,
+                        "glucose",
+                        "chemical"
+                    ],
+                    [
+                        94,
+                        96,
+                        "C5",
+                        "chemical"
+                    ],
+                    [
+                        129,
+                        138,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        158,
+                        169,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        172,
+                        177,
+                        "fifth",
+                        "residue_number"
+                    ],
+                    [
+                        178,
+                        185,
+                        "glucose",
+                        "chemical"
+                    ],
+                    [
+                        227,
+                        245,
+                        "catalytic residues",
+                        "site"
+                    ],
+                    [
+                        281,
+                        285,
+                        "R234",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 51,
+                "sent": "The ability of BlCel5B to cleave C5 into glucose and C4 molecules in solution was demonstrated by enzymatic product profile mass spectrometry analysis (Fig. 2A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        15,
+                        22,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        33,
+                        35,
+                        "C5",
+                        "chemical"
+                    ],
+                    [
+                        41,
+                        48,
+                        "glucose",
+                        "chemical"
+                    ],
+                    [
+                        53,
+                        55,
+                        "C4",
+                        "chemical"
+                    ],
+                    [
+                        98,
+                        141,
+                        "enzymatic product profile mass spectrometry",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 52,
+                "sent": "The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-\u03c0 interaction with residue W47 (Fig. 1D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        6,
+                        "C4",
+                        "chemical"
+                    ],
+                    [
+                        23,
+                        30,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        31,
+                        43,
+                        "binding site",
+                        "site"
+                    ],
+                    [
+                        47,
+                        58,
+                        "coordinated",
+                        "bond_interaction"
+                    ],
+                    [
+                        62,
+                        76,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        89,
+                        92,
+                        "N36",
+                        "residue_name_number"
+                    ],
+                    [
+                        94,
+                        98,
+                        "H113",
+                        "residue_name_number"
+                    ],
+                    [
+                        100,
+                        104,
+                        "H114",
+                        "residue_name_number"
+                    ],
+                    [
+                        106,
+                        110,
+                        "N158",
+                        "residue_name_number"
+                    ],
+                    [
+                        112,
+                        116,
+                        "W301",
+                        "residue_name_number"
+                    ],
+                    [
+                        122,
+                        126,
+                        "N303",
+                        "residue_name_number"
+                    ],
+                    [
+                        136,
+                        152,
+                        "CH-\u03c0 interaction",
+                        "bond_interaction"
+                    ],
+                    [
+                        166,
+                        169,
+                        "W47",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 53,
+                "sent": "These residues belong to the CD and are conserved in the GH5 family.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        31,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        40,
+                        49,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        57,
+                        60,
+                        "GH5",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 54,
+                "sent": "BlCel5B enzymatic activity",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 55,
+                "sent": "BlCel5B exhibits optimum activity toward carboxymethylcellulose (CMC; 8.7\u2009U/mg) at a pH of 4.0 and 55\u2009\u00b0C and retains approximately half of its maximum activity at 80\u2009\u00b0C, demonstrating considerable thermal stability (Fig. 2B,C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        41,
+                        63,
+                        "carboxymethylcellulose",
+                        "chemical"
+                    ],
+                    [
+                        65,
+                        68,
+                        "CMC",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 56,
+                "sent": "BlCel5B is also active on \u03b2-glucan (34\u2009U/mg), lichenan (17.8\u2009U/mg) and xyloglucan (15.7\u2009U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-\u03b2-mannan or the insoluble substrate Azo-Avicel.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        16,
+                        22,
+                        "active",
+                        "protein_state"
+                    ],
+                    [
+                        26,
+                        34,
+                        "\u03b2-glucan",
+                        "chemical"
+                    ],
+                    [
+                        46,
+                        54,
+                        "lichenan",
+                        "chemical"
+                    ],
+                    [
+                        71,
+                        81,
+                        "xyloglucan",
+                        "chemical"
+                    ],
+                    [
+                        152,
+                        165,
+                        "galactomannan",
+                        "chemical"
+                    ],
+                    [
+                        167,
+                        170,
+                        "rye",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        171,
+                        183,
+                        "arabinoxylan",
+                        "chemical"
+                    ],
+                    [
+                        185,
+                        197,
+                        "1,4-\u03b2-mannan",
+                        "chemical"
+                    ],
+                    [
+                        225,
+                        235,
+                        "Azo-Avicel",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 57,
+                "sent": "Kinetic parameters were calculated assuming Michaelis-Menten behavior with CMC as substrate: KM\u2009=\u20091.78\u2009g L\u22121 and Vmax\u2009=\u20091.41\u2009\u00d7\u200910\u22124 g s\u22121 mg protein\u22121 (Fig. 2D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        44,
+                        69,
+                        "Michaelis-Menten behavior",
+                        "experimental_method"
+                    ],
+                    [
+                        75,
+                        78,
+                        "CMC",
+                        "chemical"
+                    ],
+                    [
+                        93,
+                        95,
+                        "KM",
+                        "evidence"
+                    ],
+                    [
+                        113,
+                        117,
+                        "Vmax",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 58,
+                "sent": "Although BlCel5B is not a highly active enzyme against one specific substrate as compared to others GH5_4, it has the advantage of being active against different substrates with \u03b2-1,3 and/or \u03b2-1,4 glycosidic linkages.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        16,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        33,
+                        39,
+                        "active",
+                        "protein_state"
+                    ],
+                    [
+                        100,
+                        105,
+                        "GH5_4",
+                        "protein_type"
+                    ],
+                    [
+                        137,
+                        143,
+                        "active",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 59,
+                "sent": "To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (\u0394CBM46) and an Ig-like\u2009+\u2009CBM46 deletion (\u0394Ig-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        36,
+                        53,
+                        "ancillary modules",
+                        "structure_element"
+                    ],
+                    [
+                        58,
+                        65,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        76,
+                        92,
+                        "enzymatic assays",
+                        "experimental_method"
+                    ],
+                    [
+                        128,
+                        135,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        139,
+                        144,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        145,
+                        153,
+                        "deletion",
+                        "experimental_method"
+                    ],
+                    [
+                        155,
+                        161,
+                        "\u0394CBM46",
+                        "mutant"
+                    ],
+                    [
+                        170,
+                        177,
+                        "Ig-like",
+                        "structure_element"
+                    ],
+                    [
+                        180,
+                        185,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        186,
+                        194,
+                        "deletion",
+                        "experimental_method"
+                    ],
+                    [
+                        196,
+                        205,
+                        "\u0394Ig-CBM46",
+                        "mutant"
+                    ],
+                    [
+                        218,
+                        233,
+                        "point mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        241,
+                        246,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        270,
+                        275,
+                        "W479A",
+                        "mutant"
+                    ],
+                    [
+                        280,
+                        285,
+                        "W481A",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 60,
+                "sent": "These mutants were expressed and purified as described for the wild-type enzyme.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        6,
+                        13,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        19,
+                        41,
+                        "expressed and purified",
+                        "experimental_method"
+                    ],
+                    [
+                        63,
+                        72,
+                        "wild-type",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 61,
+                "sent": "Strikingly, neither of the deletion variants exhibited detectable activity toward any of the substrates tested using full-length BlCel5B (Table 1), demonstrating that the Ig-like module and the CBM46 are essential for BlCel5B activity.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        27,
+                        44,
+                        "deletion variants",
+                        "protein_state"
+                    ],
+                    [
+                        117,
+                        128,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        129,
+                        136,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        171,
+                        185,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        194,
+                        199,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        218,
+                        225,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 62,
+                "sent": "Thermal shift assays were conducted to confirm structural stability of the mutants (Supplementary Fig. 1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        20,
+                        "Thermal shift assays",
+                        "experimental_method"
+                    ],
+                    [
+                        75,
+                        82,
+                        "mutants",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 63,
+                "sent": "All of the constructs showed similar melting temperatures: 62\u2009\u00b0C for BlCel5B, 58\u2009\u00b0C for BlCel5B\u0394CBM46, 56\u2009\u00b0C for BlCel5B\u0394Ig-CBM46, 65\u2009\u00b0C for BlCel5BW479A and 59\u2009\u00b0C for BlCel5BW479A, thus confirming their proper overall fold.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        37,
+                        57,
+                        "melting temperatures",
+                        "evidence"
+                    ],
+                    [
+                        69,
+                        76,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        88,
+                        101,
+                        "BlCel5B\u0394CBM46",
+                        "mutant"
+                    ],
+                    [
+                        113,
+                        129,
+                        "BlCel5B\u0394Ig-CBM46",
+                        "mutant"
+                    ],
+                    [
+                        141,
+                        153,
+                        "BlCel5BW479A",
+                        "mutant"
+                    ],
+                    [
+                        168,
+                        180,
+                        "BlCel5BW479A",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 64,
+                "sent": "We also examined the function of the CBM46 inner surface residues W479 and W481 (Fig. 1A) in BlCel5B activity by performing enzymatic assays with W479A and W481A mutants.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        37,
+                        42,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        49,
+                        56,
+                        "surface",
+                        "site"
+                    ],
+                    [
+                        66,
+                        70,
+                        "W479",
+                        "residue_name_number"
+                    ],
+                    [
+                        75,
+                        79,
+                        "W481",
+                        "residue_name_number"
+                    ],
+                    [
+                        93,
+                        100,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        124,
+                        140,
+                        "enzymatic assays",
+                        "experimental_method"
+                    ],
+                    [
+                        146,
+                        151,
+                        "W479A",
+                        "mutant"
+                    ],
+                    [
+                        156,
+                        161,
+                        "W481A",
+                        "mutant"
+                    ],
+                    [
+                        162,
+                        169,
+                        "mutants",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 65,
+                "sent": "Both mutations reduced enzymatic activity toward all tested substrates (Table 1), with W481A having a stronger effect than W479A (~64% vs. 79% activity relative to wt BlCel5B using \u03b2-glucan and ~10% vs. 50% using CMC).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        14,
+                        "mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        87,
+                        92,
+                        "W481A",
+                        "mutant"
+                    ],
+                    [
+                        123,
+                        128,
+                        "W479A",
+                        "mutant"
+                    ],
+                    [
+                        164,
+                        166,
+                        "wt",
+                        "protein_state"
+                    ],
+                    [
+                        167,
+                        174,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        181,
+                        189,
+                        "\u03b2-glucan",
+                        "chemical"
+                    ],
+                    [
+                        213,
+                        216,
+                        "CMC",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 66,
+                "sent": "This indicates that CBM46 must interact with the substrate via residues W479 and W481.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        20,
+                        25,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        72,
+                        76,
+                        "W479",
+                        "residue_name_number"
+                    ],
+                    [
+                        81,
+                        85,
+                        "W481",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 67,
+                "sent": "However, since the BlCel5B crystal structure exhibits no close contact between these residues and the substrate, these results suggest the existence of large-amplitude interdomain motions that may enable direct interactions between CBM46 and the carbohydrate.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        26,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        27,
+                        44,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        57,
+                        62,
+                        "close",
+                        "protein_state"
+                    ],
+                    [
+                        232,
+                        237,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        246,
+                        258,
+                        "carbohydrate",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 68,
+                "sent": "BlCelB5 dynamics and binding-site architecture",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "BlCelB5",
+                        "protein"
+                    ],
+                    [
+                        21,
+                        33,
+                        "binding-site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 69,
+                "sent": "Molecular dynamics (MD) simulations were performed to investigate the conformational mobility of BlCel5B.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        18,
+                        "Molecular dynamics",
+                        "experimental_method"
+                    ],
+                    [
+                        20,
+                        22,
+                        "MD",
+                        "experimental_method"
+                    ],
+                    [
+                        24,
+                        35,
+                        "simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        97,
+                        104,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 70,
+                "sent": "In the simulations of the crystal structure for BlCel5B bound to C4, the substrate dissociates from the protein within the first 100\u2009ns of the simulation time (Supplementary Fig. 2A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        7,
+                        18,
+                        "simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        26,
+                        43,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        48,
+                        55,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        56,
+                        64,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        65,
+                        67,
+                        "C4",
+                        "chemical"
+                    ],
+                    [
+                        143,
+                        153,
+                        "simulation",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 71,
+                "sent": "This observation suggests that cellotetraose does not exhibit detectable affinity for this specific BlCel5B conformation in solution, as one might otherwise expect for a reaction product.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        31,
+                        44,
+                        "cellotetraose",
+                        "chemical"
+                    ],
+                    [
+                        100,
+                        107,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 72,
+                "sent": "No changes beyond local fluctuations were observed in any of the three BlCel5B domains within the time scale of these runs (400\u2009ns; Supplementary Fig. 2B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        71,
+                        78,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 73,
+                "sent": "However, the CBM46 and Ig-like domains did exhibit rigid body-like motions relative to the CD, with rmsd values around 2.3\u2009\u00c5 and 1.8\u2009\u00c5, respectively, suggesting that BlCel5B may execute large-amplitude interdomain motions over longer time scales (Supplementary Fig. 2B,C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        18,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        23,
+                        38,
+                        "Ig-like domains",
+                        "structure_element"
+                    ],
+                    [
+                        91,
+                        93,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        100,
+                        104,
+                        "rmsd",
+                        "evidence"
+                    ],
+                    [
+                        166,
+                        173,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 74,
+                "sent": "Accordingly, simulations were then performed using accelerated molecular dynamics (aMD) techniques to probe BlCel5B interdomain motions.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        24,
+                        "simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        51,
+                        81,
+                        "accelerated molecular dynamics",
+                        "experimental_method"
+                    ],
+                    [
+                        83,
+                        86,
+                        "aMD",
+                        "experimental_method"
+                    ],
+                    [
+                        108,
+                        115,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 75,
+                "sent": "aMD enhances conformational sampling by raising the basins of the dihedral potential energy surface without affecting the general form of the atomistic potential, thereby increasing transition rates between different local minima.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "aMD",
+                        "experimental_method"
+                    ],
+                    [
+                        66,
+                        99,
+                        "dihedral potential energy surface",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 76,
+                "sent": "aMD trajectories corresponding to more than 1.0\u2009\u03bcs of conventional MD runs were generated.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "aMD",
+                        "experimental_method"
+                    ],
+                    [
+                        4,
+                        16,
+                        "trajectories",
+                        "evidence"
+                    ],
+                    [
+                        67,
+                        69,
+                        "MD",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 77,
+                "sent": "During these simulations, we observed occlusive conformations between CBM46 and CD that resulted in a rearrangement of the enzyme\u2019s architecture around the active site (Video S1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        24,
+                        "simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        70,
+                        75,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        80,
+                        82,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        156,
+                        167,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 78,
+                "sent": "Figure 3A shows BlCel5B in the crystallographic conformation (red) and in a selected configuration obtained with aMD (blue) in the absence of the substrate.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        16,
+                        23,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        31,
+                        47,
+                        "crystallographic",
+                        "experimental_method"
+                    ],
+                    [
+                        113,
+                        116,
+                        "aMD",
+                        "experimental_method"
+                    ],
+                    [
+                        131,
+                        141,
+                        "absence of",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 79,
+                "sent": "Interdomain motions were gauged by the time evolution of the distance between the \u03b1 carbons of residues I120 and E477 (represented as spheres in Fig. 3A), belonging to the CD and CBM46, respectively.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        61,
+                        69,
+                        "distance",
+                        "evidence"
+                    ],
+                    [
+                        104,
+                        108,
+                        "I120",
+                        "residue_name_number"
+                    ],
+                    [
+                        113,
+                        117,
+                        "E477",
+                        "residue_name_number"
+                    ],
+                    [
+                        172,
+                        174,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        179,
+                        184,
+                        "CBM46",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 80,
+                "sent": "Figure 3C shows that the I120-E477 distance (red curve) gradually decreases from ~35\u2009\u00c5 to ~7\u2009\u00c5 within the first half of the 1.0\u2009\u03bcs aMD trajectory, indicating a transition between the semi-open (crystallographic) and occluded (aMD sampled) configurations.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        29,
+                        "I120",
+                        "residue_name_number"
+                    ],
+                    [
+                        30,
+                        34,
+                        "E477",
+                        "residue_name_number"
+                    ],
+                    [
+                        35,
+                        43,
+                        "distance",
+                        "evidence"
+                    ],
+                    [
+                        131,
+                        134,
+                        "aMD",
+                        "experimental_method"
+                    ],
+                    [
+                        135,
+                        145,
+                        "trajectory",
+                        "evidence"
+                    ],
+                    [
+                        183,
+                        192,
+                        "semi-open",
+                        "protein_state"
+                    ],
+                    [
+                        194,
+                        210,
+                        "crystallographic",
+                        "experimental_method"
+                    ],
+                    [
+                        216,
+                        224,
+                        "occluded",
+                        "protein_state"
+                    ],
+                    [
+                        226,
+                        229,
+                        "aMD",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 81,
+                "sent": "During the second half of the aMD simulation, the full-length enzyme remained in the closed conformation, with the CBM46 covering the carbohydrate-binding site.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        30,
+                        44,
+                        "aMD simulation",
+                        "experimental_method"
+                    ],
+                    [
+                        50,
+                        61,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        85,
+                        91,
+                        "closed",
+                        "protein_state"
+                    ],
+                    [
+                        115,
+                        120,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        134,
+                        159,
+                        "carbohydrate-binding site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 82,
+                "sent": "These results suggest that BlCel5B undergoes large-scale interdomain movements that enable interactions between CBM46 and the substrate bound to the CD.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        27,
+                        34,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        112,
+                        117,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        136,
+                        144,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        149,
+                        151,
+                        "CD",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 83,
+                "sent": "To study the interactions of BlCel5B with a non-hydrolyzed glucan chain, we built a model structure with a cellooctaose (C8) chain spanning the entire positive (+1 to +4) and negative (\u22124 to \u22121) subsites of the enzyme.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        36,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        59,
+                        65,
+                        "glucan",
+                        "chemical"
+                    ],
+                    [
+                        90,
+                        99,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        107,
+                        119,
+                        "cellooctaose",
+                        "chemical"
+                    ],
+                    [
+                        121,
+                        123,
+                        "C8",
+                        "chemical"
+                    ],
+                    [
+                        151,
+                        170,
+                        "positive (+1 to +4)",
+                        "site"
+                    ],
+                    [
+                        175,
+                        194,
+                        "negative (\u22124 to \u22121)",
+                        "site"
+                    ],
+                    [
+                        195,
+                        203,
+                        "subsites",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 84,
+                "sent": "Starting from the crystallographic BlCel5B conformation, the C8 molecule deviated significantly from the active site and assumed a non-productive binding mode (Supplementary Fig. 2D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        35,
+                        42,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        61,
+                        63,
+                        "C8",
+                        "chemical"
+                    ],
+                    [
+                        105,
+                        116,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 85,
+                "sent": "This observation suggests that the open conformation of BlCel5B is not able to hold the substrate in a position suitable for hydrolysis (Supplementary Fig. 2E).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        35,
+                        39,
+                        "open",
+                        "protein_state"
+                    ],
+                    [
+                        56,
+                        63,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 86,
+                "sent": "However, after subjecting the BlCel5B-C8 complex to a 0.5\u2009\u03bcs aMD simulation with harmonic restraints on the C8 chain to prevent it from deviating from the productive binding mode, the CBM46 readily closed over the CD and trapped the C8 chain in position for hydrolysis (Fig. 3B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        30,
+                        40,
+                        "BlCel5B-C8",
+                        "complex_assembly"
+                    ],
+                    [
+                        61,
+                        75,
+                        "aMD simulation",
+                        "experimental_method"
+                    ],
+                    [
+                        108,
+                        110,
+                        "C8",
+                        "chemical"
+                    ],
+                    [
+                        184,
+                        189,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        198,
+                        204,
+                        "closed",
+                        "protein_state"
+                    ],
+                    [
+                        214,
+                        216,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        233,
+                        235,
+                        "C8",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 87,
+                "sent": "In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20\u2009\u00c5 in the closed configuration that traps the C8 molecule (in contrast to ~7\u2009\u00c5 for substrate-free BlCel5B) (Fig. 3C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        7,
+                        18,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        34,
+                        39,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        93,
+                        119,
+                        "crystallographic structure",
+                        "evidence"
+                    ],
+                    [
+                        145,
+                        159,
+                        "substrate-free",
+                        "protein_state"
+                    ],
+                    [
+                        160,
+                        167,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        168,
+                        183,
+                        "aMD simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        212,
+                        216,
+                        "I120",
+                        "residue_name_number"
+                    ],
+                    [
+                        217,
+                        221,
+                        "E477",
+                        "residue_name_number"
+                    ],
+                    [
+                        222,
+                        230,
+                        "distance",
+                        "evidence"
+                    ],
+                    [
+                        266,
+                        272,
+                        "closed",
+                        "protein_state"
+                    ],
+                    [
+                        302,
+                        304,
+                        "C8",
+                        "chemical"
+                    ],
+                    [
+                        339,
+                        353,
+                        "substrate-free",
+                        "protein_state"
+                    ],
+                    [
+                        354,
+                        361,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 88,
+                "sent": "This BlCel5B-C8 configuration remains stable over an additional 500 ns of conventional MD simulation with no restraints (Fig. 3C cyan line, Supplementary Fig. 2E,F).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        15,
+                        "BlCel5B-C8",
+                        "complex_assembly"
+                    ],
+                    [
+                        87,
+                        100,
+                        "MD simulation",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 89,
+                "sent": "A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        81,
+                        92,
+                        "simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        110,
+                        115,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        116,
+                        126,
+                        "tryptophan",
+                        "residue_name"
+                    ],
+                    [
+                        136,
+                        140,
+                        "W479",
+                        "residue_name_number"
+                    ],
+                    [
+                        145,
+                        149,
+                        "W481",
+                        "residue_name_number"
+                    ],
+                    [
+                        162,
+                        164,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        165,
+                        175,
+                        "tryptophan",
+                        "residue_name"
+                    ],
+                    [
+                        210,
+                        222,
+                        "carbohydrate",
+                        "chemical"
+                    ],
+                    [
+                        265,
+                        271,
+                        "tunnel",
+                        "site"
+                    ],
+                    [
+                        296,
+                        303,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        304,
+                        317,
+                        "binding cleft",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 90,
+                "sent": "Together, these results indicate that CBM46 is a key component of the catalytic active complex, providing an explanation as to why CBM46 is essential for the enzymatic activity of BlCel5B.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        38,
+                        43,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        70,
+                        86,
+                        "catalytic active",
+                        "protein_state"
+                    ],
+                    [
+                        131,
+                        136,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        180,
+                        187,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 91,
+                "sent": "To enable substantially longer time scales compared to atomistic simulations, we further explored the dynamics of BlCel5B using coarse-grained MD (CG-MD) simulations.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        55,
+                        76,
+                        "atomistic simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        114,
+                        121,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        128,
+                        145,
+                        "coarse-grained MD",
+                        "experimental_method"
+                    ],
+                    [
+                        147,
+                        152,
+                        "CG-MD",
+                        "experimental_method"
+                    ],
+                    [
+                        154,
+                        165,
+                        "simulations",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 92,
+                "sent": "We performed three independent ~120\u2009\u03bcs CG-MD simulations, for a total of approximately 360\u2009\u03bcs of sampling.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        39,
+                        56,
+                        "CG-MD simulations",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 93,
+                "sent": "The distance between the \u03b1 carbons of two residues centrally positioned in the CD and CBM46 (Fig. 4A) was monitored, and the results shown in Fig. 4B indicate that the wide-amplitude events described above frequently appear in this time scale.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        12,
+                        "distance",
+                        "evidence"
+                    ],
+                    [
+                        79,
+                        81,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        86,
+                        91,
+                        "CBM46",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 94,
+                "sent": "The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        34,
+                        "computed distance distribution",
+                        "evidence"
+                    ],
+                    [
+                        115,
+                        121,
+                        "closed",
+                        "protein_state"
+                    ],
+                    [
+                        172,
+                        186,
+                        "substrate-free",
+                        "protein_state"
+                    ],
+                    [
+                        187,
+                        202,
+                        "aMD simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        213,
+                        218,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        238,
+                        240,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        254,
+                        276,
+                        "substrate binding site",
+                        "site"
+                    ],
+                    [
+                        286,
+                        295,
+                        "semi-open",
+                        "protein_state"
+                    ],
+                    [
+                        325,
+                        351,
+                        "crystallographic structure",
+                        "evidence"
+                    ],
+                    [
+                        363,
+                        371,
+                        "extended",
+                        "protein_state"
+                    ],
+                    [
+                        372,
+                        379,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        407,
+                        409,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        414,
+                        419,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        455,
+                        472,
+                        "crystal structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 95,
+                "sent": "BlCel5B conformers fit the SAXS envelope",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        27,
+                        31,
+                        "SAXS",
+                        "experimental_method"
+                    ],
+                    [
+                        32,
+                        40,
+                        "envelope",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 96,
+                "sent": "SAXS experiments were conducted to assess BlCel5B conformational states in solution, and the results revealed the enzyme in its monomeric form, with average values of Rg\u2009=\u200927.17\u2009\u00c5 and Dmax\u2009=\u200987.59\u2009\u00c5 (Supplementary Table 2).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "SAXS",
+                        "experimental_method"
+                    ],
+                    [
+                        42,
+                        49,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        128,
+                        137,
+                        "monomeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        167,
+                        169,
+                        "Rg",
+                        "evidence"
+                    ],
+                    [
+                        184,
+                        188,
+                        "Dmax",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 97,
+                "sent": "The ab initio dummy atom model (DAM) demonstrated that the SAXS-derived BlCel5B molecular envelope could not be single-handedly filled by any of the main conformational states encountered in the simulations (Fig. 4D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        30,
+                        "ab initio dummy atom model",
+                        "experimental_method"
+                    ],
+                    [
+                        32,
+                        35,
+                        "DAM",
+                        "experimental_method"
+                    ],
+                    [
+                        59,
+                        63,
+                        "SAXS",
+                        "experimental_method"
+                    ],
+                    [
+                        72,
+                        79,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        90,
+                        98,
+                        "envelope",
+                        "evidence"
+                    ],
+                    [
+                        195,
+                        206,
+                        "simulations",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 98,
+                "sent": "It is known that a Kratky plot exhibits a peak with an elevated baseline at high q for a monodisperse system composed of multi-domain particles with flexible extensions.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        30,
+                        "Kratky plot",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 99,
+                "sent": "Indeed, an elevation of the baseline toward a hyperbolic-like curve was observed for BlCel5B, indicating a considerable degree of molecular mobility in solution (Supplementary Fig. 3).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        85,
+                        92,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 100,
+                "sent": "Thus, the conformational heterogeneity of the enzyme can be decomposed in structural terms as a combination of conformational states identified in our crystallographic and MD studies.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        151,
+                        182,
+                        "crystallographic and MD studies",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 101,
+                "sent": "We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        22,
+                        "SAXS",
+                        "experimental_method"
+                    ],
+                    [
+                        23,
+                        31,
+                        "envelope",
+                        "evidence"
+                    ],
+                    [
+                        75,
+                        90,
+                        "superimposition",
+                        "experimental_method"
+                    ],
+                    [
+                        152,
+                        159,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        173,
+                        179,
+                        "closed",
+                        "protein_state"
+                    ],
+                    [
+                        183,
+                        188,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        189,
+                        191,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        192,
+                        200,
+                        "occluded",
+                        "protein_state"
+                    ],
+                    [
+                        233,
+                        244,
+                        "simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        278,
+                        287,
+                        "semi-open",
+                        "protein_state"
+                    ],
+                    [
+                        320,
+                        337,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        367,
+                        375,
+                        "extended",
+                        "protein_state"
+                    ],
+                    [
+                        398,
+                        409,
+                        "simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        445,
+                        449,
+                        "SAXS",
+                        "experimental_method"
+                    ],
+                    [
+                        450,
+                        458,
+                        "envelope",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 102,
+                "sent": "The resulting average scattering curve from this model fits the experimental protein scattering intensity, with \u03c7\u2009=\u20091.89 (Supplementary Fig. 3).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        38,
+                        "average scattering curve",
+                        "evidence"
+                    ],
+                    [
+                        85,
+                        105,
+                        "scattering intensity",
+                        "evidence"
+                    ],
+                    [
+                        112,
+                        113,
+                        "\u03c7",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 103,
+                "sent": "GH5_4 phylogenetic analysis",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "GH5_4",
+                        "protein_type"
+                    ],
+                    [
+                        6,
+                        27,
+                        "phylogenetic analysis",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 104,
+                "sent": "After the exclusion of partial sequences and the suppression of highly identical members (higher than 90% identity), 144 sequences containing between 277 and 400 residues were aligned and used to construct a phylogenetic tree (Supplementary Fig. 4A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        150,
+                        161,
+                        "277 and 400",
+                        "residue_range"
+                    ],
+                    [
+                        176,
+                        183,
+                        "aligned",
+                        "experimental_method"
+                    ],
+                    [
+                        208,
+                        225,
+                        "phylogenetic tree",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 105,
+                "sent": "According to PFAM database conserved domain classification, 128 GH5 enzymes have an architecture consisting of an N-terminal catalytic module, a CBM_X2 module and an unknown module of approximately 100 residues at the C-terminus (Supplementary Fig. 4B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        64,
+                        67,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        125,
+                        141,
+                        "catalytic module",
+                        "structure_element"
+                    ],
+                    [
+                        145,
+                        151,
+                        "CBM_X2",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 106,
+                "sent": "Of these, 12 enzymes have an additional CBM1, and 5 have a CBM2 at the N-terminal region.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        40,
+                        44,
+                        "CBM1",
+                        "structure_element"
+                    ],
+                    [
+                        59,
+                        63,
+                        "CBM2",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 107,
+                "sent": "Based on this PFAM architecture and CAZy subfamily classification, all the 144 enzymes (including BlCel5B) belong to the GH5_4 subfamily and group together in the same branch of the phylogenetic tree, evidencing a common ancestor.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        98,
+                        105,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        121,
+                        126,
+                        "GH5_4",
+                        "protein_type"
+                    ],
+                    [
+                        182,
+                        199,
+                        "phylogenetic tree",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 108,
+                "sent": "These results support the hypothesis that the enzymes may employ the same mechanism by which ligand binding is mediated by an extensive conformational breathing of the enzyme that involves the large-scale movement of CBM46 around the Ig-like module (CBM_X2) as a structural hinge.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        217,
+                        222,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        234,
+                        248,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        250,
+                        256,
+                        "CBM_X2",
+                        "structure_element"
+                    ],
+                    [
+                        263,
+                        279,
+                        "structural hinge",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 109,
+                "sent": "Here, we elucidate the trimodular molecular architecture of the full-length BlCel5B, a member of the GH5_4 subfamily, for which large-scale conformational dynamics appears to play a central role in its enzymatic activity.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        23,
+                        33,
+                        "trimodular",
+                        "protein_state"
+                    ],
+                    [
+                        64,
+                        75,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        76,
+                        83,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        101,
+                        106,
+                        "GH5_4",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 110,
+                "sent": "Full-length BlCel5B is active on both cellulosic and hemicellulosic substrates and auxiliary modules are crucial for its activity.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Full-length",
+                        "protein_state"
+                    ],
+                    [
+                        12,
+                        19,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        23,
+                        29,
+                        "active",
+                        "protein_state"
+                    ],
+                    [
+                        38,
+                        48,
+                        "cellulosic",
+                        "chemical"
+                    ],
+                    [
+                        53,
+                        67,
+                        "hemicellulosic",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 111,
+                "sent": "Most carbohydrate-active enzymes are modular and consist of a catalytic domain appended to one or more separate AMs.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        5,
+                        32,
+                        "carbohydrate-active enzymes",
+                        "protein_type"
+                    ],
+                    [
+                        62,
+                        78,
+                        "catalytic domain",
+                        "structure_element"
+                    ],
+                    [
+                        112,
+                        115,
+                        "AMs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 112,
+                "sent": "AMs, such as CBMs, typically recognize carbohydrates and target their cognate catalytic domains toward the substrate.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "AMs",
+                        "structure_element"
+                    ],
+                    [
+                        13,
+                        17,
+                        "CBMs",
+                        "structure_element"
+                    ],
+                    [
+                        39,
+                        52,
+                        "carbohydrates",
+                        "chemical"
+                    ],
+                    [
+                        78,
+                        95,
+                        "catalytic domains",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 113,
+                "sent": "Because the structural analysis of the protein is challenging if the linkers connecting the structural subunits of the enzyme are long and flexible, the standard approach is to study the domains separately.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        12,
+                        31,
+                        "structural analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        69,
+                        76,
+                        "linkers",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 114,
+                "sent": "In this work, a combination of protein crystallography, computational molecular dynamics, and SAXS analyses enabled the identification of a new conformational selection-based molecular mechanism that involves GH5 catalytic domain and two AMs in full-length BlCel5B.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        31,
+                        54,
+                        "protein crystallography",
+                        "experimental_method"
+                    ],
+                    [
+                        56,
+                        88,
+                        "computational molecular dynamics",
+                        "experimental_method"
+                    ],
+                    [
+                        94,
+                        98,
+                        "SAXS",
+                        "experimental_method"
+                    ],
+                    [
+                        209,
+                        212,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        213,
+                        229,
+                        "catalytic domain",
+                        "structure_element"
+                    ],
+                    [
+                        238,
+                        241,
+                        "AMs",
+                        "structure_element"
+                    ],
+                    [
+                        245,
+                        256,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        257,
+                        264,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 115,
+                "sent": "We observed that the BlCel5B distal CBM46 is directly involved in shaping the local architecture of the substrate-binding site.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        21,
+                        28,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        36,
+                        41,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        104,
+                        126,
+                        "substrate-binding site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 116,
+                "sent": "Although the CD alone appears unable to bind the substrate for catalysis, the AMs exhibit open-close motions that allow the substrate to be captured in a suitable position for hydrolysis.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        13,
+                        15,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        16,
+                        21,
+                        "alone",
+                        "protein_state"
+                    ],
+                    [
+                        78,
+                        81,
+                        "AMs",
+                        "structure_element"
+                    ],
+                    [
+                        90,
+                        94,
+                        "open",
+                        "protein_state"
+                    ],
+                    [
+                        95,
+                        100,
+                        "close",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 117,
+                "sent": "Here, we advocate that large-amplitude motions of AMs are crucial for assembling the enzyme into its active conformation, highlighting a new function of CBMs.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        50,
+                        53,
+                        "AMs",
+                        "structure_element"
+                    ],
+                    [
+                        101,
+                        107,
+                        "active",
+                        "protein_state"
+                    ],
+                    [
+                        153,
+                        157,
+                        "CBMs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 118,
+                "sent": "This mechanism of substrate binding closely resembles the extended conformational selection model, with the induced-fit mechanism of reaction as its limiting case.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        58,
+                        66,
+                        "extended",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 119,
+                "sent": "To the best of our knowledge, this enzymatic mechanism has not been proposed previously for any GH.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        96,
+                        98,
+                        "GH",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 120,
+                "sent": "The CD binding site of BlCel5B is open and relatively flat and is thus barely able to properly hold the substrate in position for catalysis without assistance from the CBM46.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        19,
+                        "CD binding site",
+                        "site"
+                    ],
+                    [
+                        23,
+                        30,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        168,
+                        173,
+                        "CBM46",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 121,
+                "sent": "In contrast, other GH5s belonging to subfamily 4 listed in the Protein Data Bank exhibit a deep binding cleft or tunnel that can effectively entrap the substrate for catalysis (Fig. 5).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        19,
+                        23,
+                        "GH5s",
+                        "protein_type"
+                    ],
+                    [
+                        96,
+                        109,
+                        "binding cleft",
+                        "site"
+                    ],
+                    [
+                        113,
+                        119,
+                        "tunnel",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 122,
+                "sent": "Due to the marked interdomain conformational rearrangement observed in our simulations, the CBM46 generates a confined binding site in BlCel5B that resembles the binding site architecture of the other GH5 enzymes that lack AMs.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        75,
+                        86,
+                        "simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        92,
+                        97,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        119,
+                        131,
+                        "binding site",
+                        "site"
+                    ],
+                    [
+                        135,
+                        142,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        162,
+                        174,
+                        "binding site",
+                        "site"
+                    ],
+                    [
+                        201,
+                        204,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        218,
+                        222,
+                        "lack",
+                        "protein_state"
+                    ],
+                    [
+                        223,
+                        226,
+                        "AMs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 123,
+                "sent": "Thus, BlCel5B appears to have adopted a strategy of CBM46-mediated interactions for proper functioning.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        6,
+                        13,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        52,
+                        57,
+                        "CBM46",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 124,
+                "sent": "Although the homologous BhCel5B has the same domain architecture of BlCel5B and belongs to the same subfamily (a comparison of the sequence and structure of BlCel5B and BhCel5B is presented in Supplementary Fig. 5), its binding site exhibits important differences that may impact the catalytic mechanism.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        24,
+                        31,
+                        "BhCel5B",
+                        "protein"
+                    ],
+                    [
+                        68,
+                        75,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        144,
+                        153,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        157,
+                        164,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        169,
+                        176,
+                        "BhCel5B",
+                        "protein"
+                    ],
+                    [
+                        220,
+                        232,
+                        "binding site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 125,
+                "sent": "The BhCel5B binding site is V-shaped and deeper than the BlCel5B binding site (Figs 5 and 6).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "BhCel5B",
+                        "protein"
+                    ],
+                    [
+                        12,
+                        24,
+                        "binding site",
+                        "site"
+                    ],
+                    [
+                        28,
+                        36,
+                        "V-shaped",
+                        "protein_state"
+                    ],
+                    [
+                        57,
+                        64,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        65,
+                        77,
+                        "binding site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 126,
+                "sent": "This is due to the loop between residues F177 and R185 from BhCel5B (absent in the BlCel5B), which contains residue W181 that forms part of the binding cleft (Fig. 6).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        19,
+                        23,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        41,
+                        45,
+                        "F177",
+                        "residue_name_number"
+                    ],
+                    [
+                        50,
+                        54,
+                        "R185",
+                        "residue_name_number"
+                    ],
+                    [
+                        60,
+                        67,
+                        "BhCel5B",
+                        "protein"
+                    ],
+                    [
+                        69,
+                        75,
+                        "absent",
+                        "protein_state"
+                    ],
+                    [
+                        83,
+                        90,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        116,
+                        120,
+                        "W181",
+                        "residue_name_number"
+                    ],
+                    [
+                        144,
+                        157,
+                        "binding cleft",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 127,
+                "sent": "Consistently, although BhCel5B CBM46 is important for \u03b2-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        23,
+                        30,
+                        "BhCel5B",
+                        "protein"
+                    ],
+                    [
+                        31,
+                        36,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        54,
+                        70,
+                        "\u03b2-1,3-1,4-glucan",
+                        "chemical"
+                    ],
+                    [
+                        83,
+                        90,
+                        "BhCel5B",
+                        "protein"
+                    ],
+                    [
+                        113,
+                        119,
+                        "active",
+                        "protein_state"
+                    ],
+                    [
+                        120,
+                        127,
+                        "without",
+                        "protein_state"
+                    ],
+                    [
+                        128,
+                        133,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        140,
+                        149,
+                        "truncated",
+                        "protein_state"
+                    ],
+                    [
+                        171,
+                        177,
+                        "active",
+                        "protein_state"
+                    ],
+                    [
+                        186,
+                        196,
+                        "xyloglucan",
+                        "chemical"
+                    ],
+                    [
+                        218,
+                        223,
+                        "CBM46",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 128,
+                "sent": "A closer inspection of results of the phylogenetic analysis, more specifically of the clade composed by GH5_4 enzymes with trimodular architecture (Supplementary Fig. 4C), reveals subclades whose main characteristic is the varying length of the loop located between residues 161 and 163 (BlCel5B residue numbering).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        38,
+                        59,
+                        "phylogenetic analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        104,
+                        109,
+                        "GH5_4",
+                        "protein_type"
+                    ],
+                    [
+                        123,
+                        133,
+                        "trimodular",
+                        "protein_state"
+                    ],
+                    [
+                        245,
+                        249,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        275,
+                        286,
+                        "161 and 163",
+                        "residue_range"
+                    ],
+                    [
+                        288,
+                        295,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 129,
+                "sent": "Therefore, our results show that BlCel5B represents a smaller group of enzymes that are completely dependent on its AMs for hydrolysis of plant cell wall polysaccharides, and that the underlying mechanism may rely on large-scale interdomain motions.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        33,
+                        40,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        116,
+                        119,
+                        "AMs",
+                        "structure_element"
+                    ],
+                    [
+                        138,
+                        143,
+                        "plant",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        154,
+                        169,
+                        "polysaccharides",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 130,
+                "sent": "The amino acid sequence of the BlCel5B Ig-like module is recognized by BLASTP as belonging to CBM_X2, a poorly described group that has been compared with CBM-like accessory modules without a defined function.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        31,
+                        38,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        39,
+                        53,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        71,
+                        77,
+                        "BLASTP",
+                        "experimental_method"
+                    ],
+                    [
+                        94,
+                        100,
+                        "CBM_X2",
+                        "structure_element"
+                    ],
+                    [
+                        155,
+                        181,
+                        "CBM-like accessory modules",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 131,
+                "sent": "Despite the similarity of BlCel5B Ig-like module to CBMs, it lacks an identifiable aromatic residue-rich carbohydrate-binding site.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        26,
+                        33,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        34,
+                        48,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        52,
+                        56,
+                        "CBMs",
+                        "structure_element"
+                    ],
+                    [
+                        105,
+                        130,
+                        "carbohydrate-binding site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 132,
+                "sent": "Nonetheless, according to our results, the Ig-like module seems to play an important function as a structural hinge, dynamically holding the CBM46 and CD in positions that are appropriate for enzymatic activity.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        43,
+                        57,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        99,
+                        115,
+                        "structural hinge",
+                        "structure_element"
+                    ],
+                    [
+                        141,
+                        146,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        151,
+                        153,
+                        "CD",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 133,
+                "sent": "Based on the results of our crystallographic, computer simulation, and SAXS structural analyses, as well as site-directed mutagenesis and activity assays, we propose a molecular mechanism for BlCel5B substrate binding, which might apply to other GH5_4 subfamily enzymes that share this tri-modular architecture.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        28,
+                        65,
+                        "crystallographic, computer simulation",
+                        "experimental_method"
+                    ],
+                    [
+                        71,
+                        95,
+                        "SAXS structural analyses",
+                        "experimental_method"
+                    ],
+                    [
+                        108,
+                        133,
+                        "site-directed mutagenesis",
+                        "experimental_method"
+                    ],
+                    [
+                        138,
+                        153,
+                        "activity assays",
+                        "experimental_method"
+                    ],
+                    [
+                        192,
+                        199,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        246,
+                        251,
+                        "GH5_4",
+                        "protein_type"
+                    ],
+                    [
+                        286,
+                        297,
+                        "tri-modular",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 134,
+                "sent": "BlCel5B can be found in several different conformational states ranging from CBM46/CD closed (or occluded) to extended conformations (Fig. 7).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        77,
+                        82,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        83,
+                        85,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        86,
+                        92,
+                        "closed",
+                        "protein_state"
+                    ],
+                    [
+                        97,
+                        105,
+                        "occluded",
+                        "protein_state"
+                    ],
+                    [
+                        110,
+                        118,
+                        "extended",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 135,
+                "sent": "In extended configurations, the substrate may dock at the shallow substrate binding site of CD in one of the semi-closed conformations of the enzyme; however, its binding is properly stabilized for hydrolysis only with the aid of induced-fit repositioning mediated by CBM46.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        3,
+                        11,
+                        "extended",
+                        "protein_state"
+                    ],
+                    [
+                        66,
+                        88,
+                        "substrate binding site",
+                        "site"
+                    ],
+                    [
+                        92,
+                        94,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        109,
+                        120,
+                        "semi-closed",
+                        "protein_state"
+                    ],
+                    [
+                        268,
+                        273,
+                        "CBM46",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 136,
+                "sent": "After cleavage, the intrinsic dynamics of BlCel5B would eventually allow the opening of the active site for product release.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        42,
+                        49,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        92,
+                        103,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 137,
+                "sent": "The proposed mechanism is consistent with our mutagenesis and enzymatic activity assays, which show that the Ig-like module and CBM46 are indispensable for BlCel5B catalytic activity and, together with the CD, form the unique catalytic domain of the enzyme.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        46,
+                        87,
+                        "mutagenesis and enzymatic activity assays",
+                        "experimental_method"
+                    ],
+                    [
+                        109,
+                        123,
+                        "Ig-like module",
+                        "structure_element"
+                    ],
+                    [
+                        128,
+                        133,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        156,
+                        163,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        206,
+                        208,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        219,
+                        225,
+                        "unique",
+                        "protein_state"
+                    ],
+                    [
+                        226,
+                        242,
+                        "catalytic domain",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 138,
+                "sent": "These experiments reveal a novel function for CBMs in which they are intimately involved in the assembly of the active site and catalytic process.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        46,
+                        50,
+                        "CBMs",
+                        "structure_element"
+                    ],
+                    [
+                        112,
+                        123,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 139,
+                "sent": "Computer simulations suggest that large-scale motions of the CBM46 and Ig-like domains mediate conformational selection and final induced-fit adjustments to trap the substrate at the active site and promote hydrolysis.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        20,
+                        "Computer simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        61,
+                        66,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        71,
+                        86,
+                        "Ig-like domains",
+                        "structure_element"
+                    ],
+                    [
+                        183,
+                        194,
+                        "active site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 140,
+                "sent": "SAXS data support the modeling results, providing compelling evidence for highly mobile domains in solution.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "SAXS",
+                        "experimental_method"
+                    ],
+                    [
+                        22,
+                        30,
+                        "modeling",
+                        "experimental_method"
+                    ],
+                    [
+                        74,
+                        87,
+                        "highly mobile",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 141,
+                "sent": "A single spectrum was obtained by averaging four independent spectra generated by 300 laser shots at 60% potency.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        9,
+                        17,
+                        "spectrum",
+                        "evidence"
+                    ],
+                    [
+                        61,
+                        68,
+                        "spectra",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 142,
+                "sent": "The missing residues were taken from the apo BlCel5B structure after structural alignment using the LovoAlign server.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        41,
+                        44,
+                        "apo",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 143,
+                "sent": "BlCel5B-cellooctaose",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        0,
+                        20,
+                        "BlCel5B-cellooctaose",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 144,
+                "sent": "To get a model of the BlCel5B-cellooctaose complex in the closed conformation, we took the configuration after 80\u2009ns of the restrained 200-ns MD simulation as the starting point for a 500-ns-long restrained aMD simulation, in which the CBM46 moved towards the CD in the presence of the harmonically-restrained cellooctaose chain.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        22,
+                        42,
+                        "BlCel5B-cellooctaose",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 145,
+                "sent": "After this procedure, we released the restraints and propagated the closed BlCel5B-cellooctaose complex for additional 500\u2009ns of conventional, restraint-free MD simulation.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        75,
+                        95,
+                        "BlCel5B-cellooctaose",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 146,
+                "sent": "Crystal models of BlCel5B.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        14,
+                        "Crystal models",
+                        "evidence"
+                    ],
+                    [
+                        18,
+                        25,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 147,
+                "sent": "Complete structure is shown as a cartoon illustration in (a) and a van der Waals surface in (b).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        9,
+                        18,
+                        "structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 148,
+                "sent": "The CD module (red) has a typical TIM-barrel fold, and its substrate-binding site is adjacent to CBM46 (blue).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        6,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        34,
+                        49,
+                        "TIM-barrel fold",
+                        "structure_element"
+                    ],
+                    [
+                        59,
+                        81,
+                        "substrate-binding site",
+                        "site"
+                    ],
+                    [
+                        97,
+                        102,
+                        "CBM46",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 149,
+                "sent": "Despite the proximity of the binding site in the crystallographic model, the CBM46 residues W479 and W481 are distant from the substrate cellotetraose (yellow).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        29,
+                        41,
+                        "binding site",
+                        "site"
+                    ],
+                    [
+                        77,
+                        82,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        92,
+                        96,
+                        "W479",
+                        "residue_name_number"
+                    ],
+                    [
+                        101,
+                        105,
+                        "W481",
+                        "residue_name_number"
+                    ],
+                    [
+                        137,
+                        150,
+                        "cellotetraose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 150,
+                "sent": "The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        18,
+                        "Ig-like domain",
+                        "structure_element"
+                    ],
+                    [
+                        86,
+                        88,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        93,
+                        98,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        106,
+                        119,
+                        "superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        127,
+                        141,
+                        "Ig-like domain",
+                        "structure_element"
+                    ],
+                    [
+                        146,
+                        151,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        248,
+                        252,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        286,
+                        299,
+                        "Cellotetraose",
+                        "chemical"
+                    ],
+                    [
+                        309,
+                        326,
+                        "subsites -1 to -3",
+                        "site"
+                    ],
+                    [
+                        344,
+                        355,
+                        "coordinated",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 151,
+                "sent": "BlCel5B enzymatic activity characterization.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        8,
+                        43,
+                        "enzymatic activity characterization",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 152,
+                "sent": "(a) MALDI/TOF-MS spectra of the products released after incubation of BlCel5B and its two deletion constructs (\u0394CBM46 and \u0394Ig-CBM46) with the substrate cellopentaose (C5).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        16,
+                        "MALDI/TOF-MS",
+                        "experimental_method"
+                    ],
+                    [
+                        17,
+                        24,
+                        "spectra",
+                        "evidence"
+                    ],
+                    [
+                        70,
+                        77,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        90,
+                        109,
+                        "deletion constructs",
+                        "experimental_method"
+                    ],
+                    [
+                        111,
+                        117,
+                        "\u0394CBM46",
+                        "mutant"
+                    ],
+                    [
+                        122,
+                        131,
+                        "\u0394Ig-CBM46",
+                        "mutant"
+                    ],
+                    [
+                        152,
+                        165,
+                        "cellopentaose",
+                        "chemical"
+                    ],
+                    [
+                        167,
+                        169,
+                        "C5",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 153,
+                "sent": "The first three spectra show the substrate, enzyme and buffer controls.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        16,
+                        23,
+                        "spectra",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 154,
+                "sent": "The forth spectrum reveals that full length BlCel5B is capable of enzymatic hydrolysis of C5 into smaller oligosaccharides such as C4, C3 and C2.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        10,
+                        18,
+                        "spectrum",
+                        "evidence"
+                    ],
+                    [
+                        32,
+                        43,
+                        "full length",
+                        "protein_state"
+                    ],
+                    [
+                        44,
+                        51,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        90,
+                        92,
+                        "C5",
+                        "chemical"
+                    ],
+                    [
+                        106,
+                        122,
+                        "oligosaccharides",
+                        "chemical"
+                    ],
+                    [
+                        131,
+                        133,
+                        "C4",
+                        "chemical"
+                    ],
+                    [
+                        135,
+                        137,
+                        "C3",
+                        "chemical"
+                    ],
+                    [
+                        142,
+                        144,
+                        "C2",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 155,
+                "sent": "The last two spectra show that the C-terminal deletions eliminate the enzyme activity.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        13,
+                        20,
+                        "spectra",
+                        "evidence"
+                    ],
+                    [
+                        56,
+                        85,
+                        "eliminate the enzyme activity",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 156,
+                "sent": "BlCel5B activities on CMC as functions of pH and temperature are shown in (b) and (c), respectively.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        22,
+                        25,
+                        "CMC",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 157,
+                "sent": "(d) Michaelis-Menten curve using CMC as a substrate.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        26,
+                        "Michaelis-Menten curve",
+                        "evidence"
+                    ],
+                    [
+                        33,
+                        36,
+                        "CMC",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 158,
+                "sent": "Open-close transitions of BlCel5B.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "Open",
+                        "protein_state"
+                    ],
+                    [
+                        5,
+                        10,
+                        "close",
+                        "protein_state"
+                    ],
+                    [
+                        26,
+                        33,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 159,
+                "sent": "(a) BlCel5B in the absence of substrate and (b) in the presence of cellooctaose, as observed in our aMD simulations.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        19,
+                        29,
+                        "absence of",
+                        "protein_state"
+                    ],
+                    [
+                        55,
+                        66,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        67,
+                        79,
+                        "cellooctaose",
+                        "chemical"
+                    ],
+                    [
+                        100,
+                        115,
+                        "aMD simulations",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 160,
+                "sent": "The distance between the \u03b1 carbon of residues I120 (CD) and E477 (CBM46), illustrated as spheres in (a), is plotted in (c), revealing a transition by the decrease in the distance from 40\u2009\u00c5 to 7\u2009\u00c5 (substrate-free) or 20\u2009\u00c5 (in presence of cellooctaose).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        12,
+                        "distance",
+                        "evidence"
+                    ],
+                    [
+                        46,
+                        50,
+                        "I120",
+                        "residue_name_number"
+                    ],
+                    [
+                        52,
+                        54,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        60,
+                        64,
+                        "E477",
+                        "residue_name_number"
+                    ],
+                    [
+                        66,
+                        71,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        170,
+                        178,
+                        "distance",
+                        "evidence"
+                    ],
+                    [
+                        197,
+                        211,
+                        "substrate-free",
+                        "protein_state"
+                    ],
+                    [
+                        225,
+                        236,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        237,
+                        249,
+                        "cellooctaose",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 161,
+                "sent": "For the substrate-free enzyme, the red line refers to a 1\u2009\u03bcs-long aMD; for the BlCel5B-cellooctaose complex, the first 500\u2009ns refers to aMD (in blue) and the second 500\u2009ns to conventional MD (in turquoise).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        8,
+                        22,
+                        "substrate-free",
+                        "protein_state"
+                    ],
+                    [
+                        66,
+                        69,
+                        "aMD",
+                        "experimental_method"
+                    ],
+                    [
+                        79,
+                        99,
+                        "BlCel5B-cellooctaose",
+                        "complex_assembly"
+                    ],
+                    [
+                        136,
+                        139,
+                        "aMD",
+                        "experimental_method"
+                    ],
+                    [
+                        188,
+                        190,
+                        "MD",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 162,
+                "sent": "(d) A snapshot of the BlCel5B-cellooctaose complex, highlighting the tryptophan residues that interact with the glucan chain in subsites \u22124 to +4.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        22,
+                        42,
+                        "BlCel5B-cellooctaose",
+                        "complex_assembly"
+                    ],
+                    [
+                        69,
+                        79,
+                        "tryptophan",
+                        "residue_name"
+                    ],
+                    [
+                        112,
+                        118,
+                        "glucan",
+                        "chemical"
+                    ],
+                    [
+                        128,
+                        145,
+                        "subsites \u22124 to +4",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 163,
+                "sent": "Residues W479 and W481 belong to CBM46 and only become available for substrate interactions in the closed configuration of BlCel5B.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        9,
+                        13,
+                        "W479",
+                        "residue_name_number"
+                    ],
+                    [
+                        18,
+                        22,
+                        "W481",
+                        "residue_name_number"
+                    ],
+                    [
+                        33,
+                        38,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        99,
+                        105,
+                        "closed",
+                        "protein_state"
+                    ],
+                    [
+                        123,
+                        130,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 164,
+                "sent": "Large-scale movements of BlCel5B modules and superposition of their representative conformations with the SAXS envelope.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        25,
+                        32,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        45,
+                        58,
+                        "superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        106,
+                        110,
+                        "SAXS",
+                        "experimental_method"
+                    ],
+                    [
+                        111,
+                        119,
+                        "envelope",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 165,
+                "sent": "(a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        12,
+                        21,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        34,
+                        42,
+                        "distance",
+                        "evidence"
+                    ],
+                    [
+                        82,
+                        86,
+                        "I120",
+                        "residue_name_number"
+                    ],
+                    [
+                        91,
+                        95,
+                        "E477",
+                        "residue_name_number"
+                    ],
+                    [
+                        128,
+                        130,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        135,
+                        140,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        246,
+                        250,
+                        "I120",
+                        "residue_name_number"
+                    ],
+                    [
+                        251,
+                        255,
+                        "E477",
+                        "residue_name_number"
+                    ],
+                    [
+                        256,
+                        264,
+                        "distance",
+                        "evidence"
+                    ],
+                    [
+                        280,
+                        297,
+                        "CG-MD simulations",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 166,
+                "sent": "Different colors separated by vertical lines correspond to independent simulations of approximately 120\u2009\u03bcs. (c) The distance distribution indicates three major peaks: closed or occluded CBM46/CD conformations (I); semi-open (II), which is similar to the crystallographic structure; and extended conformers (III).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        71,
+                        82,
+                        "simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        116,
+                        137,
+                        "distance distribution",
+                        "evidence"
+                    ],
+                    [
+                        167,
+                        173,
+                        "closed",
+                        "protein_state"
+                    ],
+                    [
+                        177,
+                        185,
+                        "occluded",
+                        "protein_state"
+                    ],
+                    [
+                        186,
+                        191,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        192,
+                        194,
+                        "CD",
+                        "structure_element"
+                    ],
+                    [
+                        214,
+                        223,
+                        "semi-open",
+                        "protein_state"
+                    ],
+                    [
+                        254,
+                        280,
+                        "crystallographic structure",
+                        "evidence"
+                    ],
+                    [
+                        286,
+                        294,
+                        "extended",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 167,
+                "sent": "(d) Superimposition of the three representative molecular conformations of BlCel5B with the SAXS model. (e) Average structures obtained from the simulation segments corresponding to population groups I-III, which are individually superposed on the SAXS envelope.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        19,
+                        "Superimposition",
+                        "experimental_method"
+                    ],
+                    [
+                        75,
+                        82,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        92,
+                        96,
+                        "SAXS",
+                        "experimental_method"
+                    ],
+                    [
+                        97,
+                        102,
+                        "model",
+                        "evidence"
+                    ],
+                    [
+                        116,
+                        126,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        145,
+                        155,
+                        "simulation",
+                        "experimental_method"
+                    ],
+                    [
+                        230,
+                        240,
+                        "superposed",
+                        "experimental_method"
+                    ],
+                    [
+                        248,
+                        252,
+                        "SAXS",
+                        "experimental_method"
+                    ],
+                    [
+                        253,
+                        261,
+                        "envelope",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 168,
+                "sent": "Comparison of the binding site shape of GH5_4 enzymes available on the Protein Data Bank.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        10,
+                        "Comparison",
+                        "experimental_method"
+                    ],
+                    [
+                        18,
+                        30,
+                        "binding site",
+                        "site"
+                    ],
+                    [
+                        40,
+                        45,
+                        "GH5_4",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 169,
+                "sent": "(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        19,
+                        35,
+                        "crystallographic",
+                        "experimental_method"
+                    ],
+                    [
+                        40,
+                        46,
+                        "closed",
+                        "protein_state"
+                    ],
+                    [
+                        66,
+                        85,
+                        "Bacillus halodurans",
+                        "species"
+                    ],
+                    [
+                        86,
+                        91,
+                        "Cel5B",
+                        "protein"
+                    ],
+                    [
+                        93,
+                        100,
+                        "BhCel5B",
+                        "protein"
+                    ],
+                    [
+                        121,
+                        142,
+                        "Piromyces rhizinflata",
+                        "species"
+                    ],
+                    [
+                        143,
+                        146,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        147,
+                        160,
+                        "endoglucanase",
+                        "protein_type"
+                    ],
+                    [
+                        181,
+                        207,
+                        "Clostridium cellulolyticum",
+                        "species"
+                    ],
+                    [
+                        208,
+                        211,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        212,
+                        225,
+                        "endoglucanase",
+                        "protein_type"
+                    ],
+                    [
+                        246,
+                        271,
+                        "Clostridium cellulovorans",
+                        "species"
+                    ],
+                    [
+                        272,
+                        275,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        276,
+                        289,
+                        "endoglucanase",
+                        "protein_type"
+                    ],
+                    [
+                        310,
+                        328,
+                        "Bacteroides ovatus",
+                        "species"
+                    ],
+                    [
+                        329,
+                        332,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        333,
+                        346,
+                        "xyloglucanase",
+                        "protein_type"
+                    ],
+                    [
+                        367,
+                        387,
+                        "Paenibacillus pabuli",
+                        "species"
+                    ],
+                    [
+                        388,
+                        391,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        392,
+                        405,
+                        "xyloglucanase",
+                        "protein_type"
+                    ],
+                    [
+                        426,
+                        445,
+                        "Prevotella bryantii",
+                        "species"
+                    ],
+                    [
+                        446,
+                        449,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        450,
+                        463,
+                        "endoglucanase",
+                        "protein_type"
+                    ],
+                    [
+                        484,
+                        514,
+                        "Ruminiclostridium thermocellum",
+                        "species"
+                    ],
+                    [
+                        531,
+                        534,
+                        "GH5",
+                        "protein_type"
+                    ],
+                    [
+                        535,
+                        544,
+                        "cellulase",
+                        "protein_type"
+                    ],
+                    [
+                        546,
+                        554,
+                        "xylanase",
+                        "protein_type"
+                    ],
+                    [
+                        559,
+                        566,
+                        "mannase",
+                        "protein_type"
+                    ],
+                    [
+                        587,
+                        610,
+                        "Bacteroidetes bacterium",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        611,
+                        615,
+                        "AC2a",
+                        "protein_type"
+                    ],
+                    [
+                        616,
+                        629,
+                        "endocellulase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 170,
+                "sent": "Comparison of the binding cleft of the BlCel5B and BhCel5B.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        10,
+                        "Comparison",
+                        "experimental_method"
+                    ],
+                    [
+                        18,
+                        31,
+                        "binding cleft",
+                        "site"
+                    ],
+                    [
+                        39,
+                        46,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        51,
+                        58,
+                        "BhCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 171,
+                "sent": "The main difference between BlCel5B and BhCel5B is that the latter exhibits a deeper cleft due to the presence of residue W181 in the loop between F177 and R185.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        28,
+                        35,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        40,
+                        47,
+                        "BhCel5B",
+                        "protein"
+                    ],
+                    [
+                        85,
+                        90,
+                        "cleft",
+                        "site"
+                    ],
+                    [
+                        102,
+                        113,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        122,
+                        126,
+                        "W181",
+                        "residue_name_number"
+                    ],
+                    [
+                        134,
+                        138,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        147,
+                        151,
+                        "F177",
+                        "residue_name_number"
+                    ],
+                    [
+                        156,
+                        160,
+                        "R185",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 172,
+                "sent": "We conjecture that this difference in the binding site architecture relates to the importance that the CBM46 plays in the BlCel5B enzymatic mechanism.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        42,
+                        54,
+                        "binding site",
+                        "site"
+                    ],
+                    [
+                        103,
+                        108,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        122,
+                        129,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 173,
+                "sent": "Proposed molecular mechanism of BlCel5B conformational selection.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        32,
+                        39,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 174,
+                "sent": "As suggested by the simulations and SAXS data, BlCel5B spans multiple conformations ranging from closed to extended CBM46/CD states.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        20,
+                        31,
+                        "simulations",
+                        "experimental_method"
+                    ],
+                    [
+                        36,
+                        40,
+                        "SAXS",
+                        "experimental_method"
+                    ],
+                    [
+                        47,
+                        54,
+                        "BlCel5B",
+                        "protein"
+                    ],
+                    [
+                        97,
+                        103,
+                        "closed",
+                        "protein_state"
+                    ],
+                    [
+                        107,
+                        115,
+                        "extended",
+                        "protein_state"
+                    ],
+                    [
+                        116,
+                        121,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        122,
+                        124,
+                        "CD",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 175,
+                "sent": "In a given open state, the substrate may reach the active site and become entrapped by the capping of CBM46 onto CD and induced-fit conformational adjustments.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        11,
+                        15,
+                        "open",
+                        "protein_state"
+                    ],
+                    [
+                        51,
+                        62,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        102,
+                        107,
+                        "CBM46",
+                        "structure_element"
+                    ],
+                    [
+                        113,
+                        115,
+                        "CD",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 176,
+                "sent": "After hydrolysis, the reaction product is released to yield apo-BlCel5B, which becomes ready for a new cycle.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        60,
+                        63,
+                        "apo",
+                        "protein_state"
+                    ],
+                    [
+                        64,
+                        71,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 177,
+                "sent": "Activity of BlCel5B constructs against tested substrates.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        12,
+                        19,
+                        "BlCel5B",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 178,
+                "sent": "Substrate (1%)\tRelative Activity (%)\t \tWT*\tW479A\tW481A\t\u0394CBM46\t\u0394Ig-CBM46\t \t\u03b2-glucan\t100\t79.1\t63.6\tnd\tnd\t \tCMC\t25.5\t12.2\t2.4\tnd\tnd\t \tLichenan\t52.4\t41\t28.6\tnd\tnd\t \tXyloglucan\t45.2\t41.2\t30.8\tnd\tnd\t \tAzo-Avicel\tnd**\tnd\tnd\tnd\tnd\t \tArabinoxylan\tnd\tnd\tnd\tnd\tnd\t \tGalactomannan\tnd\tnd\tnd\tnd\tnd\t \t1,4-\u03b2-mannan\tnd\tnd\tnd\tnd\tnd\t \t",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        39,
+                        41,
+                        "WT",
+                        "protein_state"
+                    ],
+                    [
+                        43,
+                        48,
+                        "W479A",
+                        "mutant"
+                    ],
+                    [
+                        49,
+                        54,
+                        "W481A",
+                        "mutant"
+                    ],
+                    [
+                        55,
+                        61,
+                        "\u0394CBM46",
+                        "mutant"
+                    ],
+                    [
+                        62,
+                        71,
+                        "\u0394Ig-CBM46",
+                        "mutant"
+                    ],
+                    [
+                        74,
+                        82,
+                        "\u03b2-glucan",
+                        "chemical"
+                    ],
+                    [
+                        105,
+                        108,
+                        "CMC",
+                        "chemical"
+                    ],
+                    [
+                        131,
+                        139,
+                        "Lichenan",
+                        "chemical"
+                    ],
+                    [
+                        161,
+                        171,
+                        "Xyloglucan",
+                        "chemical"
+                    ],
+                    [
+                        195,
+                        205,
+                        "Azo-Avicel",
+                        "chemical"
+                    ],
+                    [
+                        225,
+                        237,
+                        "Arabinoxylan",
+                        "chemical"
+                    ],
+                    [
+                        255,
+                        268,
+                        "Galactomannan",
+                        "chemical"
+                    ],
+                    [
+                        286,
+                        298,
+                        "1,4-\u03b2-mannan",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 179,
+                "sent": "*WT\u2009=\u2009wild type.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        1,
+                        3,
+                        "WT",
+                        "protein_state"
+                    ],
+                    [
+                        6,
+                        15,
+                        "wild type",
+                        "protein_state"
+                    ]
+                ]
+            }
+        ]
+    },
+    "PMC5012862": {
+        "annotations": [
+            {
+                "sid": 0,
+                "sent": "Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments",
+                "section": "TITLE",
+                "ner": [
+                    [
+                        0,
+                        27,
+                        "Structural characterization",
+                        "experimental_method"
+                    ],
+                    [
+                        31,
+                        43,
+                        "encapsulated",
+                        "protein_state"
+                    ],
+                    [
+                        44,
+                        52,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        75,
+                        79,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        91,
+                        100,
+                        "bacterial",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        101,
+                        117,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 1,
+                "sent": "Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        57,
+                        61,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        79,
+                        84,
+                        "shell",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 2,
+                "sent": "We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        26,
+                        35,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        72,
+                        80,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        123,
+                        133,
+                        "encapsulin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 3,
+                "sent": "We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        18,
+                        30,
+                        "encapsulated",
+                        "protein_state"
+                    ],
+                    [
+                        31,
+                        39,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        41,
+                        47,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        57,
+                        75,
+                        "main alpha helices",
+                        "structure_element"
+                    ],
+                    [
+                        97,
+                        112,
+                        "metal dependent",
+                        "protein_state"
+                    ],
+                    [
+                        130,
+                        148,
+                        "ferroxidase center",
+                        "site"
+                    ],
+                    [
+                        154,
+                        169,
+                        "dimer interface",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 4,
+                "sent": "EncFtn adopts an open decameric structure that is topologically distinct from other ferritins.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        6,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        17,
+                        21,
+                        "open",
+                        "protein_state"
+                    ],
+                    [
+                        22,
+                        31,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        32,
+                        41,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        84,
+                        93,
+                        "ferritins",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 5,
+                "sent": "While EncFtn acts as a ferroxidase, it cannot mineralize iron.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        6,
+                        12,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        23,
+                        34,
+                        "ferroxidase",
+                        "protein_type"
+                    ],
+                    [
+                        57,
+                        61,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 6,
+                "sent": "Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        16,
+                        26,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        27,
+                        32,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        49,
+                        53,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        62,
+                        86,
+                        "not enzymatically active",
+                        "protein_state"
+                    ],
+                    [
+                        112,
+                        118,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        145,
+                        155,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        160,
+                        164,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 7,
+                "sent": "This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        5,
+                        15,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        16,
+                        31,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ],
+                    [
+                        57,
+                        65,
+                        "bacteria",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        70,
+                        77,
+                        "archaea",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        113,
+                        117,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        176,
+                        180,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 8,
+                "sent": "Iron is essential for life as it is a key component of many different enzymes that participate in processes such as energy production and metabolism.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "Iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 9,
+                "sent": "However, iron can also be highly toxic to cells because it readily reacts with oxygen.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        9,
+                        13,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        79,
+                        85,
+                        "oxygen",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 10,
+                "sent": "To balance the cell\u2019s need for iron against its potential damaging effects, organisms have evolved iron storage proteins known as ferritins that form cage-like structures.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        31,
+                        35,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        99,
+                        120,
+                        "iron storage proteins",
+                        "protein_type"
+                    ],
+                    [
+                        130,
+                        139,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        150,
+                        170,
+                        "cage-like structures",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 11,
+                "sent": "The ferritins convert iron into a less reactive form that is mineralised and safely stored in the central cavity of the ferritin cage and is available for cells when they need it.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        22,
+                        26,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        98,
+                        112,
+                        "central cavity",
+                        "site"
+                    ],
+                    [
+                        120,
+                        128,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 12,
+                "sent": "Recently, a new family of ferritins known as encapsulated ferritins have been found in some microorganisms.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        26,
+                        35,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        45,
+                        57,
+                        "encapsulated",
+                        "protein_state"
+                    ],
+                    [
+                        58,
+                        67,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        92,
+                        106,
+                        "microorganisms",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 13,
+                "sent": "These ferritins are found in bacterial genomes with a gene that codes for a protein cage called an encapsulin.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        6,
+                        15,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        29,
+                        38,
+                        "bacterial",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        99,
+                        109,
+                        "encapsulin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 14,
+                "sent": "Although the structure of the encapsulin cage is known to look like the shell of a virus, the structure that the encapsulated ferritin itself forms is not known.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        13,
+                        22,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        30,
+                        40,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        72,
+                        77,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        83,
+                        88,
+                        "virus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        94,
+                        103,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        113,
+                        125,
+                        "encapsulated",
+                        "protein_state"
+                    ],
+                    [
+                        126,
+                        134,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 15,
+                "sent": "It is also not clear how encapsulin and the encapsulated ferritin work together to store iron.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        25,
+                        35,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        44,
+                        56,
+                        "encapsulated",
+                        "protein_state"
+                    ],
+                    [
+                        57,
+                        65,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        89,
+                        93,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 16,
+                "sent": "He et al. have now used the techniques of X-ray crystallography and mass spectrometry to determine the structure of the encapsulated ferritin found in some bacteria.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        42,
+                        63,
+                        "X-ray crystallography",
+                        "experimental_method"
+                    ],
+                    [
+                        68,
+                        85,
+                        "mass spectrometry",
+                        "experimental_method"
+                    ],
+                    [
+                        103,
+                        112,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        120,
+                        132,
+                        "encapsulated",
+                        "protein_state"
+                    ],
+                    [
+                        133,
+                        141,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        156,
+                        164,
+                        "bacteria",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 17,
+                "sent": "The encapsulated ferritin forms a ring-shaped doughnut in which ten subunits of ferritin are arranged in a ring; this is totally different from the enclosed cages that other ferritins form.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        4,
+                        16,
+                        "encapsulated",
+                        "protein_state"
+                    ],
+                    [
+                        17,
+                        25,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        34,
+                        45,
+                        "ring-shaped",
+                        "structure_element"
+                    ],
+                    [
+                        46,
+                        54,
+                        "doughnut",
+                        "structure_element"
+                    ],
+                    [
+                        68,
+                        76,
+                        "subunits",
+                        "structure_element"
+                    ],
+                    [
+                        80,
+                        88,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        107,
+                        111,
+                        "ring",
+                        "structure_element"
+                    ],
+                    [
+                        157,
+                        162,
+                        "cages",
+                        "structure_element"
+                    ],
+                    [
+                        174,
+                        183,
+                        "ferritins",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 18,
+                "sent": "Biochemical studies revealed that the encapsulated ferritin is able to convert iron into a less reactive form, but it cannot store iron on its own since it does not form a cage.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        19,
+                        "Biochemical studies",
+                        "experimental_method"
+                    ],
+                    [
+                        38,
+                        50,
+                        "encapsulated",
+                        "protein_state"
+                    ],
+                    [
+                        51,
+                        59,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        79,
+                        83,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        131,
+                        135,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 19,
+                "sent": "Thus, the encapsulated ferritin needs to be housed within the encapsulin cage to store iron.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        10,
+                        22,
+                        "encapsulated",
+                        "protein_state"
+                    ],
+                    [
+                        23,
+                        31,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        62,
+                        72,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        87,
+                        91,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 20,
+                "sent": "Further work is needed to investigate how iron moves into the encapsulin cage to reach the ferritin proteins.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        42,
+                        46,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        62,
+                        72,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        91,
+                        99,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 21,
+                "sent": "Some organisms have both standard ferritin cages and encapsulated ferritins; why this is the case also remains to be discovered.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        34,
+                        42,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        53,
+                        65,
+                        "encapsulated",
+                        "protein_state"
+                    ],
+                    [
+                        66,
+                        75,
+                        "ferritins",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 22,
+                "sent": "Encapsulin nanocompartments are a family of proteinaceous metabolic compartments that are widely distributed in bacteria and archaea.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        10,
+                        "Encapsulin",
+                        "protein_type"
+                    ],
+                    [
+                        11,
+                        27,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ],
+                    [
+                        112,
+                        120,
+                        "bacteria",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        125,
+                        132,
+                        "archaea",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 23,
+                "sent": "They share a common architecture, comprising an icosahedral shell formed by the oligomeric assembly of a protein, encapsulin, that is structurally related to the HK97 bacteriophage capsid protein gp5.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        48,
+                        59,
+                        "icosahedral",
+                        "protein_state"
+                    ],
+                    [
+                        60,
+                        65,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        114,
+                        124,
+                        "encapsulin",
+                        "protein_type"
+                    ],
+                    [
+                        162,
+                        180,
+                        "HK97 bacteriophage",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        196,
+                        199,
+                        "gp5",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 24,
+                "sent": "Gp5 is known to assemble as a 66 nm diameter icosahedral shell of 420 subunits.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "Gp5",
+                        "protein"
+                    ],
+                    [
+                        45,
+                        56,
+                        "icosahedral",
+                        "protein_state"
+                    ],
+                    [
+                        57,
+                        62,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        70,
+                        78,
+                        "subunits",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 25,
+                "sent": "In contrast, both the Pyrococcus furiosus and\u00a0Myxococcus xanthus encapsulin shell-proteins form 32 nm icosahedra with 180 subunits; while the Thermotoga maritima encapsulin is smaller still with a 25 nm, 60-subunit icosahedron.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        22,
+                        41,
+                        "Pyrococcus furiosus",
+                        "species"
+                    ],
+                    [
+                        46,
+                        64,
+                        "Myxococcus xanthus",
+                        "species"
+                    ],
+                    [
+                        65,
+                        75,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        76,
+                        81,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        102,
+                        112,
+                        "icosahedra",
+                        "structure_element"
+                    ],
+                    [
+                        122,
+                        130,
+                        "subunits",
+                        "structure_element"
+                    ],
+                    [
+                        142,
+                        161,
+                        "Thermotoga maritima",
+                        "species"
+                    ],
+                    [
+                        162,
+                        172,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        215,
+                        226,
+                        "icosahedron",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 26,
+                "sent": "The high structural similarity of the encapsulin shell-proteins to gp5 suggests a common evolutionary origin for these proteins.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        38,
+                        48,
+                        "encapsulin",
+                        "protein_type"
+                    ],
+                    [
+                        49,
+                        54,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        67,
+                        70,
+                        "gp5",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 27,
+                "sent": "The genes encoding encapsulin proteins are found downstream of genes for dye-dependent peroxidase (DyP) family enzymes, or encapsulin-associated ferritins (EncFtn).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        19,
+                        29,
+                        "encapsulin",
+                        "protein_type"
+                    ],
+                    [
+                        73,
+                        97,
+                        "dye-dependent peroxidase",
+                        "protein_type"
+                    ],
+                    [
+                        99,
+                        102,
+                        "DyP",
+                        "protein_type"
+                    ],
+                    [
+                        123,
+                        154,
+                        "encapsulin-associated ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        156,
+                        162,
+                        "EncFtn",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 28,
+                "sent": "Enzymes in the DyP family are active against polyphenolic compounds such as azo dyes and lignin breakdown products; although their physiological function and natural substrates are not known.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        15,
+                        25,
+                        "DyP family",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 29,
+                "sent": "Ferritin family proteins are found in all kingdoms and have a wide range of activities, including ribonucleotide reductase, protecting DNA from oxidative damage, and iron storage.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "Ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        42,
+                        50,
+                        "kingdoms",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        98,
+                        122,
+                        "ribonucleotide reductase",
+                        "protein_type"
+                    ],
+                    [
+                        166,
+                        170,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 30,
+                "sent": "The classical iron storage ferritin nanocages are found in all kingdoms and are essential in eukaryotes; they play a central role in iron homeostasis, where they protect the cell from toxic free Fe2+ by oxidizing it and storing the resulting Fe3+ as ferrihydrite minerals within their central cavity.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        14,
+                        45,
+                        "iron storage ferritin nanocages",
+                        "complex_assembly"
+                    ],
+                    [
+                        63,
+                        71,
+                        "kingdoms",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        93,
+                        103,
+                        "eukaryotes",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        133,
+                        137,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        195,
+                        199,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        242,
+                        246,
+                        "Fe3+",
+                        "chemical"
+                    ],
+                    [
+                        250,
+                        262,
+                        "ferrihydrite",
+                        "chemical"
+                    ],
+                    [
+                        285,
+                        299,
+                        "central cavity",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 31,
+                "sent": "The encapsulin-associated enzymes are sequestered within the icosahedral shell through interactions between the shell\u2019s inner surface and a short localization sequence (Gly-Ser-Leu-Lys) appended to their C-termini.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        14,
+                        "encapsulin",
+                        "protein_type"
+                    ],
+                    [
+                        61,
+                        72,
+                        "icosahedral",
+                        "protein_state"
+                    ],
+                    [
+                        73,
+                        78,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        112,
+                        117,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        140,
+                        167,
+                        "short localization sequence",
+                        "structure_element"
+                    ],
+                    [
+                        169,
+                        184,
+                        "Gly-Ser-Leu-Lys",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 32,
+                "sent": "This motif is well-conserved, and the\u00a0addition of this sequence to heterologous proteins is sufficient to direct them to the interior of encapsulins.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        10,
+                        "This motif",
+                        "structure_element"
+                    ],
+                    [
+                        14,
+                        28,
+                        "well-conserved",
+                        "protein_state"
+                    ],
+                    [
+                        137,
+                        148,
+                        "encapsulins",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 33,
+                "sent": "A recent study of the Myxococcus xanthus encapsulin showed that it sequesters a number of different EncFtn proteins and acts as an \u2018iron-megastore\u2019 to protect these bacteria from oxidative stress.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        22,
+                        40,
+                        "Myxococcus xanthus",
+                        "species"
+                    ],
+                    [
+                        41,
+                        51,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        100,
+                        106,
+                        "EncFtn",
+                        "protein_type"
+                    ],
+                    [
+                        132,
+                        136,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        165,
+                        173,
+                        "bacteria",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 34,
+                "sent": "At 32 nm in diameter, it is much larger than other members of the ferritin superfamily, such as the 12 nm 24-subunit classical ferritin nanocage and the 8 nm 12-subunit Dps (DNA-binding protein from starved cells) complex; and is thus capable of sequestering up to ten times more iron than these ferritins.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        66,
+                        74,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        117,
+                        126,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        127,
+                        135,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        136,
+                        144,
+                        "nanocage",
+                        "complex_assembly"
+                    ],
+                    [
+                        169,
+                        172,
+                        "Dps",
+                        "protein_type"
+                    ],
+                    [
+                        174,
+                        193,
+                        "DNA-binding protein",
+                        "protein_type"
+                    ],
+                    [
+                        280,
+                        284,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        296,
+                        305,
+                        "ferritins",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 35,
+                "sent": "The primary sequences of EncFtn proteins have Glu-X-X-His metal coordination sites, which are shared features of the ferritin family proteins.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        25,
+                        31,
+                        "EncFtn",
+                        "protein_type"
+                    ],
+                    [
+                        46,
+                        57,
+                        "Glu-X-X-His",
+                        "structure_element"
+                    ],
+                    [
+                        58,
+                        82,
+                        "metal coordination sites",
+                        "site"
+                    ],
+                    [
+                        117,
+                        125,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 36,
+                "sent": "Secondary structure prediction identifies two major \u03b1-helical regions in these proteins; this is in contrast to other members of the ferritin superfamily, which have four major \u03b1-helices (Supplementary file 1).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        30,
+                        "Secondary structure prediction",
+                        "experimental_method"
+                    ],
+                    [
+                        46,
+                        69,
+                        "major \u03b1-helical regions",
+                        "structure_element"
+                    ],
+                    [
+                        133,
+                        141,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        171,
+                        186,
+                        "major \u03b1-helices",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 37,
+                "sent": "The \u2018half-ferritin\u2019 primary sequence of the EncFtn family and their association with encapsulin nanocompartments suggests a distinct biochemical and structural organization to other ferritin family proteins.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        10,
+                        18,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        44,
+                        50,
+                        "EncFtn",
+                        "protein_type"
+                    ],
+                    [
+                        85,
+                        95,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        96,
+                        112,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ],
+                    [
+                        182,
+                        190,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 38,
+                "sent": "The Rhodospirillum\u00a0rubrum EncFtn protein (Rru_A0973) shares 33% protein sequence identity with the M. xanthus (MXAN_4464), 53% with the T. maritima (Tmari_0787), and 29% with the P. furiosus (PF1192) homologues.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        25,
+                        "Rhodospirillum\u00a0rubrum",
+                        "species"
+                    ],
+                    [
+                        26,
+                        32,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        42,
+                        51,
+                        "Rru_A0973",
+                        "gene"
+                    ],
+                    [
+                        99,
+                        109,
+                        "M. xanthus",
+                        "species"
+                    ],
+                    [
+                        111,
+                        120,
+                        "MXAN_4464",
+                        "gene"
+                    ],
+                    [
+                        136,
+                        147,
+                        "T. maritima",
+                        "species"
+                    ],
+                    [
+                        149,
+                        159,
+                        "Tmari_0787",
+                        "gene"
+                    ],
+                    [
+                        179,
+                        190,
+                        "P. furiosus",
+                        "species"
+                    ],
+                    [
+                        192,
+                        198,
+                        "PF1192",
+                        "gene"
+                    ]
+                ]
+            },
+            {
+                "sid": 39,
+                "sent": "The GXXH motifs are strictly conserved in each of these species (Supplementary file 1).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "GXXH",
+                        "structure_element"
+                    ],
+                    [
+                        20,
+                        38,
+                        "strictly conserved",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 40,
+                "sent": "Here we investigate the structure and biochemistry of EncFtn in order to understand iron storage within the encapsulin nanocompartment.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        24,
+                        33,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        54,
+                        60,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        84,
+                        88,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        108,
+                        118,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        119,
+                        134,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 41,
+                "sent": "We have produced recombinant encapsulin (Enc) and EncFtn from the aquatic purple-sulfur bacterium R.\u00a0rubrum, which serves as a model organism for the study of the control of the bacterial nitrogen fixation machinery, in Escherichia coli.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        29,
+                        39,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        41,
+                        44,
+                        "Enc",
+                        "protein"
+                    ],
+                    [
+                        50,
+                        56,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        66,
+                        73,
+                        "aquatic",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        74,
+                        97,
+                        "purple-sulfur bacterium",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        98,
+                        107,
+                        "R.\u00a0rubrum",
+                        "species"
+                    ],
+                    [
+                        178,
+                        187,
+                        "bacterial",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        220,
+                        236,
+                        "Escherichia coli",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 42,
+                "sent": "Analysis by transmission electron microscopy (TEM) indicates that their co-expression leads to the production of an icosahedral nanocompartment with encapsulated EncFtn.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        12,
+                        44,
+                        "transmission electron microscopy",
+                        "experimental_method"
+                    ],
+                    [
+                        46,
+                        49,
+                        "TEM",
+                        "experimental_method"
+                    ],
+                    [
+                        72,
+                        85,
+                        "co-expression",
+                        "experimental_method"
+                    ],
+                    [
+                        116,
+                        127,
+                        "icosahedral",
+                        "protein_state"
+                    ],
+                    [
+                        128,
+                        143,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ],
+                    [
+                        149,
+                        161,
+                        "encapsulated",
+                        "protein_state"
+                    ],
+                    [
+                        162,
+                        168,
+                        "EncFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 43,
+                "sent": "The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular \u2018ring-doughnut\u2019 topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        21,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        27,
+                        36,
+                        "truncated",
+                        "protein_state"
+                    ],
+                    [
+                        37,
+                        57,
+                        "hexahistidine-tagged",
+                        "protein_state"
+                    ],
+                    [
+                        73,
+                        79,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        89,
+                        97,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        121,
+                        130,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        131,
+                        140,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        158,
+                        171,
+                        "ring-doughnut",
+                        "structure_element"
+                    ],
+                    [
+                        210,
+                        230,
+                        "four-helical bundles",
+                        "structure_element"
+                    ],
+                    [
+                        238,
+                        245,
+                        "24meric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        246,
+                        255,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        260,
+                        272,
+                        "dodecahedral",
+                        "oligomeric_state"
+                    ],
+                    [
+                        273,
+                        276,
+                        "DPS",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 44,
+                "sent": "We identify a symmetrical iron bound ferroxidase center (FOC) formed between subunits in the decamer and additional metal-binding sites close to the center of the ring and on the outer surface.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        26,
+                        36,
+                        "iron bound",
+                        "protein_state"
+                    ],
+                    [
+                        37,
+                        55,
+                        "ferroxidase center",
+                        "site"
+                    ],
+                    [
+                        57,
+                        60,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        77,
+                        85,
+                        "subunits",
+                        "structure_element"
+                    ],
+                    [
+                        93,
+                        100,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        116,
+                        135,
+                        "metal-binding sites",
+                        "site"
+                    ],
+                    [
+                        163,
+                        167,
+                        "ring",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 45,
+                "sent": "We also demonstrate the metal-dependent assembly of EncFtn decamers using native PAGE, analytical gel-filtration, and native mass spectrometry.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        52,
+                        58,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        59,
+                        67,
+                        "decamers",
+                        "oligomeric_state"
+                    ],
+                    [
+                        74,
+                        85,
+                        "native PAGE",
+                        "experimental_method"
+                    ],
+                    [
+                        87,
+                        112,
+                        "analytical gel-filtration",
+                        "experimental_method"
+                    ],
+                    [
+                        118,
+                        142,
+                        "native mass spectrometry",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 46,
+                "sent": "Biochemical assays show that EncFtn is active as a ferroxidase enzyme.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        18,
+                        "Biochemical assays",
+                        "experimental_method"
+                    ],
+                    [
+                        29,
+                        35,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        39,
+                        45,
+                        "active",
+                        "protein_state"
+                    ],
+                    [
+                        51,
+                        62,
+                        "ferroxidase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 47,
+                "sent": "Through site-directed mutagenesis we show that the conserved glutamic acid and histidine residues in the FOC influence protein assembly and activity.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        8,
+                        33,
+                        "site-directed mutagenesis",
+                        "experimental_method"
+                    ],
+                    [
+                        51,
+                        60,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        61,
+                        74,
+                        "glutamic acid",
+                        "residue_name"
+                    ],
+                    [
+                        79,
+                        88,
+                        "histidine",
+                        "residue_name"
+                    ],
+                    [
+                        105,
+                        108,
+                        "FOC",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 48,
+                "sent": "We use our combined structural and biochemical data to propose a model for the EncFtn-catalyzed sequestration of iron within the encapsulin shell.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        20,
+                        51,
+                        "structural and biochemical data",
+                        "evidence"
+                    ],
+                    [
+                        79,
+                        85,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        113,
+                        117,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        129,
+                        139,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        140,
+                        145,
+                        "shell",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 49,
+                "sent": "Assembly of R. rubrum EncFtn encapsulin nanocompartments in E. coli",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        12,
+                        21,
+                        "R. rubrum",
+                        "species"
+                    ],
+                    [
+                        22,
+                        28,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        29,
+                        39,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        40,
+                        56,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ],
+                    [
+                        60,
+                        67,
+                        "E. coli",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 50,
+                "sent": "Full-frame transmission electron micrographs of R. rubrum nanocompartments.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        44,
+                        "Full-frame transmission electron micrographs",
+                        "evidence"
+                    ],
+                    [
+                        48,
+                        57,
+                        "R. rubrum",
+                        "species"
+                    ],
+                    [
+                        58,
+                        74,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 51,
+                "sent": "(A/B) Negative stain TEM image of recombinant R. rubrum encapsulin and EncFtn-Enc nanocompartments.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        6,
+                        24,
+                        "Negative stain TEM",
+                        "experimental_method"
+                    ],
+                    [
+                        25,
+                        30,
+                        "image",
+                        "evidence"
+                    ],
+                    [
+                        46,
+                        55,
+                        "R. rubrum",
+                        "species"
+                    ],
+                    [
+                        56,
+                        66,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        71,
+                        81,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        82,
+                        98,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 52,
+                "sent": "All samples were imaged at 143,000 x magnification; the scale bar length corresponds to 50 nm. (C) Histogram showing the distribution of nanocompartment diameters.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        99,
+                        108,
+                        "Histogram",
+                        "evidence"
+                    ],
+                    [
+                        137,
+                        152,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 53,
+                "sent": "A model Gaussian nonlinear least square function was fitted to the data to obtain a mean diameter of 24.6 nm with a\u00a0standard deviation of 2.0 nm for encapsulin (grey) and a mean value of 23.9 nm with a\u00a0standard deviation of 2.2 nm for co-expressed EncFtn and encapsulin (EncFtn-Enc, black).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        8,
+                        48,
+                        "Gaussian nonlinear least square function",
+                        "experimental_method"
+                    ],
+                    [
+                        149,
+                        159,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        235,
+                        247,
+                        "co-expressed",
+                        "experimental_method"
+                    ],
+                    [
+                        248,
+                        254,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        259,
+                        269,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        271,
+                        281,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 54,
+                "sent": "Purification of recombinant R. rubrum encapsulin nanocompartments.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        28,
+                        37,
+                        "R. rubrum",
+                        "species"
+                    ],
+                    [
+                        38,
+                        48,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        49,
+                        65,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 55,
+                "sent": "(A) Recombinantly expressed encapsulin (Enc) and co-expressed EncFtn-Enc were purified by sucrose gradient ultracentrifugation from E. coli B834(DE3) grown in SeMet medium.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        27,
+                        "Recombinantly expressed",
+                        "experimental_method"
+                    ],
+                    [
+                        28,
+                        38,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        40,
+                        43,
+                        "Enc",
+                        "protein"
+                    ],
+                    [
+                        49,
+                        61,
+                        "co-expressed",
+                        "experimental_method"
+                    ],
+                    [
+                        62,
+                        72,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        90,
+                        126,
+                        "sucrose gradient ultracentrifugation",
+                        "experimental_method"
+                    ],
+                    [
+                        132,
+                        139,
+                        "E. coli",
+                        "species"
+                    ],
+                    [
+                        159,
+                        164,
+                        "SeMet",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 56,
+                "sent": "Samples were resolved by 18% acrylamide SDS-PAGE; the position of the proteins found in the complexes as resolved on the gel are shown with arrows.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        40,
+                        48,
+                        "SDS-PAGE",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 57,
+                "sent": "(B/C) Negative stain TEM image of recombinant encapsulin and EncFtn-Enc nanocompartments.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        6,
+                        24,
+                        "Negative stain TEM",
+                        "experimental_method"
+                    ],
+                    [
+                        46,
+                        56,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        61,
+                        71,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        72,
+                        88,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 58,
+                "sent": "Representative encapsulin and EncFtn-Enc complexes are indicated with red arrows.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        15,
+                        25,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        30,
+                        40,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 59,
+                "sent": "We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        24,
+                        33,
+                        "R. rubrum",
+                        "species"
+                    ],
+                    [
+                        34,
+                        44,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        45,
+                        61,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ],
+                    [
+                        65,
+                        72,
+                        "E. coli",
+                        "species"
+                    ],
+                    [
+                        76,
+                        89,
+                        "co-expression",
+                        "experimental_method"
+                    ],
+                    [
+                        97,
+                        107,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        109,
+                        118,
+                        "Rru_A0974",
+                        "gene"
+                    ],
+                    [
+                        124,
+                        130,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        132,
+                        141,
+                        "Rru_A0973",
+                        "gene"
+                    ],
+                    [
+                        175,
+                        212,
+                        "sucrose gradient ultra-centrifugation",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 60,
+                "sent": "TEM imaging of uranyl acetate-stained samples revealed that, when expressed in isolation, the encapsulin protein forms empty compartments with an average diameter of 24 nm (Figure 1B and Figure 1\u2014figure supplement 1A/C), consistent with the appearance and size of the T. maritima encapsulin.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "TEM",
+                        "experimental_method"
+                    ],
+                    [
+                        66,
+                        88,
+                        "expressed in isolation",
+                        "experimental_method"
+                    ],
+                    [
+                        94,
+                        104,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        119,
+                        124,
+                        "empty",
+                        "protein_state"
+                    ],
+                    [
+                        125,
+                        137,
+                        "compartments",
+                        "complex_assembly"
+                    ],
+                    [
+                        268,
+                        279,
+                        "T. maritima",
+                        "species"
+                    ],
+                    [
+                        280,
+                        290,
+                        "encapsulin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 61,
+                "sent": "We were not able to resolve any higher-order structures of EncFtn by TEM.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        59,
+                        65,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        69,
+                        72,
+                        "TEM",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 62,
+                "sent": "Protein purified from co-expression of the encapsulin and EncFtn resulted in 24 nm compartments with regions in the center that exclude stain, consistent with the presence of the EncFtn within the encapsulin shell (Figure 1C and Figure 1\u2014figure supplement 1B/C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        22,
+                        35,
+                        "co-expression",
+                        "experimental_method"
+                    ],
+                    [
+                        43,
+                        53,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        58,
+                        64,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        163,
+                        174,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        179,
+                        185,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        197,
+                        207,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        208,
+                        213,
+                        "shell",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 63,
+                "sent": "R.\u00a0rubrum EncFtn forms a metal-ion stabilized decamer in solution",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "R.\u00a0rubrum",
+                        "species"
+                    ],
+                    [
+                        10,
+                        16,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        46,
+                        53,
+                        "decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 64,
+                "sent": "Purification of recombinant R. rubrum EncFtnsH.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        27,
+                        "Purification of recombinant",
+                        "experimental_method"
+                    ],
+                    [
+                        28,
+                        37,
+                        "R. rubrum",
+                        "species"
+                    ],
+                    [
+                        38,
+                        46,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 65,
+                "sent": "(A) Recombinant SeMet-labeled EncFtnsH produced with 1 mM Fe(NH4)2(SO4)2 in the growth medium was purified by nickel affinity chromatography and size-exclusion chromatography using a\u00a0Superdex\u00a0200 16/60 column (GE Healthcare).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        16,
+                        29,
+                        "SeMet-labeled",
+                        "protein_state"
+                    ],
+                    [
+                        30,
+                        38,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        58,
+                        72,
+                        "Fe(NH4)2(SO4)2",
+                        "chemical"
+                    ],
+                    [
+                        110,
+                        140,
+                        "nickel affinity chromatography",
+                        "experimental_method"
+                    ],
+                    [
+                        145,
+                        174,
+                        "size-exclusion chromatography",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 66,
+                "sent": "Chromatogram traces measured at 280 nm and 315 nm are shown with the results from ICP-MS analysis of the iron content of the fractions collected during the experiment.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        12,
+                        "Chromatogram",
+                        "evidence"
+                    ],
+                    [
+                        82,
+                        88,
+                        "ICP-MS",
+                        "experimental_method"
+                    ],
+                    [
+                        105,
+                        109,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 67,
+                "sent": "The peak around 73 ml corresponds to a molecular weight of around 130 kDa when compared to calibration standards; this is consistent with a decamer of EncFtnsH. The small peak at 85 ml corresponds to the 13 kDa monomer compared to the standards.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        39,
+                        55,
+                        "molecular weight",
+                        "evidence"
+                    ],
+                    [
+                        140,
+                        147,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        151,
+                        159,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        211,
+                        218,
+                        "monomer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 68,
+                "sent": "Only the decamer peak contains significant amounts of iron as indicated by the ICP-MS analysis.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        9,
+                        16,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        54,
+                        58,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        79,
+                        85,
+                        "ICP-MS",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 69,
+                "sent": "(B) Peak fractions from the gel filtration run were resolved by 15% acrylamide SDS-PAGE and stained with Coomassie blue stain.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        28,
+                        42,
+                        "gel filtration",
+                        "experimental_method"
+                    ],
+                    [
+                        79,
+                        87,
+                        "SDS-PAGE",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 70,
+                "sent": "The bands around 13 kDa and 26 kDa correspond to EncFtnsH, as\u00a0identified by MALDI peptide mass fingerprinting.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        49,
+                        57,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        76,
+                        109,
+                        "MALDI peptide mass fingerprinting",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 71,
+                "sent": "The band at 13 kDa is consistent with the monomer mass, while the band at 26 kDa is consistent with a dimer of EncFtnsH. The dimer species only appears in the decamer fractions.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        42,
+                        49,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        102,
+                        107,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        111,
+                        119,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        125,
+                        130,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        159,
+                        166,
+                        "decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 72,
+                "sent": "(C) SEC-MALLS analysis of EncFtnsH from decamer fractions and monomer fractions allows assignment of an average mass of 132 kDa to decamer fractions and 13 kDa to monomer fractions, consistent with decamer and monomer species (Table 2).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "SEC-MALLS",
+                        "experimental_method"
+                    ],
+                    [
+                        26,
+                        34,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        40,
+                        47,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        62,
+                        69,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        131,
+                        138,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        163,
+                        170,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        198,
+                        205,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        210,
+                        217,
+                        "monomer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 73,
+                "sent": "Determination of the Fe/EncFtnsH protein ratio by ICP-MS.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        21,
+                        23,
+                        "Fe",
+                        "chemical"
+                    ],
+                    [
+                        24,
+                        32,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        50,
+                        56,
+                        "ICP-MS",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 74,
+                "sent": "EncFtnsH was purified as a SeMet derivative from E. coli B834(DE3) cells grown in SeMet medium with 1 mM Fe(NH4)2(SO4)2.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        27,
+                        32,
+                        "SeMet",
+                        "chemical"
+                    ],
+                    [
+                        49,
+                        66,
+                        "E. coli B834(DE3)",
+                        "species"
+                    ],
+                    [
+                        82,
+                        87,
+                        "SeMet",
+                        "chemical"
+                    ],
+                    [
+                        105,
+                        119,
+                        "Fe(NH4)2(SO4)2",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 75,
+                "sent": "Fractions from SEC were collected, acidified and analysed by ICP-MS.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        15,
+                        18,
+                        "SEC",
+                        "experimental_method"
+                    ],
+                    [
+                        61,
+                        67,
+                        "ICP-MS",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 76,
+                "sent": "EncFtnsH concentration was calculated based on the presence of two SeMet per mature monomer.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        51,
+                        62,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        67,
+                        72,
+                        "SeMet",
+                        "chemical"
+                    ],
+                    [
+                        77,
+                        83,
+                        "mature",
+                        "protein_state"
+                    ],
+                    [
+                        84,
+                        91,
+                        "monomer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 77,
+                "sent": "These data were collected from EncFtnsH fractions from a single gel-filtration run.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        31,
+                        39,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        64,
+                        78,
+                        "gel-filtration",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 78,
+                "sent": "Peak\tEncFtnsHretention volume (ml)\tElement concentration (\u00b5M)\tDerived EncFtnsHconcentration (\u00b5M)\tDerived Fe/ EncFtnsH monomer\t \tCa\tFe\tZn\tSe\t \tDecamer\t66.5\tn.d.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        5,
+                        13,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        70,
+                        78,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        105,
+                        107,
+                        "Fe",
+                        "chemical"
+                    ],
+                    [
+                        109,
+                        117,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        118,
+                        125,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        128,
+                        130,
+                        "Ca",
+                        "chemical"
+                    ],
+                    [
+                        131,
+                        133,
+                        "Fe",
+                        "chemical"
+                    ],
+                    [
+                        134,
+                        136,
+                        "Zn",
+                        "chemical"
+                    ],
+                    [
+                        137,
+                        139,
+                        "Se",
+                        "chemical"
+                    ],
+                    [
+                        142,
+                        149,
+                        "Decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 79,
+                "sent": "Estimates of EncFtnsH molecular weight from SEC-MALLS analysis.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        13,
+                        21,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        22,
+                        38,
+                        "molecular weight",
+                        "evidence"
+                    ],
+                    [
+                        44,
+                        53,
+                        "SEC-MALLS",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 80,
+                "sent": "EncFtnsH was purified from E. coli BL21(DE3) grown in minimal medium\u00a0(MM) by nickel affinity chromatography\u00a0and size-exclusion chromatography.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        27,
+                        44,
+                        "E. coli BL21(DE3)",
+                        "species"
+                    ],
+                    [
+                        54,
+                        68,
+                        "minimal medium",
+                        "experimental_method"
+                    ],
+                    [
+                        70,
+                        72,
+                        "MM",
+                        "experimental_method"
+                    ],
+                    [
+                        77,
+                        107,
+                        "nickel affinity chromatography",
+                        "experimental_method"
+                    ],
+                    [
+                        112,
+                        141,
+                        "size-exclusion chromatography",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 81,
+                "sent": "Fractions from two peaks (decamer and monomer) were pooled separately (Figure 1C) and analysed by SEC-MALLS using a Superdex\u00a0200 10/300 GL column\u00a0(GE\u00a0Healthcare) and Viscotek SEC-MALLS instruments\u00a0(Malvern\u00a0Instruments) (Figure 2C).",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        19,
+                        24,
+                        "peaks",
+                        "evidence"
+                    ],
+                    [
+                        26,
+                        33,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        38,
+                        45,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        98,
+                        107,
+                        "SEC-MALLS",
+                        "experimental_method"
+                    ],
+                    [
+                        175,
+                        184,
+                        "SEC-MALLS",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 82,
+                "sent": "The decamer and monomer peaks were both symmetric and monodisperse, allowing the estimation of the molecular weight of the species in these fractions.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        16,
+                        23,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        24,
+                        29,
+                        "peaks",
+                        "evidence"
+                    ],
+                    [
+                        99,
+                        115,
+                        "molecular weight",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 83,
+                "sent": "The proteins analyzed by SEC-MALLS came from single protein preparation.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        25,
+                        34,
+                        "SEC-MALLS",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 84,
+                "sent": "Molecular Weight (kDa)\tDecamer peak\tMonomer peak\t \tTheoretical\t133\t13\t \tEncFtnsH-decamer fractions\t132\t15\t \tEncFtnsH-monomer fractions\t126\t13\t \t",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        16,
+                        "Molecular Weight",
+                        "evidence"
+                    ],
+                    [
+                        23,
+                        30,
+                        "Decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        36,
+                        43,
+                        "Monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        72,
+                        80,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        81,
+                        88,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        108,
+                        116,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        117,
+                        124,
+                        "monomer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 85,
+                "sent": "We purified recombinant R. rubrum EncFtn as both the full-length sequence (140 amino acids) and a truncated C-terminal hexahistidine-tagged variant (amino acids 1\u201396 plus the tag; herein EncFtnsH).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        24,
+                        33,
+                        "R. rubrum",
+                        "species"
+                    ],
+                    [
+                        34,
+                        40,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        53,
+                        64,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        75,
+                        90,
+                        "140 amino acids",
+                        "residue_range"
+                    ],
+                    [
+                        98,
+                        107,
+                        "truncated",
+                        "protein_state"
+                    ],
+                    [
+                        119,
+                        139,
+                        "hexahistidine-tagged",
+                        "protein_state"
+                    ],
+                    [
+                        161,
+                        165,
+                        "1\u201396",
+                        "residue_range"
+                    ],
+                    [
+                        187,
+                        195,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 86,
+                "sent": "In both cases the elution profile from size-exclusion chromatography (SEC) displayed two peaks (Figure 2A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        33,
+                        "elution profile",
+                        "evidence"
+                    ],
+                    [
+                        39,
+                        68,
+                        "size-exclusion chromatography",
+                        "experimental_method"
+                    ],
+                    [
+                        70,
+                        73,
+                        "SEC",
+                        "experimental_method"
+                    ],
+                    [
+                        89,
+                        94,
+                        "peaks",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 87,
+                "sent": "SDS-PAGE analysis of fractions from these peaks showed that the high molecular weight peak was partially resistant to SDS and heat-induced denaturation; in contrast, the low molecular weight peak was consistent with monomeric mass of 13 kDa (Figure 2B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "SDS-PAGE",
+                        "experimental_method"
+                    ],
+                    [
+                        42,
+                        47,
+                        "peaks",
+                        "evidence"
+                    ],
+                    [
+                        69,
+                        85,
+                        "molecular weight",
+                        "evidence"
+                    ],
+                    [
+                        174,
+                        190,
+                        "molecular weight",
+                        "evidence"
+                    ],
+                    [
+                        216,
+                        225,
+                        "monomeric",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 88,
+                "sent": "MALDI peptide mass fingerprinting of these bands confirmed the identity of both as EncFtn.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        33,
+                        "MALDI peptide mass fingerprinting",
+                        "experimental_method"
+                    ],
+                    [
+                        83,
+                        89,
+                        "EncFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 89,
+                "sent": "Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the SEC fractions showed 100 times more iron in the oligomeric fraction than the monomer (Figure 2A, blue scatter points; Table 1), suggesting that EncFtn oligomerization is associated with iron binding.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        44,
+                        "Inductively coupled plasma mass spectrometry",
+                        "experimental_method"
+                    ],
+                    [
+                        46,
+                        52,
+                        "ICP-MS",
+                        "experimental_method"
+                    ],
+                    [
+                        70,
+                        73,
+                        "SEC",
+                        "experimental_method"
+                    ],
+                    [
+                        106,
+                        110,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        147,
+                        154,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        214,
+                        220,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        256,
+                        260,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 90,
+                "sent": "In order to determine the iron-loading stoichiometry in the EncFtn complex, further ICP-MS experiments were performed using selenomethionine\u00a0(SeMet)-labelled protein EncFtn (Table 1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        26,
+                        30,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        60,
+                        66,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        84,
+                        90,
+                        "ICP-MS",
+                        "experimental_method"
+                    ],
+                    [
+                        124,
+                        140,
+                        "selenomethionine",
+                        "chemical"
+                    ],
+                    [
+                        142,
+                        147,
+                        "SeMet",
+                        "chemical"
+                    ],
+                    [
+                        166,
+                        172,
+                        "EncFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 91,
+                "sent": "In these experiments, we observed sub-stoichiometric metal binding, which is in contrast to the classical ferritins.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        96,
+                        105,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        106,
+                        115,
+                        "ferritins",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 92,
+                "sent": "Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) analysis of samples taken from each peak gave calculated molecular weights consistent with a decamer for the high molecular weight peak and a monomer for the low molecular weight peak (Figure 2C, Table 2).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        29,
+                        "Size-exclusion chromatography",
+                        "experimental_method"
+                    ],
+                    [
+                        35,
+                        69,
+                        "multi-angle laser light scattering",
+                        "experimental_method"
+                    ],
+                    [
+                        71,
+                        80,
+                        "SEC-MALLS",
+                        "experimental_method"
+                    ],
+                    [
+                        175,
+                        182,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        196,
+                        212,
+                        "molecular weight",
+                        "evidence"
+                    ],
+                    [
+                        224,
+                        231,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        244,
+                        260,
+                        "molecular weight",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 93,
+                "sent": "Effect of metal ions on the oligomeric state of EncFtnsH\u00a0in solution.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        48,
+                        56,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 94,
+                "sent": "(A/B) EncFtnsH-monomer was incubated with one mole equivalent of various metal salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        6,
+                        14,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        15,
+                        22,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        27,
+                        36,
+                        "incubated",
+                        "experimental_method"
+                    ],
+                    [
+                        108,
+                        133,
+                        "analytical gel-filtration",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 95,
+                "sent": "Co2+ and Zn2+ induced the formation of the decameric form of EncFtnsH; while Mn2+, Mg2+ and Fe3+ did not significantly alter the oligomeric state of EncFtnsH.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "Co2+",
+                        "chemical"
+                    ],
+                    [
+                        9,
+                        13,
+                        "Zn2+",
+                        "chemical"
+                    ],
+                    [
+                        43,
+                        52,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        61,
+                        69,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        77,
+                        81,
+                        "Mn2+",
+                        "chemical"
+                    ],
+                    [
+                        83,
+                        87,
+                        "Mg2+",
+                        "chemical"
+                    ],
+                    [
+                        92,
+                        96,
+                        "Fe3+",
+                        "chemical"
+                    ],
+                    [
+                        149,
+                        157,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 96,
+                "sent": "PAGE analysis of the effect of metal ions on the oligomeric state of EncFtnsH.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "PAGE",
+                        "experimental_method"
+                    ],
+                    [
+                        69,
+                        77,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 97,
+                "sent": "50 \u00b5M EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        6,
+                        14,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        15,
+                        22,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        26,
+                        33,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        91,
+                        96,
+                        "Fe2+,",
+                        "chemical"
+                    ],
+                    [
+                        97,
+                        102,
+                        "Co2+,",
+                        "chemical"
+                    ],
+                    [
+                        103,
+                        108,
+                        "Zn2+,",
+                        "chemical"
+                    ],
+                    [
+                        109,
+                        114,
+                        "Mn2+,",
+                        "chemical"
+                    ],
+                    [
+                        115,
+                        120,
+                        "Ca2+,",
+                        "chemical"
+                    ],
+                    [
+                        121,
+                        125,
+                        "Mg2+",
+                        "chemical"
+                    ],
+                    [
+                        130,
+                        135,
+                        "Fe3+,",
+                        "chemical"
+                    ],
+                    [
+                        159,
+                        170,
+                        "Native PAGE",
+                        "experimental_method"
+                    ],
+                    [
+                        181,
+                        189,
+                        "SDS-PAGE",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 98,
+                "sent": "\u00a0(A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        9,
+                        20,
+                        "Native PAGE",
+                        "experimental_method"
+                    ],
+                    [
+                        33,
+                        41,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        42,
+                        49,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        104,
+                        115,
+                        "Native PAGE",
+                        "experimental_method"
+                    ],
+                    [
+                        128,
+                        136,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        137,
+                        144,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        199,
+                        207,
+                        "SDS-PAGE",
+                        "experimental_method"
+                    ],
+                    [
+                        236,
+                        244,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        245,
+                        252,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        292,
+                        300,
+                        "SDS-PAGE",
+                        "experimental_method"
+                    ],
+                    [
+                        329,
+                        337,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        338,
+                        345,
+                        "decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 99,
+                "sent": "Effect of Fe2+ and protein concentration on the oligomeric state of EncFtnsH in solution.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        10,
+                        14,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        68,
+                        76,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 100,
+                "sent": "(A) Recombinant EncFtnsH was purified by Gel filtration Superdex\u00a0200 chromatography from E. coli BL21(DE3) grown in MM or in MM supplemented with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        16,
+                        24,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        41,
+                        55,
+                        "Gel filtration",
+                        "experimental_method"
+                    ],
+                    [
+                        89,
+                        106,
+                        "E. coli BL21(DE3)",
+                        "species"
+                    ],
+                    [
+                        116,
+                        118,
+                        "MM",
+                        "experimental_method"
+                    ],
+                    [
+                        125,
+                        127,
+                        "MM",
+                        "experimental_method"
+                    ],
+                    [
+                        151,
+                        165,
+                        "Fe(NH4)2(SO4)2",
+                        "chemical"
+                    ],
+                    [
+                        167,
+                        169,
+                        "MM",
+                        "experimental_method"
+                    ],
+                    [
+                        170,
+                        174,
+                        "Fe2+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 101,
+                "sent": "A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column\u00a0(GE\u00a0Healthcare).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        23,
+                        30,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        95,
+                        97,
+                        "MM",
+                        "experimental_method"
+                    ],
+                    [
+                        98,
+                        102,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        115,
+                        123,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        124,
+                        126,
+                        "MM",
+                        "experimental_method"
+                    ],
+                    [
+                        144,
+                        148,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        184,
+                        192,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        206,
+                        214,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        215,
+                        222,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        266,
+                        270,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        300,
+                        325,
+                        "analytical gel-filtration",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 102,
+                "sent": "Both Fe2+ salts tested induced the formation of decamer indicated by the peak between 1.2 and 1.6 ml.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        5,
+                        9,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        48,
+                        55,
+                        "decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 103,
+                "sent": "Monomeric and decameric samples of EncFtnsH\u00a0are shown as controls.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Monomeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        14,
+                        23,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        35,
+                        43,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 104,
+                "sent": "Peaks around 0.8 ml were seen as protein aggregation.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "Peaks",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 105,
+                "sent": "(C) Analytical gel filtration of EncFtn monomer at different concentrations to illustrate the effect of protein concentration on multimerization.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        29,
+                        "Analytical gel filtration",
+                        "experimental_method"
+                    ],
+                    [
+                        33,
+                        39,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        40,
+                        47,
+                        "monomer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 106,
+                "sent": "The major peak shows a shift towards a dimer species at high concentration of protein, but the ratio of this peak (1.5\u20131.8 ml) to the decamer peak (1.2\u20131.5 ml) does not change when compared to the low concentration sample.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        39,
+                        44,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        134,
+                        141,
+                        "decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 107,
+                "sent": "Gel-filtration peak area ratios for EncFtnsH decamer and monomer on addition of different metal ions.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        14,
+                        "Gel-filtration",
+                        "experimental_method"
+                    ],
+                    [
+                        15,
+                        31,
+                        "peak area ratios",
+                        "evidence"
+                    ],
+                    [
+                        36,
+                        44,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        45,
+                        52,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        57,
+                        64,
+                        "monomer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 108,
+                "sent": "EncFtnsH was produced in E. coli BL21(DE3) cultured in MM and MM with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+) and purified by gel-filtration chromatography using an Superdex\u00a0200 16/60\u00a0column (GE Healthcare).",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        25,
+                        42,
+                        "E. coli BL21(DE3)",
+                        "species"
+                    ],
+                    [
+                        55,
+                        57,
+                        "MM",
+                        "experimental_method"
+                    ],
+                    [
+                        62,
+                        64,
+                        "MM",
+                        "experimental_method"
+                    ],
+                    [
+                        75,
+                        89,
+                        "Fe(NH4)2(SO4)2",
+                        "chemical"
+                    ],
+                    [
+                        91,
+                        93,
+                        "MM",
+                        "experimental_method"
+                    ],
+                    [
+                        94,
+                        98,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        116,
+                        145,
+                        "gel-filtration chromatography",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 109,
+                "sent": "Monomer fractions of EncFtnsH purified from MM were pooled and run in subsequent analytical gel-filtration runs over the course of three days.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "Monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        21,
+                        29,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        44,
+                        46,
+                        "MM",
+                        "experimental_method"
+                    ],
+                    [
+                        81,
+                        106,
+                        "analytical gel-filtration",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 110,
+                "sent": "Samples of EncFtnsH monomer were incubated with one molar equivalent of metal ion salts at room temperature for two hours before analysis by analytical gel\u00a0filtration chromatography (AGF) using a Superdex 200\u00a010/300 GL column.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        11,
+                        19,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        20,
+                        27,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        141,
+                        181,
+                        "analytical gel\u00a0filtration chromatography",
+                        "experimental_method"
+                    ],
+                    [
+                        183,
+                        186,
+                        "AGF",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 111,
+                "sent": "The area for resulting protein peaks were calculated using the Unicorn software (GE Healthcare); peak ratios were calculated to quantify the propensity of EncFtnsH to multimerize in the presence of the different metal ions.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        31,
+                        36,
+                        "peaks",
+                        "evidence"
+                    ],
+                    [
+                        97,
+                        108,
+                        "peak ratios",
+                        "evidence"
+                    ],
+                    [
+                        155,
+                        163,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        186,
+                        197,
+                        "presence of",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 112,
+                "sent": "The change in the\u00a0ratios of monomer to decamer over the three days of experiments may be a consequence of experimental variability, or the propensity of this protein to equilibrate towards decamer over time.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        28,
+                        35,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        39,
+                        46,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        189,
+                        196,
+                        "decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 113,
+                "sent": "The increased decamer: monomer ratio seen in the presence of Fe2+, Co2+, and Zn2+ indicates that these metal ions facilitate multimerization of the EncFtnsH protein, while the other metal ions tested do not appear to induce multimerization.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        14,
+                        21,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        23,
+                        30,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        49,
+                        60,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        61,
+                        65,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        67,
+                        71,
+                        "Co2+",
+                        "chemical"
+                    ],
+                    [
+                        77,
+                        81,
+                        "Zn2+",
+                        "chemical"
+                    ],
+                    [
+                        148,
+                        156,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 114,
+                "sent": "The analytical gel filtration experiment was repeated twice using two independent preparations of protein, of which values calculated from one sample are presented here.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        4,
+                        29,
+                        "analytical gel filtration",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 115,
+                "sent": "Method\tSample\tMonomer area\tDecamer area\tDecamer/Monomer\t \tGel filtration Superdex\u00a0200 chromatography\tEncFtnsH-MM\t64.3\t583.6\t0.1\t \tEncFtnsH-MM+Fe2+\t1938.4\t426.4\t4.5\t \tAnalytical Gel filtration Day1\tEncFtnsH-decamer fractions\t20.2\t1.8\t11.2\t \tEncFtnsH-monomer fractions\t2.9\t21.9\t0.1\t \tFe(NH4)2(SO4)2/EncFtnsH-monomer\t11.0\t13.0\t0.8\t \tFeSO4-HCl/EncFtnsH-monomer\t11.3\t11.4\t1.0\t \tAnalytical Gel filtration Day2\tEncFtnsH-monomer fractions\t8.3\t22.8\t0.4\t \tCoCl2/EncFtnsH-monomer\t17.7\t14.5\t1.2\t \tMnCl2/EncFtnsH-monomer\t3.1\t30.5\t0.1\t \tZnSO4/EncFtnsH-monomer\t20.4\t9.0\t2.3\t \tFeCl3/EncFtnsH-monomer\t3.9\t28.6\t0.1\t \tAnalytical Gel filtration Day3\tEncFtnsH-monomer fractions\t6.3\t23.4\t0.3\t \tMgSO4/EncFtnsH-monomer\t5.8\t30.2\t0.2\t \tCa acetate/EncFtnsH-monomer\t5.6\t25.2\t0.2\t \t",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        14,
+                        21,
+                        "Monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        27,
+                        34,
+                        "Decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        40,
+                        47,
+                        "Decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        48,
+                        55,
+                        "Monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        58,
+                        72,
+                        "Gel filtration",
+                        "experimental_method"
+                    ],
+                    [
+                        101,
+                        109,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        110,
+                        112,
+                        "MM",
+                        "experimental_method"
+                    ],
+                    [
+                        130,
+                        138,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        139,
+                        141,
+                        "MM",
+                        "experimental_method"
+                    ],
+                    [
+                        142,
+                        146,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        166,
+                        191,
+                        "Analytical Gel filtration",
+                        "experimental_method"
+                    ],
+                    [
+                        197,
+                        205,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        206,
+                        213,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        240,
+                        248,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        249,
+                        256,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        282,
+                        296,
+                        "Fe(NH4)2(SO4)2",
+                        "chemical"
+                    ],
+                    [
+                        297,
+                        305,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        306,
+                        313,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        330,
+                        339,
+                        "FeSO4-HCl",
+                        "chemical"
+                    ],
+                    [
+                        340,
+                        348,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        349,
+                        356,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        373,
+                        398,
+                        "Analytical Gel filtration",
+                        "experimental_method"
+                    ],
+                    [
+                        404,
+                        412,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        413,
+                        420,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        446,
+                        451,
+                        "CoCl2",
+                        "chemical"
+                    ],
+                    [
+                        452,
+                        460,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        461,
+                        468,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        485,
+                        490,
+                        "MnCl2",
+                        "chemical"
+                    ],
+                    [
+                        491,
+                        499,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        500,
+                        507,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        523,
+                        528,
+                        "ZnSO4",
+                        "chemical"
+                    ],
+                    [
+                        529,
+                        537,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        538,
+                        545,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        561,
+                        566,
+                        "FeCl3",
+                        "chemical"
+                    ],
+                    [
+                        567,
+                        575,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        576,
+                        583,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        599,
+                        624,
+                        "Analytical Gel filtration",
+                        "experimental_method"
+                    ],
+                    [
+                        630,
+                        638,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        639,
+                        646,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        672,
+                        677,
+                        "MgSO4",
+                        "chemical"
+                    ],
+                    [
+                        678,
+                        686,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        687,
+                        694,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        710,
+                        720,
+                        "Ca acetate",
+                        "chemical"
+                    ],
+                    [
+                        721,
+                        729,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        730,
+                        737,
+                        "monomer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 116,
+                "sent": "We purified EncFtnsH from E. coli grown in MM with or without the addition of 1 mM Fe(NH4)2(SO4)2.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        12,
+                        20,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        26,
+                        33,
+                        "E. coli",
+                        "species"
+                    ],
+                    [
+                        43,
+                        45,
+                        "MM",
+                        "experimental_method"
+                    ],
+                    [
+                        83,
+                        97,
+                        "Fe(NH4)2(SO4)2",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 117,
+                "sent": "The decamer to monomer ratio in the sample purified from cells grown in iron-supplemented media was 4.5, while that from the iron-free media was 0.11, suggesting that iron induces the oligomerization of EncFtnsH in vivo\u00a0(Figure 3A, Table 3).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        15,
+                        22,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        72,
+                        76,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        125,
+                        134,
+                        "iron-free",
+                        "protein_state"
+                    ],
+                    [
+                        167,
+                        171,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        203,
+                        211,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 118,
+                "sent": "To test the metal-dependent oligomerization of EncFtnsH in vitro, we incubated the protein with various metal cations and subjected samples to analytical SEC and non-denaturing PAGE.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        47,
+                        55,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        69,
+                        78,
+                        "incubated",
+                        "experimental_method"
+                    ],
+                    [
+                        143,
+                        157,
+                        "analytical SEC",
+                        "experimental_method"
+                    ],
+                    [
+                        162,
+                        181,
+                        "non-denaturing PAGE",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 119,
+                "sent": "Of the metals tested, only Fe2+, Zn2+ and Co2+ induced the formation of significant amounts of the decamer (Figure 3B, Figure 3\u2014figure supplement 1/2).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        27,
+                        32,
+                        "Fe2+,",
+                        "chemical"
+                    ],
+                    [
+                        33,
+                        37,
+                        "Zn2+",
+                        "chemical"
+                    ],
+                    [
+                        42,
+                        46,
+                        "Co2+",
+                        "chemical"
+                    ],
+                    [
+                        99,
+                        106,
+                        "decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 120,
+                "sent": "While Fe2+ induces the multimerization of EncFtnsH, Fe3+ in the form of FeCl3 does not have this effect on the protein, highlighting the apparent preference this protein has for the ferrous form of iron.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        6,
+                        10,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        42,
+                        50,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        52,
+                        56,
+                        "Fe3+",
+                        "chemical"
+                    ],
+                    [
+                        72,
+                        77,
+                        "FeCl3",
+                        "chemical"
+                    ],
+                    [
+                        182,
+                        202,
+                        "ferrous form of iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 121,
+                "sent": "To determine if the oligomerization of EncFtnsH was concentration dependent we performed analytical SEC at 90 and 700 \u00b5M protein concentration (Figure 3C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        39,
+                        47,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        89,
+                        103,
+                        "analytical SEC",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 122,
+                "sent": "At the higher concentration, no increase in the decameric form of EncFtn was observed; however, the shift in the major peak from the position of the monomer species indicated a tendency to dimerize at high concentration.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        48,
+                        57,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        66,
+                        72,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        149,
+                        156,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        189,
+                        197,
+                        "dimerize",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 123,
+                "sent": "Crystal structure of EncFtnsH",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        17,
+                        "Crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        29,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 124,
+                "sent": "Electrostatic surface of EncFtnsH.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        25,
+                        33,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 125,
+                "sent": "The solvent accessible surface of EncFtnsH is shown, colored by electrostatic potential as calculated using the APBS plugin in PyMOL.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        34,
+                        42,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 126,
+                "sent": "Negatively charged regions are colored red and positive regions in blue, neutral regions in grey. (A) View of the surface of the EncFtnsH decamer looking down the central axis.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        129,
+                        137,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        138,
+                        145,
+                        "decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 127,
+                "sent": "(B) Orthogonal view of (A). (C) Cutaway view of (B) showing the charge distribution within the central cavity.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        95,
+                        109,
+                        "central cavity",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 128,
+                "sent": "Crystal structure of EncFtnsH.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        17,
+                        "Crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        29,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 129,
+                "sent": "(A) Overall architecture of EncFtnsH. Transparent solvent accessible surface view with \u03b1-helices shown as tubes and bound metal ions as spheres.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        28,
+                        36,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        87,
+                        96,
+                        "\u03b1-helices",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 130,
+                "sent": "Alternating subunits are colored blue and green for clarity.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        12,
+                        20,
+                        "subunits",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 131,
+                "sent": "The doughnut-like decamer is 7 nm in diameter and 4.5 nm thick. (B) Monomer of EncFtnsH shown as a secondary structure cartoon. (C/D) Dimer interfaces formed in the decameric ring of EncFtnsH. Subunits are shown as secondary structure cartoons and colored blue and green for clarity.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        17,
+                        "doughnut-like",
+                        "structure_element"
+                    ],
+                    [
+                        18,
+                        25,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        68,
+                        75,
+                        "Monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        79,
+                        87,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        134,
+                        150,
+                        "Dimer interfaces",
+                        "site"
+                    ],
+                    [
+                        165,
+                        174,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        175,
+                        179,
+                        "ring",
+                        "structure_element"
+                    ],
+                    [
+                        183,
+                        191,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        193,
+                        201,
+                        "Subunits",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 132,
+                "sent": "Bound metal ions are shown as orange spheres for Fe3+ and grey and white spheres for Ca2+.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        49,
+                        53,
+                        "Fe3+",
+                        "chemical"
+                    ],
+                    [
+                        85,
+                        89,
+                        "Ca2+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 133,
+                "sent": "We determined the crystal structure of EncFtnsH by molecular replacement to 2.0 \u00c5 resolution (see Table 1 for X-ray data collection and refinement statistics).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        35,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        39,
+                        47,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        51,
+                        72,
+                        "molecular replacement",
+                        "experimental_method"
+                    ],
+                    [
+                        110,
+                        157,
+                        "X-ray data collection and refinement statistics",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 134,
+                "sent": "The crystallographic asymmetric unit contained thirty monomers of EncFtn with visible electron density for residues 7 \u2013 96 in each chain.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        54,
+                        62,
+                        "monomers",
+                        "oligomeric_state"
+                    ],
+                    [
+                        66,
+                        72,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        86,
+                        102,
+                        "electron density",
+                        "evidence"
+                    ],
+                    [
+                        116,
+                        122,
+                        "7 \u2013 96",
+                        "residue_range"
+                    ]
+                ]
+            },
+            {
+                "sid": 135,
+                "sent": "The protein chains were arranged as three identical annular decamers, each with D5 symmetry.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        52,
+                        59,
+                        "annular",
+                        "structure_element"
+                    ],
+                    [
+                        60,
+                        68,
+                        "decamers",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 136,
+                "sent": "The decamer has a diameter of 7 nm and thickness of 4 nm (Figure 4A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 137,
+                "sent": "The monomer of EncFtn has an N-terminal 310-helix that precedes two 4 nm long antiparallel \u03b1-helices arranged with their long axes at 25\u00b0 to each other; these helices are followed by a shorter 1.4 nm helix projecting at 70\u00b0 from \u03b12 (Figure 4B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        15,
+                        21,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        40,
+                        49,
+                        "310-helix",
+                        "structure_element"
+                    ],
+                    [
+                        78,
+                        100,
+                        "antiparallel \u03b1-helices",
+                        "structure_element"
+                    ],
+                    [
+                        159,
+                        166,
+                        "helices",
+                        "structure_element"
+                    ],
+                    [
+                        200,
+                        205,
+                        "helix",
+                        "structure_element"
+                    ],
+                    [
+                        229,
+                        231,
+                        "\u03b12",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 138,
+                "sent": "The C-terminal region of the crystallized construct extends from the outer circumference of the ring, indicating that the encapsulin localization sequence in the full-length protein is on the exterior of the ring and is thus free to interact with its binding site on the encapsulin shell protein.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        21,
+                        "C-terminal region",
+                        "structure_element"
+                    ],
+                    [
+                        96,
+                        100,
+                        "ring",
+                        "structure_element"
+                    ],
+                    [
+                        122,
+                        154,
+                        "encapsulin localization sequence",
+                        "site"
+                    ],
+                    [
+                        162,
+                        173,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        208,
+                        212,
+                        "ring",
+                        "structure_element"
+                    ],
+                    [
+                        251,
+                        263,
+                        "binding site",
+                        "site"
+                    ],
+                    [
+                        271,
+                        281,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        282,
+                        287,
+                        "shell",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 139,
+                "sent": "The monomer of EncFtnsH forms two distinct dimer interfaces within the decamer (Figure 4 C/D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        15,
+                        23,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        43,
+                        59,
+                        "dimer interfaces",
+                        "site"
+                    ],
+                    [
+                        71,
+                        78,
+                        "decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 140,
+                "sent": "The first dimer is formed from two monomers arranged antiparallel to each other, with \u03b11 from each monomer interacting along their lengths and \u03b13 interdigitating with \u03b12 and \u03b13 of the partner chain.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        10,
+                        15,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        35,
+                        43,
+                        "monomers",
+                        "oligomeric_state"
+                    ],
+                    [
+                        86,
+                        88,
+                        "\u03b11",
+                        "structure_element"
+                    ],
+                    [
+                        99,
+                        106,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        143,
+                        145,
+                        "\u03b13",
+                        "structure_element"
+                    ],
+                    [
+                        167,
+                        169,
+                        "\u03b12",
+                        "structure_element"
+                    ],
+                    [
+                        174,
+                        176,
+                        "\u03b13",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 141,
+                "sent": "This interface buries one third of the surface area from each partner and is stabilized by thirty hydrogen bonds and fourteen salt bridges (Figure 4C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        14,
+                        "interface",
+                        "site"
+                    ],
+                    [
+                        98,
+                        112,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        126,
+                        138,
+                        "salt bridges",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 142,
+                "sent": "The second dimer interface forms an antiparallel four-helix bundle between helices 1 and 2 from each monomer (Figure 4D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        26,
+                        "dimer interface",
+                        "site"
+                    ],
+                    [
+                        36,
+                        66,
+                        "antiparallel four-helix bundle",
+                        "structure_element"
+                    ],
+                    [
+                        75,
+                        90,
+                        "helices 1 and 2",
+                        "structure_element"
+                    ],
+                    [
+                        101,
+                        108,
+                        "monomer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 143,
+                "sent": "This interface is less extensive than the first and is stabilized by twenty-one hydrogen bonds, six salt bridges, and a number of metal ions.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        14,
+                        "interface",
+                        "site"
+                    ],
+                    [
+                        80,
+                        94,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        100,
+                        112,
+                        "salt bridges",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 144,
+                "sent": "The arrangement of ten monomers in alternating orientation forms the decamer of EncFtn, which assembles as a pentamer of dimers (Figure 4A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        23,
+                        31,
+                        "monomers",
+                        "oligomeric_state"
+                    ],
+                    [
+                        69,
+                        76,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        80,
+                        86,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        109,
+                        117,
+                        "pentamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        121,
+                        127,
+                        "dimers",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 145,
+                "sent": "Each monomer lies at 45\u00b0 relative to the vertical central-axis of the ring, with the N-termini of alternating subunits capping the center of the ring at each end, while the C-termini are arranged around the circumference.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        12,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        70,
+                        74,
+                        "ring",
+                        "structure_element"
+                    ],
+                    [
+                        110,
+                        118,
+                        "subunits",
+                        "structure_element"
+                    ],
+                    [
+                        145,
+                        149,
+                        "ring",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 146,
+                "sent": "The central hole in the ring is 2.5 nm at its widest in the center of the complex, and 1.5 nm at its narrowest point near the outer surface, although it should be noted that a number of residues at the N-terminus are not visible in the crystallographic electron density and these may occupy the central channel.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        16,
+                        "central hole",
+                        "site"
+                    ],
+                    [
+                        24,
+                        28,
+                        "ring",
+                        "structure_element"
+                    ],
+                    [
+                        236,
+                        269,
+                        "crystallographic electron density",
+                        "evidence"
+                    ],
+                    [
+                        295,
+                        310,
+                        "central channel",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 147,
+                "sent": "The surface of the decamer has distinct negatively charged patches, both within the central hole and on the outer circumference, which form spokes through the radius of the complex (Figure 4\u2014figure supplement 1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        26,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        40,
+                        66,
+                        "negatively charged patches",
+                        "site"
+                    ],
+                    [
+                        84,
+                        96,
+                        "central hole",
+                        "site"
+                    ],
+                    [
+                        140,
+                        146,
+                        "spokes",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 148,
+                "sent": "EncFtn ferroxidase center",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        6,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        7,
+                        25,
+                        "ferroxidase center",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 149,
+                "sent": "Putative ligand-binding site in EncFtnsH.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        9,
+                        28,
+                        "ligand-binding site",
+                        "site"
+                    ],
+                    [
+                        32,
+                        40,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 150,
+                "sent": "(A) Wall-eyed stereo view of the dimer interface of EncFtn.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        33,
+                        48,
+                        "dimer interface",
+                        "site"
+                    ],
+                    [
+                        52,
+                        58,
+                        "EncFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 151,
+                "sent": "Protein chains are shown as sticks, with 2mFo-DFc electron density shown in blue mesh and contoured at 1.5 \u03c3 and mFo-DFc shown in green mesh and contoured at 3 \u03c3. (B) Wall-eyed stereo view of putative metal binding site at the external surface of EncFtnsH. Protein chains and electron density maps are shown as in (A).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        41,
+                        66,
+                        "2mFo-DFc electron density",
+                        "evidence"
+                    ],
+                    [
+                        113,
+                        120,
+                        "mFo-DFc",
+                        "evidence"
+                    ],
+                    [
+                        201,
+                        219,
+                        "metal binding site",
+                        "site"
+                    ],
+                    [
+                        247,
+                        255,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        276,
+                        297,
+                        "electron density maps",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 152,
+                "sent": "EncFtnsH metal binding sites.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        9,
+                        28,
+                        "metal binding sites",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 153,
+                "sent": "(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH. Protein residues are shown as sticks with blue and green carbons for the different subunits, iron ions are shown as orange spheres and calcium as grey spheres, and the glycolic acid ligand is shown with yellow carbon atoms coordinated above the di-iron center.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        33,
+                        69,
+                        "metal-binding dimerization interface",
+                        "site"
+                    ],
+                    [
+                        73,
+                        81,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        166,
+                        174,
+                        "subunits",
+                        "structure_element"
+                    ],
+                    [
+                        176,
+                        180,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        218,
+                        225,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        251,
+                        264,
+                        "glycolic acid",
+                        "chemical"
+                    ],
+                    [
+                        328,
+                        342,
+                        "di-iron center",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 154,
+                "sent": "The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 \u03c3 and the NCS-averaged\u00a0anomalous difference map is shown as an orange mesh and contoured at 10 \u03c3. (B) Iron coordination within the FOC including residues Glu32, Glu62, His65 and Tyr39 from two chains.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        33,
+                        "2mFo-DFc electron density map",
+                        "evidence"
+                    ],
+                    [
+                        85,
+                        122,
+                        "NCS-averaged\u00a0anomalous difference map",
+                        "evidence"
+                    ],
+                    [
+                        177,
+                        181,
+                        "Iron",
+                        "chemical"
+                    ],
+                    [
+                        182,
+                        194,
+                        "coordination",
+                        "bond_interaction"
+                    ],
+                    [
+                        206,
+                        209,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        229,
+                        234,
+                        "Glu32",
+                        "residue_name_number"
+                    ],
+                    [
+                        236,
+                        241,
+                        "Glu62",
+                        "residue_name_number"
+                    ],
+                    [
+                        243,
+                        248,
+                        "His65",
+                        "residue_name_number"
+                    ],
+                    [
+                        253,
+                        258,
+                        "Tyr39",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 155,
+                "sent": "Protein and metal ions are shown as in A. Coordination between the protein and iron ions is shown as yellow dashed lines with distances indicated. (C) Coordination of calcium within the dimer interface by four glutamic acid residues (E31 and E34 from two chains).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        42,
+                        54,
+                        "Coordination",
+                        "bond_interaction"
+                    ],
+                    [
+                        79,
+                        83,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        151,
+                        163,
+                        "Coordination",
+                        "bond_interaction"
+                    ],
+                    [
+                        167,
+                        174,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        186,
+                        201,
+                        "dimer interface",
+                        "site"
+                    ],
+                    [
+                        210,
+                        223,
+                        "glutamic acid",
+                        "residue_name"
+                    ],
+                    [
+                        234,
+                        237,
+                        "E31",
+                        "residue_name_number"
+                    ],
+                    [
+                        242,
+                        245,
+                        "E34",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 156,
+                "sent": "The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 \u00c5 away from the FOC iron.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        46,
+                        51,
+                        "water",
+                        "chemical"
+                    ],
+                    [
+                        78,
+                        90,
+                        "coordination",
+                        "bond_interaction"
+                    ],
+                    [
+                        98,
+                        105,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        136,
+                        159,
+                        "Metal coordination site",
+                        "site"
+                    ],
+                    [
+                        184,
+                        192,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        202,
+                        209,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        219,
+                        233,
+                        "coordinated by",
+                        "bond_interaction"
+                    ],
+                    [
+                        243,
+                        248,
+                        "His57",
+                        "residue_name_number"
+                    ],
+                    [
+                        250,
+                        255,
+                        "Glu61",
+                        "residue_name_number"
+                    ],
+                    [
+                        260,
+                        265,
+                        "Glu64",
+                        "residue_name_number"
+                    ],
+                    [
+                        293,
+                        296,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        297,
+                        302,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        387,
+                        390,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        391,
+                        395,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 157,
+                "sent": "The electron density maps of the initial EncFtnsH model displayed significant positive peaks in the mFo-DFc map at the center of the 4-helix bundle dimer (Figure 5\u2014figure supplement 1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        25,
+                        "electron density maps",
+                        "evidence"
+                    ],
+                    [
+                        41,
+                        49,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        100,
+                        111,
+                        "mFo-DFc map",
+                        "evidence"
+                    ],
+                    [
+                        133,
+                        147,
+                        "4-helix bundle",
+                        "structure_element"
+                    ],
+                    [
+                        148,
+                        153,
+                        "dimer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 158,
+                "sent": "Informed by the ICP-MS data indicating the presence of iron in the protein we collected diffraction data at the experimentally determined iron absorption edge (1.74 \u00c5) and calculated an anomalous difference Fourier map using this data.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        16,
+                        22,
+                        "ICP-MS",
+                        "experimental_method"
+                    ],
+                    [
+                        43,
+                        54,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        55,
+                        59,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        88,
+                        104,
+                        "diffraction data",
+                        "evidence"
+                    ],
+                    [
+                        138,
+                        142,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        186,
+                        218,
+                        "anomalous difference Fourier map",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 159,
+                "sent": "Inspection of this map showed two 10-sigma peaks between residues Glu32, Glu62 and His65 of two adjacent chains, and a statistically smaller 5-sigma peak between residues Glu31 and Glu34 of the two chains.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        22,
+                        "map",
+                        "evidence"
+                    ],
+                    [
+                        43,
+                        48,
+                        "peaks",
+                        "evidence"
+                    ],
+                    [
+                        66,
+                        71,
+                        "Glu32",
+                        "residue_name_number"
+                    ],
+                    [
+                        73,
+                        78,
+                        "Glu62",
+                        "residue_name_number"
+                    ],
+                    [
+                        83,
+                        88,
+                        "His65",
+                        "residue_name_number"
+                    ],
+                    [
+                        171,
+                        176,
+                        "Glu31",
+                        "residue_name_number"
+                    ],
+                    [
+                        181,
+                        186,
+                        "Glu34",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 160,
+                "sent": "Modeling metal ions into these peaks and refinement of the anomalous scattering parameters allowed us to identify these as two iron ions and a calcium ion respectively (Figure 5A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        41,
+                        51,
+                        "refinement",
+                        "experimental_method"
+                    ],
+                    [
+                        59,
+                        90,
+                        "anomalous scattering parameters",
+                        "evidence"
+                    ],
+                    [
+                        127,
+                        131,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        143,
+                        150,
+                        "calcium",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 161,
+                "sent": "An additional region of asymmetric electron density near the di-iron binding site in the mFo-DFc map was modeled as glycolic acid, presumably a breakdown product of the PEG 3350 used for crystallization.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        35,
+                        51,
+                        "electron density",
+                        "evidence"
+                    ],
+                    [
+                        61,
+                        81,
+                        "di-iron binding site",
+                        "site"
+                    ],
+                    [
+                        89,
+                        100,
+                        "mFo-DFc map",
+                        "evidence"
+                    ],
+                    [
+                        116,
+                        129,
+                        "glycolic acid",
+                        "chemical"
+                    ],
+                    [
+                        169,
+                        177,
+                        "PEG 3350",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 162,
+                "sent": "This di-iron center has an Fe-Fe distance of 3.5 \u00c5, Fe-Glu-O distances between 2.3 and 2.5 \u00c5, and Fe-His-N distances of 2.5 \u00c5 (Figure 5B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        19,
+                        "di-iron center",
+                        "site"
+                    ],
+                    [
+                        27,
+                        41,
+                        "Fe-Fe distance",
+                        "evidence"
+                    ],
+                    [
+                        52,
+                        70,
+                        "Fe-Glu-O distances",
+                        "evidence"
+                    ],
+                    [
+                        98,
+                        116,
+                        "Fe-His-N distances",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 163,
+                "sent": "This coordination geometry is consistent with the di-nuclear ferroxidase center (FOC) found in ferritin.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        17,
+                        "coordination",
+                        "bond_interaction"
+                    ],
+                    [
+                        50,
+                        79,
+                        "di-nuclear ferroxidase center",
+                        "site"
+                    ],
+                    [
+                        81,
+                        84,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        95,
+                        103,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 164,
+                "sent": "It is interesting to note that although we did not add any additional iron to the crystallization trials, the FOC was fully occupied with iron in the final structure, implying that this site has a very high affinity for iron.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        70,
+                        74,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        82,
+                        104,
+                        "crystallization trials",
+                        "experimental_method"
+                    ],
+                    [
+                        110,
+                        113,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        138,
+                        142,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        156,
+                        165,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        207,
+                        215,
+                        "affinity",
+                        "evidence"
+                    ],
+                    [
+                        220,
+                        224,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 165,
+                "sent": "The calcium ion coordinated by Glu31 and Glu34 adopts heptacoordinate geometry, with coordination distances of 2.5 \u00c5 between the metal ion and carboxylate oxygens of Glu31 and Glu34 (E31/34-site).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        16,
+                        30,
+                        "coordinated by",
+                        "bond_interaction"
+                    ],
+                    [
+                        31,
+                        36,
+                        "Glu31",
+                        "residue_name_number"
+                    ],
+                    [
+                        41,
+                        46,
+                        "Glu34",
+                        "residue_name_number"
+                    ],
+                    [
+                        54,
+                        69,
+                        "heptacoordinate",
+                        "protein_state"
+                    ],
+                    [
+                        85,
+                        97,
+                        "coordination",
+                        "bond_interaction"
+                    ],
+                    [
+                        166,
+                        171,
+                        "Glu31",
+                        "residue_name_number"
+                    ],
+                    [
+                        176,
+                        181,
+                        "Glu34",
+                        "residue_name_number"
+                    ],
+                    [
+                        183,
+                        194,
+                        "E31/34-site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 166,
+                "sent": "A number of ordered solvent molecules are also coordinated to this metal ion at a distance of 2.5 \u00c5. This heptacoordinate geometry is common in crystal structures with calcium ions (Figure 5C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        47,
+                        58,
+                        "coordinated",
+                        "bond_interaction"
+                    ],
+                    [
+                        106,
+                        121,
+                        "heptacoordinate",
+                        "protein_state"
+                    ],
+                    [
+                        144,
+                        162,
+                        "crystal structures",
+                        "evidence"
+                    ],
+                    [
+                        168,
+                        175,
+                        "calcium",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 167,
+                "sent": "While ICP-MS indicated that there were negligible amounts of calcium in the purified protein, the presence of 140 mM calcium acetate in the crystallization mother liquor favors the coordination of calcium at this site.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        6,
+                        12,
+                        "ICP-MS",
+                        "experimental_method"
+                    ],
+                    [
+                        61,
+                        68,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        98,
+                        109,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        117,
+                        132,
+                        "calcium acetate",
+                        "chemical"
+                    ],
+                    [
+                        181,
+                        193,
+                        "coordination",
+                        "bond_interaction"
+                    ],
+                    [
+                        197,
+                        204,
+                        "calcium",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 168,
+                "sent": "The fact that the protein does not multimerize in solution in the presence of Fe3+ may indicate that these metal binding sites have a lower affinity for the ferric form of iron, which is the product of the ferroxidase reaction.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        66,
+                        77,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        78,
+                        82,
+                        "Fe3+",
+                        "chemical"
+                    ],
+                    [
+                        107,
+                        126,
+                        "metal binding sites",
+                        "site"
+                    ],
+                    [
+                        172,
+                        176,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        206,
+                        217,
+                        "ferroxidase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 169,
+                "sent": "A number of additional metal-ions were present at the outer circumference of at least one decamer in the asymmetric unit (Figure 5D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        90,
+                        97,
+                        "decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 170,
+                "sent": "These ions are coordinated by His57, Glu61 and Glu64 from both chains in the FOC dimer and are 4.5 \u00c5 apart; Fe-Glu-O distances are between 2.5 and 3.5 \u00c5 and the Fe-His-N distances are 4 and 4.5 \u00c5.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        15,
+                        29,
+                        "coordinated by",
+                        "bond_interaction"
+                    ],
+                    [
+                        30,
+                        35,
+                        "His57",
+                        "residue_name_number"
+                    ],
+                    [
+                        37,
+                        42,
+                        "Glu61",
+                        "residue_name_number"
+                    ],
+                    [
+                        47,
+                        52,
+                        "Glu64",
+                        "residue_name_number"
+                    ],
+                    [
+                        77,
+                        80,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        81,
+                        86,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        108,
+                        116,
+                        "Fe-Glu-O",
+                        "evidence"
+                    ],
+                    [
+                        161,
+                        179,
+                        "Fe-His-N distances",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 171,
+                "sent": "Comparison of quaternary structure of EncFtnsH and ferritin.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        38,
+                        46,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        51,
+                        59,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 172,
+                "sent": "(A) Aligned FOC of EncFtnsH and Pseudo-nitzschia multiseries ferritin (PmFtn).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "Aligned",
+                        "experimental_method"
+                    ],
+                    [
+                        12,
+                        15,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        19,
+                        27,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        32,
+                        60,
+                        "Pseudo-nitzschia multiseries",
+                        "species"
+                    ],
+                    [
+                        61,
+                        69,
+                        "ferritin",
+                        "protein"
+                    ],
+                    [
+                        71,
+                        76,
+                        "PmFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 173,
+                "sent": "The metal binding site residues from two EncFtnsH chains are shown in green and blue, while the PmFtn is shown in orange.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        22,
+                        "metal binding site",
+                        "site"
+                    ],
+                    [
+                        41,
+                        49,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        96,
+                        101,
+                        "PmFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 174,
+                "sent": "Fe2+ in the FOC is shown as orange spheres and Ca2+ in EncFtnsH is shown as a grey sphere.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        12,
+                        15,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        47,
+                        51,
+                        "Ca2+",
+                        "chemical"
+                    ],
+                    [
+                        55,
+                        63,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 175,
+                "sent": "The two-fold symmetry axis of the EncFtn FOC is shown with a grey arrow (B) Cross-section surface view of quaternary structure of EncFtnsH and PmFtn as aligned in (A) (dashed black box).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        34,
+                        40,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        41,
+                        44,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        130,
+                        138,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        143,
+                        148,
+                        "PmFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 176,
+                "sent": "The central channel of EncFtnsH is spatially equivalent to the outer surface of ferritin and its outer surface corresponds to the mineralization surface within ferritin.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        19,
+                        "central channel",
+                        "site"
+                    ],
+                    [
+                        23,
+                        31,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        80,
+                        88,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        130,
+                        152,
+                        "mineralization surface",
+                        "site"
+                    ],
+                    [
+                        160,
+                        168,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 177,
+                "sent": "Comparison of the symmetric metal ion binding site of EncFtnsH and the ferritin FOC.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        10,
+                        "Comparison",
+                        "experimental_method"
+                    ],
+                    [
+                        28,
+                        50,
+                        "metal ion binding site",
+                        "site"
+                    ],
+                    [
+                        54,
+                        62,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        71,
+                        79,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        80,
+                        83,
+                        "FOC",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 178,
+                "sent": "(A) Structural alignment of the FOC residues in a dimer of EncFtnsH (green/blue) with a monomer of Pseudo-nitzschia multiseries ferritin (PmFtn) (PDBID: 4ITW) (orange).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        24,
+                        "Structural alignment",
+                        "experimental_method"
+                    ],
+                    [
+                        32,
+                        35,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        50,
+                        55,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        59,
+                        67,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        88,
+                        95,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        99,
+                        127,
+                        "Pseudo-nitzschia multiseries",
+                        "species"
+                    ],
+                    [
+                        128,
+                        136,
+                        "ferritin",
+                        "protein"
+                    ],
+                    [
+                        138,
+                        143,
+                        "PmFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 179,
+                "sent": "Iron ions are shown as orange spheres and a single calcium ion as a grey sphere.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "Iron",
+                        "chemical"
+                    ],
+                    [
+                        51,
+                        58,
+                        "calcium",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 180,
+                "sent": "Residues within the FOC are conserved between EncFtn and ferritin PmFtn, with the exception of residues in the position equivalent to H65\u2019 in the second subunit in the dimer (blue).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        20,
+                        23,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        28,
+                        37,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        46,
+                        52,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        57,
+                        65,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        66,
+                        71,
+                        "PmFtn",
+                        "protein"
+                    ],
+                    [
+                        134,
+                        137,
+                        "H65",
+                        "residue_name_number"
+                    ],
+                    [
+                        153,
+                        160,
+                        "subunit",
+                        "oligomeric_state"
+                    ],
+                    [
+                        168,
+                        173,
+                        "dimer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 181,
+                "sent": "The site in EncFtn with bound calcium is not present in other family members.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        12,
+                        18,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        24,
+                        29,
+                        "bound",
+                        "protein_state"
+                    ],
+                    [
+                        30,
+                        37,
+                        "calcium",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 182,
+                "sent": "(B) Secondary structure of aligned dimeric EncFtnsH and monomeric ferritin highlighting the conserved four-helix bundle.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        27,
+                        34,
+                        "aligned",
+                        "experimental_method"
+                    ],
+                    [
+                        35,
+                        42,
+                        "dimeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        43,
+                        51,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        56,
+                        65,
+                        "monomeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        66,
+                        74,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        92,
+                        101,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        102,
+                        119,
+                        "four-helix bundle",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 183,
+                "sent": "EncFtnsH monomers are shown in green and blue and aligned PmFtn monomer in orange as in A. (C) Cartoon of secondary structure elements in EncFtn dimer and ferritin.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        9,
+                        17,
+                        "monomers",
+                        "oligomeric_state"
+                    ],
+                    [
+                        50,
+                        57,
+                        "aligned",
+                        "experimental_method"
+                    ],
+                    [
+                        58,
+                        63,
+                        "PmFtn",
+                        "protein"
+                    ],
+                    [
+                        64,
+                        71,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        138,
+                        144,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        145,
+                        150,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        155,
+                        163,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 184,
+                "sent": "In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between \u03b12 and \u03b13 in ferritin PmFtn.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        7,
+                        12,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        16,
+                        22,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        38,
+                        41,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        71,
+                        78,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        116,
+                        123,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        165,
+                        176,
+                        "long linker",
+                        "structure_element"
+                    ],
+                    [
+                        185,
+                        187,
+                        "\u03b12",
+                        "structure_element"
+                    ],
+                    [
+                        192,
+                        194,
+                        "\u03b13",
+                        "structure_element"
+                    ],
+                    [
+                        198,
+                        206,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        207,
+                        212,
+                        "PmFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 185,
+                "sent": "Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins\u00a0\u00a0(Figure 6A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        20,
+                        "Structural alignment",
+                        "experimental_method"
+                    ],
+                    [
+                        28,
+                        48,
+                        "di-iron binding site",
+                        "site"
+                    ],
+                    [
+                        52,
+                        60,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        68,
+                        71,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        75,
+                        103,
+                        "Pseudo-nitzschia multiseries",
+                        "species"
+                    ],
+                    [
+                        104,
+                        112,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        114,
+                        119,
+                        "PmFtn",
+                        "protein"
+                    ],
+                    [
+                        177,
+                        196,
+                        "metal binding sites",
+                        "site"
+                    ],
+                    [
+                        200,
+                        208,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        217,
+                        226,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        227,
+                        236,
+                        "ferritins",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 186,
+                "sent": "The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65\u2019) (Figure 6A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        16,
+                        "di-iron site",
+                        "site"
+                    ],
+                    [
+                        20,
+                        28,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        84,
+                        99,
+                        "dimer interface",
+                        "site"
+                    ],
+                    [
+                        111,
+                        114,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        118,
+                        126,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        219,
+                        224,
+                        "His65",
+                        "residue_name_number"
+                    ],
+                    [
+                        239,
+                        245,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        246,
+                        253,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        261,
+                        274,
+                        "FOC interface",
+                        "site"
+                    ],
+                    [
+                        276,
+                        281,
+                        "His65",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 187,
+                "sent": "Structural superimposition of the FOCs of ferritin and EncFtn brings the four-helix bundle of the ferritin fold into close alignment with the EncFtn dimer, showing that the two families of proteins have essentially the same architecture around the di-iron center (Figure 6B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        26,
+                        "Structural superimposition",
+                        "experimental_method"
+                    ],
+                    [
+                        34,
+                        38,
+                        "FOCs",
+                        "site"
+                    ],
+                    [
+                        42,
+                        50,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        55,
+                        61,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        73,
+                        90,
+                        "four-helix bundle",
+                        "structure_element"
+                    ],
+                    [
+                        98,
+                        106,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        142,
+                        148,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        149,
+                        154,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        248,
+                        262,
+                        "di-iron center",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 188,
+                "sent": "The linker connecting helices 2 and 3 of ferritin is congruent with the start of the C-terminal helix of one EncFtn monomer and the N-terminal 310 helix of the second monomer (Figure 6C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        10,
+                        "linker",
+                        "structure_element"
+                    ],
+                    [
+                        22,
+                        37,
+                        "helices 2 and 3",
+                        "structure_element"
+                    ],
+                    [
+                        41,
+                        49,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        96,
+                        101,
+                        "helix",
+                        "structure_element"
+                    ],
+                    [
+                        109,
+                        115,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        116,
+                        123,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        143,
+                        152,
+                        "310 helix",
+                        "structure_element"
+                    ],
+                    [
+                        167,
+                        174,
+                        "monomer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 189,
+                "sent": "Mass spectrometry of the EncFtn assembly",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        17,
+                        "Mass spectrometry",
+                        "experimental_method"
+                    ],
+                    [
+                        25,
+                        31,
+                        "EncFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 190,
+                "sent": "Native IM-MS analysis of the apo-EncFtnsH monomer.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        12,
+                        "Native IM-MS",
+                        "experimental_method"
+                    ],
+                    [
+                        29,
+                        32,
+                        "apo",
+                        "protein_state"
+                    ],
+                    [
+                        33,
+                        41,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        42,
+                        49,
+                        "monomer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 191,
+                "sent": "(A) Mass spectrum of apo-EncFtnsH acquired from 100 mM ammonium acetate pH 8.0 under native MS conditions.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        17,
+                        "Mass spectrum",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        24,
+                        "apo",
+                        "protein_state"
+                    ],
+                    [
+                        25,
+                        33,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        85,
+                        94,
+                        "native MS",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 192,
+                "sent": "The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored\u2010box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, \u03a9 (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2\u00a0using IMPACT v. 0.9.1.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        16,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        56,
+                        61,
+                        "peaks",
+                        "evidence"
+                    ],
+                    [
+                        93,
+                        106,
+                        "charge states",
+                        "evidence"
+                    ],
+                    [
+                        110,
+                        113,
+                        "apo",
+                        "protein_state"
+                    ],
+                    [
+                        114,
+                        121,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        122,
+                        130,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        175,
+                        201,
+                        "arrival time distributions",
+                        "evidence"
+                    ],
+                    [
+                        203,
+                        220,
+                        "ion mobility data",
+                        "evidence"
+                    ],
+                    [
+                        241,
+                        244,
+                        "apo",
+                        "protein_state"
+                    ],
+                    [
+                        245,
+                        253,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        254,
+                        266,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        355,
+                        380,
+                        "arrival time distribution",
+                        "evidence"
+                    ],
+                    [
+                        415,
+                        427,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        485,
+                        511,
+                        "arrival time distributions",
+                        "evidence"
+                    ],
+                    [
+                        567,
+                        590,
+                        "collision cross section",
+                        "evidence"
+                    ],
+                    [
+                        592,
+                        593,
+                        "\u03a9",
+                        "evidence"
+                    ],
+                    [
+                        609,
+                        632,
+                        "collision cross section",
+                        "evidence"
+                    ],
+                    [
+                        645,
+                        652,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        667,
+                        684,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        692,
+                        701,
+                        "Fe-loaded",
+                        "protein_state"
+                    ],
+                    [
+                        702,
+                        710,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        711,
+                        718,
+                        "decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 193,
+                "sent": "The +8 to +15 protein charge states have observed CCS between 20\u201326 nm2, which is significantly higher than the calculated CCS for an EncFtnsH monomer taken from the decameric assembly crystal structure (15.8 nm2).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        22,
+                        35,
+                        "charge states",
+                        "evidence"
+                    ],
+                    [
+                        50,
+                        53,
+                        "CCS",
+                        "evidence"
+                    ],
+                    [
+                        123,
+                        126,
+                        "CCS",
+                        "evidence"
+                    ],
+                    [
+                        134,
+                        142,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        143,
+                        150,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        166,
+                        175,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        185,
+                        202,
+                        "crystal structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 194,
+                "sent": "The mobility of the +7 charge state displays broad drift-time distribution with maxima consistent with CCS of 15.9 and 17.9 nm2.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        12,
+                        "mobility",
+                        "evidence"
+                    ],
+                    [
+                        23,
+                        35,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        51,
+                        74,
+                        "drift-time distribution",
+                        "evidence"
+                    ],
+                    [
+                        103,
+                        106,
+                        "CCS",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 195,
+                "sent": "Finally, the 6+ charge state of EncFtnsH has mobility consistent with a CCS of 12.3 nm2, indicating a more compact/collapsed structure.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        16,
+                        28,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        32,
+                        40,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        45,
+                        53,
+                        "mobility",
+                        "evidence"
+                    ],
+                    [
+                        72,
+                        75,
+                        "CCS",
+                        "evidence"
+                    ],
+                    [
+                        107,
+                        114,
+                        "compact",
+                        "protein_state"
+                    ],
+                    [
+                        115,
+                        124,
+                        "collapsed",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 196,
+                "sent": "It is clear from this data that apo-EncFtnsH exists in several gas phase conformations.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        32,
+                        35,
+                        "apo",
+                        "protein_state"
+                    ],
+                    [
+                        36,
+                        44,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 197,
+                "sent": "The range of charge states occupied by the protein (6+ to 15+) and the range of CCS in which the protein is observed (12.3 nm2 \u2013 26 nm2) are both large.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        13,
+                        26,
+                        "charge states",
+                        "evidence"
+                    ],
+                    [
+                        80,
+                        83,
+                        "CCS",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 198,
+                "sent": "In addition, many of the charge states observed have higher charge than the theoretical maximal charge on spherical globular protein, as determined by the De La Mora relationship (ZR = 0.0778m; for the EncFtnsH monomer ZR = 8.9) Fernandez.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        25,
+                        38,
+                        "charge states",
+                        "evidence"
+                    ],
+                    [
+                        116,
+                        124,
+                        "globular",
+                        "protein_state"
+                    ],
+                    [
+                        155,
+                        178,
+                        "De La Mora relationship",
+                        "experimental_method"
+                    ],
+                    [
+                        180,
+                        182,
+                        "ZR",
+                        "evidence"
+                    ],
+                    [
+                        202,
+                        210,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        211,
+                        218,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        219,
+                        221,
+                        "ZR",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 199,
+                "sent": "As described by Beveridge et al., all these factors are indicative of a disordered protein.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        72,
+                        82,
+                        "disordered",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 200,
+                "sent": "Gas-phase disassembly of the holo-EncFtnsH decameric assembly.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        29,
+                        33,
+                        "holo",
+                        "protein_state"
+                    ],
+                    [
+                        34,
+                        42,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        43,
+                        52,
+                        "decameric",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 201,
+                "sent": "The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the \u2018stripped\u2019 complex (a 9mer; 118.7 kDa; yellow circles).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        11,
+                        23,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        44,
+                        53,
+                        "Fe-loaded",
+                        "protein_state"
+                    ],
+                    [
+                        54,
+                        58,
+                        "holo",
+                        "protein_state"
+                    ],
+                    [
+                        60,
+                        68,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        109,
+                        141,
+                        "collisional-induced dissociation",
+                        "experimental_method"
+                    ],
+                    [
+                        143,
+                        146,
+                        "CID",
+                        "experimental_method"
+                    ],
+                    [
+                        239,
+                        242,
+                        "CID",
+                        "experimental_method"
+                    ],
+                    [
+                        243,
+                        256,
+                        "mass spectrum",
+                        "evidence"
+                    ],
+                    [
+                        295,
+                        299,
+                        "holo",
+                        "protein_state"
+                    ],
+                    [
+                        301,
+                        309,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        310,
+                        317,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        370,
+                        377,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        407,
+                        415,
+                        "stripped",
+                        "protein_state"
+                    ],
+                    [
+                        428,
+                        432,
+                        "9mer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 202,
+                "sent": "The mass of the ejected-monomer is consistent with apo- EncFtnsH (13.2 kDa), suggesting unfolding of the monomer (and loss of Fe) occurs during ejection from the complex.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        24,
+                        31,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        51,
+                        54,
+                        "apo",
+                        "protein_state"
+                    ],
+                    [
+                        56,
+                        64,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        105,
+                        112,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        118,
+                        125,
+                        "loss of",
+                        "protein_state"
+                    ],
+                    [
+                        126,
+                        128,
+                        "Fe",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 203,
+                "sent": "This observation of asymmetric charge partitioning of the sub-complexes with respect to the mass of the complex is consistent with the 'typical' pathway of dissociation of protein assemblies by CID, as described by.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        194,
+                        197,
+                        "CID",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 204,
+                "sent": "In addition, a third, lower abundance, charge state distribution is observed which overlaps the EncFtn ejected monomer charge state distribution; this region of the spectrum is highlighted in (B).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        39,
+                        51,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        96,
+                        102,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        111,
+                        118,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        119,
+                        131,
+                        "charge state",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 205,
+                "sent": "This distribution is consistent with an ejected EncFtnsH dimer (orange circles).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        48,
+                        56,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        57,
+                        62,
+                        "dimer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 206,
+                "sent": "Interestingly, closer analysis of the individual charge state of this dimeric CID product shows that this sub-complex exists in three forms \u2013 displaying mass consistent with an EncFtnsH dimer binding 0, 1, and 2 Fe ions.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        49,
+                        61,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        70,
+                        77,
+                        "dimeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        78,
+                        81,
+                        "CID",
+                        "experimental_method"
+                    ],
+                    [
+                        177,
+                        185,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        186,
+                        191,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        212,
+                        214,
+                        "Fe",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 207,
+                "sent": "This is highlighted in (C), where the 15+ charge state of the EncFtnsH dimer is shown; 3 peaks are observed with m/z 1760.5, 1763.8, and 1767.0 Th \u2013 the lowest peak corresponds to neutral masses of 26392.5 Da [predicted EncFtnsH dimer, (C572H884N172O185S2)2;\u00a026388.6 Da].",
+                "section": "FIG",
+                "ner": [
+                    [
+                        42,
+                        54,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        62,
+                        70,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        71,
+                        76,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        89,
+                        94,
+                        "peaks",
+                        "evidence"
+                    ],
+                    [
+                        220,
+                        228,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        229,
+                        234,
+                        "dimer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 208,
+                "sent": "The two further peaks have a delta-mass of ~+50 Da, consistent with Fe binding.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        16,
+                        21,
+                        "peaks",
+                        "evidence"
+                    ],
+                    [
+                        68,
+                        70,
+                        "Fe",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 209,
+                "sent": "We interpret these observations as partial \u2018atypical\u2019 CID fragmentation of the decameric complex \u2013 i.e. fragmentation of the initial complex with retention of subunit and ligand interactions.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        54,
+                        57,
+                        "CID",
+                        "experimental_method"
+                    ],
+                    [
+                        79,
+                        88,
+                        "decameric",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 210,
+                "sent": "We postulate the high stability of this iron-bound dimer sub-complex is due to the metal coordination at the dimer interface, increasing the strength of the dimer interface.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        40,
+                        50,
+                        "iron-bound",
+                        "protein_state"
+                    ],
+                    [
+                        51,
+                        56,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        83,
+                        88,
+                        "metal",
+                        "chemical"
+                    ],
+                    [
+                        89,
+                        101,
+                        "coordination",
+                        "bond_interaction"
+                    ],
+                    [
+                        109,
+                        124,
+                        "dimer interface",
+                        "site"
+                    ],
+                    [
+                        157,
+                        172,
+                        "dimer interface",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 211,
+                "sent": "Taken together, these observations support our findings that the topology of the decameric EncFtnsH assembly is arranged as a pentamer of dimers, with two Fe ions at each dimer interface.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        81,
+                        90,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        91,
+                        99,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        126,
+                        134,
+                        "pentamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        138,
+                        144,
+                        "dimers",
+                        "oligomeric_state"
+                    ],
+                    [
+                        155,
+                        157,
+                        "Fe",
+                        "chemical"
+                    ],
+                    [
+                        171,
+                        186,
+                        "dimer interface",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 212,
+                "sent": "Native mass spectrometry and ion mobility analysis of iron loading in EncFtnsH.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        24,
+                        "Native mass spectrometry",
+                        "experimental_method"
+                    ],
+                    [
+                        29,
+                        50,
+                        "ion mobility analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        54,
+                        58,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        70,
+                        78,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 213,
+                "sent": "All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 \u00b5M. (A) Native nanoelectrospray ionization (nESI) mass spectrometry of EncFtnsH at varying iron concentrations.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "spectra",
+                        "evidence"
+                    ],
+                    [
+                        45,
+                        52,
+                        "acetate",
+                        "chemical"
+                    ],
+                    [
+                        103,
+                        137,
+                        "Native nanoelectrospray ionization",
+                        "experimental_method"
+                    ],
+                    [
+                        139,
+                        143,
+                        "nESI",
+                        "experimental_method"
+                    ],
+                    [
+                        145,
+                        162,
+                        "mass spectrometry",
+                        "experimental_method"
+                    ],
+                    [
+                        166,
+                        174,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        186,
+                        190,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 214,
+                "sent": "A1, nESI spectrum of iron-free EncFtnsH displays a charge state distribution consistent with EncFtnsH monomer (blue circles, 13,194 Da).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "nESI",
+                        "experimental_method"
+                    ],
+                    [
+                        9,
+                        17,
+                        "spectrum",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        30,
+                        "iron-free",
+                        "protein_state"
+                    ],
+                    [
+                        31,
+                        39,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        51,
+                        63,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        93,
+                        101,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        102,
+                        109,
+                        "monomer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 215,
+                "sent": "Addition of 100 \u00b5M (A2) and 300 \u00b5M (A3) Fe2+ results in the appearance of a second higher molecular weight charge state distribution consistent with a decameric assembly of EncFtnsH (green circles, 132.6 kDa).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        40,
+                        44,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        90,
+                        106,
+                        "molecular weight",
+                        "evidence"
+                    ],
+                    [
+                        107,
+                        119,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        151,
+                        160,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        173,
+                        181,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 216,
+                "sent": "(B) Ion mobility (IM)-MS of the iron-bound holo-EncFtnsH decamer.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        24,
+                        "Ion mobility (IM)-MS",
+                        "experimental_method"
+                    ],
+                    [
+                        32,
+                        42,
+                        "iron-bound",
+                        "protein_state"
+                    ],
+                    [
+                        43,
+                        47,
+                        "holo",
+                        "protein_state"
+                    ],
+                    [
+                        48,
+                        56,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        57,
+                        64,
+                        "decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 217,
+                "sent": "Top, Peaks corresponding to the 22+ to 26+ charge states of a homo-decameric assembly of EncFtnsH are observed (132.6 kDa).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        5,
+                        10,
+                        "Peaks",
+                        "evidence"
+                    ],
+                    [
+                        43,
+                        56,
+                        "charge states",
+                        "evidence"
+                    ],
+                    [
+                        62,
+                        76,
+                        "homo-decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        89,
+                        97,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 218,
+                "sent": "Top Insert, Analysis of the 24+ charge state of the assembly at m/z 5528.2 Th.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        32,
+                        44,
+                        "charge state",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 219,
+                "sent": "The theoretical average m/z of the 24+ charge state with no additional metals bound is marked by a red line (5498.7 Th); the observed m/z of the 24+ charge state indicates that the EncFtnsH assembly binds between 10 (green line, 5521.1 Th) and 15 Fe ions (blue line, 5532.4 Th) per decamer.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        39,
+                        51,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        149,
+                        161,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        181,
+                        189,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        247,
+                        249,
+                        "Fe",
+                        "chemical"
+                    ],
+                    [
+                        282,
+                        289,
+                        "decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 220,
+                "sent": "Bottom, The arrival time distributions (ion mobility data) of all ions in the EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        12,
+                        38,
+                        "arrival time distributions",
+                        "evidence"
+                    ],
+                    [
+                        40,
+                        57,
+                        "ion mobility data",
+                        "evidence"
+                    ],
+                    [
+                        78,
+                        86,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        87,
+                        99,
+                        "charge state",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 221,
+                "sent": "Bottom right, The arrival time distribution of the 24+ charge state (dashed blue box) has been extracted and plotted.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        18,
+                        43,
+                        "arrival time distribution",
+                        "evidence"
+                    ],
+                    [
+                        55,
+                        67,
+                        "charge state",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 222,
+                "sent": "The drift time for this ion is shown (ms), along with the calibrated collision cross section (CCS), \u03a9 (nm2).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        14,
+                        "drift time",
+                        "evidence"
+                    ],
+                    [
+                        69,
+                        92,
+                        "collision cross section",
+                        "evidence"
+                    ],
+                    [
+                        94,
+                        97,
+                        "CCS",
+                        "evidence"
+                    ],
+                    [
+                        100,
+                        101,
+                        "\u03a9",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 223,
+                "sent": "In order to confirm the assignment of the oligomeric state of EncFtnsH and investigate further the Fe2+-dependent assembly, we used native nano-electrospray ionization (nESI) and ion-mobility mass spectrometry (IM-MS).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        62,
+                        70,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        99,
+                        103,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        132,
+                        167,
+                        "native nano-electrospray ionization",
+                        "experimental_method"
+                    ],
+                    [
+                        169,
+                        173,
+                        "nESI",
+                        "experimental_method"
+                    ],
+                    [
+                        179,
+                        209,
+                        "ion-mobility mass spectrometry",
+                        "experimental_method"
+                    ],
+                    [
+                        211,
+                        216,
+                        "IM-MS",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 224,
+                "sent": "As described above, by recombinant production of EncFtnsH in minimal media we were able to limit the bioavailability of iron.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        23,
+                        45,
+                        "recombinant production",
+                        "experimental_method"
+                    ],
+                    [
+                        49,
+                        57,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        120,
+                        124,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 225,
+                "sent": "Native MS analysis of EncFtnsH produced in this way displayed a charge state distribution consistent with an EncFtnsH monomer (blue circles, Figure 7A1) with an average neutral mass of 13,194 Da, in agreement with the predicted mass of the EncFtnsH protein (13,194.53 Da).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Native MS",
+                        "experimental_method"
+                    ],
+                    [
+                        22,
+                        30,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        64,
+                        76,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        109,
+                        117,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        118,
+                        125,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        240,
+                        248,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 226,
+                "sent": "Titration with Fe2+ directly before native MS analysis resulted in the appearance of a new charge state distribution, consistent with an EncFtnsH decameric assembly (+22 to +26; 132.65 kDa) (Figure 7A2/3).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Titration",
+                        "experimental_method"
+                    ],
+                    [
+                        15,
+                        19,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        36,
+                        45,
+                        "native MS",
+                        "experimental_method"
+                    ],
+                    [
+                        91,
+                        103,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        137,
+                        145,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        146,
+                        155,
+                        "decameric",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 227,
+                "sent": "After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (\u0394mass observed ~0.67 kDa).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        90,
+                        94,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        139,
+                        141,
+                        "Fe",
+                        "chemical"
+                    ],
+                    [
+                        151,
+                        158,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        209,
+                        220,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        224,
+                        229,
+                        "irons",
+                        "chemical"
+                    ],
+                    [
+                        237,
+                        240,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        249,
+                        261,
+                        "coordination",
+                        "bond_interaction"
+                    ],
+                    [
+                        265,
+                        269,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        277,
+                        290,
+                        "Glu31/34-site",
+                        "site"
+                    ],
+                    [
+                        303,
+                        310,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        318,
+                        335,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        337,
+                        342,
+                        "\u0394mass",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 228,
+                "sent": "MS analysis of EncFtnsH after addition of further Fe2+ did not result in iron loading above this stoichiometry.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        2,
+                        "MS",
+                        "experimental_method"
+                    ],
+                    [
+                        15,
+                        23,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        50,
+                        54,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        73,
+                        77,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 229,
+                "sent": "Therefore, the extent of iron binding seen is limited to the FOC and Glu31/34 secondary metal binding site.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        29,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        61,
+                        64,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        69,
+                        106,
+                        "Glu31/34 secondary metal binding site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 230,
+                "sent": "These data suggest that the decameric assembly of EncFtnsH does not accrue iron in the same manner as classical ferritin, which is able to sequester around 4500 iron ions within its nanocage.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        28,
+                        37,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        50,
+                        58,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        75,
+                        79,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        102,
+                        111,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        112,
+                        120,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        161,
+                        165,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        182,
+                        190,
+                        "nanocage",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 231,
+                "sent": "Ion mobility analysis of the EncFtnsH decameric assembly, collected with minimal collisional activation, suggested that it consists of a single conformation with a collision cross section (CCS) of 58.2 nm2\u00a0(Figure 7B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        21,
+                        "Ion mobility analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        29,
+                        37,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        38,
+                        47,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        164,
+                        187,
+                        "collision cross section",
+                        "evidence"
+                    ],
+                    [
+                        189,
+                        192,
+                        "CCS",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 232,
+                "sent": "This observation is in agreement with the calculated CCS of 58.7 nm2derived from our crystal structure of the EncFtnsH decamer.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        53,
+                        56,
+                        "CCS",
+                        "evidence"
+                    ],
+                    [
+                        85,
+                        102,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        110,
+                        118,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        119,
+                        126,
+                        "decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 233,
+                "sent": "By contrast, IM-MS measurements of the monomeric EncFtnsH at pH 8.0 under the same instrumental conditions revealed that the metal-free protein monomer exists in a wide range of charge states (+6 to +16) and adopts many conformations in the gas phase with collision cross sections ranging from 12 nm2\u00a0to 26 nm2 (Figure 7\u2014figure supplement 1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        18,
+                        "IM-MS",
+                        "experimental_method"
+                    ],
+                    [
+                        39,
+                        48,
+                        "monomeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        49,
+                        57,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        61,
+                        67,
+                        "pH 8.0",
+                        "protein_state"
+                    ],
+                    [
+                        125,
+                        135,
+                        "metal-free",
+                        "protein_state"
+                    ],
+                    [
+                        136,
+                        143,
+                        "protein",
+                        "protein"
+                    ],
+                    [
+                        144,
+                        151,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        178,
+                        191,
+                        "charge states",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 234,
+                "sent": "Thus, IM-MS studies highlight that higher order structure in EncFtnsH is mediated/stabilized by metal binding, an observation that is in agreement with our solution studies.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        6,
+                        11,
+                        "IM-MS",
+                        "experimental_method"
+                    ],
+                    [
+                        61,
+                        69,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 235,
+                "sent": "Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        46,
+                        50,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        72,
+                        75,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        79,
+                        87,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        118,
+                        132,
+                        "4-helix bundle",
+                        "structure_element"
+                    ],
+                    [
+                        133,
+                        148,
+                        "dimer interface",
+                        "site"
+                    ],
+                    [
+                        181,
+                        190,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        191,
+                        199,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        234,
+                        240,
+                        "dimers",
+                        "oligomeric_state"
+                    ],
+                    [
+                        309,
+                        318,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        352,
+                        369,
+                        "crystal structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 236,
+                "sent": "We subsequently performed gas phase disassembly of the decameric EncFtnsH using collision-induced dissociation (CID) tandem mass spectrometry.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        55,
+                        64,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        65,
+                        73,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        80,
+                        110,
+                        "collision-induced dissociation",
+                        "experimental_method"
+                    ],
+                    [
+                        112,
+                        115,
+                        "CID",
+                        "experimental_method"
+                    ],
+                    [
+                        117,
+                        141,
+                        "tandem mass spectrometry",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 237,
+                "sent": "Under the correct CID conditions, protein assemblies can dissociate with retention of subunit and ligand interactions, and thus provide structurally-informative evidence as to the topology of the original assembly; this has been termed \u2018atypical\u2019 dissociation.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        21,
+                        "CID",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 238,
+                "sent": "For EncFtnsH, this atypical dissociation pathway was clearly evident; CID of the EncFtnsH decamer resulted in the appearance of a dimeric EncFtnsH subcomplex containing 0, 1, or 2 iron ions (Figure 7\u2014figure supplement 2).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        12,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        70,
+                        73,
+                        "CID",
+                        "experimental_method"
+                    ],
+                    [
+                        81,
+                        89,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        90,
+                        97,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        130,
+                        137,
+                        "dimeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        138,
+                        146,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        180,
+                        184,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 239,
+                "sent": "In light of the crystal structure, this observation can be rationalized as dissociation of the EncFtnsH decamer by disruption of the non-FOC interface with at least partial retention of the FOC interface and the FOC-Fe.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        16,
+                        33,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        95,
+                        103,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        104,
+                        111,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        133,
+                        150,
+                        "non-FOC interface",
+                        "site"
+                    ],
+                    [
+                        190,
+                        203,
+                        "FOC interface",
+                        "site"
+                    ],
+                    [
+                        212,
+                        215,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        216,
+                        218,
+                        "Fe",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 240,
+                "sent": "Thus, this observation supports our crystallographic assignment of the overall topology of the EncFtnsH assembly as a pentameric assembly of dimers with two iron ions located at the FOC dimer interface.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        95,
+                        103,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        118,
+                        128,
+                        "pentameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        141,
+                        147,
+                        "dimers",
+                        "oligomeric_state"
+                    ],
+                    [
+                        157,
+                        161,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        182,
+                        201,
+                        "FOC dimer interface",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 241,
+                "sent": "In addition, this analysis provides evidence that the overall architecture of the complex is consistent in the crystal, solution and gas phases.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        111,
+                        118,
+                        "crystal",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 242,
+                "sent": "Ferroxidase activity",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Ferroxidase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 243,
+                "sent": "TEM visualization of iron-loaded bacterial nanocompartments and ferritin.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "TEM",
+                        "experimental_method"
+                    ],
+                    [
+                        21,
+                        32,
+                        "iron-loaded",
+                        "protein_state"
+                    ],
+                    [
+                        33,
+                        42,
+                        "bacterial",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        43,
+                        59,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ],
+                    [
+                        64,
+                        72,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 244,
+                "sent": "Decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin, at 8.5 \u00b5M, were mixed with 147 \u00b5M, 1 mM, 1 mM and 215 \u00b5M acidic Fe(NH4)2(SO4)2, respectively.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        10,
+                        18,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        20,
+                        30,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        32,
+                        42,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        47,
+                        58,
+                        "apoferritin",
+                        "protein_state"
+                    ],
+                    [
+                        124,
+                        138,
+                        "Fe(NH4)2(SO4)2",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 245,
+                "sent": "Protein mixtures were incubated at room temperature for 1 hr prior to TEM analysis with or without uranyl acetate stain.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        70,
+                        73,
+                        "TEM",
+                        "experimental_method"
+                    ],
+                    [
+                        99,
+                        113,
+                        "uranyl acetate",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 246,
+                "sent": "(A\u2013D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F\u2013I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        16,
+                        24,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        26,
+                        36,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        38,
+                        48,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        50,
+                        61,
+                        "apoferritin",
+                        "protein_state"
+                    ],
+                    [
+                        62,
+                        73,
+                        "loaded with",
+                        "protein_state"
+                    ],
+                    [
+                        74,
+                        78,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        198,
+                        205,
+                        "Stained",
+                        "experimental_method"
+                    ],
+                    [
+                        206,
+                        214,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        216,
+                        226,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        228,
+                        238,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        240,
+                        251,
+                        "apoferritin",
+                        "protein_state"
+                    ],
+                    [
+                        252,
+                        263,
+                        "loaded with",
+                        "protein_state"
+                    ],
+                    [
+                        264,
+                        268,
+                        "Fe2+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 247,
+                "sent": "Spectroscopic evidence for the ferroxidase activity and comparison of iron loading capacity of apoferritin, EncFtnsH, encapsulin, and EncFtn-Enc.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        31,
+                        42,
+                        "ferroxidase",
+                        "protein_type"
+                    ],
+                    [
+                        70,
+                        74,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        95,
+                        106,
+                        "apoferritin",
+                        "protein_state"
+                    ],
+                    [
+                        108,
+                        116,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        118,
+                        128,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        134,
+                        144,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 248,
+                "sent": "(A) Apoferritin (10 \u03bcM monomer concentration) and EncFtnsH decamer fractions (20 \u03bcM monomer concentration, 10 \u03bcM FOC concentration) were incubated with 20 and 100 \u03bcM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        15,
+                        "Apoferritin",
+                        "protein_state"
+                    ],
+                    [
+                        23,
+                        30,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        50,
+                        58,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        59,
+                        66,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        84,
+                        91,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        113,
+                        116,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        166,
+                        170,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        204,
+                        208,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        213,
+                        216,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        222,
+                        237,
+                        "progress curves",
+                        "evidence"
+                    ],
+                    [
+                        258,
+                        262,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        266,
+                        270,
+                        "Fe3+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 249,
+                "sent": "The background oxidation of iron at 20 and 100 \u03bcM in enzyme-free controls are shown for reference. (B) Encapsulin and EncFtn-Enc complexes at 10 \u03bcM asymmetric unit concentration were incubated with Fe2+ at 20 and 100 \u03bcM and progress curves for iron oxidation at A315 were measured in a UV/visible spectrophotometer.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        28,
+                        32,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        103,
+                        113,
+                        "Encapsulin",
+                        "protein"
+                    ],
+                    [
+                        118,
+                        128,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        183,
+                        192,
+                        "incubated",
+                        "experimental_method"
+                    ],
+                    [
+                        198,
+                        202,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        224,
+                        239,
+                        "progress curves",
+                        "evidence"
+                    ],
+                    [
+                        244,
+                        248,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        286,
+                        314,
+                        "UV/visible spectrophotometer",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 250,
+                "sent": "Enzyme free controls for background oxidation of Fe2+ are shown for reference. (C) Histogram of the iron loading capacity per biological assembly of EncFtnsH, encapsulin, EncFtn-Enc and apoferritin.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        49,
+                        53,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        100,
+                        104,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        149,
+                        157,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        159,
+                        169,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        171,
+                        181,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        186,
+                        197,
+                        "apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 251,
+                "sent": "The results shown are for three technical replicates and represent the optimal iron loading by the complexes after three hours when incubated with Fe2+.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        79,
+                        83,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        147,
+                        151,
+                        "Fe2+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 252,
+                "sent": "In light of the identification of an iron-loaded FOC in the crystal structure of EncFtn and our native mass spectrometry data, we performed ferroxidase and peroxidase assays to demonstrate the catalytic activity of this protein.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        37,
+                        48,
+                        "iron-loaded",
+                        "protein_state"
+                    ],
+                    [
+                        49,
+                        52,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        60,
+                        77,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        81,
+                        87,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        96,
+                        120,
+                        "native mass spectrometry",
+                        "experimental_method"
+                    ],
+                    [
+                        140,
+                        173,
+                        "ferroxidase and peroxidase assays",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 253,
+                "sent": "In addition, we also assayed equine apoferritin, an example of a classical ferritin enzyme, as a positive control.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        35,
+                        "equine",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        36,
+                        47,
+                        "apoferritin",
+                        "protein_state"
+                    ],
+                    [
+                        65,
+                        74,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        75,
+                        83,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 254,
+                "sent": "Unlike the Dps family of ferritin-like proteins, EncFtn showed no peroxidase activity when assayed with the substrate ortho-phenylenediamine.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        21,
+                        "Dps family",
+                        "protein_type"
+                    ],
+                    [
+                        25,
+                        47,
+                        "ferritin-like proteins",
+                        "protein_type"
+                    ],
+                    [
+                        49,
+                        55,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        118,
+                        140,
+                        "ortho-phenylenediamine",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 255,
+                "sent": "The ferroxidase activity of EncFtnsH was measured by recording the progress curve of Fe2+ oxidation to Fe3+ at 315 nm after addition of 20 and 100 \u00b5M Fe2+ (2 and 10 times molar ratio Fe2+/FOC).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        15,
+                        "ferroxidase",
+                        "protein_type"
+                    ],
+                    [
+                        28,
+                        36,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        67,
+                        81,
+                        "progress curve",
+                        "evidence"
+                    ],
+                    [
+                        85,
+                        89,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        103,
+                        107,
+                        "Fe3+",
+                        "chemical"
+                    ],
+                    [
+                        150,
+                        154,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        183,
+                        187,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        188,
+                        191,
+                        "FOC",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 256,
+                "sent": "In both experiments the rate of oxidation was faster than background oxidation of Fe2+ by molecular oxygen, and was highest for 100 \u00b5M Fe2+ (Figure 8A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        82,
+                        86,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        100,
+                        106,
+                        "oxygen",
+                        "chemical"
+                    ],
+                    [
+                        135,
+                        139,
+                        "Fe2+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 257,
+                "sent": "These data show that recombinant EncFtnsH acts as an active ferroxidase enzyme.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        33,
+                        41,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        53,
+                        59,
+                        "active",
+                        "protein_state"
+                    ],
+                    [
+                        60,
+                        71,
+                        "ferroxidase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 258,
+                "sent": "When compared to apoferritin, EncFtnsH oxidized Fe2+ at a slower rate and the reaction did not run to completion over the 1800 s of the experiment.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        17,
+                        28,
+                        "apoferritin",
+                        "protein_state"
+                    ],
+                    [
+                        30,
+                        38,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        48,
+                        52,
+                        "Fe2+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 259,
+                "sent": "Addition of higher quantities of iron resulted in the formation of a yellow/red precipitate at the end of the reaction.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        33,
+                        37,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 260,
+                "sent": "We also performed these assays on purified recombinant encapsulin; which, when assayed alone, did not display ferroxidase activity above background Fe2+ oxidation (Figure 8B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        55,
+                        65,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        110,
+                        121,
+                        "ferroxidase",
+                        "protein_type"
+                    ],
+                    [
+                        148,
+                        152,
+                        "Fe2+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 261,
+                "sent": "In contrast, complexes of the full EncFtn encapsulin nanocompartment (i.e. the EncFtn-Enc protein complex) displayed ferroxidase activity comparable to apoferritin without the formation of precipitates (Figure 8B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        30,
+                        34,
+                        "full",
+                        "protein_state"
+                    ],
+                    [
+                        35,
+                        41,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        42,
+                        52,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        53,
+                        68,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ],
+                    [
+                        79,
+                        89,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        117,
+                        128,
+                        "ferroxidase",
+                        "protein_type"
+                    ],
+                    [
+                        152,
+                        163,
+                        "apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 262,
+                "sent": "We attributed the precipitates observed in the EncFtnsH ferroxidase assay to the production of insoluble Fe3+ complexes, which led us to propose that EncFtn does not directly store Fe3+ in a mineral form.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        47,
+                        55,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        56,
+                        73,
+                        "ferroxidase assay",
+                        "experimental_method"
+                    ],
+                    [
+                        105,
+                        109,
+                        "Fe3+",
+                        "chemical"
+                    ],
+                    [
+                        150,
+                        156,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        181,
+                        185,
+                        "Fe3+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 263,
+                "sent": "This observation agrees with native MS results, which indicates a maximum iron loading of 10\u201315 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        38,
+                        "native MS",
+                        "experimental_method"
+                    ],
+                    [
+                        74,
+                        78,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        96,
+                        100,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        110,
+                        119,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        120,
+                        126,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        136,
+                        145,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        183,
+                        202,
+                        "iron-storage cavity",
+                        "site"
+                    ],
+                    [
+                        221,
+                        230,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        231,
+                        240,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        245,
+                        264,
+                        "Dps family proteins",
+                        "protein_type"
+                    ],
+                    [
+                        302,
+                        306,
+                        "Fe3+",
+                        "chemical"
+                    ],
+                    [
+                        320,
+                        335,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ],
+                    [
+                        336,
+                        346,
+                        "structures",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 264,
+                "sent": "To analyze the products of these reactions and determine whether the EncFtn and encapsulin were able to store iron in a mineral form, we performed TEM on the reaction mixtures from the ferroxidase assay.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        69,
+                        75,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        80,
+                        90,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        110,
+                        114,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        147,
+                        150,
+                        "TEM",
+                        "experimental_method"
+                    ],
+                    [
+                        185,
+                        202,
+                        "ferroxidase assay",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 265,
+                "sent": "The EncFtnsH reaction mixture showed the formation of large, irregular electron-dense precipitates (Figure 8\u2014figure supplement 1A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        12,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 266,
+                "sent": "A similar distribution of particles was observed after addition of Fe2+ to the encapsulin protein (Figure 8\u2014figure supplement 1B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        67,
+                        71,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        79,
+                        89,
+                        "encapsulin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 267,
+                "sent": "In contrast, addition of Fe2+ to the EncFtn-Enc nanocompartment resulted in small, highly regular, electron dense particles of approximately 5 nm in diameter (Figure 8\u2014figure supplement 1C); we interpret these observations as controlled mineralization of iron within the nanocompartment.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        29,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        37,
+                        47,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        48,
+                        63,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ],
+                    [
+                        255,
+                        259,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        271,
+                        286,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 268,
+                "sent": "Addition of Fe2+ to apoferritin resulted in a mixture of large particles and small (~2 nm) particles consistent with partial mineralization by the ferritin and some background oxidation of the iron (Figure 8\u2014figure supplement 1D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        12,
+                        16,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        20,
+                        31,
+                        "apoferritin",
+                        "protein_state"
+                    ],
+                    [
+                        147,
+                        155,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        193,
+                        197,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 269,
+                "sent": "Negative stain TEM of these samples revealed that upon addition of iron, the EncFtnsH\u00a0protein showed significant aggregation (Figure 8\u2014figure supplement 1F); while the encapsulin, EncFtn-Enc system, and apoferritin are present as distinct nanocompartments without significant protein aggregation (Figure\u00a08\u2014figure supplement 1G\u2013I).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        18,
+                        "Negative stain TEM",
+                        "experimental_method"
+                    ],
+                    [
+                        67,
+                        71,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        77,
+                        85,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        168,
+                        178,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        180,
+                        190,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        203,
+                        214,
+                        "apoferritin",
+                        "protein_state"
+                    ],
+                    [
+                        239,
+                        255,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 270,
+                "sent": "Iron storage in encapsulin nanocompartments",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "Iron",
+                        "chemical"
+                    ],
+                    [
+                        16,
+                        26,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        27,
+                        43,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 271,
+                "sent": "The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        36,
+                        "ferroxidase assay",
+                        "experimental_method"
+                    ],
+                    [
+                        41,
+                        52,
+                        "micrographs",
+                        "evidence"
+                    ],
+                    [
+                        140,
+                        149,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        150,
+                        159,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        182,
+                        188,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        193,
+                        203,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        223,
+                        229,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        242,
+                        253,
+                        "ferroxidase",
+                        "protein_type"
+                    ],
+                    [
+                        262,
+                        272,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        273,
+                        278,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        321,
+                        325,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 272,
+                "sent": "To investigate this further, we added Fe2+ at various concentrations to samples of apo-ferritin, EncFtn, isolated encapsulin, and the EncFtn-Enc protein complex, and subjected these samples to a ferrozine assay to quantify the amount of iron associated with the proteins after three hours of incubation.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        38,
+                        42,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        83,
+                        86,
+                        "apo",
+                        "protein_state"
+                    ],
+                    [
+                        87,
+                        95,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        97,
+                        103,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        114,
+                        124,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        134,
+                        144,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        195,
+                        210,
+                        "ferrozine assay",
+                        "experimental_method"
+                    ],
+                    [
+                        237,
+                        241,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 273,
+                "sent": "The maximum iron loading capacity of these systems was calculated as the quantity of iron per biological assembly (Figure 8C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        12,
+                        16,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        85,
+                        89,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 274,
+                "sent": "In this assay, the EncFtnsH\u00a0decamer binds a maximum of around 48 iron ions before excess iron induces protein precipitation.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        27,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        28,
+                        35,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        65,
+                        69,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        89,
+                        93,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 275,
+                "sent": "The encapsulin shell protein can sequester about 2200 iron ions before significant protein loss occurs, and the reconstituted EncFtn-Enc nanocompartment sequestered about 4150 iron ions.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        14,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        15,
+                        20,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        54,
+                        58,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        126,
+                        136,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        137,
+                        152,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ],
+                    [
+                        176,
+                        180,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 276,
+                "sent": "This latter result is significantly more than the apoferritin used in our assay, which sequesters approximately 570 iron ions in this assay (Figure 8C, Table 5).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        50,
+                        61,
+                        "apoferritin",
+                        "protein_state"
+                    ],
+                    [
+                        116,
+                        120,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 277,
+                "sent": "Consideration of the functional oligomeric states of these proteins, where EncFtn is a decamer and encapsulin forms an icosahedral cage, and estimation of the iron loading capacity of these complexes gives insight into the role of the two proteins in iron storage and mineralization.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        75,
+                        81,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        87,
+                        94,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        99,
+                        109,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        119,
+                        130,
+                        "icosahedral",
+                        "protein_state"
+                    ],
+                    [
+                        131,
+                        135,
+                        "cage",
+                        "complex_assembly"
+                    ],
+                    [
+                        159,
+                        163,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        251,
+                        255,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 278,
+                "sent": "EncFtn decamers bind up to 48 iron ions (Figure 8C), which is significantly higher than the stoichiometry of fifteen metal ions visible in the FOC and E31/34-site of the crystal structure of the EncFtnsH decamer and our MS analysis.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        6,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        7,
+                        15,
+                        "decamers",
+                        "oligomeric_state"
+                    ],
+                    [
+                        30,
+                        34,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        143,
+                        146,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        151,
+                        162,
+                        "E31/34-site",
+                        "site"
+                    ],
+                    [
+                        170,
+                        187,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        195,
+                        203,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        204,
+                        211,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        220,
+                        222,
+                        "MS",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 279,
+                "sent": "The discrepancy between these solution measurements and our MS analysis may indicate that there are additional metal-binding sites on the interior channel and exterior faces of the protein; this is consistent with our identification of a number of weak metal-binding sites at the surface of the protein in the crystal structure (Figure 5D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        30,
+                        51,
+                        "solution measurements",
+                        "experimental_method"
+                    ],
+                    [
+                        60,
+                        62,
+                        "MS",
+                        "experimental_method"
+                    ],
+                    [
+                        111,
+                        130,
+                        "metal-binding sites",
+                        "site"
+                    ],
+                    [
+                        147,
+                        154,
+                        "channel",
+                        "site"
+                    ],
+                    [
+                        253,
+                        272,
+                        "metal-binding sites",
+                        "site"
+                    ],
+                    [
+                        310,
+                        327,
+                        "crystal structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 280,
+                "sent": "These observations are consistent with hydrated Fe2+ ions being channeled to the active site from the E31/34-site and the subsequent exit of Fe3+ products on the outer surface, as is seen in other ferritin family proteins.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        48,
+                        52,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        81,
+                        92,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        102,
+                        113,
+                        "E31/34-site",
+                        "site"
+                    ],
+                    [
+                        141,
+                        145,
+                        "Fe3+",
+                        "chemical"
+                    ],
+                    [
+                        197,
+                        205,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 281,
+                "sent": "While the isolated encapsulin shell does not display any ferroxidase activity, it binds around 2200 iron ions in our assay (Table 5).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        29,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        30,
+                        35,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        57,
+                        68,
+                        "ferroxidase",
+                        "protein_type"
+                    ],
+                    [
+                        100,
+                        104,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 282,
+                "sent": "This implies that the shell can bind a significant amount of iron on its outer and inner surfaces.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        22,
+                        27,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        61,
+                        65,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 283,
+                "sent": "While the maximum reported loading capacity of classical ferritins is approximately 4500 iron ions, in our assay system we were only able to load apoferritin with around 570 iron ions.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        47,
+                        56,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        57,
+                        66,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        89,
+                        93,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        146,
+                        157,
+                        "apoferritin",
+                        "protein_state"
+                    ],
+                    [
+                        174,
+                        178,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 284,
+                "sent": "However, the recombinant EncFtn-Enc nanocompartment was able to bind over 4100 iron ions in the same time period, over seven times the amount seen for the apoferritin.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        35,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        36,
+                        51,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ],
+                    [
+                        79,
+                        83,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        155,
+                        166,
+                        "apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 285,
+                "sent": "We note we do not reach the experimental maximum iron\u00a0loading\u00a0for apoferritin and therefore the total iron-loading capacity of our system may be significantly higher than in this experimental system.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        49,
+                        53,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        66,
+                        77,
+                        "apoferritin",
+                        "protein_state"
+                    ],
+                    [
+                        102,
+                        106,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 286,
+                "sent": "Taken together, our data show that EncFtn can catalytically oxidize Fe2+ to Fe3+; however, iron binding in EncFtn is limited to the FOC and several surface metal binding sites.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        35,
+                        41,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        68,
+                        72,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        76,
+                        80,
+                        "Fe3+",
+                        "chemical"
+                    ],
+                    [
+                        91,
+                        95,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        107,
+                        113,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        132,
+                        135,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        156,
+                        175,
+                        "metal binding sites",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 287,
+                "sent": "In contrast, the encapsulin protein displays no catalytic activity, but has the ability to bind a considerable amount of iron.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        17,
+                        27,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        121,
+                        125,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 288,
+                "sent": "Finally, the EncFtn-Enc nanocompartment complex retains the catalytic activity of EncFtn, and sequesters iron within the encapsulin shell at a higher level than the isolated components of the system, and at a significantly higher level than the classical ferritins.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        23,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        24,
+                        39,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ],
+                    [
+                        82,
+                        88,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        105,
+                        109,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        121,
+                        131,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        132,
+                        137,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        245,
+                        254,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        255,
+                        264,
+                        "ferritins",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 289,
+                "sent": "\u00a0Furthermore, our recombinant nanocompartments may not have the physiological subunit stoichiometry, and the iron-loading capacity of native nanocompartments is potentially much higher than the level we have observed.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        30,
+                        46,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ],
+                    [
+                        109,
+                        113,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        134,
+                        140,
+                        "native",
+                        "protein_state"
+                    ],
+                    [
+                        141,
+                        157,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 290,
+                "sent": "Mutagenesis of the EncFtnsHferroxidase center",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Mutagenesis",
+                        "experimental_method"
+                    ],
+                    [
+                        19,
+                        27,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        27,
+                        45,
+                        "ferroxidase center",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 291,
+                "sent": "Purification of recombinant R. rubrum EncFtnsH FOC mutants.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        28,
+                        37,
+                        "R. rubrum",
+                        "species"
+                    ],
+                    [
+                        38,
+                        46,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        47,
+                        50,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        51,
+                        58,
+                        "mutants",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 292,
+                "sent": "Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex\u00a0200 size-exclusion\u00a0chromatography.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        7,
+                        14,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        15,
+                        19,
+                        "E32A",
+                        "mutant"
+                    ],
+                    [
+                        21,
+                        25,
+                        "E62A",
+                        "mutant"
+                    ],
+                    [
+                        31,
+                        35,
+                        "H65A",
+                        "mutant"
+                    ],
+                    [
+                        39,
+                        47,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        62,
+                        79,
+                        "E. coli BL21(DE3)",
+                        "species"
+                    ],
+                    [
+                        95,
+                        97,
+                        "MM",
+                        "experimental_method"
+                    ],
+                    [
+                        102,
+                        104,
+                        "MM",
+                        "experimental_method"
+                    ],
+                    [
+                        123,
+                        127,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        159,
+                        188,
+                        "size-exclusion\u00a0chromatography",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 293,
+                "sent": "(A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH\u00a0resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy\u00a0of the E62A mutant form of EncFtnsH\u00a0resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography\u00a0of the H65A mutant form of EncFtnsH\u00a0resulted in a single peak corresponding to the protein monomer.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        31,
+                        "Gel-filtration chromatogram",
+                        "evidence"
+                    ],
+                    [
+                        39,
+                        43,
+                        "E32A",
+                        "mutant"
+                    ],
+                    [
+                        44,
+                        50,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        59,
+                        67,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        83,
+                        98,
+                        "elution profile",
+                        "evidence"
+                    ],
+                    [
+                        145,
+                        154,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        201,
+                        208,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        214,
+                        242,
+                        "Gel-filtration chromatograhy",
+                        "experimental_method"
+                    ],
+                    [
+                        250,
+                        254,
+                        "E62A",
+                        "mutant"
+                    ],
+                    [
+                        255,
+                        261,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        270,
+                        278,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        294,
+                        309,
+                        "elution profile",
+                        "evidence"
+                    ],
+                    [
+                        330,
+                        339,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        350,
+                        379,
+                        "Gel-filtration chromatography",
+                        "experimental_method"
+                    ],
+                    [
+                        387,
+                        391,
+                        "H65A",
+                        "mutant"
+                    ],
+                    [
+                        392,
+                        398,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        407,
+                        415,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        471,
+                        478,
+                        "monomer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 294,
+                "sent": "To investigate the structural and biochemical role played by the metal binding residues in the di-iron FOC of EncFtnsH we produced alanine mutations in each of these residues: Glu32, Glu62, and His65.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        65,
+                        87,
+                        "metal binding residues",
+                        "site"
+                    ],
+                    [
+                        95,
+                        106,
+                        "di-iron FOC",
+                        "site"
+                    ],
+                    [
+                        110,
+                        118,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        131,
+                        148,
+                        "alanine mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        176,
+                        181,
+                        "Glu32",
+                        "residue_name_number"
+                    ],
+                    [
+                        183,
+                        188,
+                        "Glu62",
+                        "residue_name_number"
+                    ],
+                    [
+                        194,
+                        199,
+                        "His65",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 295,
+                "sent": "These EncFtnsH mutants were produced in E. coli cells grown in MM, both in the absence and presence of additional iron.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        6,
+                        14,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        15,
+                        22,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        40,
+                        47,
+                        "E. coli",
+                        "species"
+                    ],
+                    [
+                        63,
+                        65,
+                        "MM",
+                        "experimental_method"
+                    ],
+                    [
+                        79,
+                        86,
+                        "absence",
+                        "protein_state"
+                    ],
+                    [
+                        91,
+                        102,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        114,
+                        118,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 296,
+                "sent": "The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "E32A",
+                        "mutant"
+                    ],
+                    [
+                        13,
+                        17,
+                        "E62A",
+                        "mutant"
+                    ],
+                    [
+                        18,
+                        25,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        38,
+                        41,
+                        "SEC",
+                        "experimental_method"
+                    ],
+                    [
+                        74,
+                        83,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        92,
+                        100,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        129,
+                        136,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        142,
+                        146,
+                        "H65A",
+                        "mutant"
+                    ],
+                    [
+                        147,
+                        153,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        193,
+                        202,
+                        "monomeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        211,
+                        219,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 297,
+                "sent": "For all of the mutants studied, no change in oligomerization state was apparent upon addition of Fe2+\u00a0in vitro.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        15,
+                        22,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        97,
+                        101,
+                        "Fe2+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 298,
+                "sent": "Native mass spectrometry of EncFtnsH mutants.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        24,
+                        "Native mass spectrometry",
+                        "experimental_method"
+                    ],
+                    [
+                        28,
+                        36,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        37,
+                        44,
+                        "mutants",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 299,
+                "sent": "All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 \u00b5M. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant\u00a0is also observed (blue circles).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "spectra",
+                        "evidence"
+                    ],
+                    [
+                        45,
+                        52,
+                        "acetate",
+                        "chemical"
+                    ],
+                    [
+                        103,
+                        112,
+                        "Wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        113,
+                        121,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        129,
+                        139,
+                        "absence of",
+                        "protein_state"
+                    ],
+                    [
+                        140,
+                        144,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        156,
+                        181,
+                        "charge state distribution",
+                        "evidence"
+                    ],
+                    [
+                        200,
+                        207,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        233,
+                        237,
+                        "E32A",
+                        "mutant"
+                    ],
+                    [
+                        238,
+                        246,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        258,
+                        271,
+                        "charge states",
+                        "evidence"
+                    ],
+                    [
+                        290,
+                        297,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        352,
+                        359,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        363,
+                        367,
+                        "E32A",
+                        "mutant"
+                    ],
+                    [
+                        368,
+                        374,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 300,
+                "sent": "(C) E62A EncFtnsH displays charge states consistent with a decamer (green circles). (D) H65A EncFtnsH displays charge states consistent with both monomer (blue circles) and dimer (purple circles).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "E62A",
+                        "mutant"
+                    ],
+                    [
+                        9,
+                        17,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        27,
+                        40,
+                        "charge states",
+                        "evidence"
+                    ],
+                    [
+                        59,
+                        66,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        88,
+                        92,
+                        "H65A",
+                        "mutant"
+                    ],
+                    [
+                        93,
+                        101,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        111,
+                        124,
+                        "charge states",
+                        "evidence"
+                    ],
+                    [
+                        146,
+                        153,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        173,
+                        178,
+                        "dimer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 301,
+                "sent": "In addition to SEC studies, native mass spectrometry of the apo-EncFtnsH mutants was performed and compared with the wild-type apo-EncFtnsH protein (Figure 10).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        15,
+                        18,
+                        "SEC",
+                        "experimental_method"
+                    ],
+                    [
+                        28,
+                        52,
+                        "native mass spectrometry",
+                        "experimental_method"
+                    ],
+                    [
+                        60,
+                        63,
+                        "apo",
+                        "protein_state"
+                    ],
+                    [
+                        64,
+                        72,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        73,
+                        80,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        117,
+                        126,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        127,
+                        130,
+                        "apo",
+                        "protein_state"
+                    ],
+                    [
+                        131,
+                        139,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 302,
+                "sent": "As described above, the apo-EncFtnsH\u00a0has a charge state distribution consistent with an unstructured monomer, and decamer formation is only initiated upon addition of ferrous iron.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        24,
+                        27,
+                        "apo",
+                        "protein_state"
+                    ],
+                    [
+                        28,
+                        36,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        43,
+                        55,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        88,
+                        100,
+                        "unstructured",
+                        "protein_state"
+                    ],
+                    [
+                        101,
+                        108,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        114,
+                        121,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        175,
+                        179,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 303,
+                "sent": "Both the E32A mutant and E62A mutant displayed charge state distributions consistent with decamers, even in the absence of Fe2+.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        13,
+                        "E32A",
+                        "mutant"
+                    ],
+                    [
+                        14,
+                        20,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        25,
+                        29,
+                        "E62A",
+                        "mutant"
+                    ],
+                    [
+                        30,
+                        36,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        47,
+                        59,
+                        "charge state",
+                        "evidence"
+                    ],
+                    [
+                        90,
+                        98,
+                        "decamers",
+                        "oligomeric_state"
+                    ],
+                    [
+                        112,
+                        122,
+                        "absence of",
+                        "protein_state"
+                    ],
+                    [
+                        123,
+                        127,
+                        "Fe2+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 304,
+                "sent": "This gas-phase observation is consistent with SEC measurements, which indicate both of these variants were also decamers in solution.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        46,
+                        49,
+                        "SEC",
+                        "experimental_method"
+                    ],
+                    [
+                        112,
+                        120,
+                        "decamers",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 305,
+                "sent": "Thus it seems that these mutations allow the decamer to form in the absence of iron in the FOC.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        45,
+                        52,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        68,
+                        78,
+                        "absence of",
+                        "protein_state"
+                    ],
+                    [
+                        79,
+                        83,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        91,
+                        94,
+                        "FOC",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 306,
+                "sent": "In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        32,
+                        "glutamic acid",
+                        "residue_name"
+                    ],
+                    [
+                        33,
+                        40,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        42,
+                        44,
+                        "MS",
+                        "experimental_method"
+                    ],
+                    [
+                        61,
+                        65,
+                        "H65A",
+                        "mutant"
+                    ],
+                    [
+                        66,
+                        72,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        87,
+                        96,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        97,
+                        100,
+                        "apo",
+                        "protein_state"
+                    ],
+                    [
+                        101,
+                        109,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        130,
+                        137,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        175,
+                        182,
+                        "dimeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        183,
+                        187,
+                        "H65A",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 307,
+                "sent": "We propose that the observed differences in the oligomerization state of the E32A and E62A mutants compared to wild-type are due to the changes in the electrostatic environment within the FOC.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        77,
+                        81,
+                        "E32A",
+                        "mutant"
+                    ],
+                    [
+                        86,
+                        90,
+                        "E62A",
+                        "mutant"
+                    ],
+                    [
+                        91,
+                        98,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        111,
+                        120,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        188,
+                        191,
+                        "FOC",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 308,
+                "sent": "At neutral pH the glutamic acid residues are negatively charged, while the histidine residues are predominantly in their uncharged state.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        3,
+                        13,
+                        "neutral pH",
+                        "protein_state"
+                    ],
+                    [
+                        18,
+                        31,
+                        "glutamic acid",
+                        "residue_name"
+                    ],
+                    [
+                        75,
+                        84,
+                        "histidine",
+                        "residue_name"
+                    ]
+                ]
+            },
+            {
+                "sid": 309,
+                "sent": "In the wild-type\u00a0(WT) EncFtnsH this leads to electrostatic repulsion between subunits in the absence of iron.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        7,
+                        16,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        18,
+                        20,
+                        "WT",
+                        "protein_state"
+                    ],
+                    [
+                        22,
+                        30,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        77,
+                        85,
+                        "subunits",
+                        "structure_element"
+                    ],
+                    [
+                        93,
+                        103,
+                        "absence of",
+                        "protein_state"
+                    ],
+                    [
+                        104,
+                        108,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 310,
+                "sent": "Coordination of Fe2+ in this site stabilizes the dimer and reconstitutes the active FOC.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        12,
+                        "Coordination",
+                        "bond_interaction"
+                    ],
+                    [
+                        16,
+                        20,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        49,
+                        54,
+                        "dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        77,
+                        83,
+                        "active",
+                        "protein_state"
+                    ],
+                    [
+                        84,
+                        87,
+                        "FOC",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 311,
+                "sent": "The geometric arrangement of Glu32 and Glu62 in the FOC explains their behavior in solution and the gas phase, where they both favor the formation of decamers due to the loss of a repulsive negative charge.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        34,
+                        "Glu32",
+                        "residue_name_number"
+                    ],
+                    [
+                        39,
+                        44,
+                        "Glu62",
+                        "residue_name_number"
+                    ],
+                    [
+                        52,
+                        55,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        150,
+                        158,
+                        "decamers",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 312,
+                "sent": "The FOC in the H65A mutant is destabilized through the loss of this metal coordinating residue and potential positive charge carrier, thus favoring the monomer in solution and the gas phase.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        15,
+                        19,
+                        "H65A",
+                        "mutant"
+                    ],
+                    [
+                        20,
+                        26,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        55,
+                        62,
+                        "loss of",
+                        "protein_state"
+                    ],
+                    [
+                        68,
+                        94,
+                        "metal coordinating residue",
+                        "site"
+                    ],
+                    [
+                        152,
+                        159,
+                        "monomer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 313,
+                "sent": "Data collection and refinement statistics.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        41,
+                        "Data collection and refinement statistics",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 314,
+                "sent": "\tWT\tE32A\tE62A\tH65A\t \tData collection\t\t\t\t\t \tWavelength (\u00c5)\t1.74\t1.73\t1.73\t1.74\t \tResolution range (\u00c5)\t49.63 - 2.06 (2.10 - 2.06)\t48.84 - 2.59 (2.683 - 2.59)\t48.87 - 2.21 (2.29 - 2.21)\t48.86 - 2.97 (3.08 - 2.97)\t \tSpace group\tP 1 21 1\tP 1 21 1\tP 1 21 1\tP 1 21 1\t \tUnit cell (\u00c5) a\u00a0b \u00a0c\u00a0\u03b2 (\u00b0)\t98.18 120.53 140.30 95.36\t97.78 120.28 140.53 95.41\t98.09 120.23 140.36 95.50\t98.03 120.29 140.43 95.39\t \tTotal reflections\t1,264,922 (41,360)\t405,488 (36,186)\t1,069,345 (95,716)\t323,853 (32,120)\t \tUnique reflections\t197,873 (8,766)\t100,067 (9,735)\t162,379 (15,817)\t66,658 (6,553)\t \tMultiplicity\t6.4 (4.7)\t4.1 (3.7)\t6.6 (6.1)\t4.9 (4.9)\t \tAnomalous multiplicity\t3.2 (2.6)\tN/A\tN/A\tN/A\t \tCompleteness (%)\t99.2 (88.6)\t99.0 (97.0)\t100 (97.0)\t100 (99.0)\t \tAnomalous completeness (%)\t96.7 (77.2)\tN/A\tN/A\tN/A\t \tMean I/sigma(I)\t10.6 (1.60)\t8.46 (1.79)\t13.74 (1.80)\t8.09 (1.74)\t \tWilson B-factor\t26.98\t40.10\t33.97\t52.20\t \tRmerge\t0.123 (0.790)\t0.171 (0.792)\t0.0979 (1.009)\t0.177 (0.863)\t \tRmeas\t0.147 (0.973)\t0.196 (0.923)\t0.1064 (1.107)\t0.199 (0.966)\t \tCC1/2\t0.995 (0.469)\t0.985 (0.557)\t0.998 (0.642)\t0.989 (0.627)\t \tCC*\t0.999 (0.846)\t0.996 (0.846)\t0.999 (0.884)\t0.997 (0.878)\t \tImage DOI\t10.7488/ds/1342\t10.7488/ds/1419\t10.7488/ds/1420\t10.7488/ds/1421\t \tRefinement\t\t\t\t\t \tRwork\t0.171 (0.318)\t0.183 (0.288)\t0.165 (0.299)\t0.186 (0.273)\t \tRfree\t0.206 (0.345)\t0.225 (0351)\t0.216 (0.364)\t0.237 (0.325)\t \tNumber of non-hydrogen atoms\t23,222\t22,366\t22,691\t22,145\t \tmacromolecules\t22,276\t22,019\t21,965\t22,066\t \tligands\t138\t8\t24\t74\t \twater\t808\t339\t702\t5\t \tProtein residues\t2,703\t2,686\t2,675\t2,700\t \tRMS(bonds) (\u00c5)\t0.012\t0.005\t0.011\t0.002\t \tRMS(angles) (\u00b0)\t1.26\t0.58\t1.02\t0.40\t \tRamachandran favored (%)\t100\t99\t100\t99\t \tRamachandran allowed (%)\t0\t1\t0\t1\t \tRamachandran outliers (%)\t0\t0\t0\t0\t \tClash score\t1.42\t1.42\t1.79\t0.97\t \tAverage B-factor (\u00c52)\t33.90\t42.31\t41.34\t47.68\t \tmacromolecules\t33.80\t42.35\t41.31\t47.60\t \tligands\t40.40\t72.80\t65.55\t72.34\t \tsolvent\t36.20\t38.95\t41.46\t33.85\t \tPDB ID\t5DA5\t5L89\t5L8B\t5L8G\t \t",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        1,
+                        3,
+                        "WT",
+                        "protein_state"
+                    ],
+                    [
+                        4,
+                        8,
+                        "E32A",
+                        "mutant"
+                    ],
+                    [
+                        9,
+                        13,
+                        "E62A",
+                        "mutant"
+                    ],
+                    [
+                        14,
+                        18,
+                        "H65A",
+                        "mutant"
+                    ],
+                    [
+                        1504,
+                        1509,
+                        "water",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 315,
+                "sent": "Iron loading capacity of EncFtn, encapsulin and ferritin.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "Iron",
+                        "chemical"
+                    ],
+                    [
+                        25,
+                        31,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        33,
+                        43,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        48,
+                        56,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 316,
+                "sent": "Protein samples (at 8.5 \u00b5M) including decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were mixed with Fe(NH4)2(SO4) (in 0.1%\u00a0(v/v) HCl) of different concentrations in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl buffer at room temperature for 3 hrs in the air.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        38,
+                        47,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        48,
+                        56,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        58,
+                        68,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        70,
+                        80,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        85,
+                        96,
+                        "apoferritin",
+                        "protein_state"
+                    ],
+                    [
+                        113,
+                        126,
+                        "Fe(NH4)2(SO4)",
+                        "chemical"
+                    ],
+                    [
+                        142,
+                        145,
+                        "HCl",
+                        "chemical"
+                    ],
+                    [
+                        210,
+                        214,
+                        "NaCl",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 317,
+                "sent": "Protein-Fe mixtures were centrifuged at 13,000 x g to remove precipitated material and desalted prior to the Fe and protein content analysis by ferrozine assay and BCA microplate assay, respectively.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        8,
+                        10,
+                        "Fe",
+                        "chemical"
+                    ],
+                    [
+                        109,
+                        111,
+                        "Fe",
+                        "chemical"
+                    ],
+                    [
+                        144,
+                        159,
+                        "ferrozine assay",
+                        "experimental_method"
+                    ],
+                    [
+                        164,
+                        184,
+                        "BCA microplate assay",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 318,
+                "sent": "Fe to protein ratio was calculated to indicate the Fe binding capacity of the protein.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        2,
+                        "Fe",
+                        "chemical"
+                    ],
+                    [
+                        51,
+                        53,
+                        "Fe",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 319,
+                "sent": "Protein stability was compromised at high iron concentrations; therefore, the highest iron loading with the least protein precipitation was used to derive the maximum iron loading capacity per biological assembly (underlined and highlighted in bold).",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        42,
+                        46,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        86,
+                        90,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        167,
+                        171,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 320,
+                "sent": "The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        37,
+                        44,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        49,
+                        57,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        61,
+                        66,
+                        "60mer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        71,
+                        81,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        85,
+                        90,
+                        "60mer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        94,
+                        104,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        105,
+                        116,
+                        "loaded with",
+                        "protein_state"
+                    ],
+                    [
+                        130,
+                        139,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        140,
+                        146,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        167,
+                        172,
+                        "24mer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        177,
+                        182,
+                        "horse",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        190,
+                        201,
+                        "apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 321,
+                "sent": "Errors are quoted as the standard deviation of three technical repeats in both the ferrozine and BCA microplate assays.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        83,
+                        118,
+                        "ferrozine and BCA microplate assays",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 322,
+                "sent": "The proteins used in Fe loading experiment came from a single preparation.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        21,
+                        23,
+                        "Fe",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 323,
+                "sent": "Protein sample\tFe(NH4)2(SO4)2 loading (\u00b5M)\tFe detected by ferrozine assay (\u00b5M)\tProtein detected by BCA microplate assay (\u00b5M)\tFe / monomeric protein\tMaximum Fe loading per biological assembly unit\t \t8.46 \u00b5M EncFtnsH-10mer\t0\t4.73 \u00b1 2.32\t5.26 \u00b1 0.64\t0.90 \u00b1 0.44\t\t \t39.9\t9.93 \u00b1 1.20\t5.36 \u00b1 0.69\t1.85 \u00b1 0.22\t\t \t84\t17.99 \u00b1 2.01\t4.96 \u00b1 0.04\t3.63 \u00b1 0.41\t\t \t147\t21.09 \u00b1 1.94\t4.44 \u00b1 0.21\t4.75 \u00b1 0.44\t48 \u00b1 4\t \t224\t28.68 \u00b1 0.30\t3.73 \u00b1 0.53\t7.68 \u00b1 0.08\t\t \t301\t11.27 \u00b1 1.10\t2.50 \u00b1 0.05\t4.51 \u00b1 0.44\t\t \t8.50 \u00b5M Encapsulin\t0\t-1.02 \u00b1 0.54\t8.63 \u00b1 0.17\t-0.12 \u00b1 0.06\t\t \t224\t62.24 \u00b1 2.49\t10.01 \u00b1 0.58\t6.22 \u00b1 0.35\t\t \t301\t67.94 \u00b1 3.15\t8.69 \u00b1 0.42\t7.81 \u00b1 0.36\t\t \t450\t107.96 \u00b1 8.88\t8.50 \u00b1 0.69\t12.71 \u00b1 1.05\t\t \t700\t97.51 \u00b1 3.19\t7.26 \u00b1 0.20\t13.44 \u00b1 0.44\t\t \t1000\t308.63 \u00b1 2.06\t8.42 \u00b1 0.34\t36.66 \u00b1 0.24\t2199 \u00b1 15\t \t1500\t57.09 \u00b1 0.90\t1.44 \u00b1 0.21\t39.77 \u00b1 0.62\t\t \t2000\t9.2 \u00b1 1.16\t0.21 \u00b1 0.14\t44.73 \u00b1 5.63\t\t \t8.70 \u00b5M EncFtn-Enc\t0\t3.31 \u00b1 1.57\t6.85 \u00b1 0.07\t0.48 \u00b1 0.23\t\t \t224\t116.27 \u00b1 3.74\t7.63 \u00b1 0.12\t15.25 \u00b1 0.49\t\t \t301\t132.86 \u00b1 4.03\t6.66 \u00b1 0.31\t19.96 \u00b1 0.61\t\t \t450\t220.57 \u00b1 27.33\t6.12 \u00b1 1.07\t36.06 \u00b1 4.47\t\t \t700\t344.03 \u00b1 40.38\t6.94 \u00b1 0.17\t49.58 \u00b1 5.82\t\t \t1000\t496.00 \u00b1 38.48\t7.19 \u00b1 0.08\t68.94 \u00b1 5.35\t4137 \u00b1 321\t \t1500\t569.98 \u00b1 73.63\t5.73 \u00b1 0.03\t99.44 \u00b1 12.84\t\t \t2000\t584.30 \u00b1 28.33\t4.88 \u00b1 0.22\t119.62 \u00b1 5.80\t\t \t8.50 \u00b5M Apoferritin\t0\t3.95 \u00b1 2.26\t9.37 \u00b1 0.24\t0.42 \u00b1 0.25\t\t \t42.5\t10.27 \u00b1 1.12\t8.27 \u00b1 0.30\t1.24 \u00b1 0.18\t\t \t212.5\t44.48 \u00b1 2.76\t7.85 \u00b1 0.77\t5.67 \u00b1 0.83\t\t \t637.5\t160.93 \u00b1 4.27\t6.76 \u00b1 0.81\t23.79 \u00b1 3.12\t571 \u00b1 75\t \t1275\t114.92 \u00b1 3.17\t3.84 \u00b1 0.30\t29.91 \u00b1 2.95\t\t \t1700\t91.40 \u00b1 3.37\t3.14 \u00b1 0.35\t29.13 \u00b1 3.86\t\t \t",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        15,
+                        29,
+                        "Fe(NH4)2(SO4)2",
+                        "chemical"
+                    ],
+                    [
+                        43,
+                        45,
+                        "Fe",
+                        "chemical"
+                    ],
+                    [
+                        58,
+                        73,
+                        "ferrozine assay",
+                        "experimental_method"
+                    ],
+                    [
+                        99,
+                        119,
+                        "BCA microplate assay",
+                        "experimental_method"
+                    ],
+                    [
+                        125,
+                        127,
+                        "Fe",
+                        "chemical"
+                    ],
+                    [
+                        156,
+                        158,
+                        "Fe",
+                        "chemical"
+                    ],
+                    [
+                        206,
+                        214,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        215,
+                        220,
+                        "10mer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        495,
+                        505,
+                        "Encapsulin",
+                        "protein"
+                    ],
+                    [
+                        883,
+                        893,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        1285,
+                        1296,
+                        "Apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 324,
+                "sent": "To understand the impact of the mutants on the organization and metal binding of the FOC, we determined the X-ray crystal structures of each of the EncFtnsH\u00a0mutants (See Table 4 for data collection and refinement statistics).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        32,
+                        39,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        85,
+                        88,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        108,
+                        132,
+                        "X-ray crystal structures",
+                        "evidence"
+                    ],
+                    [
+                        148,
+                        156,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        157,
+                        164,
+                        "mutants",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 325,
+                "sent": "The crystal packing of all of the mutants in this study is essentially isomorphous to the EncFtnsH structure.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        41,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        90,
+                        98,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        99,
+                        108,
+                        "structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 326,
+                "sent": "All of the mutants display the same decameric arrangement in the crystals as the EncFtnsH structure, and the monomers superimpose with an average RMSDC\u03b1 of less than 0.2 \u00c5.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        18,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        36,
+                        45,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        65,
+                        73,
+                        "crystals",
+                        "evidence"
+                    ],
+                    [
+                        81,
+                        89,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        90,
+                        99,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        109,
+                        117,
+                        "monomers",
+                        "oligomeric_state"
+                    ],
+                    [
+                        118,
+                        129,
+                        "superimpose",
+                        "experimental_method"
+                    ],
+                    [
+                        146,
+                        152,
+                        "RMSDC\u03b1",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 327,
+                "sent": "FOC dimer interface of EncFtnsH-E32A mutant.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        4,
+                        19,
+                        "dimer interface",
+                        "site"
+                    ],
+                    [
+                        23,
+                        36,
+                        "EncFtnsH-E32A",
+                        "mutant"
+                    ],
+                    [
+                        37,
+                        43,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 328,
+                "sent": "(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-E32A.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        33,
+                        69,
+                        "metal-binding dimerization interface",
+                        "site"
+                    ],
+                    [
+                        73,
+                        86,
+                        "EncFtnsH-E32A",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 329,
+                "sent": "Protein residues are shown as sticks with blue and green carbons for the different subunits.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        83,
+                        91,
+                        "subunits",
+                        "structure_element"
+                    ],
+                    [
+                        83,
+                        91,
+                        "subunits",
+                        "structure_element"
+                    ],
+                    [
+                        83,
+                        91,
+                        "subunits",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 330,
+                "sent": "The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 \u03c3.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        33,
+                        "2mFo-DFc electron density map",
+                        "evidence"
+                    ],
+                    [
+                        4,
+                        33,
+                        "2mFo-DFc electron density map",
+                        "evidence"
+                    ],
+                    [
+                        4,
+                        33,
+                        "2mFo-DFc electron density map",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 331,
+                "sent": "(B) Views of the FOC of the EncFtnsH-E32Amutant.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        17,
+                        20,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        28,
+                        41,
+                        "EncFtnsH-E32A",
+                        "mutant"
+                    ],
+                    [
+                        41,
+                        47,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 332,
+                "sent": "FOC dimer interface of EncFtnsH-E62A mutant.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        19,
+                        "FOC dimer interface",
+                        "site"
+                    ],
+                    [
+                        23,
+                        36,
+                        "EncFtnsH-E62A",
+                        "mutant"
+                    ],
+                    [
+                        37,
+                        43,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 333,
+                "sent": "(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-E62A.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        33,
+                        69,
+                        "metal-binding dimerization interface",
+                        "site"
+                    ],
+                    [
+                        73,
+                        86,
+                        "EncFtnsH-E62A",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 334,
+                "sent": "The single coordinated calcium ion is shown as a grey sphere. (B) Views of the FOC of the EncFtnsH-E62A mutant.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        23,
+                        30,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        79,
+                        82,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        90,
+                        103,
+                        "EncFtnsH-E62A",
+                        "mutant"
+                    ],
+                    [
+                        104,
+                        110,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 335,
+                "sent": "FOC dimer interface of EncFtnsH-H65A mutant.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        19,
+                        "FOC dimer interface",
+                        "site"
+                    ],
+                    [
+                        23,
+                        36,
+                        "EncFtnsH-H65A",
+                        "mutant"
+                    ],
+                    [
+                        37,
+                        43,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 336,
+                "sent": "(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-H65A.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        33,
+                        69,
+                        "metal-binding dimerization interface",
+                        "site"
+                    ],
+                    [
+                        73,
+                        86,
+                        "EncFtnsH-H65A",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 337,
+                "sent": "The coordinated calcium ions are shown as a grey spheres with coordination distances in the FOC highlighted with yellow dashed lines.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        16,
+                        23,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        62,
+                        74,
+                        "coordination",
+                        "bond_interaction"
+                    ],
+                    [
+                        92,
+                        95,
+                        "FOC",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 338,
+                "sent": "(B) Views of the FOC of the EncFtnsH-H65A mutant.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        17,
+                        20,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        28,
+                        41,
+                        "EncFtnsH-H65A",
+                        "mutant"
+                    ],
+                    [
+                        42,
+                        48,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 339,
+                "sent": "Comparison of the EncFtnsH FOC mutants vs wild type.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        18,
+                        26,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        27,
+                        30,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        31,
+                        38,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        42,
+                        51,
+                        "wild type",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 340,
+                "sent": "The structures of the three EncFtnsH\u00a0mutants were all determined by X-ray crystallography.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        14,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        28,
+                        36,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        37,
+                        44,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        68,
+                        89,
+                        "X-ray crystallography",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 341,
+                "sent": "The E32A, E62A and H65A mutants were crystallized in identical conditions to the wild type.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "E32A",
+                        "mutant"
+                    ],
+                    [
+                        10,
+                        14,
+                        "E62A",
+                        "mutant"
+                    ],
+                    [
+                        19,
+                        23,
+                        "H65A",
+                        "mutant"
+                    ],
+                    [
+                        24,
+                        31,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        37,
+                        49,
+                        "crystallized",
+                        "experimental_method"
+                    ],
+                    [
+                        81,
+                        90,
+                        "wild type",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 342,
+                "sent": "EncFtnsH structure and were essentially isomorphous in terms of their unit cell dimensions.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        9,
+                        18,
+                        "structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 343,
+                "sent": "The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        24,
+                        31,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        36,
+                        42,
+                        "native",
+                        "protein_state"
+                    ],
+                    [
+                        43,
+                        51,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        52,
+                        62,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        88,
+                        99,
+                        "coordinated",
+                        "bond_interaction"
+                    ],
+                    [
+                        100,
+                        104,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        119,
+                        123,
+                        "Ca2+",
+                        "chemical"
+                    ],
+                    [
+                        168,
+                        177,
+                        "wild type",
+                        "protein_state"
+                    ],
+                    [
+                        185,
+                        189,
+                        "E32A",
+                        "mutant"
+                    ],
+                    [
+                        197,
+                        201,
+                        "E62A",
+                        "mutant"
+                    ],
+                    [
+                        210,
+                        214,
+                        "H65A",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 344,
+                "sent": "Of the mutants, only H65A has any coordinated metal ions, which appear to be calcium ions from the crystallization condition.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        7,
+                        14,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        21,
+                        25,
+                        "H65A",
+                        "mutant"
+                    ],
+                    [
+                        34,
+                        45,
+                        "coordinated",
+                        "bond_interaction"
+                    ],
+                    [
+                        77,
+                        84,
+                        "calcium",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 345,
+                "sent": "The overall organization of FOC residues is retained in the mutants, with almost no backbone movements.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        28,
+                        31,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        60,
+                        67,
+                        "mutants",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 346,
+                "sent": "Significant differences center around Tyr39, which moves to coordinate the bound calcium ions in the H65A mutant; and Glu32, which moves away from the metal ions in this structure.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        38,
+                        43,
+                        "Tyr39",
+                        "residue_name_number"
+                    ],
+                    [
+                        60,
+                        70,
+                        "coordinate",
+                        "bond_interaction"
+                    ],
+                    [
+                        75,
+                        80,
+                        "bound",
+                        "protein_state"
+                    ],
+                    [
+                        81,
+                        88,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        101,
+                        105,
+                        "H65A",
+                        "mutant"
+                    ],
+                    [
+                        106,
+                        112,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        118,
+                        123,
+                        "Glu32",
+                        "residue_name_number"
+                    ],
+                    [
+                        170,
+                        179,
+                        "structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 347,
+                "sent": "Close inspection of the region of the protein around the FOC in each of the mutants highlights their effect on metal binding (Figure 11 and Figure 11\u2014figure supplement 1\u20133).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        57,
+                        60,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        76,
+                        83,
+                        "mutants",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 348,
+                "sent": "In the E32A mutant the position of the side chains of the remaining iron coordinating residues in the FOC is essentially unchanged, but the absence of the axial-metal coordinating ligand provided by the Glu32 side chain abrogates metal binding in this site.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        7,
+                        11,
+                        "E32A",
+                        "mutant"
+                    ],
+                    [
+                        12,
+                        18,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        68,
+                        94,
+                        "iron coordinating residues",
+                        "site"
+                    ],
+                    [
+                        102,
+                        105,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        140,
+                        150,
+                        "absence of",
+                        "protein_state"
+                    ],
+                    [
+                        167,
+                        179,
+                        "coordinating",
+                        "bond_interaction"
+                    ],
+                    [
+                        203,
+                        208,
+                        "Glu32",
+                        "residue_name_number"
+                    ],
+                    [
+                        220,
+                        243,
+                        "abrogates metal binding",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 349,
+                "sent": "The Glu31/34-site also lacks metal, with the side chain of Glu31 rotated by 180\u00b0\u00a0at the C\u03b2 in the absence of metal (Figure 11\u2014figure supplement 1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        17,
+                        "Glu31/34-site",
+                        "site"
+                    ],
+                    [
+                        23,
+                        28,
+                        "lacks",
+                        "protein_state"
+                    ],
+                    [
+                        29,
+                        34,
+                        "metal",
+                        "chemical"
+                    ],
+                    [
+                        59,
+                        64,
+                        "Glu31",
+                        "residue_name_number"
+                    ],
+                    [
+                        98,
+                        108,
+                        "absence of",
+                        "protein_state"
+                    ],
+                    [
+                        109,
+                        114,
+                        "metal",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 350,
+                "sent": "The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11\u2014figure supplement 2).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "E62A",
+                        "mutant"
+                    ],
+                    [
+                        9,
+                        15,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        44,
+                        47,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        55,
+                        59,
+                        "E32A",
+                        "mutant"
+                    ],
+                    [
+                        60,
+                        66,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        80,
+                        90,
+                        "entry site",
+                        "site"
+                    ],
+                    [
+                        103,
+                        110,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        115,
+                        126,
+                        "coordinated",
+                        "bond_interaction"
+                    ],
+                    [
+                        144,
+                        149,
+                        "Glu31",
+                        "residue_name_number"
+                    ],
+                    [
+                        154,
+                        159,
+                        "Glu34",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 351,
+                "sent": "The H65A mutant diverges significantly from the wild type in the position of the residues Glu32 and Tyr39 in the FOC.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "H65A",
+                        "mutant"
+                    ],
+                    [
+                        9,
+                        15,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        48,
+                        57,
+                        "wild type",
+                        "protein_state"
+                    ],
+                    [
+                        90,
+                        95,
+                        "Glu32",
+                        "residue_name_number"
+                    ],
+                    [
+                        100,
+                        105,
+                        "Tyr39",
+                        "residue_name_number"
+                    ],
+                    [
+                        113,
+                        116,
+                        "FOC",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 352,
+                "sent": "E32 appears in either the original orientation as the wild type and coordinates Ca2+ in this position, or it is flipped by 180\u00b0 at the C\u03b2, moving away from the coordinated calcium ion in the FOC.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "E32",
+                        "residue_name_number"
+                    ],
+                    [
+                        54,
+                        63,
+                        "wild type",
+                        "protein_state"
+                    ],
+                    [
+                        68,
+                        79,
+                        "coordinates",
+                        "bond_interaction"
+                    ],
+                    [
+                        80,
+                        84,
+                        "Ca2+",
+                        "chemical"
+                    ],
+                    [
+                        160,
+                        171,
+                        "coordinated",
+                        "bond_interaction"
+                    ],
+                    [
+                        172,
+                        179,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        191,
+                        194,
+                        "FOC",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 353,
+                "sent": "Tyr39 moves closer to Ca2+ compared to the wild-type and coordinates the calcium ion (Figure 11\u2014figure supplement 3).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "Tyr39",
+                        "residue_name_number"
+                    ],
+                    [
+                        22,
+                        26,
+                        "Ca2+",
+                        "chemical"
+                    ],
+                    [
+                        43,
+                        52,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        57,
+                        68,
+                        "coordinates",
+                        "bond_interaction"
+                    ],
+                    [
+                        73,
+                        80,
+                        "calcium",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 354,
+                "sent": "A single calcium ion is present in the entry site of this mutant; however, Glu31 of one chain is rotated away from the metal ion and is not involved in coordination.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        16,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        39,
+                        49,
+                        "entry site",
+                        "site"
+                    ],
+                    [
+                        58,
+                        64,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        75,
+                        80,
+                        "Glu31",
+                        "residue_name_number"
+                    ],
+                    [
+                        152,
+                        164,
+                        "coordination",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 355,
+                "sent": "Taken together the results of our data show that these changes to the FOC of EncFtn still permit the formation of the decameric form of the protein.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        70,
+                        73,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        77,
+                        83,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        118,
+                        127,
+                        "decameric",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 356,
+                "sent": "While the proteins all appear decameric in crystals, their solution and gas-phase behavior differs considerably and the mutants no longer show metal-dependent oligomerization.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        30,
+                        39,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        43,
+                        51,
+                        "crystals",
+                        "evidence"
+                    ],
+                    [
+                        120,
+                        127,
+                        "mutants",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 357,
+                "sent": "These results highlight the importance of metal coordination in the FOC for the stability and assembly of the EncFtn protein.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        42,
+                        47,
+                        "metal",
+                        "chemical"
+                    ],
+                    [
+                        48,
+                        60,
+                        "coordination",
+                        "bond_interaction"
+                    ],
+                    [
+                        68,
+                        71,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        110,
+                        116,
+                        "EncFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 358,
+                "sent": "Progress curves recording ferroxidase activity of EncFtnsH mutants.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        15,
+                        "Progress curves",
+                        "evidence"
+                    ],
+                    [
+                        26,
+                        37,
+                        "ferroxidase",
+                        "protein_type"
+                    ],
+                    [
+                        50,
+                        58,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        59,
+                        66,
+                        "mutants",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 359,
+                "sent": "20 \u00b5M wild-type\u00a0EncFtnsH, E32A, E62A and H65A mutants\u00a0were mixed with 20 \u00b5M or 100 \u00b5M acidic Fe(NH4)2(SO4)2, respectively.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        6,
+                        15,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        16,
+                        24,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        26,
+                        30,
+                        "E32A",
+                        "mutant"
+                    ],
+                    [
+                        32,
+                        36,
+                        "E62A",
+                        "mutant"
+                    ],
+                    [
+                        41,
+                        45,
+                        "H65A",
+                        "mutant"
+                    ],
+                    [
+                        46,
+                        53,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        93,
+                        107,
+                        "Fe(NH4)2(SO4)2",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 360,
+                "sent": "Absorbance at 315 nm was recorded for 1800 s at 25\u00b0C as an indication of Fe3+ formation.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        73,
+                        77,
+                        "Fe3+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 361,
+                "sent": "Protein free samples (dashed and dotted lines) were measured for Fe2+ background oxidation as controls.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        65,
+                        69,
+                        "Fe2+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 362,
+                "sent": "Relative ferroxidase activity of EncFtnsH mutants.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        9,
+                        20,
+                        "ferroxidase",
+                        "protein_type"
+                    ],
+                    [
+                        33,
+                        41,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        42,
+                        49,
+                        "mutants",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 363,
+                "sent": "EncFtnsH, and the mutant forms E32A, E62A and H65A, each at 20 \u00b5M, were mixed with 100 \u00b5M acidic Fe(NH4)2(SO4)2.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        18,
+                        24,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        31,
+                        35,
+                        "E32A",
+                        "mutant"
+                    ],
+                    [
+                        37,
+                        41,
+                        "E62A",
+                        "mutant"
+                    ],
+                    [
+                        46,
+                        50,
+                        "H65A",
+                        "mutant"
+                    ],
+                    [
+                        97,
+                        111,
+                        "Fe(NH4)2(SO4)2",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 364,
+                "sent": "Ferroxidase activity of the mutant forms is determined by measuring the absorbance at 315 nm for 1800 s at 25\u00a0\u00b0C as an indication of Fe3+ formation.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Ferroxidase",
+                        "protein_type"
+                    ],
+                    [
+                        28,
+                        34,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        58,
+                        92,
+                        "measuring the absorbance at 315 nm",
+                        "experimental_method"
+                    ],
+                    [
+                        133,
+                        137,
+                        "Fe3+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 365,
+                "sent": "The relative ferroxidase activity of mutants is plotted as a proportion of the activity of the wild-type protein using the endpoint measurement of A315.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        13,
+                        24,
+                        "ferroxidase",
+                        "protein_type"
+                    ],
+                    [
+                        37,
+                        44,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        95,
+                        104,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        132,
+                        151,
+                        "measurement of A315",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 366,
+                "sent": "The FOC mutants showed reduced ferroxidase activity to varied extents, among which E62A significantly abrogated the ferroxidase activity.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        8,
+                        15,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        31,
+                        42,
+                        "ferroxidase",
+                        "protein_type"
+                    ],
+                    [
+                        83,
+                        87,
+                        "E62A",
+                        "mutant"
+                    ],
+                    [
+                        116,
+                        127,
+                        "ferroxidase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 367,
+                "sent": "To address the question of how mutagenesis of the iron coordinating residues affects the enzymatic activity of the EncFtnsH protein we recorded progress curves for the oxidation of Fe2+ to Fe3+ by the different mutants as before.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        31,
+                        42,
+                        "mutagenesis",
+                        "experimental_method"
+                    ],
+                    [
+                        50,
+                        76,
+                        "iron coordinating residues",
+                        "site"
+                    ],
+                    [
+                        115,
+                        123,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        144,
+                        159,
+                        "progress curves",
+                        "evidence"
+                    ],
+                    [
+                        181,
+                        185,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        189,
+                        193,
+                        "Fe3+",
+                        "chemical"
+                    ],
+                    [
+                        211,
+                        218,
+                        "mutants",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 368,
+                "sent": "Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%;\u00a0the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Mutagenesis",
+                        "experimental_method"
+                    ],
+                    [
+                        15,
+                        19,
+                        "E32A",
+                        "mutant"
+                    ],
+                    [
+                        24,
+                        28,
+                        "H65A",
+                        "mutant"
+                    ],
+                    [
+                        53,
+                        61,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        84,
+                        88,
+                        "E62A",
+                        "mutant"
+                    ],
+                    [
+                        89,
+                        95,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        150,
+                        157,
+                        "loss of",
+                        "protein_state"
+                    ],
+                    [
+                        171,
+                        183,
+                        "coordination",
+                        "bond_interaction"
+                    ],
+                    [
+                        209,
+                        231,
+                        "di-nuclear iron center",
+                        "site"
+                    ],
+                    [
+                        239,
+                        242,
+                        "FOC",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 369,
+                "sent": "Collectively, the effect of mutating these residues in the FOC confirms the importance of the iron coordinating residues for the ferroxidase activity of the EncFtnsH protein.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        28,
+                        36,
+                        "mutating",
+                        "experimental_method"
+                    ],
+                    [
+                        59,
+                        62,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        94,
+                        120,
+                        "iron coordinating residues",
+                        "site"
+                    ],
+                    [
+                        129,
+                        140,
+                        "ferroxidase",
+                        "protein_type"
+                    ],
+                    [
+                        157,
+                        165,
+                        "EncFtnsH",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 370,
+                "sent": "Phylogenetic tree of ferritin family proteins.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        17,
+                        "Phylogenetic tree",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        29,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 371,
+                "sent": "The tree was built using the Neighbor-Joining method based on step-wise amino acid sequence alignment of the four-helical bundle portions of ferritin family proteins (Supplementary file 1).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        29,
+                        52,
+                        "Neighbor-Joining method",
+                        "experimental_method"
+                    ],
+                    [
+                        62,
+                        101,
+                        "step-wise amino acid sequence alignment",
+                        "experimental_method"
+                    ],
+                    [
+                        109,
+                        128,
+                        "four-helical bundle",
+                        "structure_element"
+                    ],
+                    [
+                        141,
+                        149,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 372,
+                "sent": "The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        26,
+                        "evolutionary distances",
+                        "evidence"
+                    ],
+                    [
+                        51,
+                        68,
+                        "p-distance method",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 373,
+                "sent": "Our study reports on a new class of ferritin-like proteins (EncFtn), which are associated with bacterial encapsulin nanocompartments (Enc).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        36,
+                        44,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        60,
+                        66,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        95,
+                        104,
+                        "bacterial",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        105,
+                        115,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        116,
+                        132,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ],
+                    [
+                        134,
+                        137,
+                        "Enc",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 374,
+                "sent": "By studying the EncFtn from R. rubrum we demonstrate that iron binding results in assembly of EncFtn decamers, which display a unique annular architecture.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        16,
+                        22,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        28,
+                        37,
+                        "R. rubrum",
+                        "species"
+                    ],
+                    [
+                        58,
+                        62,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        94,
+                        100,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        101,
+                        109,
+                        "decamers",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 375,
+                "sent": "Despite a radically different quaternary structure to the classical ferritins, the four-helical bundle scaffold and FOC of EncFtnsH are strikingly similar to ferritin (Figure 6A).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        58,
+                        67,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        68,
+                        77,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        83,
+                        111,
+                        "four-helical bundle scaffold",
+                        "structure_element"
+                    ],
+                    [
+                        116,
+                        119,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        123,
+                        131,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        158,
+                        166,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 376,
+                "sent": "A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        2,
+                        34,
+                        "sequence-based phylogenetic tree",
+                        "experimental_method"
+                    ],
+                    [
+                        55,
+                        63,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        107,
+                        116,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        117,
+                        126,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        128,
+                        145,
+                        "bacterioferritins",
+                        "protein_type"
+                    ],
+                    [
+                        150,
+                        153,
+                        "Dps",
+                        "protein_type"
+                    ],
+                    [
+                        190,
+                        234,
+                        "encapsulin-associated ferritin-like proteins",
+                        "protein_type"
+                    ],
+                    [
+                        236,
+                        243,
+                        "EncFtns",
+                        "protein_type"
+                    ],
+                    [
+                        291,
+                        301,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        312,
+                        322,
+                        "Non-EncFtn",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 377,
+                "sent": "The analysis revealed that the EncFtn and Non-EncFtn proteins form groups distinct from the other clearly delineated groups of ferritins, and represent outliers in the tree (Figure 13).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        31,
+                        37,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        42,
+                        52,
+                        "Non-EncFtn",
+                        "protein_type"
+                    ],
+                    [
+                        127,
+                        136,
+                        "ferritins",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 378,
+                "sent": "While it is difficult to infer ancestral lineages in protein families, the similarity seen in the active site scaffold of these proteins highlights a shared evolutionary relationship between EncFtn proteins and other members of the ferritin superfamily that has been noted in previous studies (;\u00a0).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        98,
+                        118,
+                        "active site scaffold",
+                        "site"
+                    ],
+                    [
+                        191,
+                        197,
+                        "EncFtn",
+                        "protein_type"
+                    ],
+                    [
+                        232,
+                        240,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 379,
+                "sent": "From this analysis, we propose that the four-helical fold of the classical ferritins may have arisen through gene duplication of an ancestor of EncFtn.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        40,
+                        57,
+                        "four-helical fold",
+                        "structure_element"
+                    ],
+                    [
+                        65,
+                        74,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        75,
+                        84,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        144,
+                        150,
+                        "EncFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 380,
+                "sent": "This gene duplication would result in the C-terminal region of one EncFtn monomer being linked to the N-terminus of another and thus stabilizing the four-helix bundle fold within a single polypeptide chain (Figure 6B).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        42,
+                        59,
+                        "C-terminal region",
+                        "structure_element"
+                    ],
+                    [
+                        67,
+                        73,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        74,
+                        81,
+                        "monomer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        149,
+                        171,
+                        "four-helix bundle fold",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 381,
+                "sent": "Linking the protein together in this way relaxes the requirement for the maintenance of a symmetrical FOC and thus provides a path to the diversity in active-site residues seen across the ferritin family (Figure 6A, residues Glu95, Gln128 and Glu131 in PmFtn, Supplementary file 1).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        102,
+                        105,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        151,
+                        171,
+                        "active-site residues",
+                        "site"
+                    ],
+                    [
+                        188,
+                        196,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        225,
+                        230,
+                        "Glu95",
+                        "residue_name_number"
+                    ],
+                    [
+                        232,
+                        238,
+                        "Gln128",
+                        "residue_name_number"
+                    ],
+                    [
+                        243,
+                        249,
+                        "Glu131",
+                        "residue_name_number"
+                    ],
+                    [
+                        253,
+                        258,
+                        "PmFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 382,
+                "sent": "Relationship between ferritin structure and activity",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        21,
+                        29,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        30,
+                        39,
+                        "structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 383,
+                "sent": "The quaternary arrangement of classical ferritins into an octahedral nanocage and Dps into a dodecamer is absolutely required for their function as iron storage compartments.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        30,
+                        39,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        40,
+                        49,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        58,
+                        68,
+                        "octahedral",
+                        "protein_state"
+                    ],
+                    [
+                        69,
+                        77,
+                        "nanocage",
+                        "complex_assembly"
+                    ],
+                    [
+                        82,
+                        85,
+                        "Dps",
+                        "protein"
+                    ],
+                    [
+                        93,
+                        102,
+                        "dodecamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        148,
+                        152,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 384,
+                "sent": "The oxidation and mineralization of iron must be spatially separated from the host cytosol to prevent the formation of damaging hydroxyl radicals in the Fenton and Haber-Weiss reactions.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        36,
+                        40,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 385,
+                "sent": "\u00a0This is achieved in all ferritins by confining the oxidation of iron to the interior of the protein complex, thus achieving sequestration of the Fe3+ mineralization product.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        25,
+                        34,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        65,
+                        69,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        146,
+                        150,
+                        "Fe3+",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 386,
+                "sent": "A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6\u2014figure supplement 1A).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        2,
+                        22,
+                        "structural alignment",
+                        "experimental_method"
+                    ],
+                    [
+                        30,
+                        33,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        37,
+                        43,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        53,
+                        62,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        63,
+                        71,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        72,
+                        77,
+                        "PmFtn",
+                        "protein"
+                    ],
+                    [
+                        93,
+                        105,
+                        "central ring",
+                        "structure_element"
+                    ],
+                    [
+                        109,
+                        115,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        155,
+                        163,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        198,
+                        204,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        233,
+                        255,
+                        "mineralization surface",
+                        "site"
+                    ],
+                    [
+                        259,
+                        267,
+                        "ferritin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 387,
+                "sent": "This overlay highlights the fact that the ferroxidase center of EncFtn faces in the opposite direction relative to the classical ferritins and is essentially inside out regarding iron storage space (Figure 6\u2014figure supplement 1B, boxed region).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        5,
+                        12,
+                        "overlay",
+                        "experimental_method"
+                    ],
+                    [
+                        42,
+                        60,
+                        "ferroxidase center",
+                        "site"
+                    ],
+                    [
+                        64,
+                        70,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        119,
+                        128,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        129,
+                        138,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        179,
+                        183,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 388,
+                "sent": "Analysis of each of the single mutations (E32A, E62A and H65A) made in the FOC highlights the importance of the iron-coordinating residues in the catalytic activity of EncFtn.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        31,
+                        40,
+                        "mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        42,
+                        46,
+                        "E32A",
+                        "mutant"
+                    ],
+                    [
+                        48,
+                        52,
+                        "E62A",
+                        "mutant"
+                    ],
+                    [
+                        57,
+                        61,
+                        "H65A",
+                        "mutant"
+                    ],
+                    [
+                        75,
+                        78,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        112,
+                        138,
+                        "iron-coordinating residues",
+                        "site"
+                    ],
+                    [
+                        168,
+                        174,
+                        "EncFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 389,
+                "sent": "Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        33,
+                        40,
+                        "calcium",
+                        "chemical"
+                    ],
+                    [
+                        45,
+                        59,
+                        "coordinated by",
+                        "bond_interaction"
+                    ],
+                    [
+                        60,
+                        65,
+                        "Glu31",
+                        "residue_name_number"
+                    ],
+                    [
+                        70,
+                        75,
+                        "Glu34",
+                        "residue_name_number"
+                    ],
+                    [
+                        88,
+                        96,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        97,
+                        106,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        119,
+                        129,
+                        "entry site",
+                        "site"
+                    ],
+                    [
+                        161,
+                        164,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        207,
+                        211,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        275,
+                        278,
+                        "FOC",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 390,
+                "sent": "The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        68,
+                        74,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        76,
+                        81,
+                        "His57",
+                        "residue_name_number"
+                    ],
+                    [
+                        83,
+                        88,
+                        "Glu61",
+                        "residue_name_number"
+                    ],
+                    [
+                        93,
+                        98,
+                        "Glu64",
+                        "residue_name_number"
+                    ],
+                    [
+                        158,
+                        180,
+                        "mineralization surface",
+                        "site"
+                    ],
+                    [
+                        192,
+                        201,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        202,
+                        210,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        211,
+                        219,
+                        "nanocage",
+                        "complex_assembly"
+                    ],
+                    [
+                        254,
+                        257,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        281,
+                        292,
+                        "exit portal",
+                        "site"
+                    ],
+                    [
+                        297,
+                        316,
+                        "mineralization site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 391,
+                "sent": "The absolute requirement for the spatial separation of oxidation and mineralization in ferritins suggests that the EncFtn family proteins are not capable of storing iron minerals due to the absence of an enclosed compartment in their structure (Figure 6\u2014figure supplement 1B).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        87,
+                        96,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        115,
+                        121,
+                        "EncFtn",
+                        "protein_type"
+                    ],
+                    [
+                        165,
+                        169,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        190,
+                        200,
+                        "absence of",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 392,
+                "sent": "Our biochemical characterization of EncFtn supports this hypothesis, indicating that while this protein is capable of oxidizing iron, it does not accrue mineralized iron in an analogous manner to classical ferritins.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        32,
+                        "biochemical characterization",
+                        "experimental_method"
+                    ],
+                    [
+                        36,
+                        42,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        128,
+                        132,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        165,
+                        169,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        196,
+                        205,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        206,
+                        215,
+                        "ferritins",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 393,
+                "sent": "While EncFtn does not store iron itself, its association with the encapsulin nanocage suggests that mineralization occurs within the cavity of the encapsulin shell.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        6,
+                        12,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        28,
+                        32,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        66,
+                        76,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        77,
+                        85,
+                        "nanocage",
+                        "complex_assembly"
+                    ],
+                    [
+                        133,
+                        139,
+                        "cavity",
+                        "site"
+                    ],
+                    [
+                        147,
+                        157,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        158,
+                        163,
+                        "shell",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 394,
+                "sent": "Our ferroxidase assay data on the recombinant EncFtn-Enc nanocompartments, which accrue over 4100 iron ions per complex and form regular nanoparticles, are consistent with the encapsulin protein acting as the store for iron oxidized by the EncFtn enzyme.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        21,
+                        "ferroxidase assay",
+                        "experimental_method"
+                    ],
+                    [
+                        46,
+                        56,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        57,
+                        73,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ],
+                    [
+                        98,
+                        102,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        137,
+                        150,
+                        "nanoparticles",
+                        "complex_assembly"
+                    ],
+                    [
+                        176,
+                        186,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        219,
+                        223,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        240,
+                        246,
+                        "EncFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 395,
+                "sent": "TEM analysis of the reaction products shows the production of homogeneous iron nanoparticles only in the EncFtn-Enc nanocompartment (Figure 8\u2014figure supplement 1).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "TEM",
+                        "experimental_method"
+                    ],
+                    [
+                        74,
+                        78,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        105,
+                        115,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        116,
+                        131,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 396,
+                "sent": "Model of iron oxidation in encapsulin nanocompartments.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        9,
+                        13,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        27,
+                        37,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        38,
+                        54,
+                        "nanocompartments",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 397,
+                "sent": "(A) Model of EncFtnsH docking to the encapsulin shell.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        13,
+                        21,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        22,
+                        29,
+                        "docking",
+                        "experimental_method"
+                    ],
+                    [
+                        37,
+                        47,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        48,
+                        53,
+                        "shell",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 398,
+                "sent": "A single pentamer of the icosahedral T. maritima encapsulin structure (PDBID: 3DKT) is shown as a blue surface with the encapsulin localization sequence of EncFtn shown as a purple surface.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        9,
+                        17,
+                        "pentamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        25,
+                        36,
+                        "icosahedral",
+                        "protein_state"
+                    ],
+                    [
+                        37,
+                        48,
+                        "T. maritima",
+                        "species"
+                    ],
+                    [
+                        49,
+                        59,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        60,
+                        69,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        120,
+                        130,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        131,
+                        152,
+                        "localization sequence",
+                        "structure_element"
+                    ],
+                    [
+                        156,
+                        162,
+                        "EncFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 399,
+                "sent": "The C-terminal regions of the EncFtn subunits correspond to the position of the localization sequences seen in 3DKT.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        30,
+                        36,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        37,
+                        45,
+                        "subunits",
+                        "structure_element"
+                    ],
+                    [
+                        80,
+                        102,
+                        "localization sequences",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 400,
+                "sent": "Alignment of EncFtnsH with 3DKT positions the central channel directly above the pore in the 3DKT pentamer axis (shown as a grey pentagon). (B) Surface view of EncFtn within the encapsulin nanocompartment (grey and blue respectively).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Alignment",
+                        "experimental_method"
+                    ],
+                    [
+                        13,
+                        21,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        46,
+                        61,
+                        "central channel",
+                        "site"
+                    ],
+                    [
+                        81,
+                        85,
+                        "pore",
+                        "site"
+                    ],
+                    [
+                        98,
+                        106,
+                        "pentamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        160,
+                        166,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        178,
+                        188,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        189,
+                        204,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 401,
+                "sent": "The lumen of the encapsulin nanocompartment is considerably larger than the interior of ferritin (shown in orange behind the encapsulin for reference) and thus allows the storage of significantly more iron.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        17,
+                        27,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        28,
+                        43,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ],
+                    [
+                        88,
+                        96,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        125,
+                        135,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        201,
+                        205,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 402,
+                "sent": "The proposed pathway for iron movement through the encapsulin shell and EncFtn FOC is shown with arrows. (C) Model ofiron oxidation within an encapsulin nanocompartment.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        25,
+                        29,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        51,
+                        61,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        62,
+                        67,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        72,
+                        78,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        79,
+                        82,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        142,
+                        152,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        153,
+                        168,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 403,
+                "sent": "As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        3,
+                        9,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        34,
+                        38,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        64,
+                        68,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        91,
+                        101,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        102,
+                        107,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        128,
+                        146,
+                        "metal binding site",
+                        "site"
+                    ],
+                    [
+                        158,
+                        173,
+                        "central channel",
+                        "site"
+                    ],
+                    [
+                        177,
+                        185,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        187,
+                        197,
+                        "entry site",
+                        "site"
+                    ],
+                    [
+                        229,
+                        232,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        248,
+                        252,
+                        "Fe3+",
+                        "chemical"
+                    ],
+                    [
+                        341,
+                        351,
+                        "encapsulin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 404,
+                "sent": "Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT) \u00a0shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "Docking",
+                        "experimental_method"
+                    ],
+                    [
+                        12,
+                        19,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        20,
+                        29,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        33,
+                        41,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        51,
+                        59,
+                        "pentamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        67,
+                        78,
+                        "T. maritima",
+                        "species"
+                    ],
+                    [
+                        79,
+                        89,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        90,
+                        100,
+                        "Tmari_0786",
+                        "gene"
+                    ],
+                    [
+                        148,
+                        169,
+                        "C-terminal extensions",
+                        "structure_element"
+                    ],
+                    [
+                        177,
+                        185,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        186,
+                        195,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        220,
+                        242,
+                        "localization sequences",
+                        "structure_element"
+                    ],
+                    [
+                        248,
+                        256,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        261,
+                        271,
+                        "encapsulin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 405,
+                "sent": "Thus, it appears that the EncFtn decamer is the physiological state of this protein.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        26,
+                        32,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        33,
+                        40,
+                        "decamer",
+                        "oligomeric_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 406,
+                "sent": "This arrangement positions the central ring of EncFtn directly above the pore at the five-fold symmetry axis of the encapsulin shell and highlights a potential route for the entry of iron into the encapsulin and towards the active site of EncFtn.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        31,
+                        43,
+                        "central ring",
+                        "structure_element"
+                    ],
+                    [
+                        47,
+                        53,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        73,
+                        77,
+                        "pore",
+                        "site"
+                    ],
+                    [
+                        116,
+                        126,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        127,
+                        132,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        183,
+                        187,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        197,
+                        207,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        224,
+                        235,
+                        "active site",
+                        "site"
+                    ],
+                    [
+                        239,
+                        245,
+                        "EncFtn",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 407,
+                "sent": "A comparison of the encapsulin nanocompartment and the ferritin nanocage highlights the size differential between the two complexes (Figure 14B) that allows the encapsulin to store significantly more iron.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        20,
+                        30,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        31,
+                        46,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ],
+                    [
+                        55,
+                        63,
+                        "ferritin",
+                        "protein_type"
+                    ],
+                    [
+                        64,
+                        72,
+                        "nanocage",
+                        "complex_assembly"
+                    ],
+                    [
+                        161,
+                        171,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        200,
+                        204,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 408,
+                "sent": "The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        15,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        21,
+                        25,
+                        "FOCs",
+                        "site"
+                    ],
+                    [
+                        30,
+                        38,
+                        "EncFtnsH",
+                        "protein"
+                    ],
+                    [
+                        39,
+                        46,
+                        "decamer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        69,
+                        80,
+                        "icosahedral",
+                        "protein_state"
+                    ],
+                    [
+                        81,
+                        91,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        92,
+                        100,
+                        "nanocage",
+                        "complex_assembly"
+                    ],
+                    [
+                        126,
+                        135,
+                        "decameric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        136,
+                        142,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        234,
+                        238,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        272,
+                        287,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 409,
+                "sent": "This arrangement of multiple reaction centers in a single protein assembly is reminiscent of classical ferritins, which has 24 FOCs distributed around the nanocage.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        93,
+                        102,
+                        "classical",
+                        "protein_state"
+                    ],
+                    [
+                        103,
+                        112,
+                        "ferritins",
+                        "protein_type"
+                    ],
+                    [
+                        127,
+                        131,
+                        "FOCs",
+                        "site"
+                    ],
+                    [
+                        155,
+                        163,
+                        "nanocage",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 410,
+                "sent": "Our structural data, coupled with biochemical and ICP-MS analysis, suggest a model for the activity of the encapsulin iron-megastore (Figure 14C).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        19,
+                        "structural data",
+                        "evidence"
+                    ],
+                    [
+                        34,
+                        56,
+                        "biochemical and ICP-MS",
+                        "experimental_method"
+                    ],
+                    [
+                        107,
+                        117,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        118,
+                        132,
+                        "iron-megastore",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 411,
+                "sent": "The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4\u2014figure supplement 1).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        21,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        29,
+                        40,
+                        "T. maritima",
+                        "species"
+                    ],
+                    [
+                        41,
+                        51,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        52,
+                        57,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        72,
+                        95,
+                        "negatively charged pore",
+                        "site"
+                    ],
+                    [
+                        131,
+                        135,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        145,
+                        155,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        199,
+                        222,
+                        "negatively charged hole",
+                        "site"
+                    ],
+                    [
+                        230,
+                        236,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        237,
+                        241,
+                        "ring",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 412,
+                "sent": "The five metal-binding sites on the interior of the ring (Glu31/34-sites) may select for the Fe2+ ion and direct it towards their cognate FOCs.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        9,
+                        28,
+                        "metal-binding sites",
+                        "site"
+                    ],
+                    [
+                        52,
+                        56,
+                        "ring",
+                        "structure_element"
+                    ],
+                    [
+                        58,
+                        72,
+                        "Glu31/34-sites",
+                        "site"
+                    ],
+                    [
+                        93,
+                        97,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        138,
+                        142,
+                        "FOCs",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 413,
+                "sent": "We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by \u00a0in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        33,
+                        37,
+                        "Fe2+",
+                        "chemical"
+                    ],
+                    [
+                        41,
+                        45,
+                        "Fe3+",
+                        "chemical"
+                    ],
+                    [
+                        64,
+                        67,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        119,
+                        122,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        133,
+                        147,
+                        "substrate site",
+                        "site"
+                    ],
+                    [
+                        162,
+                        166,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        196,
+                        221,
+                        "weakly coordinating sites",
+                        "site"
+                    ],
+                    [
+                        265,
+                        270,
+                        "His57",
+                        "residue_name_number"
+                    ],
+                    [
+                        272,
+                        277,
+                        "Glu61",
+                        "residue_name_number"
+                    ],
+                    [
+                        282,
+                        287,
+                        "Glu64",
+                        "residue_name_number"
+                    ],
+                    [
+                        315,
+                        327,
+                        "ferrihydrite",
+                        "chemical"
+                    ],
+                    [
+                        391,
+                        401,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        402,
+                        417,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 414,
+                "sent": "Here we describe for the first time the structure and biochemistry of a new class of encapsulin-associated ferritin-like protein and demonstrate that it has an absolute requirement for compartmentalization within an encapsulin nanocage to act as an iron store.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        40,
+                        49,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        85,
+                        128,
+                        "encapsulin-associated ferritin-like protein",
+                        "protein_type"
+                    ],
+                    [
+                        216,
+                        226,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        227,
+                        235,
+                        "nanocage",
+                        "complex_assembly"
+                    ],
+                    [
+                        249,
+                        253,
+                        "iron",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 415,
+                "sent": "Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        20,
+                        30,
+                        "EncFtn-Enc",
+                        "complex_assembly"
+                    ],
+                    [
+                        31,
+                        46,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ],
+                    [
+                        103,
+                        107,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        120,
+                        130,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        131,
+                        136,
+                        "shell",
+                        "structure_element"
+                    ],
+                    [
+                        155,
+                        159,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        177,
+                        183,
+                        "EncFtn",
+                        "protein"
+                    ],
+                    [
+                        184,
+                        187,
+                        "FOC",
+                        "site"
+                    ],
+                    [
+                        235,
+                        245,
+                        "encapsulin",
+                        "protein"
+                    ],
+                    [
+                        246,
+                        261,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 416,
+                "sent": "TEM imaging was performed on purified encapsulin, EncFtn, and EncFtn-Enc and apoferritin.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        77,
+                        88,
+                        "apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 417,
+                "sent": "To observe iron mineral formation by TEM, protein samples at 8.5 \u00b5M concentration including EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were supplemented with acidic Fe(NH4)2(SO4)2 at their maximum iron loading ratio in room temperature for 1 hr.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        129,
+                        140,
+                        "apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 418,
+                "sent": "Horse spleen apoferritin preparation",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        13,
+                        24,
+                        "apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 419,
+                "sent": "Horse spleen apoferritin purchased from Sigma Aldrich (UK)\u00a0was dissolved in deaerated MOPS buffer (100 mM MOPS, 100 mM NaCl, 3 g/100 ml Na2S2O4 and 0.5 M EDTA, pH 6.5).",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        13,
+                        24,
+                        "apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 420,
+                "sent": "Fe content of apoferritin was detected using ferrozine assay.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        14,
+                        25,
+                        "apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 421,
+                "sent": "Apoferritin containing less than 0.5 Fe per 24-mer was used in the ferroxidase assay.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 422,
+                "sent": "Apoferritin used in the Fe loading capacity experiment was prepared in the same way with 5\u201315 Fe per 24-mer.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 423,
+                "sent": "In order to determine the maximum iron loading capacity, around 8.5 \u00b5M proteins including decameric EncFtnsH, Encapsulin, EncFtn-Enc and apoferritin were loaded with various amount of acidic Fe(NH4)2(SO4)2 ranging from 0 to 1700 \u00b5M. Protein mixtures were incubated in room temperature for 3 hrs before desalting in Zebra spin desalting columns (7 kDa cut-off, Thermo Fisher Scientific,\u00a0UK) to remove free iron ions.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        137,
+                        148,
+                        "apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 424,
+                "sent": "Both monomer and decamer fractions of EncFtnsH left at room temperature for 2 hrs, or overnight, were also analysed as controls to show the stability of the protein samples in the absence of additional metal ions.",
+                "section": "METHODS",
+                "ner": [
+                    [
+                        180,
+                        190,
+                        "absence of",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 425,
+                "sent": "Characterization of a Mycobacterium tuberculosis nanocompartment and its potential cargo proteins",
+                "section": "REF",
+                "ner": [
+                    [
+                        49,
+                        64,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 426,
+                "sent": "A virus capsid-like nanocompartment that stores iron and protects bacteria from oxidative stress",
+                "section": "REF",
+                "ner": [
+                    [
+                        20,
+                        35,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 427,
+                "sent": "Self-sorting of foreign proteins in a bacterial nanocompartment",
+                "section": "REF",
+                "ner": [
+                    [
+                        48,
+                        63,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 428,
+                "sent": "Structural basis of enzyme encapsulation into a bacterial nanocompartment",
+                "section": "REF",
+                "ner": [
+                    [
+                        58,
+                        73,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 429,
+                "sent": "1) Methods: What procedures and analyses did the author use to assess whether the iron added to the various ferritin derivatives was protein coated or was simply balls of rust attached to protein fragments? If the latter, it could easily generate reactive oxygen species in air under physiological conditions.",
+                "section": "REVIEW_INFO",
+                "ner": [
+                    [
+                        256,
+                        262,
+                        "oxygen",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 430,
+                "sent": "Even an experimental situation: 24 subunit (monomer) ferritin with a biomineral prepared experimentally from apoferritin and containing, on average, only 1000 iron atoms/24 subunit cage, the equivalent parameter appears to be 1000/24 = 42.",
+                "section": "REVIEW_INFO",
+                "ner": [
+                    [
+                        109,
+                        120,
+                        "apoferritin",
+                        "protein_state"
+                    ],
+                    [
+                        109,
+                        120,
+                        "apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 431,
+                "sent": "Missing are data for the starting material, 24 subunit ferritin or apoferritin (ferritin with the iron removed, by reduction and chelation, as a control.)",
+                "section": "REVIEW_INFO",
+                "ner": [
+                    [
+                        67,
+                        78,
+                        "apoferritin",
+                        "protein_state"
+                    ],
+                    [
+                        67,
+                        78,
+                        "apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 432,
+                "sent": "4) I would have liked to see some mutagenesis experiments to test the models of assembly, iron binding and ferroxidase activity.",
+                "section": "REVIEW_INFO",
+                "ner": [
+                    [
+                        34,
+                        45,
+                        "mutagenesis",
+                        "experimental_method"
+                    ],
+                    [
+                        107,
+                        118,
+                        "ferroxidase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 433,
+                "sent": "These results show the production of ROS by apoferritin, which is consistent with the published data on the reaction mechanism of certain ferritins; however, no significant ROS were detected for the EncFtn or encapsulin proteins.",
+                "section": "REVIEW_INFO",
+                "ner": [
+                    [
+                        44,
+                        55,
+                        "apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 434,
+                "sent": "We have clarified this key difference in the discussion of the iron storage function of the encapsulin nanocompartment (subsection \u201cIron storage in encapsulin nanocompartments\u201d, second paragraph).",
+                "section": "REVIEW_INFO",
+                "ner": [
+                    [
+                        103,
+                        118,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 435,
+                "sent": "The key conclusion of the paper is that the iron storage and iron oxidation functions that are combined in classical ferritins are split between the encapsulin nanocompartment and the EncFtn protein.",
+                "section": "REVIEW_INFO",
+                "ner": [
+                    [
+                        160,
+                        175,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 436,
+                "sent": "Control data for apoferritin have been added to this table and are illustrated in Figure 8.",
+                "section": "REVIEW_INFO",
+                "ner": [
+                    [
+                        17,
+                        28,
+                        "apoferritin",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 437,
+                "sent": "We note that we do not reach the experimental maximum loading capacity for apoferritin; however, we also note that the EncFtn-encapsulin nanocompartment sequesters five times more iron than the ferritin under the same reaction conditions, supporting the published observations that these nanocompartments can store more iron than classical ferritin nanocages.",
+                "section": "REVIEW_INFO",
+                "ner": [
+                    [
+                        75,
+                        86,
+                        "apoferritin",
+                        "protein_state"
+                    ],
+                    [
+                        137,
+                        152,
+                        "nanocompartment",
+                        "complex_assembly"
+                    ]
+                ]
+            }
+        ]
+    },
+    "PMC4981400": {
+        "annotations": [
+            {
+                "sid": 0,
+                "sent": "Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA",
+                "section": "TITLE",
+                "ner": [
+                    [
+                        0,
+                        17,
+                        "Crystal Structure",
+                        "evidence"
+                    ],
+                    [
+                        25,
+                        29,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        44,
+                        55,
+                        "Arabidopsis",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        56,
+                        75,
+                        "Flowering Regulator",
+                        "protein_type"
+                    ],
+                    [
+                        76,
+                        79,
+                        "FPA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 1,
+                "sent": "The Arabidopsis protein FPA controls flowering time by regulating the alternative 3\u2032-end processing of the FLOWERING LOCUS (FLC) antisense RNA.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        4,
+                        15,
+                        "Arabidopsis",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        24,
+                        27,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        107,
+                        122,
+                        "FLOWERING LOCUS",
+                        "gene"
+                    ],
+                    [
+                        124,
+                        127,
+                        "FLC",
+                        "gene"
+                    ],
+                    [
+                        129,
+                        142,
+                        "antisense RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 2,
+                "sent": "FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        19,
+                        29,
+                        "split ends",
+                        "protein_type"
+                    ],
+                    [
+                        31,
+                        35,
+                        "SPEN",
+                        "protein_type"
+                    ],
+                    [
+                        82,
+                        104,
+                        "RNA recognition motifs",
+                        "structure_element"
+                    ],
+                    [
+                        106,
+                        110,
+                        "RRMs",
+                        "structure_element"
+                    ],
+                    [
+                        118,
+                        154,
+                        "SPEN paralog and ortholog C-terminal",
+                        "structure_element"
+                    ],
+                    [
+                        156,
+                        160,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 3,
+                "sent": "The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        19,
+                        35,
+                        "highly conserved",
+                        "protein_state"
+                    ],
+                    [
+                        42,
+                        45,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        58,
+                        64,
+                        "plants",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        112,
+                        116,
+                        "SPEN",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 4,
+                "sent": "We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 \u00c5 resolution.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        23,
+                        40,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        44,
+                        64,
+                        "Arabidopsis thaliana",
+                        "species"
+                    ],
+                    [
+                        65,
+                        68,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        69,
+                        73,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 5,
+                "sent": "The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        12,
+                        21,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        48,
+                        52,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        63,
+                        68,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        69,
+                        108,
+                        "SMRT/HDAC1 Associated Repressor Protein",
+                        "protein"
+                    ],
+                    [
+                        110,
+                        115,
+                        "SHARP",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 6,
+                "sent": "Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        32,
+                        "Structural and sequence analyses",
+                        "experimental_method"
+                    ],
+                    [
+                        44,
+                        57,
+                        "surface patch",
+                        "site"
+                    ],
+                    [
+                        66,
+                        75,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        82,
+                        87,
+                        "plant",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        88,
+                        91,
+                        "FPA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 7,
+                "sent": "Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3\u2032-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        34,
+                        47,
+                        "surface patch",
+                        "site"
+                    ],
+                    [
+                        64,
+                        67,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        106,
+                        110,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        150,
+                        153,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        168,
+                        171,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        213,
+                        216,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        217,
+                        221,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        319,
+                        324,
+                        "SHARP",
+                        "protein"
+                    ],
+                    [
+                        325,
+                        329,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 8,
+                "sent": "Eukaryotic messenger RNAs (mRNAs) are made as precursors through transcription by RNA polymerase II (Pol II), and these primary transcripts undergo extensive processing, including 3\u2032-end cleavage and polyadenylation.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        10,
+                        "Eukaryotic",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        11,
+                        25,
+                        "messenger RNAs",
+                        "chemical"
+                    ],
+                    [
+                        27,
+                        32,
+                        "mRNAs",
+                        "chemical"
+                    ],
+                    [
+                        82,
+                        99,
+                        "RNA polymerase II",
+                        "complex_assembly"
+                    ],
+                    [
+                        101,
+                        107,
+                        "Pol II",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 9,
+                "sent": "In addition, alternative 3\u2032-end cleavage and polyadenylation is an essential and ubiquitous process in eukaryotes.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        103,
+                        113,
+                        "eukaryotes",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 10,
+                "sent": "Recently, the split ends (SPEN) family of proteins was identified as RNA binding proteins that regulate alternative 3\u2032-end cleavage and polyadenylation.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        14,
+                        24,
+                        "split ends",
+                        "protein_type"
+                    ],
+                    [
+                        26,
+                        30,
+                        "SPEN",
+                        "protein_type"
+                    ],
+                    [
+                        69,
+                        89,
+                        "RNA binding proteins",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 11,
+                "sent": "They are characterized by possessing N-terminal RNA recognition motifs (RRMs) and a conserved SPEN paralog and ortholog C-terminal (SPOC) domain (Fig 1A).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        48,
+                        70,
+                        "RNA recognition motifs",
+                        "structure_element"
+                    ],
+                    [
+                        72,
+                        76,
+                        "RRMs",
+                        "structure_element"
+                    ],
+                    [
+                        84,
+                        93,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        94,
+                        130,
+                        "SPEN paralog and ortholog C-terminal",
+                        "structure_element"
+                    ],
+                    [
+                        132,
+                        136,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 12,
+                "sent": "The SPOC domain is believed to mediate protein-protein interactions and has diverse functions among SPEN family proteins, but the molecular mechanism of these functions is not well understood.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        100,
+                        104,
+                        "SPEN",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 13,
+                "sent": "Sequence conservation of SPOC domains.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        21,
+                        "Sequence conservation",
+                        "evidence"
+                    ],
+                    [
+                        25,
+                        29,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 14,
+                "sent": "Domain organization of A. thaliana FPA. (B).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        23,
+                        34,
+                        "A. thaliana",
+                        "species"
+                    ],
+                    [
+                        35,
+                        38,
+                        "FPA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 15,
+                "sent": "Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        18,
+                        "Sequence alignment",
+                        "experimental_method"
+                    ],
+                    [
+                        26,
+                        30,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        42,
+                        62,
+                        "Arabidopsis thaliana",
+                        "species"
+                    ],
+                    [
+                        63,
+                        66,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        68,
+                        73,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        74,
+                        79,
+                        "RBM15",
+                        "protein"
+                    ],
+                    [
+                        81,
+                        91,
+                        "Drosophila",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        92,
+                        96,
+                        "SPEN",
+                        "protein_type"
+                    ],
+                    [
+                        98,
+                        103,
+                        "mouse",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        104,
+                        108,
+                        "MINT",
+                        "protein"
+                    ],
+                    [
+                        114,
+                        119,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        120,
+                        125,
+                        "SHARP",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 16,
+                "sent": "Residues in surface patch 1 are indicated with the orange dots, and those in surface patch 2 with the green dots.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        12,
+                        27,
+                        "surface patch 1",
+                        "site"
+                    ],
+                    [
+                        77,
+                        92,
+                        "surface patch 2",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 17,
+                "sent": "The secondary structure elements in the structure of FPA SPOC are labeled.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        40,
+                        49,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        53,
+                        56,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        57,
+                        61,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 18,
+                "sent": "Residues that are strictly conserved among the five proteins are shown in white with a red background, and those that are mostly conserved in red.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        18,
+                        36,
+                        "strictly conserved",
+                        "protein_state"
+                    ],
+                    [
+                        122,
+                        138,
+                        "mostly conserved",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 19,
+                "sent": "FPA, a SPEN family protein in Arabidopsis thaliana and other plants, was found to regulate the 3\u2032-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        7,
+                        11,
+                        "SPEN",
+                        "protein_type"
+                    ],
+                    [
+                        30,
+                        50,
+                        "Arabidopsis thaliana",
+                        "species"
+                    ],
+                    [
+                        61,
+                        67,
+                        "plants",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        150,
+                        164,
+                        "antisense RNAs",
+                        "chemical"
+                    ],
+                    [
+                        168,
+                        183,
+                        "FLOWERING LOCUS",
+                        "gene"
+                    ],
+                    [
+                        185,
+                        188,
+                        "FLC",
+                        "gene"
+                    ]
+                ]
+            },
+            {
+                "sid": 20,
+                "sent": "FPA promotes the 3\u2032-end processing of class I FLC antisense RNAs, which includes the proximal polyadenylation site.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        46,
+                        49,
+                        "FLC",
+                        "gene"
+                    ],
+                    [
+                        50,
+                        64,
+                        "antisense RNAs",
+                        "chemical"
+                    ],
+                    [
+                        94,
+                        114,
+                        "polyadenylation site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 21,
+                "sent": "This is associated with histone demethylase activity and down-regulation of FLC transcription.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        24,
+                        43,
+                        "histone demethylase",
+                        "protein_type"
+                    ],
+                    [
+                        76,
+                        79,
+                        "FLC",
+                        "gene"
+                    ]
+                ]
+            },
+            {
+                "sid": 22,
+                "sent": "Although a SPOC domain is found in all the SPEN family proteins, its sequence conservation is rather low.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        11,
+                        15,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        43,
+                        47,
+                        "SPEN",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 23,
+                "sent": "For example, the sequence identity between the SPOC domains of A. thaliana FPA and human SMRT/HDAC1 Associated Repressor Protein (SHARP) is only 19% (Fig 1B).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        47,
+                        51,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        63,
+                        74,
+                        "A. thaliana",
+                        "species"
+                    ],
+                    [
+                        75,
+                        78,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        83,
+                        88,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        89,
+                        128,
+                        "SMRT/HDAC1 Associated Repressor Protein",
+                        "protein"
+                    ],
+                    [
+                        130,
+                        135,
+                        "SHARP",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 24,
+                "sent": "Currently, the SHARP SPOC domain is the only one with structural information.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        15,
+                        20,
+                        "SHARP",
+                        "protein"
+                    ],
+                    [
+                        21,
+                        25,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 25,
+                "sent": "As a first step toward understanding the molecular basis for the regulation of alternative 3\u2032-end processing and flowering by FPA, we have determined the crystal structure of the SPOC domain of A. thaliana FPA at 2.7 \u00c5 resolution.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        126,
+                        129,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        154,
+                        171,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        179,
+                        183,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        194,
+                        205,
+                        "A. thaliana",
+                        "species"
+                    ],
+                    [
+                        206,
+                        209,
+                        "FPA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 26,
+                "sent": "The overall structure is similar to that of the SHARP SPOC domain, although there are also substantial conformational differences between them.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        12,
+                        21,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        48,
+                        53,
+                        "SHARP",
+                        "protein"
+                    ],
+                    [
+                        54,
+                        58,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 27,
+                "sent": "The structure reveals a surface patch that is conserved among FPA homologs.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        24,
+                        37,
+                        "surface patch",
+                        "site"
+                    ],
+                    [
+                        46,
+                        55,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        62,
+                        65,
+                        "FPA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 28,
+                "sent": "Structure of FPA SPOC domain",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Structure",
+                        "evidence"
+                    ],
+                    [
+                        13,
+                        16,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        17,
+                        21,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 29,
+                "sent": "The crystal structure of the SPOC domain of A. thaliana FPA has been determined at 2.7 \u00c5 resolution using the selenomethionyl single-wavelength anomalous dispersion method.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        21,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        29,
+                        33,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        44,
+                        55,
+                        "A. thaliana",
+                        "species"
+                    ],
+                    [
+                        56,
+                        59,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        110,
+                        171,
+                        "selenomethionyl single-wavelength anomalous dispersion method",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 30,
+                "sent": "The expression construct contained residues 433\u2013565 of FPA, but only residues 439\u2013460 and 465\u2013565 are ordered in the crystal.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        44,
+                        51,
+                        "433\u2013565",
+                        "residue_range"
+                    ],
+                    [
+                        55,
+                        58,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        78,
+                        85,
+                        "439\u2013460",
+                        "residue_range"
+                    ],
+                    [
+                        90,
+                        97,
+                        "465\u2013565",
+                        "residue_range"
+                    ],
+                    [
+                        117,
+                        124,
+                        "crystal",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 31,
+                "sent": "The atomic model has good agreement with the X-ray diffraction data and the expected bond lengths, bond angles and other geometric parameters (Table 1).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        16,
+                        "atomic model",
+                        "evidence"
+                    ],
+                    [
+                        45,
+                        67,
+                        "X-ray diffraction data",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 32,
+                "sent": "All the residues are located in the favored regions of the Ramachandran plot (data not shown).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        59,
+                        76,
+                        "Ramachandran plot",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 33,
+                "sent": "The structure has been deposited in the Protein Data Bank, with accession code 5KXF.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 34,
+                "sent": "Resolution range (\u00c5)1\t50\u20132.7 (2.8\u20132.7)\t \tNumber of observations\t78,008\t \tRmerge (%)\t10.5 (45.3)\t \tI/\u03c3I\t24.1 (6.3)\t \tRedundancy\t\t \tCompleteness (%)\t100 (100)\t \tR factor (%)\t19.2 (25.0)\t \tFree R factor (%)\t25.4 (35.4)\t \tRms deviation in bond lengths (\u00c5)\t0.017\t \tRms deviation in bond angles (\u00b0)\t1.9\t \t",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        159,
+                        167,
+                        "R factor",
+                        "evidence"
+                    ],
+                    [
+                        186,
+                        199,
+                        "Free R factor",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 35,
+                "sent": "The crystal structure of the FPA SPOC domain contains a seven-stranded, mostly anti-parallel \u03b2-barrel (\u03b21-\u03b27) and three helices (\u03b1A-\u03b1C) (Fig 2A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        21,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        29,
+                        32,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        33,
+                        37,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        56,
+                        101,
+                        "seven-stranded, mostly anti-parallel \u03b2-barrel",
+                        "structure_element"
+                    ],
+                    [
+                        103,
+                        108,
+                        "\u03b21-\u03b27",
+                        "structure_element"
+                    ],
+                    [
+                        120,
+                        127,
+                        "helices",
+                        "structure_element"
+                    ],
+                    [
+                        129,
+                        134,
+                        "\u03b1A-\u03b1C",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 36,
+                "sent": "Only two of the neighboring strands, \u03b21 and \u03b23, are parallel to each other.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        28,
+                        35,
+                        "strands",
+                        "structure_element"
+                    ],
+                    [
+                        37,
+                        39,
+                        "\u03b21",
+                        "structure_element"
+                    ],
+                    [
+                        44,
+                        46,
+                        "\u03b23",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 37,
+                "sent": "Helix \u03b1B covers one end of the barrel, while helices \u03b1A and \u03b1C are located next to each other at one side of the barrel (Fig 2B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "Helix",
+                        "structure_element"
+                    ],
+                    [
+                        6,
+                        8,
+                        "\u03b1B",
+                        "structure_element"
+                    ],
+                    [
+                        31,
+                        37,
+                        "barrel",
+                        "structure_element"
+                    ],
+                    [
+                        45,
+                        52,
+                        "helices",
+                        "structure_element"
+                    ],
+                    [
+                        53,
+                        55,
+                        "\u03b1A",
+                        "structure_element"
+                    ],
+                    [
+                        60,
+                        62,
+                        "\u03b1C",
+                        "structure_element"
+                    ],
+                    [
+                        113,
+                        119,
+                        "barrel",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 38,
+                "sent": "The other end of the \u03b2-barrel is covered by the loop connecting strands \u03b22 and \u03b23, which contains the disordered 461\u2013464 segment.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        21,
+                        29,
+                        "\u03b2-barrel",
+                        "structure_element"
+                    ],
+                    [
+                        48,
+                        52,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        71,
+                        "strands",
+                        "structure_element"
+                    ],
+                    [
+                        72,
+                        74,
+                        "\u03b22",
+                        "structure_element"
+                    ],
+                    [
+                        79,
+                        81,
+                        "\u03b23",
+                        "structure_element"
+                    ],
+                    [
+                        102,
+                        112,
+                        "disordered",
+                        "protein_state"
+                    ],
+                    [
+                        113,
+                        120,
+                        "461\u2013464",
+                        "residue_range"
+                    ]
+                ]
+            },
+            {
+                "sid": 39,
+                "sent": "The center of the barrel is filled with hydrophobic side chains and is not accessible to the solvent.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        24,
+                        "barrel",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 40,
+                "sent": "Crystal structure of the SPOC domain of A. thaliana FPA.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        17,
+                        "Crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        25,
+                        29,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        40,
+                        51,
+                        "A. thaliana",
+                        "species"
+                    ],
+                    [
+                        52,
+                        55,
+                        "FPA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 41,
+                "sent": "Schematic drawing of the structure of FPA SPOC domain, colored from blue at the N terminus to red at the C terminus.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        25,
+                        34,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        38,
+                        41,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        42,
+                        46,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 42,
+                "sent": "The view is from the side of the \u03b2-barrel.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        33,
+                        41,
+                        "\u03b2-barrel",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 43,
+                "sent": "The disordered segment (residues 460\u2013465) is indicated with the dotted line.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        14,
+                        "disordered",
+                        "protein_state"
+                    ],
+                    [
+                        33,
+                        40,
+                        "460\u2013465",
+                        "residue_range"
+                    ]
+                ]
+            },
+            {
+                "sid": 44,
+                "sent": "Structure of the FPA SPOC domain, viewed from the end of the \u03b2-barrel, after 90\u00b0 rotation around the horizontal axis from panel A. All structure figures were produced with PyMOL (www.pymol.org).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Structure",
+                        "evidence"
+                    ],
+                    [
+                        17,
+                        20,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        21,
+                        25,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        61,
+                        69,
+                        "\u03b2-barrel",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 45,
+                "sent": "Comparisons to structural homologs of the SPOC domain",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        34,
+                        "Comparisons to structural homologs",
+                        "experimental_method"
+                    ],
+                    [
+                        42,
+                        46,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 46,
+                "sent": "Only five structural homologs of the FPA SPOC domain were found in the Protein Data Bank with the DaliLite server, suggesting that the SPOC domain structure is relatively unique.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        37,
+                        40,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        41,
+                        45,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        98,
+                        113,
+                        "DaliLite server",
+                        "experimental_method"
+                    ],
+                    [
+                        135,
+                        139,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        147,
+                        156,
+                        "structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 47,
+                "sent": "The top hit is the SPOC domain of human SHARP (Fig 3A), with a Z score of 12.3.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        23,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        34,
+                        39,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        40,
+                        45,
+                        "SHARP",
+                        "protein"
+                    ],
+                    [
+                        63,
+                        70,
+                        "Z score",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 48,
+                "sent": "The other four structural homologs include the \u03b2-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        47,
+                        55,
+                        "\u03b2-barrel",
+                        "structure_element"
+                    ],
+                    [
+                        79,
+                        83,
+                        "Ku70",
+                        "protein"
+                    ],
+                    [
+                        88,
+                        92,
+                        "Ku80",
+                        "protein"
+                    ],
+                    [
+                        94,
+                        101,
+                        "Z score",
+                        "evidence"
+                    ],
+                    [
+                        134,
+                        154,
+                        "chromodomain protein",
+                        "protein_type"
+                    ],
+                    [
+                        155,
+                        159,
+                        "Chp1",
+                        "protein"
+                    ],
+                    [
+                        161,
+                        168,
+                        "Z score",
+                        "evidence"
+                    ],
+                    [
+                        193,
+                        221,
+                        "activator interacting domain",
+                        "structure_element"
+                    ],
+                    [
+                        223,
+                        227,
+                        "ACID",
+                        "structure_element"
+                    ],
+                    [
+                        236,
+                        241,
+                        "Med25",
+                        "protein"
+                    ],
+                    [
+                        275,
+                        282,
+                        "Z score",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 49,
+                "sent": "The next structural homolog has a Z score of 3.0.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        41,
+                        "Z score",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 50,
+                "sent": "Structural homologs of the FPA SPOC domain.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        27,
+                        30,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        31,
+                        35,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 51,
+                "sent": "Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "Overlay",
+                        "experimental_method"
+                    ],
+                    [
+                        15,
+                        25,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        33,
+                        36,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        37,
+                        41,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        69,
+                        "SHARP",
+                        "protein"
+                    ],
+                    [
+                        70,
+                        74,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 52,
+                "sent": "The bound position of a doubly-phosphorylated peptide from SMRT is shown in magenta.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        24,
+                        45,
+                        "doubly-phosphorylated",
+                        "protein_state"
+                    ],
+                    [
+                        46,
+                        53,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        59,
+                        63,
+                        "SMRT",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 53,
+                "sent": "Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 \u03b2-barrel domain (gray).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "Overlay",
+                        "experimental_method"
+                    ],
+                    [
+                        15,
+                        25,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        33,
+                        36,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        37,
+                        41,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        68,
+                        "Ku70",
+                        "protein"
+                    ],
+                    [
+                        69,
+                        77,
+                        "\u03b2-barrel",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 54,
+                "sent": "Ku80 contains a homologous domain (green), which forms a hetero-dimer with that in Ku70.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "Ku80",
+                        "protein"
+                    ],
+                    [
+                        57,
+                        69,
+                        "hetero-dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        83,
+                        87,
+                        "Ku70",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 55,
+                "sent": "The two domains, and inserted segments on them, mediate the binding of dsDNA (orange).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        71,
+                        76,
+                        "dsDNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 56,
+                "sent": "The red rectangle highlights the region of contact between the two \u03b2-barrel domains.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        67,
+                        75,
+                        "\u03b2-barrel",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 57,
+                "sent": "Overlay of the structures of the FPA SPOC domain (cyan) and the homologous domain in Chp1 (gray).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "Overlay",
+                        "experimental_method"
+                    ],
+                    [
+                        15,
+                        25,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        33,
+                        36,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        37,
+                        41,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        85,
+                        89,
+                        "Chp1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 58,
+                "sent": "The binding partner of Chp1, Tas3, is shown in green.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        23,
+                        27,
+                        "Chp1",
+                        "protein"
+                    ],
+                    [
+                        29,
+                        33,
+                        "Tas3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 59,
+                "sent": "The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (D).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        57,
+                        69,
+                        "binding site",
+                        "site"
+                    ],
+                    [
+                        77,
+                        82,
+                        "SMART",
+                        "protein"
+                    ],
+                    [
+                        83,
+                        97,
+                        "phosphopeptide",
+                        "ptm"
+                    ],
+                    [
+                        101,
+                        106,
+                        "SHARP",
+                        "protein"
+                    ],
+                    [
+                        107,
+                        111,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        128,
+                        132,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        136,
+                        140,
+                        "Tas3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 60,
+                "sent": "Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "Overlay",
+                        "experimental_method"
+                    ],
+                    [
+                        15,
+                        25,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        33,
+                        36,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        37,
+                        41,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        69,
+                        "Med25",
+                        "protein"
+                    ],
+                    [
+                        70,
+                        74,
+                        "ACID",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 61,
+                "sent": "SHARP is a transcriptional co-repressor in the nuclear receptor and Notch/RBP-J\u03ba signaling pathways.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "SHARP",
+                        "protein"
+                    ],
+                    [
+                        11,
+                        39,
+                        "transcriptional co-repressor",
+                        "protein_type"
+                    ],
+                    [
+                        47,
+                        63,
+                        "nuclear receptor",
+                        "protein_type"
+                    ],
+                    [
+                        68,
+                        73,
+                        "Notch",
+                        "protein"
+                    ],
+                    [
+                        74,
+                        80,
+                        "RBP-J\u03ba",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 62,
+                "sent": "The SPOC domain of SHARP interacts directly with silencing mediator for retinoid and thyroid receptor (SMRT), nuclear receptor co-repressor (N-CoR), HDAC, and other components to represses transcription.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        19,
+                        24,
+                        "SHARP",
+                        "protein"
+                    ],
+                    [
+                        49,
+                        101,
+                        "silencing mediator for retinoid and thyroid receptor",
+                        "protein"
+                    ],
+                    [
+                        103,
+                        107,
+                        "SMRT",
+                        "protein"
+                    ],
+                    [
+                        110,
+                        139,
+                        "nuclear receptor co-repressor",
+                        "protein_type"
+                    ],
+                    [
+                        141,
+                        146,
+                        "N-CoR",
+                        "protein_type"
+                    ],
+                    [
+                        149,
+                        153,
+                        "HDAC",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 63,
+                "sent": "While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the \u03b2-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        27,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        35,
+                        38,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        39,
+                        43,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        77,
+                        82,
+                        "SHARP",
+                        "protein"
+                    ],
+                    [
+                        83,
+                        87,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        155,
+                        164,
+                        "\u03b2-strands",
+                        "structure_element"
+                    ],
+                    [
+                        173,
+                        180,
+                        "helices",
+                        "structure_element"
+                    ],
+                    [
+                        198,
+                        203,
+                        "loops",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 64,
+                "sent": "In addition, the SHARP SPOC domain has three extra helices.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        17,
+                        22,
+                        "SHARP",
+                        "protein"
+                    ],
+                    [
+                        23,
+                        27,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        51,
+                        58,
+                        "helices",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 65,
+                "sent": "One of them covers the other end of the \u03b2-barrel, and the other two shield an additional surface of the side of the \u03b2-barrel from solvent.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        40,
+                        48,
+                        "\u03b2-barrel",
+                        "structure_element"
+                    ],
+                    [
+                        116,
+                        124,
+                        "\u03b2-barrel",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 66,
+                "sent": "A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands \u03b21 and \u03b23 (Fig 3A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        2,
+                        23,
+                        "doubly-phosphorylated",
+                        "protein_state"
+                    ],
+                    [
+                        24,
+                        31,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        37,
+                        41,
+                        "SMRT",
+                        "protein"
+                    ],
+                    [
+                        45,
+                        53,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        70,
+                        76,
+                        "barrel",
+                        "structure_element"
+                    ],
+                    [
+                        83,
+                        90,
+                        "strands",
+                        "structure_element"
+                    ],
+                    [
+                        91,
+                        93,
+                        "\u03b21",
+                        "structure_element"
+                    ],
+                    [
+                        98,
+                        100,
+                        "\u03b23",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 67,
+                "sent": "Such a binding mode probably would not be possible in FPA, as the peptide would clash with the \u03b21-\u03b22 loop.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        54,
+                        57,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        66,
+                        73,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        95,
+                        105,
+                        "\u03b21-\u03b22 loop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 68,
+                "sent": "The Ku70-Ku80 hetero-dimer is involved in DNA double-strand break repair and the \u03b2-barrel domain contributes to DNA binding.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "Ku70-Ku80",
+                        "complex_assembly"
+                    ],
+                    [
+                        14,
+                        26,
+                        "hetero-dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        81,
+                        89,
+                        "\u03b2-barrel",
+                        "structure_element"
+                    ],
+                    [
+                        112,
+                        115,
+                        "DNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 69,
+                "sent": "In fact, the \u03b2-barrel domains of Ku70 and Ku80 form a hetero-dimer, primarily through interactions between the loops connecting the third and fourth strands of the barrel (Fig 3B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        21,
+                        "\u03b2-barrel",
+                        "structure_element"
+                    ],
+                    [
+                        33,
+                        37,
+                        "Ku70",
+                        "protein"
+                    ],
+                    [
+                        42,
+                        46,
+                        "Ku80",
+                        "protein"
+                    ],
+                    [
+                        54,
+                        66,
+                        "hetero-dimer",
+                        "oligomeric_state"
+                    ],
+                    [
+                        111,
+                        116,
+                        "loops",
+                        "structure_element"
+                    ],
+                    [
+                        132,
+                        156,
+                        "third and fourth strands",
+                        "structure_element"
+                    ],
+                    [
+                        164,
+                        170,
+                        "barrel",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 70,
+                "sent": "The open ends of the two \u03b2-barrels face the DNA binding sites, and contact the phosphodiester backbone of the dsDNA.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        34,
+                        "\u03b2-barrels",
+                        "structure_element"
+                    ],
+                    [
+                        44,
+                        61,
+                        "DNA binding sites",
+                        "site"
+                    ],
+                    [
+                        110,
+                        115,
+                        "dsDNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 71,
+                "sent": "In addition, a long insert connecting strands \u03b22 and \u03b23 in the two domains form an arch-like structure, encircling the dsDNA.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        15,
+                        26,
+                        "long insert",
+                        "structure_element"
+                    ],
+                    [
+                        38,
+                        45,
+                        "strands",
+                        "structure_element"
+                    ],
+                    [
+                        46,
+                        48,
+                        "\u03b22",
+                        "structure_element"
+                    ],
+                    [
+                        53,
+                        55,
+                        "\u03b23",
+                        "structure_element"
+                    ],
+                    [
+                        83,
+                        102,
+                        "arch-like structure",
+                        "structure_element"
+                    ],
+                    [
+                        119,
+                        124,
+                        "dsDNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 72,
+                "sent": "Chp1 is a subunit of the RNA-induced initiation of transcriptional gene silencing (RITS) complex.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "Chp1",
+                        "protein"
+                    ],
+                    [
+                        25,
+                        81,
+                        "RNA-induced initiation of transcriptional gene silencing",
+                        "complex_assembly"
+                    ],
+                    [
+                        83,
+                        87,
+                        "RITS",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 73,
+                "sent": "The partner of Chp1, Tas3, is bound between the barrel domain and the second domain of Chp1, and the linker between the two domains is also crucial for this interaction (Fig 3C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        15,
+                        19,
+                        "Chp1",
+                        "protein"
+                    ],
+                    [
+                        21,
+                        25,
+                        "Tas3",
+                        "protein"
+                    ],
+                    [
+                        48,
+                        61,
+                        "barrel domain",
+                        "structure_element"
+                    ],
+                    [
+                        70,
+                        83,
+                        "second domain",
+                        "structure_element"
+                    ],
+                    [
+                        87,
+                        91,
+                        "Chp1",
+                        "protein"
+                    ],
+                    [
+                        101,
+                        107,
+                        "linker",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 74,
+                "sent": "It is probably unlikely that the \u03b2-barrel itself is sufficient to bind Tas3.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        33,
+                        41,
+                        "\u03b2-barrel",
+                        "structure_element"
+                    ],
+                    [
+                        71,
+                        75,
+                        "Tas3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 75,
+                "sent": "Interestingly, a loop in Tas3 contacts strand \u03b23 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        17,
+                        21,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        25,
+                        29,
+                        "Tas3",
+                        "protein"
+                    ],
+                    [
+                        39,
+                        45,
+                        "strand",
+                        "structure_element"
+                    ],
+                    [
+                        46,
+                        48,
+                        "\u03b23",
+                        "structure_element"
+                    ],
+                    [
+                        56,
+                        69,
+                        "barrel domain",
+                        "structure_element"
+                    ],
+                    [
+                        143,
+                        147,
+                        "SMRT",
+                        "protein"
+                    ],
+                    [
+                        148,
+                        155,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        156,
+                        171,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        172,
+                        177,
+                        "SHARP",
+                        "protein"
+                    ],
+                    [
+                        178,
+                        182,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 76,
+                "sent": "Mediator is a coactivator complex that promotes transcription by Pol II.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "Mediator",
+                        "protein_type"
+                    ],
+                    [
+                        65,
+                        71,
+                        "Pol II",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 77,
+                "sent": "The Med25 subunit ACID is the target of the potent activator VP16 of the herpes simplex virus.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        9,
+                        "Med25",
+                        "protein"
+                    ],
+                    [
+                        18,
+                        22,
+                        "ACID",
+                        "structure_element"
+                    ],
+                    [
+                        61,
+                        65,
+                        "VP16",
+                        "protein"
+                    ],
+                    [
+                        73,
+                        93,
+                        "herpes simplex virus",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 78,
+                "sent": "The structure of ACID contains a helix at the C-terminus as well as an extended \u03b21-\u03b22 loop.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        17,
+                        21,
+                        "ACID",
+                        "structure_element"
+                    ],
+                    [
+                        33,
+                        38,
+                        "helix",
+                        "structure_element"
+                    ],
+                    [
+                        80,
+                        90,
+                        "\u03b21-\u03b22 loop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 79,
+                "sent": "Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands \u03b21 and \u03b23, that is used by the SHARP and Tas3 SPOC domains for binding their partners.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        17,
+                        29,
+                        "binding site",
+                        "site"
+                    ],
+                    [
+                        34,
+                        38,
+                        "VP16",
+                        "protein"
+                    ],
+                    [
+                        75,
+                        88,
+                        "surface patch",
+                        "site"
+                    ],
+                    [
+                        95,
+                        102,
+                        "strands",
+                        "structure_element"
+                    ],
+                    [
+                        103,
+                        105,
+                        "\u03b21",
+                        "structure_element"
+                    ],
+                    [
+                        110,
+                        112,
+                        "\u03b23",
+                        "structure_element"
+                    ],
+                    [
+                        134,
+                        139,
+                        "SHARP",
+                        "protein"
+                    ],
+                    [
+                        144,
+                        148,
+                        "Tas3",
+                        "protein"
+                    ],
+                    [
+                        149,
+                        153,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 80,
+                "sent": "A conserved surface patch in the FPA SPOC domain",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        2,
+                        11,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        12,
+                        25,
+                        "surface patch",
+                        "site"
+                    ],
+                    [
+                        33,
+                        36,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        37,
+                        41,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 81,
+                "sent": "An analysis of the SPOC domain indicates a large surface patch near strands \u03b21, \u03b23, \u03b25 and \u03b26 that is conserved among plant FPA homologs (Fig 4A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        23,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        49,
+                        62,
+                        "surface patch",
+                        "site"
+                    ],
+                    [
+                        68,
+                        75,
+                        "strands",
+                        "structure_element"
+                    ],
+                    [
+                        76,
+                        78,
+                        "\u03b21",
+                        "structure_element"
+                    ],
+                    [
+                        80,
+                        82,
+                        "\u03b23",
+                        "structure_element"
+                    ],
+                    [
+                        84,
+                        86,
+                        "\u03b25",
+                        "structure_element"
+                    ],
+                    [
+                        91,
+                        93,
+                        "\u03b26",
+                        "structure_element"
+                    ],
+                    [
+                        102,
+                        111,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        118,
+                        123,
+                        "plant",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        124,
+                        127,
+                        "FPA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 82,
+                "sent": "This surface patch can be broken into two sub-patches, with residues Lys447 (in strand \u03b21), Arg477 (\u03b23), Tyr515 (\u03b1B) and Arg521 (\u03b25) in one sub-patch, and residues His486 (\u03b1A), Thr478 (\u03b23), Val524 (\u03b25) and Phe534 (\u03b26) in the other sub-patch (Fig 4B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        18,
+                        "surface patch",
+                        "site"
+                    ],
+                    [
+                        42,
+                        53,
+                        "sub-patches",
+                        "site"
+                    ],
+                    [
+                        69,
+                        75,
+                        "Lys447",
+                        "residue_name_number"
+                    ],
+                    [
+                        80,
+                        86,
+                        "strand",
+                        "structure_element"
+                    ],
+                    [
+                        87,
+                        89,
+                        "\u03b21",
+                        "structure_element"
+                    ],
+                    [
+                        92,
+                        98,
+                        "Arg477",
+                        "residue_name_number"
+                    ],
+                    [
+                        100,
+                        102,
+                        "\u03b23",
+                        "structure_element"
+                    ],
+                    [
+                        105,
+                        111,
+                        "Tyr515",
+                        "residue_name_number"
+                    ],
+                    [
+                        113,
+                        115,
+                        "\u03b1B",
+                        "structure_element"
+                    ],
+                    [
+                        121,
+                        127,
+                        "Arg521",
+                        "residue_name_number"
+                    ],
+                    [
+                        129,
+                        131,
+                        "\u03b25",
+                        "structure_element"
+                    ],
+                    [
+                        140,
+                        149,
+                        "sub-patch",
+                        "site"
+                    ],
+                    [
+                        164,
+                        170,
+                        "His486",
+                        "residue_name_number"
+                    ],
+                    [
+                        172,
+                        174,
+                        "\u03b1A",
+                        "structure_element"
+                    ],
+                    [
+                        177,
+                        183,
+                        "Thr478",
+                        "residue_name_number"
+                    ],
+                    [
+                        185,
+                        187,
+                        "\u03b23",
+                        "structure_element"
+                    ],
+                    [
+                        190,
+                        196,
+                        "Val524",
+                        "residue_name_number"
+                    ],
+                    [
+                        198,
+                        200,
+                        "\u03b25",
+                        "structure_element"
+                    ],
+                    [
+                        206,
+                        212,
+                        "Phe534",
+                        "residue_name_number"
+                    ],
+                    [
+                        214,
+                        216,
+                        "\u03b26",
+                        "structure_element"
+                    ],
+                    [
+                        231,
+                        240,
+                        "sub-patch",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 83,
+                "sent": "The first surface patch is electropositive in nature (Fig 4C), and residues Arg477 and Tyr515 are also conserved in the SHARP SPOC domain (Fig 1B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        23,
+                        "first surface patch",
+                        "site"
+                    ],
+                    [
+                        27,
+                        42,
+                        "electropositive",
+                        "protein_state"
+                    ],
+                    [
+                        76,
+                        82,
+                        "Arg477",
+                        "residue_name_number"
+                    ],
+                    [
+                        87,
+                        93,
+                        "Tyr515",
+                        "residue_name_number"
+                    ],
+                    [
+                        103,
+                        112,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        120,
+                        125,
+                        "SHARP",
+                        "protein"
+                    ],
+                    [
+                        126,
+                        130,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 84,
+                "sent": "In fact, one of the phosphorylated residues of the SMRT peptide interacts with this surface patch (Fig 3A), suggesting that the FPA SPOC domain might also interact with a phosphorylated segment here.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        20,
+                        34,
+                        "phosphorylated",
+                        "protein_state"
+                    ],
+                    [
+                        51,
+                        55,
+                        "SMRT",
+                        "protein"
+                    ],
+                    [
+                        56,
+                        63,
+                        "peptide",
+                        "chemical"
+                    ],
+                    [
+                        84,
+                        97,
+                        "surface patch",
+                        "site"
+                    ],
+                    [
+                        128,
+                        131,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        132,
+                        136,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        171,
+                        185,
+                        "phosphorylated",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 85,
+                "sent": "In comparison, the second surface patch is more hydrophobic in nature (Fig 4C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        39,
+                        "second surface patch",
+                        "site"
+                    ],
+                    [
+                        48,
+                        59,
+                        "hydrophobic",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 86,
+                "sent": "A conserved surface patch of FPA SPOC domain.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        2,
+                        11,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        12,
+                        25,
+                        "surface patch",
+                        "site"
+                    ],
+                    [
+                        29,
+                        32,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        33,
+                        37,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 87,
+                "sent": "Two views of the molecular surface of FPA SPOC domain colored based on sequence conservation among plant FPA homologs.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        38,
+                        41,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        42,
+                        46,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        99,
+                        104,
+                        "plant",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        105,
+                        108,
+                        "FPA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 88,
+                "sent": "Residues in the conserved surface patch of FPA SPOC domain.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        16,
+                        25,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        26,
+                        39,
+                        "surface patch",
+                        "site"
+                    ],
+                    [
+                        43,
+                        46,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        47,
+                        51,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 89,
+                "sent": "The side chains of the residues are shown in stick models, colored orange in the first sub-patch and green in the second. (C).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        81,
+                        96,
+                        "first sub-patch",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 90,
+                "sent": "Molecular surface of FPA SPOC domain colored based on electrostatic potential.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        21,
+                        24,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        25,
+                        29,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 91,
+                "sent": "Testing the requirement of specific conserved amino acids for FPA functions",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        62,
+                        65,
+                        "FPA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 92,
+                "sent": "We next examined the potential impact of the conserved surface patch on FPA function in vivo.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        45,
+                        54,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        55,
+                        68,
+                        "surface patch",
+                        "site"
+                    ],
+                    [
+                        72,
+                        75,
+                        "FPA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 93,
+                "sent": "We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain (Fig 1B) and were found to be functionally important.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        3,
+                        10,
+                        "mutated",
+                        "experimental_method"
+                    ],
+                    [
+                        25,
+                        31,
+                        "Arg477",
+                        "residue_name_number"
+                    ],
+                    [
+                        36,
+                        42,
+                        "Tyr515",
+                        "residue_name_number"
+                    ],
+                    [
+                        51,
+                        64,
+                        "surface patch",
+                        "site"
+                    ],
+                    [
+                        81,
+                        90,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        98,
+                        103,
+                        "SHARP",
+                        "protein"
+                    ],
+                    [
+                        104,
+                        108,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 94,
+                "sent": "The mutations were introduced into a transgene designed to express FPA from its native control elements (promoter, introns and 3\u2032 UTR).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        19,
+                        29,
+                        "introduced",
+                        "experimental_method"
+                    ],
+                    [
+                        67,
+                        70,
+                        "FPA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 95,
+                "sent": "The resulting transgenes were then stably transformed into an fpa-8 mutant background so that the impact of the mutations on FPA function could be assessed.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        35,
+                        53,
+                        "stably transformed",
+                        "experimental_method"
+                    ],
+                    [
+                        62,
+                        67,
+                        "fpa-8",
+                        "gene"
+                    ],
+                    [
+                        68,
+                        74,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        112,
+                        121,
+                        "mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        125,
+                        128,
+                        "FPA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 96,
+                "sent": "Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3\u2032-end formation, for example in FPA pre-mRNA (panel B in S1 Fig).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        35,
+                        56,
+                        "expression constructs",
+                        "experimental_method"
+                    ],
+                    [
+                        62,
+                        67,
+                        "fpa-8",
+                        "gene"
+                    ],
+                    [
+                        88,
+                        97,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        98,
+                        101,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        119,
+                        122,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        131,
+                        148,
+                        "expression levels",
+                        "evidence"
+                    ],
+                    [
+                        157,
+                        166,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        222,
+                        225,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        241,
+                        244,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        278,
+                        281,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        282,
+                        290,
+                        "pre-mRNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 97,
+                "sent": "We examined independent transgenic lines expressing each R477A and Y515A mutation.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        57,
+                        62,
+                        "R477A",
+                        "mutant"
+                    ],
+                    [
+                        67,
+                        72,
+                        "Y515A",
+                        "mutant"
+                    ],
+                    [
+                        73,
+                        81,
+                        "mutation",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 98,
+                "sent": "In each case, we confirmed that detectable levels of FPA protein expression were restored close to wild-type levels in protein blot analyses using antibodies that specifically recognize FPA (S2 Fig).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        53,
+                        56,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        99,
+                        108,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        119,
+                        131,
+                        "protein blot",
+                        "experimental_method"
+                    ],
+                    [
+                        186,
+                        189,
+                        "FPA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 99,
+                "sent": "We then examined the impact of the surface patch mutations on FPA\u2019s function in controlling RNA 3\u2032-end formation by determining whether the mutant proteins functioned in FPA autoregulation and the repression of FLC expression.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        35,
+                        48,
+                        "surface patch",
+                        "site"
+                    ],
+                    [
+                        49,
+                        58,
+                        "mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        62,
+                        65,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        140,
+                        146,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        170,
+                        173,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        211,
+                        214,
+                        "FLC",
+                        "gene"
+                    ]
+                ]
+            },
+            {
+                "sid": 100,
+                "sent": "FPA autoregulates its expression by promoting cleavage and polyadenylation within intron 1 of its own pre-mRNA, resulting in a truncated transcript that does not encode functional protein.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        102,
+                        110,
+                        "pre-mRNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 101,
+                "sent": "We used RNA gel blot analyses to reveal that in each of three independent transgenic lines for each single mutant, rescue of proximally polyadenylated FPA pre-mRNA can be detected (Fig 5A and 5B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        29,
+                        "RNA gel blot analyses",
+                        "experimental_method"
+                    ],
+                    [
+                        107,
+                        113,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        151,
+                        154,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        155,
+                        163,
+                        "pre-mRNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 102,
+                "sent": "We therefore conclude that neither of these mutations disrupted the ability of FPA to promote RNA 3\u2032-end formation in its own transcript.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        79,
+                        82,
+                        "FPA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 103,
+                "sent": "Impact of individual FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        21,
+                        24,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        25,
+                        29,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        37,
+                        46,
+                        "mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        81,
+                        84,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        85,
+                        93,
+                        "pre-mRNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 104,
+                "sent": "RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A (A), or FPA::FPA Y515A (B) using poly(A)+ purified mRNAs.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        12,
+                        "RNA gel blot",
+                        "experimental_method"
+                    ],
+                    [
+                        25,
+                        27,
+                        "WT",
+                        "protein_state"
+                    ],
+                    [
+                        28,
+                        39,
+                        "A. thaliana",
+                        "species"
+                    ],
+                    [
+                        67,
+                        73,
+                        "plants",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        74,
+                        79,
+                        "fpa-8",
+                        "gene"
+                    ],
+                    [
+                        84,
+                        89,
+                        "fpa-8",
+                        "gene"
+                    ],
+                    [
+                        90,
+                        97,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        116,
+                        119,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        121,
+                        130,
+                        "FPA R477A",
+                        "mutant"
+                    ],
+                    [
+                        139,
+                        142,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        144,
+                        153,
+                        "FPA Y515A",
+                        "mutant"
+                    ],
+                    [
+                        182,
+                        187,
+                        "mRNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 105,
+                "sent": "A probe corresponding to the 5\u2019UTR region of FPA mRNA was used to detect FPA specific mRNAs.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        45,
+                        48,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        49,
+                        53,
+                        "mRNA",
+                        "chemical"
+                    ],
+                    [
+                        73,
+                        76,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        86,
+                        91,
+                        "mRNAs",
+                        "chemical"
+                    ],
+                    [
+                        45,
+                        48,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        49,
+                        53,
+                        "mRNA",
+                        "chemical"
+                    ],
+                    [
+                        73,
+                        76,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        86,
+                        91,
+                        "mRNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 106,
+                "sent": "Proximally and distally polyadenylated FPA transcripts are marked with arrows.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        39,
+                        42,
+                        "FPA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 107,
+                "sent": "The ratio of distal:proximal polyadenylated forms is given under each lane. (C,D) Impact of individual FPA SPOC domain mutations on FLC transcript levels.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        103,
+                        106,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        107,
+                        111,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        119,
+                        128,
+                        "mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        132,
+                        135,
+                        "FLC",
+                        "gene"
+                    ]
+                ]
+            },
+            {
+                "sid": 108,
+                "sent": "qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A (C), FPA::FPA Y515A (D) plants.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "qRT-PCR",
+                        "experimental_method"
+                    ],
+                    [
+                        42,
+                        45,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        67,
+                        72,
+                        "fpa-8",
+                        "gene"
+                    ],
+                    [
+                        79,
+                        82,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        83,
+                        86,
+                        "YFP",
+                        "experimental_method"
+                    ],
+                    [
+                        91,
+                        94,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        96,
+                        105,
+                        "FPA R477A",
+                        "mutant"
+                    ],
+                    [
+                        111,
+                        114,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        116,
+                        125,
+                        "FPA Y515A",
+                        "mutant"
+                    ],
+                    [
+                        130,
+                        136,
+                        "plants",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 109,
+                "sent": "Histograms show mean values \u00b1SE for three independent PCR amplifications of three biological replicates.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        10,
+                        "Histograms",
+                        "evidence"
+                    ],
+                    [
+                        54,
+                        57,
+                        "PCR",
+                        "experimental_method"
+                    ],
+                    [
+                        0,
+                        10,
+                        "Histograms",
+                        "evidence"
+                    ],
+                    [
+                        54,
+                        57,
+                        "PCR",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 110,
+                "sent": "We next examined whether the corresponding mutations disrupted the ability of FPA to control FLC expression.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        78,
+                        81,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        93,
+                        96,
+                        "FLC",
+                        "gene"
+                    ]
+                ]
+            },
+            {
+                "sid": 111,
+                "sent": "We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        15,
+                        "RT-qPCR",
+                        "experimental_method"
+                    ],
+                    [
+                        45,
+                        48,
+                        "FLC",
+                        "gene"
+                    ],
+                    [
+                        49,
+                        53,
+                        "mRNA",
+                        "chemical"
+                    ],
+                    [
+                        119,
+                        126,
+                        "mutated",
+                        "protein_state"
+                    ],
+                    [
+                        127,
+                        130,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        163,
+                        166,
+                        "FLC",
+                        "gene"
+                    ],
+                    [
+                        179,
+                        184,
+                        "fpa-8",
+                        "gene"
+                    ],
+                    [
+                        185,
+                        192,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        215,
+                        224,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        253,
+                        256,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        257,
+                        261,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        262,
+                        271,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        272,
+                        277,
+                        "patch",
+                        "site"
+                    ],
+                    [
+                        278,
+                        284,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 112,
+                "sent": "Since each surface patch mutation appeared to be insufficient to disrupt FPA functions on its own, we combined both mutations into the same transgene.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        24,
+                        "surface patch",
+                        "site"
+                    ],
+                    [
+                        25,
+                        33,
+                        "mutation",
+                        "experimental_method"
+                    ],
+                    [
+                        73,
+                        76,
+                        "FPA",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 113,
+                "sent": "We could again confirm that near wild-type levels of FPA protein were expressed from three independent transgenic lines expressing the FPA R477A;Y515A doubly mutated protein in an fpa-8 mutant background (S3 Fig).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        33,
+                        42,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        53,
+                        56,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        135,
+                        150,
+                        "FPA R477A;Y515A",
+                        "mutant"
+                    ],
+                    [
+                        151,
+                        165,
+                        "doubly mutated",
+                        "protein_state"
+                    ],
+                    [
+                        180,
+                        185,
+                        "fpa-8",
+                        "gene"
+                    ],
+                    [
+                        186,
+                        192,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 114,
+                "sent": "We found that FPA R477A;Y515A protein functioned like wild-type FPA to restore FPA pre-mRNA proximal polyadenylation (Fig 6A) and FLC expression to wild-type levels (Fig 6B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        29,
+                        "FPA R477A;Y515A",
+                        "mutant"
+                    ],
+                    [
+                        54,
+                        63,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        64,
+                        67,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        79,
+                        82,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        83,
+                        91,
+                        "pre-mRNA",
+                        "chemical"
+                    ],
+                    [
+                        130,
+                        133,
+                        "FLC",
+                        "gene"
+                    ],
+                    [
+                        148,
+                        157,
+                        "wild-type",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 115,
+                "sent": "Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and FLC expression.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        17,
+                        20,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        21,
+                        25,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        33,
+                        42,
+                        "mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        77,
+                        80,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        81,
+                        89,
+                        "pre-mRNA",
+                        "chemical"
+                    ],
+                    [
+                        94,
+                        97,
+                        "FLC",
+                        "gene"
+                    ]
+                ]
+            },
+            {
+                "sid": 116,
+                "sent": "(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        16,
+                        "RNA gel blot",
+                        "experimental_method"
+                    ],
+                    [
+                        29,
+                        31,
+                        "WT",
+                        "protein_state"
+                    ],
+                    [
+                        32,
+                        43,
+                        "A. thaliana",
+                        "species"
+                    ],
+                    [
+                        71,
+                        77,
+                        "plants",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        78,
+                        83,
+                        "fpa-8",
+                        "gene"
+                    ],
+                    [
+                        88,
+                        93,
+                        "fpa-8",
+                        "gene"
+                    ],
+                    [
+                        94,
+                        101,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        113,
+                        116,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        118,
+                        133,
+                        "FPA R477A;Y515A",
+                        "mutant"
+                    ],
+                    [
+                        158,
+                        163,
+                        "mRNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 117,
+                "sent": "Black arrows indicate the proximally and distally polyadenylated FPA mRNAs.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        65,
+                        68,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        69,
+                        74,
+                        "mRNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 118,
+                "sent": "qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, and FPA::FPA R477A;Y515A plants.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        7,
+                        "qRT-PCR",
+                        "experimental_method"
+                    ],
+                    [
+                        42,
+                        45,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        67,
+                        72,
+                        "fpa-8",
+                        "gene"
+                    ],
+                    [
+                        78,
+                        81,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        83,
+                        98,
+                        "FPA R477A;Y515A",
+                        "mutant"
+                    ],
+                    [
+                        99,
+                        105,
+                        "plants",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 119,
+                "sent": "Together our findings suggest that either the SPOC domain is not required for the role of FPA in regulating RNA 3\u2032-end formation, or that this combination of mutations is not sufficient to critically disrupt the function of the FPA SPOC domain.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        46,
+                        50,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        90,
+                        93,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        108,
+                        111,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        158,
+                        167,
+                        "mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        228,
+                        231,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        232,
+                        236,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 120,
+                "sent": "Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        24,
+                        33,
+                        "mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        41,
+                        46,
+                        "SHARP",
+                        "protein"
+                    ],
+                    [
+                        47,
+                        51,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        89,
+                        105,
+                        "unphosphorylated",
+                        "protein_state"
+                    ],
+                    [
+                        106,
+                        110,
+                        "SMRT",
+                        "protein"
+                    ],
+                    [
+                        111,
+                        119,
+                        "peptides",
+                        "chemical"
+                    ],
+                    [
+                        202,
+                        205,
+                        "FPA",
+                        "protein"
+                    ],
+                    [
+                        206,
+                        210,
+                        "SPOC",
+                        "structure_element"
+                    ],
+                    [
+                        273,
+                        277,
+                        "SPOC",
+                        "structure_element"
+                    ]
+                ]
+            }
+        ]
+    },
+    "PMC4806292": {
+        "annotations": [
+            {
+                "sid": 0,
+                "sent": "Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein",
+                "section": "TITLE",
+                "ner": [
+                    [
+                        24,
+                        47,
+                        "in vitro reconstitution",
+                        "experimental_method"
+                    ],
+                    [
+                        88,
+                        93,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        94,
+                        99,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        107,
+                        112,
+                        "ACBD3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 1,
+                "sent": "Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        34,
+                        "Phosphatidylinositol 4-kinase beta",
+                        "protein"
+                    ],
+                    [
+                        36,
+                        41,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        58,
+                        63,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        64,
+                        68,
+                        "PI4K",
+                        "protein_type"
+                    ],
+                    [
+                        91,
+                        123,
+                        "phosphatidylinositol 4-phosphate",
+                        "chemical"
+                    ],
+                    [
+                        125,
+                        129,
+                        "PI4P",
+                        "chemical"
+                    ],
+                    [
+                        184,
+                        194,
+                        "eukaryotic",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 2,
+                "sent": "To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        35,
+                        40,
+                        "PI4Ks",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 3,
+                "sent": "PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        81,
+                        85,
+                        "PI4P",
+                        "chemical"
+                    ],
+                    [
+                        136,
+                        141,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 4,
+                "sent": "Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3).",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        20,
+                        23,
+                        "NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        24,
+                        33,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        52,
+                        57,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        87,
+                        108,
+                        "Golgi adaptor protein",
+                        "protein_type"
+                    ],
+                    [
+                        109,
+                        160,
+                        "acyl-coenzyme A binding domain containing protein 3",
+                        "protein"
+                    ],
+                    [
+                        162,
+                        167,
+                        "ACBD3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 5,
+                "sent": "We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        13,
+                        18,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        44,
+                        49,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        123,
+                        128,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        132,
+                        137,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        152,
+                        170,
+                        "enzymatic activity",
+                        "evidence"
+                    ],
+                    [
+                        184,
+                        195,
+                        "ACBD3:PI4KB",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 6,
+                "sent": "Phosphatidylinositol 4-kinase beta (PI4KB, also known as PI4K III\u03b2) is a soluble cytosolic protein yet its function is to phosphorylate membrane lipids.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        34,
+                        "Phosphatidylinositol 4-kinase beta",
+                        "protein"
+                    ],
+                    [
+                        36,
+                        41,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        57,
+                        66,
+                        "PI4K III\u03b2",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 7,
+                "sent": "It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        18,
+                        23,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        24,
+                        28,
+                        "PI4K",
+                        "protein_type"
+                    ],
+                    [
+                        56,
+                        76,
+                        "phosphatidylinositol",
+                        "chemical"
+                    ],
+                    [
+                        78,
+                        80,
+                        "PI",
+                        "chemical"
+                    ],
+                    [
+                        94,
+                        126,
+                        "phosphatidylinositol 4-phosphate",
+                        "chemical"
+                    ],
+                    [
+                        128,
+                        132,
+                        "PI4P",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 8,
+                "sent": "PI4P is an essential lipid found in various membrane compartments including the Golgi and trans-Golgi network (TGN), the plasma membrane and the endocytic compartments.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "PI4P",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 9,
+                "sent": "In these locations, PI4P plays an important role in cell signaling and lipid transport, and serves as a precursor for higher phosphoinositides or as a docking site for clathrin adaptor or lipid transfer proteins.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        20,
+                        24,
+                        "PI4P",
+                        "chemical"
+                    ],
+                    [
+                        125,
+                        142,
+                        "phosphoinositides",
+                        "chemical"
+                    ],
+                    [
+                        168,
+                        176,
+                        "clathrin",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 10,
+                "sent": "A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        16,
+                        58,
+                        "positive-sense single-stranded RNA viruses",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        60,
+                        72,
+                        "+RNA viruses",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        109,
+                        114,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        133,
+                        138,
+                        "human",
+                        "species"
+                    ],
+                    [
+                        139,
+                        144,
+                        "PI4KA",
+                        "protein"
+                    ],
+                    [
+                        148,
+                        153,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        183,
+                        187,
+                        "PI4P",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 11,
+                "sent": "These structures are essential for effective viral replication.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        6,
+                        16,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        45,
+                        50,
+                        "viral",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 12,
+                "sent": "Recently, highly specific PI4KB inhibitors were developed as potential antivirals.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        26,
+                        31,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 13,
+                "sent": "PI4K kinases must be recruited to the correct membrane type to fulfill their enzymatic functions.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "PI4K",
+                        "protein_type"
+                    ],
+                    [
+                        5,
+                        12,
+                        "kinases",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 14,
+                "sent": "Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        13,
+                        "Type II PI4Ks",
+                        "protein_type"
+                    ],
+                    [
+                        15,
+                        21,
+                        "PI4K2A",
+                        "protein"
+                    ],
+                    [
+                        26,
+                        32,
+                        "PI4K2B",
+                        "protein"
+                    ],
+                    [
+                        38,
+                        59,
+                        "heavily palmitoylated",
+                        "protein_state"
+                    ],
+                    [
+                        79,
+                        96,
+                        "membrane proteins",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 15,
+                "sent": "In contrast, type III PI4Ks (PI4KA and PI4KB) are soluble cytosolic proteins that are recruited to appropriate membranes indirectly via protein-protein interactions.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        13,
+                        27,
+                        "type III PI4Ks",
+                        "protein_type"
+                    ],
+                    [
+                        29,
+                        34,
+                        "PI4KA",
+                        "protein"
+                    ],
+                    [
+                        39,
+                        44,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 16,
+                "sent": "The recruitment of PI4KA to the plasma membrane by EFR3 and TTC7 is relatively well understood even at the structural level, but, the actual molecular mechanism of PI4KB recruitment to the Golgi is still poorly understood.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        19,
+                        24,
+                        "PI4KA",
+                        "protein"
+                    ],
+                    [
+                        51,
+                        55,
+                        "EFR3",
+                        "protein"
+                    ],
+                    [
+                        60,
+                        64,
+                        "TTC7",
+                        "protein"
+                    ],
+                    [
+                        164,
+                        169,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 17,
+                "sent": "Acyl-coenzyme A binding domain containing protein 3 (ACBD3, also known as GCP60 and PAP7) is a Golgi resident protein.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        51,
+                        "Acyl-coenzyme A binding domain containing protein 3",
+                        "protein"
+                    ],
+                    [
+                        53,
+                        58,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        74,
+                        79,
+                        "GCP60",
+                        "protein"
+                    ],
+                    [
+                        84,
+                        88,
+                        "PAP7",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 18,
+                "sent": "Its membrane localization is mediated by the interaction with the Golgi integral protein golgin B1/giantin.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        89,
+                        98,
+                        "golgin B1",
+                        "protein"
+                    ],
+                    [
+                        99,
+                        106,
+                        "giantin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 19,
+                "sent": "ACBD3 functions as an adaptor protein and signaling hub across cellular signaling pathways.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "ACBD3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 20,
+                "sent": "ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        55,
+                        64,
+                        "golgin A3",
+                        "protein"
+                    ],
+                    [
+                        65,
+                        75,
+                        "golgin-160",
+                        "protein"
+                    ],
+                    [
+                        99,
+                        112,
+                        "Numb proteins",
+                        "protein_type"
+                    ],
+                    [
+                        179,
+                        196,
+                        "metal transporter",
+                        "protein_type"
+                    ],
+                    [
+                        197,
+                        201,
+                        "DMT1",
+                        "protein"
+                    ],
+                    [
+                        206,
+                        215,
+                        "monomeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        216,
+                        225,
+                        "G protein",
+                        "protein_type"
+                    ],
+                    [
+                        226,
+                        233,
+                        "Dexras1",
+                        "protein"
+                    ],
+                    [
+                        246,
+                        250,
+                        "iron",
+                        "chemical"
+                    ],
+                    [
+                        272,
+                        284,
+                        "lipid kinase",
+                        "protein_type"
+                    ],
+                    [
+                        285,
+                        290,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 21,
+                "sent": "ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington\u2019s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        138,
+                        158,
+                        "polyglutamine repeat",
+                        "structure_element"
+                    ],
+                    [
+                        170,
+                        176,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        177,
+                        187,
+                        "huntingtin",
+                        "protein"
+                    ],
+                    [
+                        215,
+                        224,
+                        "monomeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        225,
+                        234,
+                        "G protein",
+                        "protein_type"
+                    ],
+                    [
+                        235,
+                        239,
+                        "Rhes",
+                        "protein"
+                    ],
+                    [
+                        240,
+                        247,
+                        "Dexras2",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 22,
+                "sent": "ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        30,
+                        35,
+                        "viral",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        36,
+                        62,
+                        "non-structural 3A proteins",
+                        "protein_type"
+                    ],
+                    [
+                        92,
+                        106,
+                        "picornaviruses",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        117,
+                        127,
+                        "poliovirus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        129,
+                        146,
+                        "coxsackievirus B3",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        152,
+                        163,
+                        "Aichi virus",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 23,
+                "sent": "We present a biochemical and structural characterization of the molecular complex composed of the ACBD3 protein and the PI4KB enzyme.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        13,
+                        56,
+                        "biochemical and structural characterization",
+                        "experimental_method"
+                    ],
+                    [
+                        98,
+                        103,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        120,
+                        125,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 24,
+                "sent": "We show that ACBD3 can recruit PI4KB to model membranes as well as redirect PI4KB to cellular membranes where it is not naturally found.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        13,
+                        18,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        31,
+                        36,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        76,
+                        81,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 25,
+                "sent": "Our data also show that ACBD3 regulates the enzymatic activity of PI4KB kinase through membrane recruitment rather than allostery.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        24,
+                        29,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        44,
+                        62,
+                        "enzymatic activity",
+                        "evidence"
+                    ],
+                    [
+                        66,
+                        71,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        72,
+                        78,
+                        "kinase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 26,
+                "sent": "ACBD3 and PI4KB interact with 1:1 stoichiometry with submicromolar affinity",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        10,
+                        15,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 27,
+                "sent": "In order to verify the interactions between ACBD3 and PI4KB we expressed and purified both proteins.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        44,
+                        49,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        54,
+                        59,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        63,
+                        85,
+                        "expressed and purified",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 28,
+                "sent": "To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423\u2013522) was removed (Fig. 1A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        22,
+                        42,
+                        "bacterial expression",
+                        "experimental_method"
+                    ],
+                    [
+                        47,
+                        78,
+                        "intrinsically disordered region",
+                        "structure_element"
+                    ],
+                    [
+                        82,
+                        87,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        98,
+                        105,
+                        "423\u2013522",
+                        "residue_range"
+                    ],
+                    [
+                        111,
+                        118,
+                        "removed",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 29,
+                "sent": "This internal deletion does not significantly affect the kinase activity(SI Fig. 1A) or interaction with ACBD3 (SI Fig. 1B,C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        22,
+                        "deletion",
+                        "experimental_method"
+                    ],
+                    [
+                        57,
+                        63,
+                        "kinase",
+                        "protein_type"
+                    ],
+                    [
+                        105,
+                        110,
+                        "ACBD3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 30,
+                "sent": "In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        6,
+                        28,
+                        "in vitro binding assay",
+                        "experimental_method"
+                    ],
+                    [
+                        30,
+                        35,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        36,
+                        74,
+                        "co-purified with the NiNTA-immobilized",
+                        "experimental_method"
+                    ],
+                    [
+                        86,
+                        100,
+                        "His6GB1-tagged",
+                        "protein_state"
+                    ],
+                    [
+                        101,
+                        106,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 31,
+                "sent": "Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        34,
+                        "mammalian two-hybrid assay",
+                        "experimental_method"
+                    ],
+                    [
+                        60,
+                        69,
+                        "localized",
+                        "evidence"
+                    ],
+                    [
+                        94,
+                        102,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        106,
+                        111,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        152,
+                        161,
+                        "glutamine",
+                        "residue_name"
+                    ],
+                    [
+                        180,
+                        197,
+                        "N-terminal region",
+                        "structure_element"
+                    ],
+                    [
+                        201,
+                        206,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        221,
+                        235,
+                        "helical domain",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 32,
+                "sent": "We expressed the Q domain of ACBD3 (residues 241\u2013308) and the N-terminal region of PI4KB (residues 1\u201368) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        3,
+                        12,
+                        "expressed",
+                        "experimental_method"
+                    ],
+                    [
+                        17,
+                        25,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        29,
+                        34,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        45,
+                        52,
+                        "241\u2013308",
+                        "residue_range"
+                    ],
+                    [
+                        62,
+                        79,
+                        "N-terminal region",
+                        "structure_element"
+                    ],
+                    [
+                        83,
+                        88,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        99,
+                        103,
+                        "1\u201368",
+                        "residue_range"
+                    ],
+                    [
+                        108,
+                        115,
+                        "E. coli",
+                        "species"
+                    ]
+                ]
+            },
+            {
+                "sid": 33,
+                "sent": "Because it has been reported that ACBD3 can dimerize in a mammalian two-hybrid assay, we were interested in determining the stoichiometry of the ACBD3:PI4KB protein complex.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        39,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        44,
+                        52,
+                        "dimerize",
+                        "oligomeric_state"
+                    ],
+                    [
+                        58,
+                        84,
+                        "mammalian two-hybrid assay",
+                        "experimental_method"
+                    ],
+                    [
+                        145,
+                        156,
+                        "ACBD3:PI4KB",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 34,
+                "sent": "The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55\u2009kDa, 80\u2009kDa, and 130\u2009kDa, respectively.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        30,
+                        "sedimentation coefficients",
+                        "evidence"
+                    ],
+                    [
+                        34,
+                        39,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        44,
+                        49,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        50,
+                        55,
+                        "alone",
+                        "protein_state"
+                    ],
+                    [
+                        60,
+                        71,
+                        "ACBD3:PI4KB",
+                        "complex_assembly"
+                    ],
+                    [
+                        99,
+                        129,
+                        "analytical ultracentrifugation",
+                        "experimental_method"
+                    ],
+                    [
+                        198,
+                        215,
+                        "molecular weights",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 35,
+                "sent": "This result suggests that both proteins are monomeric and the stoichiometry of the ACBD3: PI4KB protein complex is 1:1 (Fig. 1C, left panel).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        44,
+                        53,
+                        "monomeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        83,
+                        95,
+                        "ACBD3: PI4KB",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 36,
+                "sent": "Similar results were obtained for the complex of the Q domain of ACBD3 and the N-terminal region of PI4KB (Fig. 1C, right panel).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        53,
+                        61,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        65,
+                        70,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        79,
+                        96,
+                        "N-terminal region",
+                        "structure_element"
+                    ],
+                    [
+                        100,
+                        105,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 37,
+                "sent": "We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        71,
+                        82,
+                        "full length",
+                        "protein_state"
+                    ],
+                    [
+                        83,
+                        88,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        93,
+                        98,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        105,
+                        130,
+                        "surface plasmon resonance",
+                        "experimental_method"
+                    ],
+                    [
+                        132,
+                        135,
+                        "SPR",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 38,
+                "sent": "SPR measurements revealed a strong interaction with a Kd value of 320\u2009+/\u2212130\u2009nM (Fig. 1D, SI Fig. 1D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "SPR",
+                        "experimental_method"
+                    ],
+                    [
+                        54,
+                        56,
+                        "Kd",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 39,
+                "sent": "We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        23,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        28,
+                        33,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        64,
+                        72,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        76,
+                        81,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        90,
+                        107,
+                        "N-terminal region",
+                        "structure_element"
+                    ],
+                    [
+                        111,
+                        116,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        146,
+                        167,
+                        "dissociation constant",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 40,
+                "sent": "Structural analysis of the ACBD3:PI4KB complex",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        19,
+                        "Structural analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        27,
+                        38,
+                        "ACBD3:PI4KB",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 41,
+                "sent": "Full length ACBD3 and PI4KB both contain large intrinsically disordered regions that impede crystallization.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Full length",
+                        "protein_state"
+                    ],
+                    [
+                        12,
+                        17,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        22,
+                        27,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        47,
+                        79,
+                        "intrinsically disordered regions",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 42,
+                "sent": "We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of the complex to determine which parts of the complex are well folded (SI Fig. 2).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        53,
+                        "hydrogen-deuterium exchange mass spectrometry",
+                        "experimental_method"
+                    ],
+                    [
+                        55,
+                        61,
+                        "HDX-MS",
+                        "experimental_method"
+                    ],
+                    [
+                        131,
+                        142,
+                        "well folded",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 43,
+                "sent": "However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        42,
+                        "crystals",
+                        "evidence"
+                    ],
+                    [
+                        73,
+                        82,
+                        "truncated",
+                        "protein_state"
+                    ],
+                    [
+                        117,
+                        122,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        123,
+                        131,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        140,
+                        157,
+                        "N-terminal region",
+                        "structure_element"
+                    ],
+                    [
+                        161,
+                        166,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 44,
+                "sent": "For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        32,
+                        52,
+                        "isotopically labeled",
+                        "protein_state"
+                    ],
+                    [
+                        53,
+                        58,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        59,
+                        67,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        72,
+                        92,
+                        "isotopically labeled",
+                        "protein_state"
+                    ],
+                    [
+                        93,
+                        98,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        99,
+                        107,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        108,
+                        113,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        114,
+                        131,
+                        "N-terminal region",
+                        "structure_element"
+                    ],
+                    [
+                        157,
+                        173,
+                        "NMR spectroscopy",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 45,
+                "sent": "As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        7,
+                        24,
+                        "N-terminal region",
+                        "structure_element"
+                    ],
+                    [
+                        57,
+                        70,
+                        "co-expression",
+                        "experimental_method"
+                    ],
+                    [
+                        152,
+                        155,
+                        "15N",
+                        "chemical"
+                    ],
+                    [
+                        156,
+                        159,
+                        "13C",
+                        "chemical"
+                    ],
+                    [
+                        160,
+                        168,
+                        "labelled",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 46,
+                "sent": "Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        68,
+                        72,
+                        "free",
+                        "protein_state"
+                    ],
+                    [
+                        73,
+                        78,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        79,
+                        87,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        127,
+                        141,
+                        "2D 15N/1H HSQC",
+                        "experimental_method"
+                    ],
+                    [
+                        142,
+                        149,
+                        "spectra",
+                        "evidence"
+                    ],
+                    [
+                        215,
+                        243,
+                        "triple-resonance experiments",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 47,
+                "sent": "Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        24,
+                        27,
+                        "15N",
+                        "chemical"
+                    ],
+                    [
+                        32,
+                        34,
+                        "1H",
+                        "chemical"
+                    ],
+                    [
+                        44,
+                        48,
+                        "free",
+                        "protein_state"
+                    ],
+                    [
+                        49,
+                        54,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        55,
+                        63,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        143,
+                        152,
+                        "Met1-Lys4",
+                        "residue_range"
+                    ],
+                    [
+                        158,
+                        163,
+                        "Gln44",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 48,
+                "sent": "Over 93% of non-exchangeable side-chain signals were assigned for the free ACBD3 Q domain.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        70,
+                        74,
+                        "free",
+                        "protein_state"
+                    ],
+                    [
+                        75,
+                        80,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        81,
+                        89,
+                        "Q domain",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 49,
+                "sent": "Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        85,
+                        88,
+                        "Gln",
+                        "residue_name"
+                    ],
+                    [
+                        96,
+                        99,
+                        "Gln",
+                        "residue_name"
+                    ],
+                    [
+                        112,
+                        117,
+                        "Gln44",
+                        "residue_name_number"
+                    ],
+                    [
+                        133,
+                        138,
+                        "Gln48",
+                        "residue_name_number"
+                    ],
+                    [
+                        234,
+                        243,
+                        "glutamine",
+                        "residue_name"
+                    ]
+                ]
+            },
+            {
+                "sid": 50,
+                "sent": "The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        53,
+                        60,
+                        "spectra",
+                        "evidence"
+                    ],
+                    [
+                        155,
+                        158,
+                        "Gln",
+                        "residue_name"
+                    ],
+                    [
+                        160,
+                        165,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        171,
+                        175,
+                        "Ala2",
+                        "residue_name_number"
+                    ],
+                    [
+                        177,
+                        182,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 51,
+                "sent": "The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        28,
+                        "15N",
+                        "chemical"
+                    ],
+                    [
+                        30,
+                        33,
+                        "13C",
+                        "chemical"
+                    ],
+                    [
+                        38,
+                        40,
+                        "1H",
+                        "chemical"
+                    ],
+                    [
+                        99,
+                        103,
+                        "NOEs",
+                        "evidence"
+                    ],
+                    [
+                        122,
+                        142,
+                        "3D 15N/1H NOESY-HSQC",
+                        "experimental_method"
+                    ],
+                    [
+                        147,
+                        164,
+                        "13C/1H HMQC-NOESY",
+                        "experimental_method"
+                    ],
+                    [
+                        165,
+                        172,
+                        "spectra",
+                        "evidence"
+                    ],
+                    [
+                        204,
+                        226,
+                        "structural calculation",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 52,
+                "sent": "Structural statistics for the final water-refined sets of structures are shown in SI Table 1.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        21,
+                        "Structural statistics",
+                        "evidence"
+                    ],
+                    [
+                        58,
+                        68,
+                        "structures",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 53,
+                "sent": "This structure revealed that the Q domain forms a two helix hairpin.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        14,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        33,
+                        41,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        50,
+                        67,
+                        "two helix hairpin",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 54,
+                "sent": "The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44\u201364, from this point on referred to as the kinase helix) (Fig. 2A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        10,
+                        15,
+                        "helix",
+                        "structure_element"
+                    ],
+                    [
+                        46,
+                        51,
+                        "helix",
+                        "structure_element"
+                    ],
+                    [
+                        84,
+                        102,
+                        "three helix bundle",
+                        "structure_element"
+                    ],
+                    [
+                        133,
+                        138,
+                        "helix",
+                        "structure_element"
+                    ],
+                    [
+                        146,
+                        151,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        173,
+                        178,
+                        "44\u201364",
+                        "residue_range"
+                    ],
+                    [
+                        218,
+                        230,
+                        "kinase helix",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 55,
+                "sent": "Preceding the kinase helix are three ordered residues (Val42, Ile43, and Asp44) that also contribute to the interaction (Fig. 2B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        26,
+                        "kinase helix",
+                        "structure_element"
+                    ],
+                    [
+                        55,
+                        60,
+                        "Val42",
+                        "residue_name_number"
+                    ],
+                    [
+                        62,
+                        67,
+                        "Ile43",
+                        "residue_name_number"
+                    ],
+                    [
+                        73,
+                        78,
+                        "Asp44",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 56,
+                "sent": "The remaining part of the PI4KB N-termini, however, is disordered (SI Fig. 5).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        26,
+                        31,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 57,
+                "sent": "Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        29,
+                        "PI4KB:ACBD3",
+                        "complex_assembly"
+                    ],
+                    [
+                        30,
+                        58,
+                        "interactions are hydrophobic",
+                        "bond_interaction"
+                    ],
+                    [
+                        81,
+                        95,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        123,
+                        128,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        129,
+                        135,
+                        "Tyr261",
+                        "residue_name_number"
+                    ],
+                    [
+                        140,
+                        145,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        146,
+                        151,
+                        "His63",
+                        "residue_name_number"
+                    ],
+                    [
+                        182,
+                        187,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        188,
+                        194,
+                        "Tyr288",
+                        "residue_name_number"
+                    ],
+                    [
+                        203,
+                        208,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        219,
+                        224,
+                        "Asp44",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 58,
+                "sent": "Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        33,
+                        38,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        39,
+                        44,
+                        "helix",
+                        "structure_element"
+                    ],
+                    [
+                        48,
+                        59,
+                        "amphipathic",
+                        "protein_state"
+                    ],
+                    [
+                        68,
+                        87,
+                        "hydrophobic surface",
+                        "site"
+                    ],
+                    [
+                        101,
+                        109,
+                        "Q domain",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 59,
+                "sent": "To corroborate the structural data, we introduced a number of point mutations and validated their effect on complex formation using an in vitro pull-down assay (Fig. 2D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        34,
+                        "structural data",
+                        "evidence"
+                    ],
+                    [
+                        39,
+                        49,
+                        "introduced",
+                        "experimental_method"
+                    ],
+                    [
+                        62,
+                        77,
+                        "point mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        135,
+                        159,
+                        "in vitro pull-down assay",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 60,
+                "sent": "Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Wild type",
+                        "protein_state"
+                    ],
+                    [
+                        10,
+                        15,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        24,
+                        35,
+                        "co-purified",
+                        "experimental_method"
+                    ],
+                    [
+                        72,
+                        83,
+                        "His6-tagged",
+                        "protein_state"
+                    ],
+                    [
+                        84,
+                        93,
+                        "wild type",
+                        "protein_state"
+                    ],
+                    [
+                        94,
+                        99,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        120,
+                        125,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        126,
+                        130,
+                        "V42A",
+                        "mutant"
+                    ],
+                    [
+                        135,
+                        139,
+                        "V47A",
+                        "mutant"
+                    ],
+                    [
+                        140,
+                        147,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        162,
+                        169,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        190,
+                        207,
+                        "binding interface",
+                        "site"
+                    ],
+                    [
+                        209,
+                        213,
+                        "I43A",
+                        "mutant"
+                    ],
+                    [
+                        215,
+                        219,
+                        "V55A",
+                        "mutant"
+                    ],
+                    [
+                        221,
+                        225,
+                        "L56A",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 61,
+                "sent": "As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        23,
+                        "wild type",
+                        "protein_state"
+                    ],
+                    [
+                        24,
+                        29,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        50,
+                        55,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        56,
+                        61,
+                        "Y266A",
+                        "mutant"
+                    ],
+                    [
+                        62,
+                        68,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        91,
+                        96,
+                        "Y285A",
+                        "mutant"
+                    ],
+                    [
+                        97,
+                        103,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        122,
+                        127,
+                        "F258A",
+                        "mutant"
+                    ],
+                    [
+                        129,
+                        134,
+                        "H284A",
+                        "mutant"
+                    ],
+                    [
+                        140,
+                        145,
+                        "Y288A",
+                        "mutant"
+                    ],
+                    [
+                        146,
+                        153,
+                        "mutants",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 62,
+                "sent": "ACBD3 efficiently recruits the PI4KB enzyme to membranes",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        31,
+                        36,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 63,
+                "sent": "We next sought to determine if the ACBD3:PI4KB interaction drives membrane localization of the PI4KB enzyme.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        35,
+                        46,
+                        "ACBD3:PI4KB",
+                        "complex_assembly"
+                    ],
+                    [
+                        95,
+                        100,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 64,
+                "sent": "To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate \u2013 the PI lipid.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        36,
+                        72,
+                        "in vitro membrane recruitment system",
+                        "experimental_method"
+                    ],
+                    [
+                        79,
+                        105,
+                        "Giant Unilamellar Vesicles",
+                        "experimental_method"
+                    ],
+                    [
+                        107,
+                        111,
+                        "GUVs",
+                        "experimental_method"
+                    ],
+                    [
+                        128,
+                        133,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        150,
+                        152,
+                        "PI",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 65,
+                "sent": "We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600\u2009nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        17,
+                        22,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        23,
+                        29,
+                        "kinase",
+                        "protein_type"
+                    ],
+                    [
+                        47,
+                        56,
+                        "localized",
+                        "evidence"
+                    ],
+                    [
+                        75,
+                        79,
+                        "GUVs",
+                        "experimental_method"
+                    ],
+                    [
+                        139,
+                        144,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        167,
+                        171,
+                        "GUVs",
+                        "experimental_method"
+                    ],
+                    [
+                        195,
+                        200,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        209,
+                        227,
+                        "DGS-NTA (Ni) lipid",
+                        "chemical"
+                    ],
+                    [
+                        246,
+                        251,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 66,
+                "sent": "We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP \u2013 Q domain construct (Fig. 3B upper panel).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        24,
+                        29,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        55,
+                        75,
+                        "localization signals",
+                        "evidence"
+                    ],
+                    [
+                        80,
+                        85,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        87,
+                        101,
+                        "overexpression",
+                        "experimental_method"
+                    ],
+                    [
+                        109,
+                        117,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        163,
+                        169,
+                        "kinase",
+                        "protein_type"
+                    ],
+                    [
+                        224,
+                        229,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        259,
+                        273,
+                        "overexpressing",
+                        "experimental_method"
+                    ],
+                    [
+                        278,
+                        281,
+                        "GFP",
+                        "experimental_method"
+                    ],
+                    [
+                        284,
+                        292,
+                        "Q domain",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 67,
+                "sent": "We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the \u201cdisappearance\u201d of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        25,
+                        31,
+                        "signal",
+                        "evidence"
+                    ],
+                    [
+                        67,
+                        73,
+                        "kinase",
+                        "protein_type"
+                    ],
+                    [
+                        179,
+                        185,
+                        "signal",
+                        "evidence"
+                    ],
+                    [
+                        194,
+                        208,
+                        "overexpression",
+                        "experimental_method"
+                    ],
+                    [
+                        212,
+                        215,
+                        "GFP",
+                        "experimental_method"
+                    ],
+                    [
+                        227,
+                        238,
+                        "non-binding",
+                        "protein_state"
+                    ],
+                    [
+                        239,
+                        247,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        248,
+                        254,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        276,
+                        288,
+                        "localization",
+                        "evidence"
+                    ],
+                    [
+                        307,
+                        312,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 68,
+                "sent": "Given this result, overexpression of the Q domain should also interfere with the PI4KB dependent Golgi functions.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        33,
+                        "overexpression",
+                        "experimental_method"
+                    ],
+                    [
+                        41,
+                        49,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        81,
+                        86,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 69,
+                "sent": "Ceramide transport and accumulation in Golgi is a well-known PI4KB dependent process.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "Ceramide",
+                        "chemical"
+                    ],
+                    [
+                        61,
+                        66,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 70,
+                "sent": "We have used fluorescently labeled ceramide and analyzed its trafficking in non-transfected cells and cell overexpressing the Q domain.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        34,
+                        "fluorescently labeled",
+                        "protein_state"
+                    ],
+                    [
+                        35,
+                        43,
+                        "ceramide",
+                        "chemical"
+                    ],
+                    [
+                        107,
+                        121,
+                        "overexpressing",
+                        "experimental_method"
+                    ],
+                    [
+                        126,
+                        134,
+                        "Q domain",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 71,
+                "sent": "As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        39,
+                        47,
+                        "ceramide",
+                        "chemical"
+                    ],
+                    [
+                        74,
+                        84,
+                        "expressing",
+                        "experimental_method"
+                    ],
+                    [
+                        89,
+                        91,
+                        "wt",
+                        "protein_state"
+                    ],
+                    [
+                        92,
+                        100,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        124,
+                        127,
+                        "RFP",
+                        "experimental_method"
+                    ],
+                    [
+                        135,
+                        141,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        142,
+                        150,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        163,
+                        171,
+                        "ceramide",
+                        "chemical"
+                    ],
+                    [
+                        207,
+                        218,
+                        "ACBD3:PI4KB",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 72,
+                "sent": "We further analyzed the function of ACBD3:PI4KB interaction in membrane recruitment of PI4KB in living cells using fluorescently tagged proteins.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        36,
+                        47,
+                        "ACBD3:PI4KB",
+                        "complex_assembly"
+                    ],
+                    [
+                        87,
+                        92,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        115,
+                        135,
+                        "fluorescently tagged",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 73,
+                "sent": "We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        12,
+                        21,
+                        "rapamycin",
+                        "chemical"
+                    ],
+                    [
+                        52,
+                        58,
+                        "FKBP12",
+                        "protein"
+                    ],
+                    [
+                        60,
+                        84,
+                        "FK506 binding protein 12",
+                        "protein"
+                    ],
+                    [
+                        90,
+                        93,
+                        "FRB",
+                        "structure_element"
+                    ],
+                    [
+                        95,
+                        103,
+                        "fragment",
+                        "structure_element"
+                    ],
+                    [
+                        107,
+                        111,
+                        "mTOR",
+                        "protein"
+                    ],
+                    [
+                        123,
+                        132,
+                        "rapamycin",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 74,
+                "sent": "We fused the FRB to residues 34\u201363 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        3,
+                        8,
+                        "fused",
+                        "experimental_method"
+                    ],
+                    [
+                        13,
+                        16,
+                        "FRB",
+                        "structure_element"
+                    ],
+                    [
+                        29,
+                        34,
+                        "34\u201363",
+                        "residue_range"
+                    ],
+                    [
+                        42,
+                        75,
+                        "mitochondrial localization signal",
+                        "structure_element"
+                    ],
+                    [
+                        81,
+                        120,
+                        "mitochondrial A-kinase anchor protein 1",
+                        "protein"
+                    ],
+                    [
+                        122,
+                        127,
+                        "AKAP1",
+                        "protein"
+                    ],
+                    [
+                        133,
+                        136,
+                        "CFP",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 75,
+                "sent": "The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        9,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        10,
+                        18,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        28,
+                        36,
+                        "fused to",
+                        "experimental_method"
+                    ],
+                    [
+                        37,
+                        43,
+                        "FKBP12",
+                        "protein"
+                    ],
+                    [
+                        48,
+                        52,
+                        "mRFP",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 76,
+                "sent": "We analyzed localization of the ACBD3 Q domain and GFP \u2013 PI4KB before and after the addition of rapamycin.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        12,
+                        24,
+                        "localization",
+                        "evidence"
+                    ],
+                    [
+                        32,
+                        37,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        38,
+                        46,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        51,
+                        54,
+                        "GFP",
+                        "experimental_method"
+                    ],
+                    [
+                        57,
+                        62,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        96,
+                        105,
+                        "rapamycin",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 77,
+                "sent": "As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        21,
+                        26,
+                        "H284A",
+                        "mutant"
+                    ],
+                    [
+                        27,
+                        33,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        41,
+                        46,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        47,
+                        55,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        89,
+                        94,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        95,
+                        101,
+                        "kinase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 78,
+                "sent": "In every case the ACDB3 Q domain was rapidly (within 5\u2009minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        23,
+                        "ACDB3",
+                        "protein"
+                    ],
+                    [
+                        24,
+                        32,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        121,
+                        130,
+                        "rapamycin",
+                        "chemical"
+                    ],
+                    [
+                        145,
+                        154,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        188,
+                        194,
+                        "kinase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 79,
+                "sent": "Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        35,
+                        38,
+                        "GFP",
+                        "experimental_method"
+                    ],
+                    [
+                        39,
+                        44,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        45,
+                        51,
+                        "kinase",
+                        "protein_type"
+                    ],
+                    [
+                        55,
+                        67,
+                        "co-expressed",
+                        "experimental_method"
+                    ],
+                    [
+                        77,
+                        86,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        87,
+                        92,
+                        "ACDB3",
+                        "protein"
+                    ],
+                    [
+                        93,
+                        101,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        129,
+                        141,
+                        "localization",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 80,
+                "sent": "However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        14,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        32,
+                        44,
+                        "localization",
+                        "evidence"
+                    ],
+                    [
+                        50,
+                        62,
+                        "co-expressed",
+                        "experimental_method"
+                    ],
+                    [
+                        72,
+                        87,
+                        "non-interacting",
+                        "protein_state"
+                    ],
+                    [
+                        88,
+                        96,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        97,
+                        103,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 81,
+                "sent": "ACBD3 increases PI4KB enzymatic activity by recruiting PI4KB to close vicinity of its substrate",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        16,
+                        21,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        22,
+                        40,
+                        "enzymatic activity",
+                        "evidence"
+                    ],
+                    [
+                        55,
+                        60,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 82,
+                "sent": "To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        16,
+                        21,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        36,
+                        41,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        42,
+                        48,
+                        "kinase",
+                        "protein_type"
+                    ],
+                    [
+                        49,
+                        67,
+                        "enzymatic activity",
+                        "evidence"
+                    ],
+                    [
+                        92,
+                        116,
+                        "luminescent kinase assay",
+                        "experimental_method"
+                    ],
+                    [
+                        123,
+                        125,
+                        "PI",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 83,
+                "sent": "We observed no effect on the kinase activity of PI4KB (Fig. 4A) suggesting that ACBD3 does not directly affect the enzyme (e.g. induction of a conformation change).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        29,
+                        35,
+                        "kinase",
+                        "protein_type"
+                    ],
+                    [
+                        48,
+                        53,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        80,
+                        85,
+                        "ACBD3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 84,
+                "sent": "However, in vivo ACBD3 is located at the Golgi membranes, whereas in this experiment, ACBD3 was located in the solution and PI is provided as micelles.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        17,
+                        22,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        86,
+                        91,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        124,
+                        126,
+                        "PI",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 85,
+                "sent": "For this, we again turned to the GUV system with ACBD3 localized to the GUV membrane.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        33,
+                        36,
+                        "GUV",
+                        "experimental_method"
+                    ],
+                    [
+                        49,
+                        54,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        55,
+                        64,
+                        "localized",
+                        "evidence"
+                    ],
+                    [
+                        72,
+                        75,
+                        "GUV",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 86,
+                "sent": "The GUVs contained 10% PI to serve as a substrate for PI4KB kinase.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "GUVs",
+                        "experimental_method"
+                    ],
+                    [
+                        23,
+                        25,
+                        "PI",
+                        "chemical"
+                    ],
+                    [
+                        54,
+                        59,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        60,
+                        66,
+                        "kinase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 87,
+                "sent": "The buffer also contained CFP-SidC, which binds to PI4P with nanomolar affinity.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        26,
+                        29,
+                        "CFP",
+                        "experimental_method"
+                    ],
+                    [
+                        30,
+                        34,
+                        "SidC",
+                        "protein"
+                    ],
+                    [
+                        51,
+                        55,
+                        "PI4P",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 88,
+                "sent": "This enabled visualization of the kinase reaction using a confocal microscope.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        40,
+                        "kinase",
+                        "protein_type"
+                    ],
+                    [
+                        58,
+                        77,
+                        "confocal microscope",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 89,
+                "sent": "We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        34,
+                        49,
+                        "phosphorylation",
+                        "ptm"
+                    ],
+                    [
+                        66,
+                        72,
+                        "kinase",
+                        "protein_type"
+                    ],
+                    [
+                        73,
+                        78,
+                        "alone",
+                        "protein_state"
+                    ],
+                    [
+                        92,
+                        98,
+                        "kinase",
+                        "protein_type"
+                    ],
+                    [
+                        131,
+                        135,
+                        "GUVs",
+                        "experimental_method"
+                    ],
+                    [
+                        139,
+                        144,
+                        "ACBD3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 90,
+                "sent": "Reaction was also performed in the absence of ATP as a negative control (Fig. 4B).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        35,
+                        45,
+                        "absence of",
+                        "protein_state"
+                    ],
+                    [
+                        46,
+                        49,
+                        "ATP",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 91,
+                "sent": "These experiments showed that PI4KB enzymatic activity increases when ACBD3 is membrane localized (Fig. 4C, SI Fig. 6).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        30,
+                        35,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        36,
+                        54,
+                        "enzymatic activity",
+                        "evidence"
+                    ],
+                    [
+                        70,
+                        75,
+                        "ACBD3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 92,
+                "sent": "Membrane recruitment of PI4KB enzyme is crucial to ensure its proper function at the Golgi and TGN.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        24,
+                        29,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 93,
+                "sent": "However, the molecular mechanism and structural basis for PI4KB interaction with the membrane is poorly understood.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        58,
+                        63,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 94,
+                "sent": "In principle, any of the binding partners of PI4KB could play a role in membrane recruitment.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        45,
+                        50,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 95,
+                "sent": "To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        17,
+                        22,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        78,
+                        91,
+                        "small GTPases",
+                        "protein_type"
+                    ],
+                    [
+                        92,
+                        97,
+                        "Rab11",
+                        "protein"
+                    ],
+                    [
+                        102,
+                        106,
+                        "Arf1",
+                        "protein"
+                    ],
+                    [
+                        127,
+                        163,
+                        "acyl-CoA binding domain containing 3",
+                        "protein"
+                    ],
+                    [
+                        165,
+                        170,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        181,
+                        206,
+                        "neuronal calcium sensor-1",
+                        "protein"
+                    ],
+                    [
+                        208,
+                        213,
+                        "NCS-1",
+                        "protein"
+                    ],
+                    [
+                        228,
+                        237,
+                        "frequenin",
+                        "protein"
+                    ],
+                    [
+                        241,
+                        246,
+                        "yeast",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        256,
+                        271,
+                        "14-3-3 proteins",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 96,
+                "sent": "The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "monomeric",
+                        "oligomeric_state"
+                    ],
+                    [
+                        14,
+                        23,
+                        "G protein",
+                        "protein_type"
+                    ],
+                    [
+                        24,
+                        29,
+                        "Rab11",
+                        "protein"
+                    ],
+                    [
+                        36,
+                        45,
+                        "mammalian",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        46,
+                        51,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        64,
+                        78,
+                        "helical domain",
+                        "structure_element"
+                    ],
+                    [
+                        86,
+                        92,
+                        "kinase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 97,
+                "sent": "Although Rab11 does not appear to be required for recruitment of PI4KB to the Golgi, PI4KB is required for Golgi recruitment of Rab11.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        9,
+                        14,
+                        "Rab11",
+                        "protein"
+                    ],
+                    [
+                        65,
+                        70,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        85,
+                        90,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        128,
+                        133,
+                        "Rab11",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 98,
+                "sent": "Arf1, the other small GTP binding protein, is known to influence the activity and localization of PI4KB, but it does not appear to interact directly with PI4KB (our unpublished data).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "Arf1",
+                        "protein"
+                    ],
+                    [
+                        16,
+                        41,
+                        "small GTP binding protein",
+                        "protein_type"
+                    ],
+                    [
+                        98,
+                        103,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        154,
+                        159,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 99,
+                "sent": "The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        9,
+                        "yeast",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        23,
+                        27,
+                        "NCS1",
+                        "protein"
+                    ],
+                    [
+                        35,
+                        44,
+                        "frequenin",
+                        "protein"
+                    ],
+                    [
+                        77,
+                        82,
+                        "Pik1p",
+                        "protein"
+                    ],
+                    [
+                        88,
+                        93,
+                        "yeast",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        108,
+                        113,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        194,
+                        199,
+                        "NCS-1",
+                        "protein"
+                    ],
+                    [
+                        203,
+                        208,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        224,
+                        233,
+                        "mammalian",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 100,
+                "sent": "NCS-1 is an N-terminally myristoylated protein that participates in exocytosis.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "NCS-1",
+                        "protein"
+                    ],
+                    [
+                        25,
+                        38,
+                        "myristoylated",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 101,
+                "sent": "It is expressed only in certain cell types, suggesting that if it contributes to PI4KB membrane recruitment, it does so in a tissues specific manner.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        81,
+                        86,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 102,
+                "sent": "The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        19,
+                        24,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        30,
+                        45,
+                        "14-3-3 proteins",
+                        "protein_type"
+                    ],
+                    [
+                        59,
+                        74,
+                        "phosphorylation",
+                        "ptm"
+                    ],
+                    [
+                        78,
+                        83,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        87,
+                        103,
+                        "protein kinase D",
+                        "protein"
+                    ],
+                    [
+                        132,
+                        137,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        157,
+                        163,
+                        "active",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 103,
+                "sent": "However, 14-3-3 proteins do not appear to interfere with membrane recruitment of this kinase.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        9,
+                        24,
+                        "14-3-3 proteins",
+                        "protein_type"
+                    ],
+                    [
+                        86,
+                        92,
+                        "kinase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 104,
+                "sent": "ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        35,
+                        44,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        51,
+                        62,
+                        "vertebrates",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        105,
+                        110,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        228,
+                        234,
+                        "kinase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 105,
+                "sent": "For these reasons we focused on the interaction of the PI4KB enzyme with the Golgi resident ACBD3 protein in this study.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        55,
+                        60,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        92,
+                        97,
+                        "ACBD3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 106,
+                "sent": "Here we present the mechanism for membrane recruitment of PI4KB by the Golgi resident ACBD3 protein.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        58,
+                        63,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        86,
+                        91,
+                        "ACBD3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 107,
+                "sent": "We show that these proteins interact directly with a Kd value in the submicromolar range.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        53,
+                        55,
+                        "Kd",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 108,
+                "sent": "The interaction is sufficient to recruit PI4KB to model membranes in vitro as well as to the mitochondria where PI4KB is never naturally found.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        41,
+                        46,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        112,
+                        117,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 109,
+                "sent": "To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44\u201364) that binds the ACBD3 Q domain.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        50,
+                        56,
+                        "solved",
+                        "experimental_method"
+                    ],
+                    [
+                        61,
+                        79,
+                        "solution structure",
+                        "evidence"
+                    ],
+                    [
+                        83,
+                        94,
+                        "ACBD3:PI4KB",
+                        "complex_assembly"
+                    ],
+                    [
+                        136,
+                        141,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        142,
+                        159,
+                        "N-terminal region",
+                        "structure_element"
+                    ],
+                    [
+                        171,
+                        193,
+                        "short amphipatic helix",
+                        "structure_element"
+                    ],
+                    [
+                        204,
+                        209,
+                        "44\u201364",
+                        "residue_range"
+                    ],
+                    [
+                        226,
+                        231,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        232,
+                        240,
+                        "Q domain",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 110,
+                "sent": "The Q domain adopts a helical hairpin fold that is further stabilized upon binding the kinase helix (Fig. 2A).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        12,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        22,
+                        42,
+                        "helical hairpin fold",
+                        "structure_element"
+                    ],
+                    [
+                        87,
+                        99,
+                        "kinase helix",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 111,
+                "sent": "Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        115,
+                        121,
+                        "kinase",
+                        "protein_type"
+                    ],
+                    [
+                        173,
+                        178,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        214,
+                        220,
+                        "kinase",
+                        "protein_type"
+                    ],
+                    [
+                        271,
+                        277,
+                        "kinase",
+                        "protein_type"
+                    ],
+                    [
+                        377,
+                        383,
+                        "kinase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 112,
+                "sent": "Based on our and previously published structures we built a pseudoatomic model of PI4KB multi-protein assembly on the membrane (Fig. 5) that illustrates how the enzyme is recruited and positioned towards its lipidic substrate and how it in turn recruits Rab11.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        38,
+                        48,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        60,
+                        78,
+                        "pseudoatomic model",
+                        "evidence"
+                    ],
+                    [
+                        82,
+                        87,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        254,
+                        259,
+                        "Rab11",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 113,
+                "sent": "+RNA viruses replicate at specific PI4P-enriched membranous compartments.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        12,
+                        "+RNA viruses",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        35,
+                        39,
+                        "PI4P",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 114,
+                "sent": "These are called replication factories (because they enhance viral replication) or membranous webs (because of their appearance under the electron microscope).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        61,
+                        66,
+                        "viral",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 115,
+                "sent": "To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        35,
+                        42,
+                        "viruses",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        85,
+                        89,
+                        "PI4K",
+                        "protein_type"
+                    ],
+                    [
+                        90,
+                        97,
+                        "kinases",
+                        "protein_type"
+                    ],
+                    [
+                        128,
+                        132,
+                        "PI4P",
+                        "chemical"
+                    ],
+                    [
+                        133,
+                        138,
+                        "lipid",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 116,
+                "sent": "Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        26,
+                        "Non-structural 3A proteins",
+                        "protein_type"
+                    ],
+                    [
+                        37,
+                        51,
+                        "picornaviruses",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        61,
+                        72,
+                        "Enterovirus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        79,
+                        89,
+                        "poliovirus",
+                        "species"
+                    ],
+                    [
+                        91,
+                        108,
+                        "coxsackievirus-B3",
+                        "species"
+                    ],
+                    [
+                        110,
+                        123,
+                        "rhinovirus-14",
+                        "species"
+                    ],
+                    [
+                        129,
+                        138,
+                        "Kobuvirus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        145,
+                        158,
+                        "Aichi virus-1",
+                        "species"
+                    ],
+                    [
+                        190,
+                        195,
+                        "ACBD3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 117,
+                "sent": "Our data suggest that they could do this via 3A:ACBD3:PI4KB complex formation.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        45,
+                        59,
+                        "3A:ACBD3:PI4KB",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 118,
+                "sent": "The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        13,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        21,
+                        26,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        27,
+                        35,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        44,
+                        56,
+                        "kinase helix",
+                        "structure_element"
+                    ],
+                    [
+                        137,
+                        142,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        144,
+                        149,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        159,
+                        170,
+                        "ACBD3:PI4KB",
+                        "complex_assembly"
+                    ],
+                    [
+                        186,
+                        198,
+                        "picornaviral",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 119,
+                "sent": "This could eventually have implications for therapeutic intervention to combat picornaviruses-mediated diseases ranging from polio to the common cold.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        79,
+                        93,
+                        "picornaviruses",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 120,
+                "sent": "Biochemical characterization of the ACBD3:PI4KB complex.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        28,
+                        "Biochemical characterization",
+                        "experimental_method"
+                    ],
+                    [
+                        36,
+                        47,
+                        "ACBD3:PI4KB",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 121,
+                "sent": "(A) Schematic representation of the ACBD3 and PI4KB constructs used for the experiments.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        36,
+                        41,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        46,
+                        51,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 122,
+                "sent": "ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        19,
+                        42,
+                        "acyl-CoA binding domain",
+                        "structure_element"
+                    ],
+                    [
+                        44,
+                        48,
+                        "ACBD",
+                        "structure_element"
+                    ],
+                    [
+                        51,
+                        77,
+                        "charged amino acids region",
+                        "structure_element"
+                    ],
+                    [
+                        79,
+                        82,
+                        "CAR",
+                        "structure_element"
+                    ],
+                    [
+                        85,
+                        106,
+                        "glutamine rich region",
+                        "structure_element"
+                    ],
+                    [
+                        108,
+                        109,
+                        "Q",
+                        "structure_element"
+                    ],
+                    [
+                        116,
+                        137,
+                        "Golgi dynamics domain",
+                        "structure_element"
+                    ],
+                    [
+                        139,
+                        143,
+                        "GOLD",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 123,
+                "sent": "PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        25,
+                        42,
+                        "N-terminal region",
+                        "structure_element"
+                    ],
+                    [
+                        44,
+                        58,
+                        "helical domain",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        77,
+                        "kinase domain",
+                        "structure_element"
+                    ],
+                    [
+                        104,
+                        127,
+                        "N- and C-terminal lobes",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 124,
+                "sent": "(B) In vitro pull-down assay.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        28,
+                        "In vitro pull-down assay",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 125,
+                "sent": "Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        16,
+                        "Pull-down assays",
+                        "experimental_method"
+                    ],
+                    [
+                        67,
+                        81,
+                        "His6GB1-tagged",
+                        "protein_state"
+                    ],
+                    [
+                        108,
+                        116,
+                        "untagged",
+                        "protein_state"
+                    ],
+                    [
+                        117,
+                        128,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        129,
+                        134,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        138,
+                        143,
+                        "ACBD3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 126,
+                "sent": "The inputs and bound proteins were analyzed on SDS gels stained with Coomassie Blue.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        47,
+                        55,
+                        "SDS gels",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 127,
+                "sent": "Please, see SI Fig. 9 for original full-length gels. (C) Analytical Ultracentrifugation.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        35,
+                        46,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        57,
+                        87,
+                        "Analytical Ultracentrifugation",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 128,
+                "sent": "AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5\u2009\u03bcM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35\u2009\u03bcM (both proteins, right panel). (D) Surface plasmon resonance.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "AUC",
+                        "experimental_method"
+                    ],
+                    [
+                        20,
+                        31,
+                        "ACBD3:PI4KB",
+                        "complex_assembly"
+                    ],
+                    [
+                        32,
+                        43,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        113,
+                        152,
+                        "ACBD3 Q domain: PI4KB N terminal region",
+                        "complex_assembly"
+                    ],
+                    [
+                        225,
+                        250,
+                        "Surface plasmon resonance",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 129,
+                "sent": "SPR analysis of the PI4KB binding to immobilized ACBD3.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "SPR",
+                        "experimental_method"
+                    ],
+                    [
+                        20,
+                        25,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        49,
+                        54,
+                        "ACBD3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 130,
+                "sent": "Sensorgrams for four concentrations of PI4KB are shown.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Sensorgrams",
+                        "evidence"
+                    ],
+                    [
+                        39,
+                        44,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 131,
+                "sent": "Structural analysis of the ACBD3:PI4KB complex.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        19,
+                        "Structural analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        27,
+                        38,
+                        "ACBD3:PI4KB",
+                        "complex_assembly"
+                    ]
+                ]
+            },
+            {
+                "sid": 132,
+                "sent": "(A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        12,
+                        21,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        29,
+                        34,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        35,
+                        43,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        58,
+                        73,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        78,
+                        83,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        84,
+                        101,
+                        "N-terminal region",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 133,
+                "sent": "Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        13,
+                        "Superposition",
+                        "experimental_method"
+                    ],
+                    [
+                        34,
+                        44,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        62,
+                        70,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        98,
+                        108,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        158,
+                        164,
+                        "folded",
+                        "protein_state"
+                    ],
+                    [
+                        173,
+                        178,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 134,
+                "sent": "The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        43,
+                        57,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        59,
+                        64,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        65,
+                        71,
+                        "Tyr261",
+                        "residue_name_number"
+                    ],
+                    [
+                        73,
+                        78,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        79,
+                        84,
+                        "His63",
+                        "residue_name_number"
+                    ],
+                    [
+                        89,
+                        94,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        95,
+                        101,
+                        "Tyr288",
+                        "residue_name_number"
+                    ],
+                    [
+                        103,
+                        108,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        109,
+                        114,
+                        "Asp44",
+                        "residue_name_number"
+                    ],
+                    [
+                        127,
+                        146,
+                        "hydrophobic surface",
+                        "site"
+                    ],
+                    [
+                        154,
+                        166,
+                        "kinase helix",
+                        "structure_element"
+                    ],
+                    [
+                        180,
+                        185,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        186,
+                        194,
+                        "Q domain",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 135,
+                "sent": "ACBD3 is shown in magenta and PI4KB in orange.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        30,
+                        35,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 136,
+                "sent": "(C) Top view of the kinase helix.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        20,
+                        32,
+                        "kinase helix",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 137,
+                "sent": "The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        16,
+                        "kinase helix",
+                        "structure_element"
+                    ],
+                    [
+                        20,
+                        31,
+                        "amphipathic",
+                        "protein_state"
+                    ],
+                    [
+                        40,
+                        59,
+                        "hydrophobic surface",
+                        "site"
+                    ],
+                    [
+                        78,
+                        83,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        84,
+                        99,
+                        "binding surface",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 138,
+                "sent": "Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        160,
+                        175,
+                        "Pull-down assay",
+                        "experimental_method"
+                    ],
+                    [
+                        214,
+                        228,
+                        "His6GB1-tagged",
+                        "protein_state"
+                    ],
+                    [
+                        229,
+                        234,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        235,
+                        241,
+                        "kinase",
+                        "protein_type"
+                    ],
+                    [
+                        246,
+                        254,
+                        "untagged",
+                        "protein_state"
+                    ],
+                    [
+                        255,
+                        260,
+                        "ACBD3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 139,
+                "sent": "Wild type proteins and selected point mutants of both PI4KB and ACBD3 were used.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Wild type",
+                        "protein_state"
+                    ],
+                    [
+                        38,
+                        45,
+                        "mutants",
+                        "protein_state"
+                    ],
+                    [
+                        54,
+                        59,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        64,
+                        69,
+                        "ACBD3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 140,
+                "sent": "Please, see SI Fig. 9 for original full-length gels.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        35,
+                        46,
+                        "full-length",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 141,
+                "sent": "ACBD3 is sufficient to recruit the PI4KB kinase to membranes.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        35,
+                        40,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        41,
+                        47,
+                        "kinase",
+                        "protein_type"
+                    ]
+                ]
+            },
+            {
+                "sid": 142,
+                "sent": "(A) GUVs recruitment assay.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        26,
+                        "GUVs recruitment assay",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 143,
+                "sent": "Top \u2013 Virtually no membrane bound kinase was observed when 600\u2009nM PI4KB was added to the GUVs.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        34,
+                        40,
+                        "kinase",
+                        "protein_type"
+                    ],
+                    [
+                        66,
+                        71,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        89,
+                        93,
+                        "GUVs",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 144,
+                "sent": "Bottom \u2013 in the presence of 600\u2009nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        16,
+                        27,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        35,
+                        47,
+                        "GUV tethered",
+                        "protein_state"
+                    ],
+                    [
+                        48,
+                        53,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        82,
+                        88,
+                        "kinase",
+                        "protein_type"
+                    ],
+                    [
+                        119,
+                        123,
+                        "GUVs",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 145,
+                "sent": "(B) Golgi displacement experiment.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        33,
+                        "Golgi displacement experiment",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 146,
+                "sent": "Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        13,
+                        18,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        19,
+                        27,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        37,
+                        40,
+                        "GFP",
+                        "experimental_method"
+                    ],
+                    [
+                        45,
+                        58,
+                        "overexpressed",
+                        "experimental_method"
+                    ],
+                    [
+                        78,
+                        83,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        88,
+                        101,
+                        "immunostained",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 147,
+                "sent": "Middle panel: The same experiment performed with GFP alone.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        49,
+                        52,
+                        "GFP",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 148,
+                "sent": "Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi \u2013 COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05\u2009\u03bcM Bodipy FL-Ceramide for 20\u2009min, then washed and depicted after 20\u2009min.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        48,
+                        54,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        55,
+                        63,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        65,
+                        70,
+                        "F258A",
+                        "mutant"
+                    ],
+                    [
+                        72,
+                        77,
+                        "H284A",
+                        "mutant"
+                    ],
+                    [
+                        79,
+                        84,
+                        "Y288A",
+                        "mutant"
+                    ],
+                    [
+                        109,
+                        114,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        120,
+                        125,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        126,
+                        134,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        135,
+                        149,
+                        "overexpression",
+                        "experimental_method"
+                    ],
+                    [
+                        159,
+                        167,
+                        "ceramide",
+                        "chemical"
+                    ],
+                    [
+                        218,
+                        227,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        228,
+                        233,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        234,
+                        242,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        243,
+                        247,
+                        "FKBP",
+                        "protein"
+                    ],
+                    [
+                        248,
+                        252,
+                        "mRFP",
+                        "experimental_method"
+                    ],
+                    [
+                        278,
+                        296,
+                        "Bodipy FL-Ceramide",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 149,
+                "sent": "Middle panel \u2013 The same experiment performed with mRFP-FKBP alone.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        50,
+                        54,
+                        "mRFP",
+                        "experimental_method"
+                    ],
+                    [
+                        55,
+                        59,
+                        "FKBP",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 150,
+                "sent": "Lower panel \u2013 The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        49,
+                        55,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        56,
+                        64,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        66,
+                        71,
+                        "F258A",
+                        "mutant"
+                    ],
+                    [
+                        73,
+                        78,
+                        "H284A",
+                        "mutant"
+                    ],
+                    [
+                        80,
+                        85,
+                        "Y288A",
+                        "mutant"
+                    ],
+                    [
+                        110,
+                        115,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        135,
+                        170,
+                        "mitochondria recruitment experiment",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 151,
+                "sent": "\u2013 The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        6,
+                        11,
+                        "AKAP1",
+                        "protein"
+                    ],
+                    [
+                        12,
+                        15,
+                        "FRB",
+                        "structure_element"
+                    ],
+                    [
+                        16,
+                        19,
+                        "CFP",
+                        "experimental_method"
+                    ],
+                    [
+                        33,
+                        42,
+                        "localized",
+                        "evidence"
+                    ],
+                    [
+                        90,
+                        93,
+                        "GFP",
+                        "experimental_method"
+                    ],
+                    [
+                        94,
+                        99,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        104,
+                        112,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        113,
+                        117,
+                        "FKBP",
+                        "protein"
+                    ],
+                    [
+                        118,
+                        122,
+                        "mRFP",
+                        "experimental_method"
+                    ],
+                    [
+                        138,
+                        147,
+                        "localized",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 152,
+                "sent": "Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        17,
+                        26,
+                        "rapamycin",
+                        "chemical"
+                    ],
+                    [
+                        31,
+                        39,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        40,
+                        44,
+                        "FKBP",
+                        "protein"
+                    ],
+                    [
+                        45,
+                        49,
+                        "mRFP",
+                        "experimental_method"
+                    ],
+                    [
+                        103,
+                        106,
+                        "GFP",
+                        "experimental_method"
+                    ],
+                    [
+                        107,
+                        112,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        126,
+                        161,
+                        "Mitochondria recruitment experiment",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 153,
+                "sent": "Left \u2013 cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        30,
+                        35,
+                        "AKAP1",
+                        "protein"
+                    ],
+                    [
+                        36,
+                        39,
+                        "FRB",
+                        "structure_element"
+                    ],
+                    [
+                        40,
+                        43,
+                        "CFP",
+                        "experimental_method"
+                    ],
+                    [
+                        45,
+                        48,
+                        "GFP",
+                        "experimental_method"
+                    ],
+                    [
+                        49,
+                        54,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        59,
+                        68,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        69,
+                        77,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        78,
+                        82,
+                        "FKBP",
+                        "protein"
+                    ],
+                    [
+                        83,
+                        87,
+                        "mRFP",
+                        "experimental_method"
+                    ],
+                    [
+                        141,
+                        150,
+                        "rapamycin",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 154,
+                "sent": "Right \u2013 The same experiment performed using the H264A Q domain mutant.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        48,
+                        53,
+                        "H264A",
+                        "mutant"
+                    ],
+                    [
+                        54,
+                        62,
+                        "Q domain",
+                        "structure_element"
+                    ],
+                    [
+                        63,
+                        69,
+                        "mutant",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 155,
+                "sent": "ACBD3 indirectly increases the activity of PI4KB.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "ACBD3",
+                        "protein"
+                    ],
+                    [
+                        43,
+                        48,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 156,
+                "sent": "(A) Micelles-based kinase assay \u2013 PI in TX100 micelles was used in a luminescent kinase assay and the production of PI4P was measured.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        31,
+                        "Micelles-based kinase assay",
+                        "experimental_method"
+                    ],
+                    [
+                        34,
+                        36,
+                        "PI",
+                        "chemical"
+                    ],
+                    [
+                        69,
+                        93,
+                        "luminescent kinase assay",
+                        "experimental_method"
+                    ],
+                    [
+                        116,
+                        120,
+                        "PI4P",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 157,
+                "sent": "Bar graph presents the mean values of PI4P generated in the presence of the proteins as indicated, normalized to the amount of PI4P generated by PI4KB alone.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        38,
+                        42,
+                        "PI4P",
+                        "chemical"
+                    ],
+                    [
+                        60,
+                        71,
+                        "presence of",
+                        "protein_state"
+                    ],
+                    [
+                        127,
+                        131,
+                        "PI4P",
+                        "chemical"
+                    ],
+                    [
+                        145,
+                        150,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 158,
+                "sent": "Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay \u2013 GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        15,
+                        42,
+                        "standard errors of the mean",
+                        "evidence"
+                    ],
+                    [
+                        44,
+                        47,
+                        "SEM",
+                        "evidence"
+                    ],
+                    [
+                        93,
+                        124,
+                        "GUV-based phosphorylation assay",
+                        "experimental_method"
+                    ],
+                    [
+                        127,
+                        131,
+                        "GUVs",
+                        "experimental_method"
+                    ],
+                    [
+                        147,
+                        149,
+                        "PI",
+                        "chemical"
+                    ],
+                    [
+                        197,
+                        201,
+                        "PI4P",
+                        "chemical"
+                    ],
+                    [
+                        225,
+                        243,
+                        "CFP-SidC biosensor",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 159,
+                "sent": "(C)\u2013Quantification of the GUV phosphorylation assay \u2013 Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        26,
+                        51,
+                        "GUV phosphorylation assay",
+                        "experimental_method"
+                    ],
+                    [
+                        54,
+                        90,
+                        "Mean membrane fluorescence intensity",
+                        "evidence"
+                    ],
+                    [
+                        98,
+                        102,
+                        "PI4P",
+                        "chemical"
+                    ],
+                    [
+                        113,
+                        117,
+                        "SidC",
+                        "protein"
+                    ],
+                    [
+                        149,
+                        152,
+                        "ATP",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 160,
+                "sent": "The mean membrane intensity value is relative to the background signal and the difference between the membrane and background signal in the reference system lacking ATP.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        27,
+                        "mean membrane intensity",
+                        "evidence"
+                    ],
+                    [
+                        165,
+                        168,
+                        "ATP",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 161,
+                "sent": "The error bars stand for SEM based on three independent experiments (also SI Fig. 6).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        25,
+                        28,
+                        "SEM",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 162,
+                "sent": "Pseudoatomic model of the PI4KB multiprotein complex assembly.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        18,
+                        "Pseudoatomic model",
+                        "evidence"
+                    ],
+                    [
+                        26,
+                        31,
+                        "PI4KB",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 163,
+                "sent": "PI4KB in orange, Rab11 in purple, ACBD3 in blue.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "PI4KB",
+                        "protein"
+                    ],
+                    [
+                        17,
+                        22,
+                        "Rab11",
+                        "protein"
+                    ],
+                    [
+                        34,
+                        39,
+                        "ACBD3",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 164,
+                "sent": "The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        26,
+                        29,
+                        "NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        30,
+                        39,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        67,
+                        84,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        88,
+                        99,
+                        "PI4KB:Rab11",
+                        "complex_assembly"
+                    ],
+                    [
+                        125,
+                        129,
+                        "ACBD",
+                        "structure_element"
+                    ],
+                    [
+                        134,
+                        138,
+                        "GOLD",
+                        "structure_element"
+                    ],
+                    [
+                        151,
+                        167,
+                        "homology modeled",
+                        "experimental_method"
+                    ],
+                    [
+                        200,
+                        210,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        227,
+                        233,
+                        "Phyre2",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 165,
+                "sent": "The GOLD domain is tethered to the membrane by GolginB1 (also known as Giantin) which is not shown for clarity.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "GOLD",
+                        "structure_element"
+                    ],
+                    [
+                        47,
+                        55,
+                        "GolginB1",
+                        "protein"
+                    ],
+                    [
+                        71,
+                        78,
+                        "Giantin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 166,
+                "sent": "Intrinsically disordered linkers are modeled in an arbitrary but physically plausible conformation.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        32,
+                        "Intrinsically disordered linkers",
+                        "structure_element"
+                    ]
+                ]
+            }
+        ]
+    },
+    "PMC5603727": {
+        "annotations": [
+            {
+                "sid": 0,
+                "sent": "Roquin recognizes a non-canonical hexaloop structure in the 3\u2032-UTR of Ox40",
+                "section": "TITLE",
+                "ner": [
+                    [
+                        0,
+                        6,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        34,
+                        42,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        60,
+                        66,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        70,
+                        74,
+                        "Ox40",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 1,
+                "sent": "The RNA-binding protein Roquin is required to prevent autoimmunity.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        4,
+                        23,
+                        "RNA-binding protein",
+                        "protein_type"
+                    ],
+                    [
+                        24,
+                        30,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 2,
+                "sent": "Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40).",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        6,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        99,
+                        122,
+                        "costimulatory receptors",
+                        "protein_type"
+                    ],
+                    [
+                        131,
+                        175,
+                        "tumor necrosis factor receptor superfamily 4",
+                        "protein"
+                    ],
+                    [
+                        177,
+                        183,
+                        "Tnfrs4",
+                        "protein"
+                    ],
+                    [
+                        187,
+                        191,
+                        "Ox40",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 3,
+                "sent": "A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        2,
+                        28,
+                        "constitutive decay element",
+                        "structure_element"
+                    ],
+                    [
+                        30,
+                        33,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        57,
+                        72,
+                        "triloop hairpin",
+                        "structure_element"
+                    ],
+                    [
+                        114,
+                        120,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 4,
+                "sent": "Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE).",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        12,
+                        24,
+                        "SELEX assays",
+                        "experimental_method"
+                    ],
+                    [
+                        45,
+                        66,
+                        "U-rich hexaloop motif",
+                        "structure_element"
+                    ],
+                    [
+                        84,
+                        109,
+                        "alternative decay element",
+                        "structure_element"
+                    ],
+                    [
+                        111,
+                        114,
+                        "ADE",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 5,
+                "sent": "Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3\u2032-UTR with identical binding modes.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        0,
+                        18,
+                        "Crystal structures",
+                        "evidence"
+                    ],
+                    [
+                        23,
+                        26,
+                        "NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        46,
+                        54,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        55,
+                        58,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        77,
+                        86,
+                        "hexaloops",
+                        "structure_element"
+                    ],
+                    [
+                        94,
+                        99,
+                        "SELEX",
+                        "experimental_method"
+                    ],
+                    [
+                        108,
+                        111,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        122,
+                        125,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        154,
+                        158,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        159,
+                        165,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 6,
+                "sent": "In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        10,
+                        13,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        23,
+                        26,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        70,
+                        74,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        78,
+                        84,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 7,
+                "sent": "Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        45,
+                        66,
+                        "hexaloop cis elements",
+                        "structure_element"
+                    ],
+                    [
+                        116,
+                        130,
+                        "messenger RNAs",
+                        "chemical"
+                    ],
+                    [
+                        134,
+                        140,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 8,
+                "sent": " Roquin is an RNA-binding protein that prevents autoimmunity by limiting expression of receptors such as Ox40.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        1,
+                        7,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        14,
+                        33,
+                        "RNA-binding protein",
+                        "protein_type"
+                    ],
+                    [
+                        105,
+                        109,
+                        "Ox40",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 9,
+                "sent": "Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques.",
+                "section": "ABSTRACT",
+                "ner": [
+                    [
+                        30,
+                        33,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        34,
+                        43,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        69,
+                        94,
+                        "alternative decay element",
+                        "structure_element"
+                    ],
+                    [
+                        139,
+                        145,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        152,
+                        189,
+                        "structural and biochemical techniques",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 10,
+                "sent": "The Roquin protein is essential in T cells for the prevention of autoimmune disease.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        10,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 11,
+                "sent": "This is evident from the so-called sanroque mutation in Roquin-1, a single amino acid exchange from Met199 to Arg that causes the development of systemic lupus erythematosus-like symptoms in homozygous mice.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        56,
+                        64,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        100,
+                        106,
+                        "Met199",
+                        "residue_name_number"
+                    ],
+                    [
+                        110,
+                        113,
+                        "Arg",
+                        "residue_name"
+                    ],
+                    [
+                        202,
+                        206,
+                        "mice",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 12,
+                "sent": "The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        9,
+                        "Rc3h1",
+                        "gene"
+                    ],
+                    [
+                        14,
+                        19,
+                        "Rc3h2",
+                        "gene"
+                    ],
+                    [
+                        40,
+                        48,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        53,
+                        61,
+                        "Roquin-2",
+                        "protein"
+                    ],
+                    [
+                        74,
+                        85,
+                        "vertebrates",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        158,
+                        162,
+                        "mice",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 13,
+                "sent": "Consistently, CD4+ and CD8+ T cells with the combined deletion of Roquin-encoding genes are spontaneously activated and CD4+ T-helper cells preferentially differentiate into the Th1, Tfh or Th17 subsets.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        54,
+                        65,
+                        "deletion of",
+                        "experimental_method"
+                    ],
+                    [
+                        66,
+                        72,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 14,
+                "sent": "Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, I\u03baBNS and I\u03baB\u03b6 (refs).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        81,
+                        105,
+                        "co-stimulatory receptors",
+                        "protein_type"
+                    ],
+                    [
+                        114,
+                        118,
+                        "Icos",
+                        "protein"
+                    ],
+                    [
+                        120,
+                        124,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        129,
+                        135,
+                        "CTLA-4",
+                        "protein"
+                    ],
+                    [
+                        141,
+                        150,
+                        "cytokines",
+                        "protein_type"
+                    ],
+                    [
+                        159,
+                        177,
+                        "interleukin (IL)-6",
+                        "protein"
+                    ],
+                    [
+                        182,
+                        204,
+                        "tumour necrosis factor",
+                        "protein"
+                    ],
+                    [
+                        212,
+                        233,
+                        "transcription factors",
+                        "protein_type"
+                    ],
+                    [
+                        242,
+                        246,
+                        "IRF4",
+                        "protein"
+                    ],
+                    [
+                        248,
+                        253,
+                        "I\u03baBNS",
+                        "protein"
+                    ],
+                    [
+                        258,
+                        262,
+                        "I\u03baB\u03b6",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 15,
+                "sent": "We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3\u2032-untranslated regions (3\u2032-UTRs) of target genes such as Tnf (ref).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        26,
+                        56,
+                        "structural and functional data",
+                        "evidence"
+                    ],
+                    [
+                        64,
+                        72,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        73,
+                        76,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        84,
+                        92,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        105,
+                        131,
+                        "constitutive decay element",
+                        "structure_element"
+                    ],
+                    [
+                        133,
+                        136,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        141,
+                        156,
+                        "short stem loop",
+                        "structure_element"
+                    ],
+                    [
+                        158,
+                        160,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        192,
+                        195,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        211,
+                        234,
+                        "3\u2032-untranslated regions",
+                        "structure_element"
+                    ],
+                    [
+                        236,
+                        243,
+                        "3\u2032-UTRs",
+                        "structure_element"
+                    ],
+                    [
+                        269,
+                        272,
+                        "Tnf",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 16,
+                "sent": "The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein\u2013RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        25,
+                        51,
+                        "extended winged helix fold",
+                        "structure_element"
+                    ],
+                    [
+                        109,
+                        112,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        171,
+                        174,
+                        "Tnf",
+                        "protein"
+                    ],
+                    [
+                        175,
+                        178,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        179,
+                        182,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 17,
+                "sent": "The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        19,
+                        "structural data",
+                        "evidence"
+                    ],
+                    [
+                        24,
+                        43,
+                        "mutational analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        120,
+                        124,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        129,
+                        133,
+                        "stem",
+                        "structure_element"
+                    ],
+                    [
+                        141,
+                        144,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        184,
+                        190,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 18,
+                "sent": "At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        43,
+                        60,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        124,
+                        127,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        133,
+                        135,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        152,
+                        171,
+                        "second binding site",
+                        "site"
+                    ],
+                    [
+                        178,
+                        197,
+                        "double-stranded RNA",
+                        "chemical"
+                    ],
+                    [
+                        199,
+                        204,
+                        "dsRNA",
+                        "chemical"
+                    ],
+                    [
+                        216,
+                        224,
+                        "extended",
+                        "protein_state"
+                    ],
+                    [
+                        225,
+                        228,
+                        "ROQ",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 19,
+                "sent": "The structural basis for CDE recognition by the Roquin-2 ROQ domain has also been recently reported.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        25,
+                        28,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        48,
+                        56,
+                        "Roquin-2",
+                        "protein"
+                    ],
+                    [
+                        57,
+                        60,
+                        "ROQ",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 20,
+                "sent": "We found that the posttranscriptional activity of Roquin-1 and Roquin-2 is regulated through cleavage by the paracaspase MALT1 (refs).",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        50,
+                        58,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        63,
+                        71,
+                        "Roquin-2",
+                        "protein"
+                    ],
+                    [
+                        109,
+                        120,
+                        "paracaspase",
+                        "protein_type"
+                    ],
+                    [
+                        121,
+                        126,
+                        "MALT1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 21,
+                "sent": "Enhanced MALT1-dependent cleavage and inactivation of Roquin, and thus less effective repression of target genes, result from increased strength of antigen recognition in T cells.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        9,
+                        14,
+                        "MALT1",
+                        "protein"
+                    ],
+                    [
+                        54,
+                        60,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 22,
+                "sent": "These findings suggest that dependent on the strength of cognate antigen recognition differential gene expression and cell fate decisions can be established in naive T cells by a graded cleavage and inactivation of Roquin.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        215,
+                        221,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 23,
+                "sent": "In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3\u2032-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        51,
+                        67,
+                        "binding affinity",
+                        "evidence"
+                    ],
+                    [
+                        86,
+                        88,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        105,
+                        112,
+                        "3\u2032-UTRs",
+                        "structure_element"
+                    ],
+                    [
+                        123,
+                        128,
+                        "mRNAs",
+                        "chemical"
+                    ],
+                    [
+                        200,
+                        206,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        221,
+                        223,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        224,
+                        227,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        262,
+                        268,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        356,
+                        362,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 24,
+                "sent": "Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3\u2032-UTR of Ox40 bound to the Roquin-1 ROQ domain.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        88,
+                        91,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        123,
+                        129,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        159,
+                        174,
+                        "U-rich hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        175,
+                        177,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        185,
+                        191,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        195,
+                        199,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        200,
+                        208,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        213,
+                        221,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        222,
+                        225,
+                        "ROQ",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 25,
+                "sent": "We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        34,
+                        38,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        73,
+                        76,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        86,
+                        94,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        95,
+                        97,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        98,
+                        102,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        128,
+                        185,
+                        "Systematic Evolution of Ligands by Exponential Enrichment",
+                        "experimental_method"
+                    ],
+                    [
+                        187,
+                        192,
+                        "SELEX",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 26,
+                "sent": "Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells.",
+                "section": "INTRO",
+                "ner": [
+                    [
+                        4,
+                        26,
+                        "X-ray crystallographic",
+                        "experimental_method"
+                    ],
+                    [
+                        28,
+                        31,
+                        "NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        33,
+                        64,
+                        "biochemical and functional data",
+                        "evidence"
+                    ],
+                    [
+                        79,
+                        98,
+                        "mutational analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        121,
+                        128,
+                        "triloop",
+                        "structure_element"
+                    ],
+                    [
+                        133,
+                        141,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        142,
+                        144,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        145,
+                        149,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        191,
+                        197,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 27,
+                "sent": "SELEX identifies novel RNA ligands of Roquin-1",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "SELEX",
+                        "experimental_method"
+                    ],
+                    [
+                        23,
+                        26,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        38,
+                        46,
+                        "Roquin-1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 28,
+                "sent": "We set out to identify Roquin-bound RNA motifs in an unbiased manner by performing SELEX experiments.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        23,
+                        35,
+                        "Roquin-bound",
+                        "protein_state"
+                    ],
+                    [
+                        36,
+                        39,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        83,
+                        88,
+                        "SELEX",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 29,
+                "sent": "A biotinylated amino-terminal protein fragment of Roquin-1 (residues 2\u2013440) was used to enrich RNAs from a library containing 47 random nucleotides over three sequential selection rounds.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        2,
+                        14,
+                        "biotinylated",
+                        "protein_state"
+                    ],
+                    [
+                        50,
+                        58,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        69,
+                        74,
+                        "2\u2013440",
+                        "residue_range"
+                    ],
+                    [
+                        95,
+                        99,
+                        "RNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 30,
+                "sent": "Next-generation sequencing (NGS) of the RNA before and after each selection round revealed that the starting pool represented about 99.6% unique reads in \u223c4.2 \u00d7 106 sequences.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        26,
+                        "Next-generation sequencing",
+                        "experimental_method"
+                    ],
+                    [
+                        28,
+                        31,
+                        "NGS",
+                        "experimental_method"
+                    ],
+                    [
+                        40,
+                        43,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 31,
+                "sent": "Bioinformatic analysis of NGS data sets derived from the starting pool and enriched selection rounds revealed that the complexity was reduced to 78.6% unique reads in 3.7 \u00d7 106 sequences that were analysed after 3 rounds of selection and enrichment.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        22,
+                        "Bioinformatic analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        26,
+                        29,
+                        "NGS",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 32,
+                "sent": "For NGS data analysis, the COMPAS software (AptaIT, Munich, Germany) was applied.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "NGS",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 33,
+                "sent": "Enriched sequences were clustered into so-called patterns with highly homologous sequences.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        9,
+                        33,
+                        "sequences were clustered",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 34,
+                "sent": "Based on this so-called co-occurrence approach, patterns on the basis of frequent motifs were generated and were searched for prominent hexamer sequences (Supplementary Fig. 1a).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        24,
+                        46,
+                        "co-occurrence approach",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 35,
+                "sent": "We identified 5\u2032-CGTTTT-3\u2032, 5\u2032-GCGTTT-3\u2032, 5\u2032-TGCGTT-3\u2032 and 5\u2032-GTTTTA-3\u2032 motifs that were also reconfirmed in an independent experiment (Supplementary Fig. 1a) and are located within highly similar sequences (Fig. 1a and Supplementary Fig. 1b).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        27,
+                        "5\u2032-CGTTTT-3\u2032,",
+                        "chemical"
+                    ],
+                    [
+                        28,
+                        40,
+                        "5\u2032-GCGTTT-3\u2032",
+                        "chemical"
+                    ],
+                    [
+                        42,
+                        54,
+                        "5\u2032-TGCGTT-3\u2032",
+                        "chemical"
+                    ],
+                    [
+                        59,
+                        71,
+                        "5\u2032-GTTTTA-3\u2032",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 36,
+                "sent": "Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        51,
+                        68,
+                        "sanroque mutation",
+                        "mutant"
+                    ],
+                    [
+                        85,
+                        88,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        100,
+                        106,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        149,
+                        154,
+                        "SELEX",
+                        "experimental_method"
+                    ],
+                    [
+                        188,
+                        196,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        221,
+                        226,
+                        "M199R",
+                        "mutant"
+                    ]
+                ]
+            },
+            {
+                "sid": 37,
+                "sent": "Notably, our SELEX approach did not reveal the previously identified CDE sequence.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        18,
+                        "SELEX",
+                        "experimental_method"
+                    ],
+                    [
+                        69,
+                        72,
+                        "CDE",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 38,
+                "sent": "We assume that the region of sequence identity in the CDE is too short for our sequence clustering algorithm.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        54,
+                        57,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        79,
+                        108,
+                        "sequence clustering algorithm",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 39,
+                "sent": "Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5\u20138\u2009nt stem, with one base in the stem not being paired (Supplementary Fig. 1c).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        45,
+                        50,
+                        "SELEX",
+                        "experimental_method"
+                    ],
+                    [
+                        86,
+                        88,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        134,
+                        138,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        160,
+                        164,
+                        "stem",
+                        "structure_element"
+                    ],
+                    [
+                        187,
+                        191,
+                        "stem",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 40,
+                "sent": "Searching the 3\u2032-UTRs of known Roquin targets with the consensus 5\u2032-TGCGTTTTAGGA-3\u2032, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3\u2032-UTR of Ox40 (Fig. 1b).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        21,
+                        "3\u2032-UTRs",
+                        "structure_element"
+                    ],
+                    [
+                        31,
+                        37,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        65,
+                        83,
+                        "5\u2032-TGCGTTTTAGGA-3\u2032",
+                        "chemical"
+                    ],
+                    [
+                        97,
+                        126,
+                        "Motif-based sequence analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        128,
+                        132,
+                        "MEME",
+                        "experimental_method"
+                    ],
+                    [
+                        195,
+                        203,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        221,
+                        227,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        231,
+                        235,
+                        "Ox40",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 41,
+                "sent": "Importantly, this motif is present across species in the 3\u2032-UTRs of respective mRNAs and showed highest conservation in the loop and the upper stem sequences with a drop of conservation towards the boundaries of the motif (Fig. 1c,d).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        57,
+                        64,
+                        "3\u2032-UTRs",
+                        "structure_element"
+                    ],
+                    [
+                        79,
+                        84,
+                        "mRNAs",
+                        "chemical"
+                    ],
+                    [
+                        124,
+                        128,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        143,
+                        147,
+                        "stem",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 42,
+                "sent": "The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5\u2032 to another CDE-like SL in the 3\u2032-UTR of Ox40 mRNA.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        16,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        35,
+                        40,
+                        "SELEX",
+                        "experimental_method"
+                    ],
+                    [
+                        84,
+                        109,
+                        "alternative decay element",
+                        "structure_element"
+                    ],
+                    [
+                        110,
+                        112,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        114,
+                        117,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        118,
+                        120,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        127,
+                        130,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        136,
+                        138,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        168,
+                        171,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        177,
+                        179,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        187,
+                        193,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        197,
+                        201,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        202,
+                        206,
+                        "mRNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 43,
+                "sent": "This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3\u2032-UTR of Tnf mRNA (CDE SL) (Fig. 1d).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        5,
+                        8,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        14,
+                        16,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        78,
+                        81,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        91,
+                        97,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        101,
+                        104,
+                        "Tnf",
+                        "protein"
+                    ],
+                    [
+                        105,
+                        109,
+                        "mRNA",
+                        "chemical"
+                    ],
+                    [
+                        111,
+                        114,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        115,
+                        117,
+                        "SL",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 44,
+                "sent": "NMR analysis of Roquin-bound SL RNAs",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        16,
+                        28,
+                        "Roquin-bound",
+                        "protein_state"
+                    ],
+                    [
+                        29,
+                        31,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        32,
+                        36,
+                        "RNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 45,
+                "sent": "We used NMR to analyse the secondary structure of Roquin-1-binding motifs derived from SELEX.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        11,
+                        "NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        50,
+                        73,
+                        "Roquin-1-binding motifs",
+                        "structure_element"
+                    ],
+                    [
+                        87,
+                        92,
+                        "SELEX",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 46,
+                "sent": "Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3\u2032-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        74,
+                        "Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy",
+                        "experimental_method"
+                    ],
+                    [
+                        76,
+                        81,
+                        "NOESY",
+                        "experimental_method"
+                    ],
+                    [
+                        83,
+                        86,
+                        "NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        87,
+                        94,
+                        "spectra",
+                        "evidence"
+                    ],
+                    [
+                        102,
+                        106,
+                        "free",
+                        "protein_state"
+                    ],
+                    [
+                        107,
+                        110,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        120,
+                        128,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        133,
+                        141,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        142,
+                        145,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        175,
+                        178,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        179,
+                        181,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        187,
+                        190,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        196,
+                        198,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        206,
+                        212,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        216,
+                        220,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        251,
+                        255,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        256,
+                        259,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        265,
+                        267,
+                        "SL",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 47,
+                "sent": "The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        20,
+                        24,
+                        "free",
+                        "protein_state"
+                    ],
+                    [
+                        25,
+                        29,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        79,
+                        91,
+                        "stem regions",
+                        "structure_element"
+                    ],
+                    [
+                        99,
+                        116,
+                        "hexa- and triloop",
+                        "structure_element"
+                    ],
+                    [
+                        117,
+                        119,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        177,
+                        181,
+                        "RNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 48,
+                "sent": "Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson\u2013Crick G\u2013G base pair at this position (Fig. 2a).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        62,
+                        65,
+                        "G15",
+                        "residue_name_number"
+                    ],
+                    [
+                        75,
+                        77,
+                        "G6",
+                        "residue_name_number"
+                    ],
+                    [
+                        86,
+                        89,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        90,
+                        92,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        107,
+                        137,
+                        "non-Watson\u2013Crick G\u2013G base pair",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 49,
+                "sent": "Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein\u2013RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        12,
+                        40,
+                        "chemical shift perturbations",
+                        "evidence"
+                    ],
+                    [
+                        42,
+                        46,
+                        "CSPs",
+                        "evidence"
+                    ],
+                    [
+                        104,
+                        107,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        156,
+                        159,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        195,
+                        198,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        213,
+                        224,
+                        "stem region",
+                        "structure_element"
+                    ],
+                    [
+                        232,
+                        235,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 50,
+                "sent": "No imino correlations are observed for the predicted Watson\u2013Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A\u2013U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        53,
+                        76,
+                        "Watson\u2013Crick base pairs",
+                        "bond_interaction"
+                    ],
+                    [
+                        98,
+                        101,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        102,
+                        104,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        113,
+                        117,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        118,
+                        121,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        127,
+                        129,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        130,
+                        134,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        155,
+                        156,
+                        "A",
+                        "residue_name"
+                    ],
+                    [
+                        157,
+                        158,
+                        "U",
+                        "residue_name"
+                    ],
+                    [
+                        182,
+                        187,
+                        "bulge",
+                        "structure_element"
+                    ],
+                    [
+                        195,
+                        199,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        200,
+                        203,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        209,
+                        211,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        212,
+                        215,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 51,
+                "sent": "In contrast, all expected base pairs are observed for the Ox40 CDE-like SL RNA (Fig. 2c; see also Supplementary Notes).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        58,
+                        62,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        63,
+                        66,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        72,
+                        74,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        75,
+                        78,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 52,
+                "sent": "Structures of ROQ bound to ADE SL RNAs",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        10,
+                        "Structures",
+                        "evidence"
+                    ],
+                    [
+                        14,
+                        17,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        18,
+                        26,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        27,
+                        30,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        31,
+                        33,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        34,
+                        38,
+                        "RNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 53,
+                "sent": "To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        17,
+                        23,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        48,
+                        50,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        78,
+                        83,
+                        "SELEX",
+                        "experimental_method"
+                    ],
+                    [
+                        97,
+                        103,
+                        "solved",
+                        "experimental_method"
+                    ],
+                    [
+                        104,
+                        122,
+                        "crystal structures",
+                        "evidence"
+                    ],
+                    [
+                        130,
+                        138,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        139,
+                        142,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        150,
+                        158,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        179,
+                        182,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 54,
+                "sent": "The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2\u2009\u00c5, respectively.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        14,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        18,
+                        21,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        22,
+                        30,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        42,
+                        45,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        46,
+                        48,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        91,
+                        95,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        96,
+                        99,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        105,
+                        107,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        108,
+                        112,
+                        "RNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 55,
+                "sent": "In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix \u03b14 and the N-terminal part of \u03b23 (Fig. 3a,b and Supplementary Fig. 2a,b).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        8,
+                        18,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        23,
+                        26,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        37,
+                        39,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        56,
+                        64,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        123,
+                        126,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        127,
+                        132,
+                        "helix",
+                        "structure_element"
+                    ],
+                    [
+                        133,
+                        135,
+                        "\u03b14",
+                        "structure_element"
+                    ],
+                    [
+                        163,
+                        165,
+                        "\u03b23",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 56,
+                "sent": "The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c\u2013e).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        9,
+                        "dsRNA",
+                        "chemical"
+                    ],
+                    [
+                        10,
+                        14,
+                        "stem",
+                        "structure_element"
+                    ],
+                    [
+                        76,
+                        79,
+                        "Tnf",
+                        "protein"
+                    ],
+                    [
+                        80,
+                        83,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        84,
+                        86,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        87,
+                        90,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 57,
+                "sent": "As may be expected, the recognition of the hexaloop is significantly different from the triloop in the CDE RNA (Fig. 3b,c and Supplementary Fig. 2b).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        43,
+                        51,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        88,
+                        95,
+                        "triloop",
+                        "structure_element"
+                    ],
+                    [
+                        103,
+                        106,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        107,
+                        110,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 58,
+                "sent": "Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein\u2013RNA contacts are virtually identical (Supplementary Fig. 2a,d,e).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        45,
+                        48,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        49,
+                        51,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        56,
+                        59,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        65,
+                        67,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        68,
+                        72,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        100,
+                        110,
+                        "structures",
+                        "evidence"
+                    ],
+                    [
+                        123,
+                        126,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 59,
+                "sent": "The only differences are a C19 bulge, the non-Watson\u2013Crick G6\u2013G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e\u2013g).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        27,
+                        30,
+                        "C19",
+                        "residue_name_number"
+                    ],
+                    [
+                        31,
+                        36,
+                        "bulge",
+                        "structure_element"
+                    ],
+                    [
+                        42,
+                        58,
+                        "non-Watson\u2013Crick",
+                        "bond_interaction"
+                    ],
+                    [
+                        59,
+                        61,
+                        "G6",
+                        "residue_name_number"
+                    ],
+                    [
+                        62,
+                        65,
+                        "G15",
+                        "residue_name_number"
+                    ],
+                    [
+                        66,
+                        75,
+                        "base pair",
+                        "bond_interaction"
+                    ],
+                    [
+                        99,
+                        101,
+                        "U1",
+                        "residue_name_number"
+                    ],
+                    [
+                        107,
+                        113,
+                        "Trp184",
+                        "residue_name_number"
+                    ],
+                    [
+                        118,
+                        124,
+                        "Phe194",
+                        "residue_name_number"
+                    ],
+                    [
+                        132,
+                        135,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        141,
+                        143,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        144,
+                        147,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 60,
+                "sent": "Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3\u2032-UTR and was solved at higher resolution.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        82,
+                        91,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        99,
+                        103,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        104,
+                        107,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        113,
+                        115,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        116,
+                        119,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        151,
+                        155,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        156,
+                        162,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 61,
+                "sent": "The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        47,
+                        67,
+                        "double-stranded stem",
+                        "structure_element"
+                    ],
+                    [
+                        75,
+                        79,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        80,
+                        83,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        89,
+                        91,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        110,
+                        113,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        114,
+                        121,
+                        "triloop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 62,
+                "sent": "Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        28,
+                        "U-rich hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        36,
+                        40,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        41,
+                        44,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        50,
+                        52,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        53,
+                        56,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        78,
+                        85,
+                        "surface",
+                        "site"
+                    ],
+                    [
+                        93,
+                        96,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        135,
+                        138,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        139,
+                        146,
+                        "triloop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 63,
+                "sent": "For example, the main chain atoms of Phe255 form two hydrogen bonds with the Watson\u2013Crick face of the U11 base (Fig. 3d).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        37,
+                        43,
+                        "Phe255",
+                        "residue_name_number"
+                    ],
+                    [
+                        53,
+                        67,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        102,
+                        105,
+                        "U11",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 64,
+                "sent": "Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d\u2013f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        16,
+                        25,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        33,
+                        36,
+                        "Tnf",
+                        "protein"
+                    ],
+                    [
+                        37,
+                        40,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        41,
+                        48,
+                        "triloop",
+                        "structure_element"
+                    ],
+                    [
+                        53,
+                        59,
+                        "Tyr250",
+                        "residue_name_number"
+                    ],
+                    [
+                        88,
+                        101,
+                        "hydrogen bond",
+                        "bond_interaction"
+                    ],
+                    [
+                        128,
+                        131,
+                        "G12",
+                        "residue_name_number"
+                    ],
+                    [
+                        183,
+                        191,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        232,
+                        238,
+                        "Tyr250",
+                        "residue_name_number"
+                    ],
+                    [
+                        271,
+                        274,
+                        "U11",
+                        "residue_name_number"
+                    ],
+                    [
+                        341,
+                        362,
+                        "stacking interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        372,
+                        375,
+                        "U10",
+                        "residue_name_number"
+                    ],
+                    [
+                        380,
+                        383,
+                        "U11",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 65,
+                "sent": "In addition, the Tyr250 main-chain carbonyl interacts with U13 imino proton (Fig. 3d,e).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        17,
+                        23,
+                        "Tyr250",
+                        "residue_name_number"
+                    ],
+                    [
+                        59,
+                        62,
+                        "U13",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 66,
+                "sent": "Val257 and Lys259 in strand \u03b23 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        6,
+                        "Val257",
+                        "residue_name_number"
+                    ],
+                    [
+                        11,
+                        17,
+                        "Lys259",
+                        "residue_name_number"
+                    ],
+                    [
+                        21,
+                        27,
+                        "strand",
+                        "structure_element"
+                    ],
+                    [
+                        28,
+                        30,
+                        "\u03b23",
+                        "structure_element"
+                    ],
+                    [
+                        58,
+                        61,
+                        "UGU",
+                        "structure_element"
+                    ],
+                    [
+                        62,
+                        69,
+                        "triloop",
+                        "structure_element"
+                    ],
+                    [
+                        77,
+                        80,
+                        "Tnf",
+                        "protein"
+                    ],
+                    [
+                        81,
+                        84,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        85,
+                        88,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        139,
+                        147,
+                        "hexaloop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 67,
+                "sent": "The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        24,
+                        "Lys259",
+                        "residue_name_number"
+                    ],
+                    [
+                        31,
+                        45,
+                        "hydrogen bonds",
+                        "bond_interaction"
+                    ],
+                    [
+                        75,
+                        78,
+                        "U10",
+                        "residue_name_number"
+                    ],
+                    [
+                        83,
+                        86,
+                        "U11",
+                        "residue_name_number"
+                    ],
+                    [
+                        133,
+                        139,
+                        "Val257",
+                        "residue_name_number"
+                    ],
+                    [
+                        140,
+                        146,
+                        "stacks",
+                        "bond_interaction"
+                    ],
+                    [
+                        156,
+                        159,
+                        "U11",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 68,
+                "sent": "The RNA stem is closed by a Watson\u2013Crick base pair (C8\u2013G15 in the hexaloop SL RNA).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        8,
+                        12,
+                        "stem",
+                        "structure_element"
+                    ],
+                    [
+                        28,
+                        50,
+                        "Watson\u2013Crick base pair",
+                        "bond_interaction"
+                    ],
+                    [
+                        52,
+                        54,
+                        "C8",
+                        "residue_name_number"
+                    ],
+                    [
+                        55,
+                        58,
+                        "G15",
+                        "residue_name_number"
+                    ],
+                    [
+                        66,
+                        74,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        75,
+                        77,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        78,
+                        81,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 69,
+                "sent": "Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        21,
+                        "G9",
+                        "residue_name_number"
+                    ],
+                    [
+                        27,
+                        33,
+                        "stacks",
+                        "bond_interaction"
+                    ],
+                    [
+                        131,
+                        134,
+                        "G12",
+                        "residue_name_number"
+                    ],
+                    [
+                        142,
+                        145,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        146,
+                        153,
+                        "triloop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 70,
+                "sent": "The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        6,
+                        "G9",
+                        "residue_name_number"
+                    ],
+                    [
+                        43,
+                        46,
+                        "A14",
+                        "residue_name_number"
+                    ],
+                    [
+                        62,
+                        65,
+                        "A14",
+                        "residue_name_number"
+                    ],
+                    [
+                        86,
+                        98,
+                        "minor groove",
+                        "site"
+                    ],
+                    [
+                        106,
+                        109,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 71,
+                "sent": "This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5\u2032-side of the RNA stem (Fig. 3e).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        38,
+                        58,
+                        "stacking interaction",
+                        "bond_interaction"
+                    ],
+                    [
+                        62,
+                        64,
+                        "G9",
+                        "residue_name_number"
+                    ],
+                    [
+                        66,
+                        69,
+                        "U10",
+                        "residue_name_number"
+                    ],
+                    [
+                        74,
+                        80,
+                        "Tyr250",
+                        "residue_name_number"
+                    ],
+                    [
+                        88,
+                        91,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        121,
+                        124,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        125,
+                        129,
+                        "stem",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 72,
+                "sent": "The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "U11",
+                        "residue_name_number"
+                    ],
+                    [
+                        12,
+                        15,
+                        "U13",
+                        "residue_name_number"
+                    ],
+                    [
+                        22,
+                        27,
+                        "stack",
+                        "bond_interaction"
+                    ],
+                    [
+                        67,
+                        70,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        78,
+                        82,
+                        "wing",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 73,
+                "sent": "This is possible by exposing the base C12 of the Ox-40 ADE-like SL towards the solvent, which accordingly does not show any contacts to the protein.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        38,
+                        41,
+                        "C12",
+                        "residue_name_number"
+                    ],
+                    [
+                        49,
+                        54,
+                        "Ox-40",
+                        "protein"
+                    ],
+                    [
+                        55,
+                        58,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        66,
+                        "SL",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 74,
+                "sent": "In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        27,
+                        30,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        31,
+                        33,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        44,
+                        47,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        48,
+                        50,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        55,
+                        58,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        66,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        67,
+                        71,
+                        "RNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 75,
+                "sent": "However, these involve an extended binding surface on the ROQ domain with a number of additional residues compared with the triloop RNA.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        58,
+                        61,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        132,
+                        135,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 76,
+                "sent": "NMR analysis of ROQ interactions with ADE SLs",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        16,
+                        19,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        38,
+                        41,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        42,
+                        45,
+                        "SLs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 77,
+                "sent": "We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        29,
+                        "NMR spectroscopy",
+                        "experimental_method"
+                    ],
+                    [
+                        45,
+                        48,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        71,
+                        74,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        84,
+                        87,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        93,
+                        95,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        96,
+                        100,
+                        "RNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 78,
+                "sent": "CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        4,
+                        "CSPs",
+                        "evidence"
+                    ],
+                    [
+                        32,
+                        35,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        61,
+                        65,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        66,
+                        69,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        75,
+                        77,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        78,
+                        81,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        150,
+                        153,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        154,
+                        157,
+                        "SLs",
+                        "structure_element"
+                    ],
+                    [
+                        167,
+                        173,
+                        "Lys220",
+                        "residue_name_number"
+                    ],
+                    [
+                        175,
+                        181,
+                        "Lys239",
+                        "residue_name_number"
+                    ],
+                    [
+                        182,
+                        188,
+                        "Thr240",
+                        "residue_name_number"
+                    ],
+                    [
+                        193,
+                        199,
+                        "Lys259",
+                        "residue_name_number"
+                    ],
+                    [
+                        200,
+                        206,
+                        "Arg260",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 79,
+                "sent": "This is fully consistent with the interactions observed in the crystal structure (Supplementary Fig. 2c\u2013e) and indicates a similar binding surface.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        63,
+                        80,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        131,
+                        146,
+                        "binding surface",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 80,
+                "sent": "However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3\u2032-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        32,
+                        47,
+                        "CSP differences",
+                        "evidence"
+                    ],
+                    [
+                        78,
+                        81,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        92,
+                        96,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        97,
+                        100,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        106,
+                        108,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        109,
+                        113,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        125,
+                        128,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        134,
+                        136,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        137,
+                        140,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        148,
+                        152,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        153,
+                        159,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        181,
+                        184,
+                        "Tnf",
+                        "protein"
+                    ],
+                    [
+                        185,
+                        188,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        189,
+                        191,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        192,
+                        195,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 81,
+                "sent": "For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        19,
+                        "Ser253",
+                        "residue_name_number"
+                    ],
+                    [
+                        64,
+                        68,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        69,
+                        72,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        78,
+                        80,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        134,
+                        142,
+                        "hexaloop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 82,
+                "sent": "On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        33,
+                        36,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        61,
+                        64,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        78,
+                        81,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        87,
+                        89,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        90,
+                        94,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        122,
+                        125,
+                        "NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        126,
+                        133,
+                        "spectra",
+                        "evidence"
+                    ],
+                    [
+                        138,
+                        142,
+                        "CSPs",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 83,
+                "sent": "This is consistent with the very similar structural features and mode of RNA recognition of the ROQ domain with these RNAs (Supplementary Fig. 2a,d,e).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        73,
+                        76,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        96,
+                        99,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        118,
+                        122,
+                        "RNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 84,
+                "sent": "Mutational analysis of the ROQ-ADE interaction",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        19,
+                        "Mutational analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        27,
+                        30,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        31,
+                        34,
+                        "ADE",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 85,
+                "sent": "To examine the individual contributions of ROQ\u2013hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        43,
+                        46,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        105,
+                        142,
+                        "electrophoretic mobility shift assays",
+                        "experimental_method"
+                    ],
+                    [
+                        144,
+                        149,
+                        "EMSAs",
+                        "experimental_method"
+                    ],
+                    [
+                        172,
+                        175,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        191,
+                        195,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        196,
+                        199,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        205,
+                        208,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 86,
+                "sent": "Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        33,
+                        42,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        43,
+                        46,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        68,
+                        76,
+                        "affinity",
+                        "evidence"
+                    ],
+                    [
+                        107,
+                        110,
+                        "Tnf",
+                        "protein"
+                    ],
+                    [
+                        111,
+                        114,
+                        "CDE",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 87,
+                "sent": "We next tested a set of mutants (Supplementary Fig. 4), which were designed based on contacts observed in the crystal structure (Fig. 3) and the NMR CSPs (Fig. 4a,b).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        110,
+                        127,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        145,
+                        148,
+                        "NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        149,
+                        153,
+                        "CSPs",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 88,
+                "sent": "In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        31,
+                        42,
+                        "ROQ-Tnf CDE",
+                        "complex_assembly"
+                    ],
+                    [
+                        109,
+                        128,
+                        "structural analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        147,
+                        153,
+                        "Lys220",
+                        "residue_name_number"
+                    ],
+                    [
+                        155,
+                        161,
+                        "Lys239",
+                        "residue_name_number"
+                    ],
+                    [
+                        163,
+                        169,
+                        "Lys259",
+                        "residue_name_number"
+                    ],
+                    [
+                        174,
+                        180,
+                        "Arg260",
+                        "residue_name_number"
+                    ],
+                    [
+                        222,
+                        233,
+                        "replacement",
+                        "experimental_method"
+                    ],
+                    [
+                        237,
+                        244,
+                        "alanine",
+                        "residue_name"
+                    ]
+                ]
+            },
+            {
+                "sid": 89,
+                "sent": "We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        58,
+                        63,
+                        "Y250A",
+                        "mutant"
+                    ],
+                    [
+                        64,
+                        70,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        78,
+                        86,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        87,
+                        89,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        90,
+                        93,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        127,
+                        130,
+                        "Tnf",
+                        "protein"
+                    ],
+                    [
+                        131,
+                        134,
+                        "CDE",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 90,
+                "sent": "This underlines the central role of Tyr250 for stabilization of the hexaloop structure and recognition by stacking interactions (Fig. 3b,e).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        36,
+                        42,
+                        "Tyr250",
+                        "residue_name_number"
+                    ],
+                    [
+                        68,
+                        76,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        106,
+                        127,
+                        "stacking interactions",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 91,
+                "sent": "Mutation of Ser253, which shows large CSPs in the NMR titrations (Fig. 4a,b), does not significantly impair complex formation (Supplementary Fig. 4).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        8,
+                        "Mutation",
+                        "experimental_method"
+                    ],
+                    [
+                        12,
+                        18,
+                        "Ser253",
+                        "residue_name_number"
+                    ],
+                    [
+                        38,
+                        42,
+                        "CSPs",
+                        "evidence"
+                    ],
+                    [
+                        50,
+                        64,
+                        "NMR titrations",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 92,
+                "sent": "The large chemical shift change is probably caused by ring current effects induced by the close proximity of the U11 and U13 bases.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        10,
+                        31,
+                        "chemical shift change",
+                        "evidence"
+                    ],
+                    [
+                        113,
+                        116,
+                        "U11",
+                        "residue_name_number"
+                    ],
+                    [
+                        121,
+                        124,
+                        "U13",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 93,
+                "sent": "Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        17,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        25,
+                        29,
+                        "wing",
+                        "structure_element"
+                    ],
+                    [
+                        37,
+                        40,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        49,
+                        54,
+                        "S265Y",
+                        "mutant"
+                    ],
+                    [
+                        152,
+                        155,
+                        "Tnf",
+                        "protein"
+                    ],
+                    [
+                        156,
+                        159,
+                        "CDE",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 94,
+                "sent": "This indicates that replacement by Tyr does not strongly affect the RNA interaction, and that some conformational variations are tolerated.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        20,
+                        31,
+                        "replacement",
+                        "experimental_method"
+                    ],
+                    [
+                        35,
+                        38,
+                        "Tyr",
+                        "residue_name"
+                    ],
+                    [
+                        68,
+                        71,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 95,
+                "sent": "Thus, the mutational analysis is fully consistent with the recognition of the hexaloop observed in our crystal structures.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        10,
+                        29,
+                        "mutational analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        78,
+                        86,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        103,
+                        121,
+                        "crystal structures",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 96,
+                "sent": "To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        45,
+                        51,
+                        "Tyr250",
+                        "residue_name_number"
+                    ],
+                    [
+                        55,
+                        63,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        67,
+                        71,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        72,
+                        76,
+                        "mRNA",
+                        "chemical"
+                    ],
+                    [
+                        117,
+                        149,
+                        "retroviral reconstitution system",
+                        "experimental_method"
+                    ],
+                    [
+                        153,
+                        159,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 97,
+                "sent": "Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        27,
+                        32,
+                        "Rc3h1",
+                        "gene"
+                    ],
+                    [
+                        33,
+                        36,
+                        "2fl",
+                        "gene"
+                    ],
+                    [
+                        37,
+                        39,
+                        "fl",
+                        "gene"
+                    ],
+                    [
+                        60,
+                        64,
+                        "mice",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        83,
+                        91,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        92,
+                        93,
+                        "2",
+                        "protein"
+                    ],
+                    [
+                        114,
+                        123,
+                        "tamoxifen",
+                        "chemical"
+                    ],
+                    [
+                        158,
+                        204,
+                        "reverse tetracycline-controlled transactivator",
+                        "protein_type"
+                    ],
+                    [
+                        205,
+                        209,
+                        "rtTA",
+                        "protein"
+                    ],
+                    [
+                        237,
+                        256,
+                        "4-hydroxy tamoxifen",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 98,
+                "sent": "The cells were then transduced with doxycycline-inducible retroviral vectors to reconstitute Roquin-1 expression (Fig. 5b).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        36,
+                        47,
+                        "doxycycline",
+                        "chemical"
+                    ],
+                    [
+                        93,
+                        101,
+                        "Roquin-1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 99,
+                "sent": "Depletion of Roquin proteins on tamoxifen treatment (Supplementary Fig. 5a) strongly increased surface expression of Ox40 and Icos (Fig. 5c).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        19,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        32,
+                        41,
+                        "tamoxifen",
+                        "chemical"
+                    ],
+                    [
+                        117,
+                        121,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        126,
+                        130,
+                        "Icos",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 100,
+                "sent": "This increase in surface expression of both costimulatory receptors was partially corrected by the doxycycline-induced reconstitution with Roquin-1 WT protein (Fig. 5c left panels).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        44,
+                        67,
+                        "costimulatory receptors",
+                        "protein_type"
+                    ],
+                    [
+                        99,
+                        110,
+                        "doxycycline",
+                        "chemical"
+                    ],
+                    [
+                        139,
+                        147,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        148,
+                        150,
+                        "WT",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 101,
+                "sent": "Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        57,
+                        62,
+                        "Y250A",
+                        "mutant"
+                    ],
+                    [
+                        63,
+                        69,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        73,
+                        81,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        89,
+                        94,
+                        "K220A",
+                        "mutant"
+                    ],
+                    [
+                        96,
+                        101,
+                        "K239A",
+                        "mutant"
+                    ],
+                    [
+                        106,
+                        110,
+                        "R260",
+                        "mutant"
+                    ],
+                    [
+                        111,
+                        117,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        149,
+                        152,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        153,
+                        155,
+                        "SL",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 102,
+                "sent": "However, it is also possible that continuous overexpression of targets following Roquin deletion induces a hyperactivated state in the T cells.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        45,
+                        59,
+                        "overexpression",
+                        "experimental_method"
+                    ],
+                    [
+                        81,
+                        87,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 103,
+                "sent": "This hyperactivation, compared with the actual posttranscriptional derepression, may contribute even stronger to the increased Icos and Ox40 expression levels.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        127,
+                        131,
+                        "Icos",
+                        "protein"
+                    ],
+                    [
+                        136,
+                        140,
+                        "Ox40",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 104,
+                "sent": "Hence, our structure\u2013function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        38,
+                        "structure\u2013function analyses",
+                        "experimental_method"
+                    ],
+                    [
+                        66,
+                        70,
+                        "Y250",
+                        "residue_name_number"
+                    ],
+                    [
+                        96,
+                        102,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        133,
+                        137,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        170,
+                        176,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        193,
+                        197,
+                        "Icos",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 105,
+                "sent": "We also investigated the role of individual nucleotides in the Ox40 ADE-like SL for complex formation with the ROQ domain.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        63,
+                        67,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        68,
+                        71,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        77,
+                        79,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        111,
+                        114,
+                        "ROQ",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 106,
+                "sent": "We designed four mutants (Mut1\u20134, see Supplementary Fig. 6) that were expected to disrupt key interactions with the protein according to our co-crystal structure (Fig. 3d\u2013f and Supplementary Fig. 2).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        141,
+                        161,
+                        "co-crystal structure",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 107,
+                "sent": "NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        32,
+                        38,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        39,
+                        43,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        78,
+                        89,
+                        "stem region",
+                        "structure_element"
+                    ],
+                    [
+                        108,
+                        117,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        118,
+                        121,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        127,
+                        129,
+                        "SL",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 108,
+                "sent": "We next used surface plasmon resonance experiments to determine dissociation constants for the ROQ-RNA interaction (Table 2 and Supplementary Fig. 7).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        38,
+                        "surface plasmon resonance",
+                        "experimental_method"
+                    ],
+                    [
+                        64,
+                        86,
+                        "dissociation constants",
+                        "evidence"
+                    ],
+                    [
+                        95,
+                        98,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        99,
+                        102,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 109,
+                "sent": "Although the replacement of a C8\u2013G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        13,
+                        24,
+                        "replacement",
+                        "experimental_method"
+                    ],
+                    [
+                        30,
+                        32,
+                        "C8",
+                        "residue_name_number"
+                    ],
+                    [
+                        33,
+                        36,
+                        "G15",
+                        "residue_name_number"
+                    ],
+                    [
+                        58,
+                        59,
+                        "A",
+                        "residue_name"
+                    ],
+                    [
+                        60,
+                        61,
+                        "U",
+                        "residue_name"
+                    ],
+                    [
+                        63,
+                        68,
+                        "Mut 4",
+                        "mutant"
+                    ],
+                    [
+                        87,
+                        95,
+                        "affinity",
+                        "evidence"
+                    ],
+                    [
+                        120,
+                        124,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        137,
+                        141,
+                        "A14C",
+                        "mutant"
+                    ],
+                    [
+                        142,
+                        148,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        150,
+                        155,
+                        "Mut 1",
+                        "mutant"
+                    ],
+                    [
+                        182,
+                        190,
+                        "affinity",
+                        "evidence"
+                    ],
+                    [
+                        222,
+                        247,
+                        "surface plasmon resonance",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 110,
+                "sent": "As intended, the mutation Mut 1 allows the formation of an additional base pair and thus leads to the formation of a tetraloop with a new G-C closing base pair (Supplementary Fig. 6a).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        26,
+                        31,
+                        "Mut 1",
+                        "mutant"
+                    ],
+                    [
+                        117,
+                        126,
+                        "tetraloop",
+                        "structure_element"
+                    ],
+                    [
+                        138,
+                        139,
+                        "G",
+                        "residue_name"
+                    ],
+                    [
+                        140,
+                        141,
+                        "C",
+                        "residue_name"
+                    ]
+                ]
+            },
+            {
+                "sid": 111,
+                "sent": "Consistent with the structural analysis, we assume that this variant alters the hexaloop conformation and thus reduces the interaction with ROQ.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        20,
+                        39,
+                        "structural analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        80,
+                        88,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        140,
+                        143,
+                        "ROQ",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 112,
+                "sent": "Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        35,
+                        "stacking interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        44,
+                        47,
+                        "G15",
+                        "residue_name_number"
+                    ],
+                    [
+                        49,
+                        51,
+                        "G9",
+                        "residue_name_number"
+                    ],
+                    [
+                        56,
+                        60,
+                        "Y250",
+                        "residue_name_number"
+                    ],
+                    [
+                        68,
+                        71,
+                        "G9C",
+                        "mutant"
+                    ],
+                    [
+                        72,
+                        78,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        80,
+                        85,
+                        "Mut 2",
+                        "mutant"
+                    ],
+                    [
+                        119,
+                        122,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        130,
+                        132,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        133,
+                        136,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 113,
+                "sent": "No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        36,
+                        44,
+                        "U11AU13G",
+                        "mutant"
+                    ],
+                    [
+                        45,
+                        58,
+                        "double mutant",
+                        "protein_state"
+                    ],
+                    [
+                        60,
+                        65,
+                        "Mut 3",
+                        "mutant"
+                    ],
+                    [
+                        153,
+                        156,
+                        "U11",
+                        "residue_name_number"
+                    ],
+                    [
+                        161,
+                        164,
+                        "U13",
+                        "residue_name_number"
+                    ],
+                    [
+                        172,
+                        180,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        186,
+                        189,
+                        "ROQ",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 114,
+                "sent": "Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        20,
+                        25,
+                        "SELEX",
+                        "experimental_method"
+                    ],
+                    [
+                        65,
+                        74,
+                        "mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        78,
+                        87,
+                        "conserved",
+                        "protein_state"
+                    ],
+                    [
+                        88,
+                        99,
+                        "nucleotides",
+                        "chemical"
+                    ],
+                    [
+                        107,
+                        111,
+                        "loop",
+                        "structure_element"
+                    ],
+                    [
+                        151,
+                        154,
+                        "ROQ",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 115,
+                "sent": "Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81\u2009nM, respectively, Table 2 and Supplementary Fig. 7).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        19,
+                        27,
+                        "affinity",
+                        "evidence"
+                    ],
+                    [
+                        35,
+                        44,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        45,
+                        48,
+                        "Tnf",
+                        "protein"
+                    ],
+                    [
+                        49,
+                        52,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        61,
+                        65,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        66,
+                        69,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        75,
+                        78,
+                        "SLs",
+                        "structure_element"
+                    ],
+                    [
+                        82,
+                        85,
+                        "ROQ",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 116,
+                "sent": "Roquin binding to different SLs in the Ox40 3\u2032-UTR",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        6,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        28,
+                        31,
+                        "SLs",
+                        "structure_element"
+                    ],
+                    [
+                        39,
+                        43,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        44,
+                        50,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 117,
+                "sent": "We have recently shown that Roquin-1 binds to a CDE-like motif in the 3\u2032-UTR of Ox40 mRNA (Figs 1d and 4c).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        28,
+                        36,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        48,
+                        51,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        70,
+                        76,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        80,
+                        84,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        85,
+                        89,
+                        "mRNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 118,
+                "sent": "We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3\u2032-UTR.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        60,
+                        63,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        77,
+                        80,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        86,
+                        88,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        89,
+                        93,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        113,
+                        121,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        152,
+                        163,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        164,
+                        168,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        169,
+                        175,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 119,
+                "sent": "The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2\u2013440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        22,
+                        "binding affinities",
+                        "evidence"
+                    ],
+                    [
+                        47,
+                        64,
+                        "N-terminal domain",
+                        "structure_element"
+                    ],
+                    [
+                        68,
+                        76,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        87,
+                        92,
+                        "2\u2013440",
+                        "residue_range"
+                    ],
+                    [
+                        127,
+                        130,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        138,
+                        143,
+                        "alone",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 120,
+                "sent": "The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81\u2009nM, respectively (Table 2).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        4,
+                        26,
+                        "dissociation constants",
+                        "evidence"
+                    ],
+                    [
+                        35,
+                        38,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        60,
+                        64,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        65,
+                        68,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        74,
+                        76,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        85,
+                        88,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        94,
+                        96,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        97,
+                        101,
+                        "RNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 121,
+                "sent": "This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3\u2032-UTR.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        37,
+                        54,
+                        "binding interface",
+                        "site"
+                    ],
+                    [
+                        101,
+                        109,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        154,
+                        162,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        163,
+                        165,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        166,
+                        169,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        177,
+                        181,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        182,
+                        188,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 122,
+                "sent": "We designed different variants of the 3\u2032-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        38,
+                        44,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        48,
+                        65,
+                        "point mutagenesis",
+                        "experimental_method"
+                    ],
+                    [
+                        97,
+                        108,
+                        "stem region",
+                        "structure_element"
+                    ],
+                    [
+                        142,
+                        144,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        145,
+                        148,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        161,
+                        168,
+                        "mutated",
+                        "experimental_method"
+                    ],
+                    [
+                        179,
+                        187,
+                        "Roquin-1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 123,
+                "sent": "These RNAs were then tested in EMSAs with the Roquin-1 N terminus (residues 2\u2013440) (Fig. 6b).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        6,
+                        10,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        31,
+                        36,
+                        "EMSAs",
+                        "experimental_method"
+                    ],
+                    [
+                        46,
+                        54,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        76,
+                        81,
+                        "2\u2013440",
+                        "residue_range"
+                    ]
+                ]
+            },
+            {
+                "sid": 124,
+                "sent": "Gel shift assays show that binding to the wild-type 3\u2032-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        16,
+                        "Gel shift assays",
+                        "experimental_method"
+                    ],
+                    [
+                        42,
+                        51,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        52,
+                        58,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        108,
+                        118,
+                        "titrations",
+                        "experimental_method"
+                    ],
+                    [
+                        165,
+                        168,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 125,
+                "sent": "Consistent with this, both bands are strongly reduced when mutations are introduced that interfere with the formation of both SLs.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        126,
+                        129,
+                        "SLs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 126,
+                "sent": "Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        82,
+                        84,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        85,
+                        88,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        117,
+                        123,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        178,
+                        187,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        188,
+                        190,
+                        "SL",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 127,
+                "sent": "We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3\u2032-UTR.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        22,
+                        28,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        53,
+                        55,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        56,
+                        59,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        89,
+                        100,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        101,
+                        105,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        106,
+                        112,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 128,
+                "sent": "Regulation of Ox40 expression via two motifs in its 3\u2032-UTR",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        18,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        52,
+                        58,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 129,
+                "sent": "To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3\u2032-UTRs in cells.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        35,
+                        38,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        60,
+                        64,
+                        "mRNA",
+                        "chemical"
+                    ],
+                    [
+                        80,
+                        90,
+                        "introduced",
+                        "experimental_method"
+                    ],
+                    [
+                        91,
+                        95,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        96,
+                        100,
+                        "mRNA",
+                        "chemical"
+                    ],
+                    [
+                        121,
+                        128,
+                        "altered",
+                        "protein_state"
+                    ],
+                    [
+                        129,
+                        136,
+                        "3\u2032-UTRs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 130,
+                "sent": "Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3\u2032-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        39,
+                        42,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        52,
+                        55,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        61,
+                        63,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        68,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        76,
+                        82,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        117,
+                        123,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        167,
+                        171,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        256,
+                        259,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        272,
+                        302,
+                        "deletion and point mutagenesis",
+                        "experimental_method"
+                    ],
+                    [
+                        303,
+                        313,
+                        "abrogating",
+                        "protein_state"
+                    ],
+                    [
+                        314,
+                        326,
+                        "base pairing",
+                        "bond_interaction"
+                    ],
+                    [
+                        334,
+                        345,
+                        "stem region",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 131,
+                "sent": "Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3\u2032-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        20,
+                        32,
+                        "retroviruses",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        47,
+                        51,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        112,
+                        119,
+                        "3\u2032-UTRs",
+                        "structure_element"
+                    ],
+                    [
+                        125,
+                        133,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        134,
+                        135,
+                        "2",
+                        "protein"
+                    ],
+                    [
+                        146,
+                        151,
+                        "mouse",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 132,
+                "sent": "Doxycycline treatment of cells from this cell line enabled ectopic Roquin-1 and co-translational mCherry expression due to the stable integration of an inducible lentiviral vector (Supplementary Fig. 8c).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        11,
+                        "Doxycycline",
+                        "chemical"
+                    ],
+                    [
+                        67,
+                        75,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        162,
+                        172,
+                        "lentiviral",
+                        "taxonomy_domain"
+                    ]
+                ]
+            },
+            {
+                "sid": 133,
+                "sent": "The expression of Ox40 in cells with and without doxycycline treatment was then quantified by flow cytometry (Supplementary Fig. 8c).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        18,
+                        22,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        49,
+                        60,
+                        "doxycycline",
+                        "chemical"
+                    ],
+                    [
+                        94,
+                        108,
+                        "flow cytometry",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 134,
+                "sent": "Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3\u2032-UTR revealed a comparable importance of both structural elements (Fig. 6c).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        23,
+                        27,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        28,
+                        57,
+                        "mean fluorescence intensities",
+                        "evidence"
+                    ],
+                    [
+                        84,
+                        95,
+                        "doxycycline",
+                        "chemical"
+                    ],
+                    [
+                        157,
+                        161,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        173,
+                        180,
+                        "without",
+                        "protein_state"
+                    ],
+                    [
+                        181,
+                        187,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 135,
+                "sent": "In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3\u2032-UTR 1\u201380 and double mut) neutralized Roquin-dependent repression of Ox40.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        14,
+                        43,
+                        "deletion or point mutagenesis",
+                        "experimental_method"
+                    ],
+                    [
+                        104,
+                        110,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        111,
+                        115,
+                        "1\u201380",
+                        "residue_range"
+                    ],
+                    [
+                        120,
+                        130,
+                        "double mut",
+                        "protein_state"
+                    ],
+                    [
+                        144,
+                        150,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        175,
+                        179,
+                        "Ox40",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 136,
+                "sent": "In contrast, individual mutations that left the hexaloop (3\u2032-UTR 1\u2013120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3\u2032-UTR (Fig. 6c).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        24,
+                        33,
+                        "mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        48,
+                        56,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        58,
+                        64,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        65,
+                        70,
+                        "1\u2013120",
+                        "residue_range"
+                    ],
+                    [
+                        74,
+                        81,
+                        "CDE mut",
+                        "mutant"
+                    ],
+                    [
+                        90,
+                        93,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        99,
+                        106,
+                        "triloop",
+                        "structure_element"
+                    ],
+                    [
+                        107,
+                        113,
+                        "intact",
+                        "protein_state"
+                    ],
+                    [
+                        128,
+                        134,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        214,
+                        225,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        226,
+                        232,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 137,
+                "sent": "To further analyse the functional consequences of Roquin binding to the 3\u2032-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        50,
+                        56,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        72,
+                        78,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        97,
+                        113,
+                        "mRNA decay rates",
+                        "evidence"
+                    ],
+                    [
+                        146,
+                        150,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        278,
+                        289,
+                        "doxycycline",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 138,
+                "sent": "Quantitative reverse transcriptase\u2013PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3\u2032-UTR (CDS t1/2=311\u2009min vs full-length t1/2=96\u2009min).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        0,
+                        38,
+                        "Quantitative reverse transcriptase\u2013PCR",
+                        "experimental_method"
+                    ],
+                    [
+                        78,
+                        82,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        83,
+                        87,
+                        "mRNA",
+                        "chemical"
+                    ],
+                    [
+                        91,
+                        102,
+                        "deletion of",
+                        "experimental_method"
+                    ],
+                    [
+                        107,
+                        113,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        115,
+                        118,
+                        "CDS",
+                        "structure_element"
+                    ],
+                    [
+                        119,
+                        123,
+                        "t1/2",
+                        "evidence"
+                    ],
+                    [
+                        135,
+                        146,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        147,
+                        151,
+                        "t1/2",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 139,
+                "sent": "A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255\u2009min).",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        43,
+                        60,
+                        "combined mutation",
+                        "experimental_method"
+                    ],
+                    [
+                        68,
+                        71,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        85,
+                        88,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        94,
+                        97,
+                        "SLs",
+                        "structure_element"
+                    ],
+                    [
+                        99,
+                        102,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        103,
+                        106,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        112,
+                        115,
+                        "mut",
+                        "protein_state"
+                    ],
+                    [
+                        116,
+                        120,
+                        "t1/2",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 140,
+                "sent": "Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170\u2009min, CDE-like mut t1/2=167\u2009min), respectively.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        11,
+                        20,
+                        "mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        35,
+                        38,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        51,
+                        54,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        60,
+                        63,
+                        "SLs",
+                        "structure_element"
+                    ],
+                    [
+                        93,
+                        96,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        102,
+                        105,
+                        "mut",
+                        "protein_state"
+                    ],
+                    [
+                        106,
+                        110,
+                        "t1/2",
+                        "evidence"
+                    ],
+                    [
+                        120,
+                        123,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        129,
+                        132,
+                        "mut",
+                        "protein_state"
+                    ],
+                    [
+                        133,
+                        137,
+                        "t1/2",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 141,
+                "sent": "These findings underscore the importance of both structural motifs and reveal that they have an additive effect on the regulation of Ox40 mRNA expression in cells.",
+                "section": "RESULTS",
+                "ner": [
+                    [
+                        133,
+                        137,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        138,
+                        142,
+                        "mRNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 142,
+                "sent": "Recent structural and functional studies have provided first insight into the RNA binding of Roquin.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        7,
+                        40,
+                        "structural and functional studies",
+                        "experimental_method"
+                    ],
+                    [
+                        78,
+                        81,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        93,
+                        99,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 143,
+                "sent": "Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine\u2013purine\u2013pyrimidine triloop.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        0,
+                        10,
+                        "Structures",
+                        "evidence"
+                    ],
+                    [
+                        14,
+                        20,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        21,
+                        29,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        30,
+                        33,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        34,
+                        36,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        37,
+                        41,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        84,
+                        86,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        87,
+                        90,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        108,
+                        114,
+                        "A-site",
+                        "site"
+                    ],
+                    [
+                        122,
+                        139,
+                        "N-terminal region",
+                        "structure_element"
+                    ],
+                    [
+                        147,
+                        153,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        221,
+                        257,
+                        "pyrimidine\u2013purine\u2013pyrimidine triloop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 144,
+                "sent": "Considering that the CDE RNA recognition is mostly structure specific and not sequence dependent, a wide spectrum of target mRNA might be recognized by Roquin.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        21,
+                        24,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        25,
+                        28,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        124,
+                        128,
+                        "mRNA",
+                        "chemical"
+                    ],
+                    [
+                        152,
+                        158,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 145,
+                "sent": "Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3\u2032-UTR and variations of which may be found in the 3\u2032-UTRs of many other genes.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        18,
+                        30,
+                        "SELEX assays",
+                        "experimental_method"
+                    ],
+                    [
+                        51,
+                        72,
+                        "RNA recognition motif",
+                        "structure_element"
+                    ],
+                    [
+                        76,
+                        84,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        110,
+                        114,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        115,
+                        121,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        166,
+                        173,
+                        "3\u2032-UTRs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 146,
+                "sent": "Our experiments show that this SELEX-derived ADE shows functional activity comparable to the previously established CDE motif.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        31,
+                        36,
+                        "SELEX",
+                        "experimental_method"
+                    ],
+                    [
+                        45,
+                        48,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        116,
+                        119,
+                        "CDE",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 147,
+                "sent": "The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        4,
+                        7,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        12,
+                        16,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        17,
+                        20,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        26,
+                        28,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        29,
+                        33,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        40,
+                        42,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        56,
+                        64,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        78,
+                        85,
+                        "triloop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 148,
+                "sent": "Notably, the recognition of the respective RNA-helical stem regions by the ROQ domain is identical for the triloop and hexaloop motifs.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        43,
+                        67,
+                        "RNA-helical stem regions",
+                        "structure_element"
+                    ],
+                    [
+                        75,
+                        78,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        107,
+                        114,
+                        "triloop",
+                        "structure_element"
+                    ],
+                    [
+                        119,
+                        127,
+                        "hexaloop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 149,
+                "sent": "However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix \u03b14 and strand \u03b23 in the ROQ domain that are absent in the triloop CDE (Fig. 3b\u2013f).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        13,
+                        29,
+                        "U-rich hexaloops",
+                        "structure_element"
+                    ],
+                    [
+                        37,
+                        40,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        45,
+                        48,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        54,
+                        56,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        57,
+                        61,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        111,
+                        116,
+                        "helix",
+                        "structure_element"
+                    ],
+                    [
+                        117,
+                        119,
+                        "\u03b14",
+                        "structure_element"
+                    ],
+                    [
+                        124,
+                        130,
+                        "strand",
+                        "structure_element"
+                    ],
+                    [
+                        131,
+                        133,
+                        "\u03b23",
+                        "structure_element"
+                    ],
+                    [
+                        141,
+                        144,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        175,
+                        182,
+                        "triloop",
+                        "structure_element"
+                    ],
+                    [
+                        183,
+                        186,
+                        "CDE",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 150,
+                "sent": "Of particular importance for the hexaloop recognition is Tyr250, which acts as a stabilizing element for the integrity of a defined loop conformation.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        33,
+                        41,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        57,
+                        63,
+                        "Tyr250",
+                        "residue_name_number"
+                    ],
+                    [
+                        132,
+                        136,
+                        "loop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 151,
+                "sent": "It stacks with nucleotides in the hexaloop but not the CDE triloop (Fig. 3b,c).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        3,
+                        9,
+                        "stacks",
+                        "bond_interaction"
+                    ],
+                    [
+                        34,
+                        42,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        55,
+                        58,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        59,
+                        66,
+                        "triloop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 152,
+                "sent": "The functional role of Tyr250 for ADE-mediated mRNA regulation by Roquin-1 is thus explained by our experiments (Fig. 5b,c).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        23,
+                        29,
+                        "Tyr250",
+                        "residue_name_number"
+                    ],
+                    [
+                        34,
+                        37,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        47,
+                        51,
+                        "mRNA",
+                        "chemical"
+                    ],
+                    [
+                        66,
+                        74,
+                        "Roquin-1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 153,
+                "sent": "The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        19,
+                        35,
+                        "U-rich hexaloops",
+                        "structure_element"
+                    ],
+                    [
+                        83,
+                        86,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        92,
+                        95,
+                        "U10",
+                        "residue_name_number"
+                    ],
+                    [
+                        97,
+                        100,
+                        "U11",
+                        "residue_name_number"
+                    ],
+                    [
+                        105,
+                        108,
+                        "U13",
+                        "residue_name_number"
+                    ],
+                    [
+                        116,
+                        120,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        121,
+                        124,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        130,
+                        132,
+                        "SL",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 154,
+                "sent": "Consistent with this, loss of ROQ binding is observed on replacement of U11 and U13 by other bases (Table 2).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        30,
+                        33,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        57,
+                        68,
+                        "replacement",
+                        "experimental_method"
+                    ],
+                    [
+                        72,
+                        75,
+                        "U11",
+                        "residue_name_number"
+                    ],
+                    [
+                        80,
+                        83,
+                        "U13",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 155,
+                "sent": "In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        53,
+                        56,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        90,
+                        96,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        122,
+                        125,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        130,
+                        133,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        139,
+                        143,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        209,
+                        212,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        213,
+                        217,
+                        "stem",
+                        "structure_element"
+                    ],
+                    [
+                        254,
+                        272,
+                        "hexa- and triloops",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 156,
+                "sent": "A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        24,
+                        29,
+                        "bound",
+                        "protein_state"
+                    ],
+                    [
+                        30,
+                        33,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        34,
+                        43,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        60,
+                        78,
+                        "tri- and hexaloops",
+                        "structure_element"
+                    ],
+                    [
+                        87,
+                        95,
+                        "stacking",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 157,
+                "sent": "Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        9,
+                        24,
+                        "structural data",
+                        "evidence"
+                    ],
+                    [
+                        79,
+                        85,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        111,
+                        113,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        114,
+                        117,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        150,
+                        155,
+                        "loops",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 158,
+                "sent": "Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        19,
+                        24,
+                        "SELEX",
+                        "experimental_method"
+                    ],
+                    [
+                        53,
+                        66,
+                        "U-rich motifs",
+                        "structure_element"
+                    ],
+                    [
+                        141,
+                        153,
+                        "U-rich loops",
+                        "structure_element"
+                    ],
+                    [
+                        190,
+                        226,
+                        "crosslinking and immunoprecipitation",
+                        "experimental_method"
+                    ],
+                    [
+                        230,
+                        238,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        245,
+                        253,
+                        "PAR-CLIP",
+                        "experimental_method"
+                    ],
+                    [
+                        306,
+                        321,
+                        "U-rich hexaloop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 159,
+                "sent": "Most probably, the experimental setup of Murakawa et al. revealed both high- and low-affinity target motifs for Roquin, whereas our structural study reports on a high-affinity binding motif.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        112,
+                        118,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        132,
+                        148,
+                        "structural study",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 160,
+                "sent": "Notably, Murakawa et al. neither found the Roquin-regulated Ox40 nor the Tnf 3\u2032-UTRs, as both genes are not expressed in HEK 293 cells.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        43,
+                        49,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        60,
+                        64,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        73,
+                        76,
+                        "Tnf",
+                        "protein"
+                    ],
+                    [
+                        77,
+                        84,
+                        "3\u2032-UTRs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 161,
+                "sent": "However, their newly identified U-rich target SL within the 3\u2032-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        46,
+                        48,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        60,
+                        66,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        70,
+                        73,
+                        "A20",
+                        "protein"
+                    ],
+                    [
+                        74,
+                        78,
+                        "mRNA",
+                        "chemical"
+                    ],
+                    [
+                        108,
+                        114,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        177,
+                        180,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        181,
+                        188,
+                        "triloop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 162,
+                "sent": "It remains to be seen which exact features govern the recognition of the A20 SL by Roquin.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        73,
+                        76,
+                        "A20",
+                        "protein"
+                    ],
+                    [
+                        77,
+                        79,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        83,
+                        89,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 163,
+                "sent": "The regulatory cis RNA elements in 3\u2032-UTRs may also be targeted by additional trans-acting factors.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        15,
+                        31,
+                        "cis RNA elements",
+                        "structure_element"
+                    ],
+                    [
+                        35,
+                        42,
+                        "3\u2032-UTRs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 164,
+                "sent": "We have recently identified the endonuclease Regnase-1 as a cofactor of Roquin function that shares an overlapping set of target mRNAs.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        32,
+                        44,
+                        "endonuclease",
+                        "protein_type"
+                    ],
+                    [
+                        45,
+                        54,
+                        "Regnase-1",
+                        "protein"
+                    ],
+                    [
+                        72,
+                        78,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        129,
+                        134,
+                        "mRNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 165,
+                "sent": "In another study, the overlap in targets was confirmed, but a mutually exclusive regulation was proposed based on studies in lipopolysaccharide (LPS)-stimulated myeloid cells.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        125,
+                        143,
+                        "lipopolysaccharide",
+                        "chemical"
+                    ],
+                    [
+                        145,
+                        148,
+                        "LPS",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 166,
+                "sent": "In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        16,
+                        22,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        31,
+                        35,
+                        "mRNA",
+                        "chemical"
+                    ],
+                    [
+                        67,
+                        75,
+                        "inactive",
+                        "protein_state"
+                    ],
+                    [
+                        76,
+                        81,
+                        "mRNAs",
+                        "chemical"
+                    ],
+                    [
+                        89,
+                        98,
+                        "Regnase-1",
+                        "protein"
+                    ],
+                    [
+                        107,
+                        111,
+                        "mRNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 167,
+                "sent": "In CD4+ T cells, Ox40 does not show derepression in individual knockouts of Roquin-1 or Roquin-2 encoding genes, but is strongly induced upon combined deficiency of both genes.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        17,
+                        21,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        76,
+                        84,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        88,
+                        96,
+                        "Roquin-2",
+                        "protein"
+                    ],
+                    [
+                        151,
+                        161,
+                        "deficiency",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 168,
+                "sent": "In addition, conditional deletion of the Regnase-1-encoding gene induced Ox40 expression in these cells.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        25,
+                        36,
+                        "deletion of",
+                        "experimental_method"
+                    ],
+                    [
+                        41,
+                        50,
+                        "Regnase-1",
+                        "protein"
+                    ],
+                    [
+                        73,
+                        77,
+                        "Ox40",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 169,
+                "sent": "Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        25,
+                        29,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        30,
+                        34,
+                        "mRNA",
+                        "chemical"
+                    ],
+                    [
+                        38,
+                        44,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        48,
+                        55,
+                        "Regnase",
+                        "protein_type"
+                    ],
+                    [
+                        243,
+                        270,
+                        "double and triple knockouts",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 170,
+                "sent": "However, in cultures of CD4+ T cells, Ox40 is translated on day 4\u20135 and is expressed much higher in T cells with combined deficiency of Roquin-1 and Roquin-2.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        38,
+                        42,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        136,
+                        144,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        149,
+                        157,
+                        "Roquin-2",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 171,
+                "sent": "At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        45,
+                        59,
+                        "reconstitution",
+                        "experimental_method"
+                    ],
+                    [
+                        65,
+                        67,
+                        "WT",
+                        "protein_state"
+                    ],
+                    [
+                        68,
+                        76,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        102,
+                        106,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        169,
+                        175,
+                        "active",
+                        "protein_state"
+                    ],
+                    [
+                        176,
+                        180,
+                        "mRNA",
+                        "chemical"
+                    ],
+                    [
+                        184,
+                        192,
+                        "Roquin-1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 172,
+                "sent": "Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3\u2032-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        44,
+                        52,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        56,
+                        64,
+                        "Roquin-2",
+                        "protein"
+                    ],
+                    [
+                        98,
+                        102,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        103,
+                        107,
+                        "mRNA",
+                        "chemical"
+                    ],
+                    [
+                        111,
+                        116,
+                        "EMSAs",
+                        "experimental_method"
+                    ],
+                    [
+                        125,
+                        131,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        135,
+                        139,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        197,
+                        218,
+                        "filter binding assays",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 173,
+                "sent": "Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3\u2032-UTR by both proteins.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        27,
+                        30,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        95,
+                        98,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        103,
+                        106,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        121,
+                        125,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        126,
+                        132,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 174,
+                "sent": "In contrast, structural details on how Regnase-1 can interact with these SL RNAs are currently missing.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        39,
+                        48,
+                        "Regnase-1",
+                        "protein"
+                    ],
+                    [
+                        73,
+                        75,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        76,
+                        80,
+                        "RNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 175,
+                "sent": "Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine\u2013purine\u2013pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        44,
+                        67,
+                        "Regnase-1-binding sites",
+                        "site"
+                    ],
+                    [
+                        71,
+                        119,
+                        "crosslinking and immunoprecipitation experiments",
+                        "experimental_method"
+                    ],
+                    [
+                        140,
+                        147,
+                        "triloop",
+                        "structure_element"
+                    ],
+                    [
+                        164,
+                        198,
+                        "pyrimidine\u2013purine\u2013pyrimidine loops",
+                        "structure_element"
+                    ],
+                    [
+                        218,
+                        223,
+                        "stems",
+                        "structure_element"
+                    ],
+                    [
+                        244,
+                        252,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        270,
+                        275,
+                        "Ptgs2",
+                        "gene"
+                    ]
+                ]
+            },
+            {
+                "sid": 176,
+                "sent": "Both were required for Regnase-1-mediated repression.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        23,
+                        32,
+                        "Regnase-1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 177,
+                "sent": "These findings therefore raise the possibility that Regnase-1 interacts with ADE-like hexaloop structures either in a direct or indirect manner.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        52,
+                        61,
+                        "Regnase-1",
+                        "protein"
+                    ],
+                    [
+                        77,
+                        80,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        86,
+                        94,
+                        "hexaloop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 178,
+                "sent": "Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        46,
+                        58,
+                        "cis-elements",
+                        "structure_element"
+                    ],
+                    [
+                        93,
+                        96,
+                        "SLs",
+                        "structure_element"
+                    ],
+                    [
+                        103,
+                        107,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        111,
+                        115,
+                        "Icos",
+                        "protein"
+                    ],
+                    [
+                        231,
+                        235,
+                        "mRNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 179,
+                "sent": "The novel 3\u2032-UTR loop motif that we have identified as a bona fide target of Roquin now expands this multilayer mode of co-regulation.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        10,
+                        16,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        17,
+                        27,
+                        "loop motif",
+                        "structure_element"
+                    ],
+                    [
+                        77,
+                        83,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 180,
+                "sent": "We suggest that differential regulation of mRNA expression is not only achieved through multiple regulators with individual preferences for a given motif or variants thereof, but that regulators may also identify and use distinct motifs, as long as they exhibit some basic features regarding shape, size and sequence.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        43,
+                        47,
+                        "mRNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 181,
+                "sent": "The presence of distinct motifs in 3\u2032-UTRs offers a broader variability for gene regulation by RNA cis elements.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        35,
+                        42,
+                        "3\u2032-UTRs",
+                        "structure_element"
+                    ],
+                    [
+                        95,
+                        98,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        99,
+                        111,
+                        "cis elements",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 182,
+                "sent": "Their accessibility can be modulated by trans-acting factors that may bind regulatory motifs, unfold higher-order structures in the RNA or maintain a preference for duplex structures as was shown recently for mRNAs that are recognized by Staufen-1 (ref.).",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        132,
+                        135,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        209,
+                        214,
+                        "mRNAs",
+                        "chemical"
+                    ],
+                    [
+                        238,
+                        247,
+                        "Staufen-1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 183,
+                "sent": "In the 3\u2032-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        7,
+                        13,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        21,
+                        25,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        26,
+                        30,
+                        "mRNA",
+                        "chemical"
+                    ],
+                    [
+                        44,
+                        47,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        61,
+                        64,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        70,
+                        72,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        102,
+                        105,
+                        "ROQ",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 184,
+                "sent": "The exact stoichiometry of Roquin bound to the Ox40 3\u2032-UTR is unknown.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        27,
+                        33,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        34,
+                        42,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        47,
+                        51,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        52,
+                        58,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 185,
+                "sent": "The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        24,
+                        46,
+                        "secondary binding site",
+                        "site"
+                    ],
+                    [
+                        51,
+                        56,
+                        "dsRNA",
+                        "chemical"
+                    ],
+                    [
+                        60,
+                        66,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        68,
+                        74,
+                        "B-site",
+                        "site"
+                    ],
+                    [
+                        128,
+                        133,
+                        "dsRNA",
+                        "chemical"
+                    ],
+                    [
+                        168,
+                        174,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        186,
+                        190,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        218,
+                        226,
+                        "affinity",
+                        "evidence"
+                    ],
+                    [
+                        227,
+                        230,
+                        "SLs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 186,
+                "sent": "In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        65,
+                        68,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        87,
+                        90,
+                        "Tnf",
+                        "protein"
+                    ],
+                    [
+                        91,
+                        94,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        99,
+                        102,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        103,
+                        105,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        106,
+                        110,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        139,
+                        154,
+                        "crystal lattice",
+                        "evidence"
+                    ],
+                    [
+                        208,
+                        213,
+                        "dsRNA",
+                        "chemical"
+                    ],
+                    [
+                        221,
+                        227,
+                        "B site",
+                        "site"
+                    ]
+                ]
+            },
+            {
+                "sid": 187,
+                "sent": "This opens the possibility that one Roquin molecule may cluster two motifs in a given 3\u2032-UTR and/or cluster motifs from distinct 3\u2032-UTRs to enhance downstream processing.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        36,
+                        42,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        86,
+                        92,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        129,
+                        136,
+                        "3\u2032-UTRs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 188,
+                "sent": "Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3\u2032-UTR of the Nfkbid mRNA.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        19,
+                        21,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        22,
+                        25,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        70,
+                        76,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        110,
+                        116,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        124,
+                        130,
+                        "Nfkbid",
+                        "protein"
+                    ],
+                    [
+                        131,
+                        135,
+                        "mRNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 189,
+                "sent": "We therefore hypothesize that the combination of multiple binding sites may be more commonly used to enhance the functional activity of Roquin.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        58,
+                        71,
+                        "binding sites",
+                        "site"
+                    ],
+                    [
+                        136,
+                        142,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 190,
+                "sent": "At the same time, the combination of cis elements may be important for differential gene regulation, as composite cis elements with lower affinity may be less sensitive to Roquin.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        37,
+                        49,
+                        "cis elements",
+                        "structure_element"
+                    ],
+                    [
+                        114,
+                        126,
+                        "cis elements",
+                        "structure_element"
+                    ],
+                    [
+                        138,
+                        146,
+                        "affinity",
+                        "evidence"
+                    ],
+                    [
+                        172,
+                        178,
+                        "Roquin",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 191,
+                "sent": "This will lead to less effective repression in T cells when antigen recognition is of moderate signal strength and only incomplete cleavage of Roquin by MALT1 occurs.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        143,
+                        149,
+                        "Roquin",
+                        "protein"
+                    ],
+                    [
+                        153,
+                        158,
+                        "MALT1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 192,
+                "sent": "For understanding the intricate complexity of 3\u2032-UTR regulation, future work will be necessary by combining large-scale approaches, such as cross-linking and immunoprecipitation experiments to identify RNA-binding sites, and structural biology to dissect the underlying molecular mechanisms.",
+                "section": "DISCUSS",
+                "ner": [
+                    [
+                        46,
+                        52,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        140,
+                        189,
+                        "cross-linking and immunoprecipitation experiments",
+                        "experimental_method"
+                    ],
+                    [
+                        202,
+                        219,
+                        "RNA-binding sites",
+                        "site"
+                    ],
+                    [
+                        225,
+                        243,
+                        "structural biology",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 193,
+                "sent": "SELEX identifies a novel SL RNA ligand of Roquin-1.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        5,
+                        "SELEX",
+                        "experimental_method"
+                    ],
+                    [
+                        25,
+                        27,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        28,
+                        31,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        42,
+                        50,
+                        "Roquin-1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 194,
+                "sent": "(a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2\u2013440) or Roquin-1 M199R N terminus (residues 2\u2013440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3\u2032-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        41,
+                        49,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        71,
+                        76,
+                        "2\u2013440",
+                        "residue_range"
+                    ],
+                    [
+                        81,
+                        95,
+                        "Roquin-1 M199R",
+                        "mutant"
+                    ],
+                    [
+                        117,
+                        122,
+                        "2\u2013440",
+                        "residue_range"
+                    ],
+                    [
+                        164,
+                        167,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        190,
+                        194,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        195,
+                        201,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        224,
+                        228,
+                        "MEME",
+                        "experimental_method"
+                    ],
+                    [
+                        244,
+                        249,
+                        "SELEX",
+                        "experimental_method"
+                    ],
+                    [
+                        259,
+                        262,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 195,
+                "sent": "(c) Conservation of the motif found in Ox40 3\u2032-UTRs for various species as indicated.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        39,
+                        43,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        44,
+                        51,
+                        "3\u2032-UTRs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 196,
+                "sent": "rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "rn5",
+                        "gene"
+                    ],
+                    [
+                        41,
+                        44,
+                        "rat",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        46,
+                        62,
+                        "Rattus novegicus",
+                        "species"
+                    ],
+                    [
+                        111,
+                        116,
+                        "SELEX",
+                        "experimental_method"
+                    ],
+                    [
+                        135,
+                        137,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        139,
+                        142,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        151,
+                        155,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        156,
+                        159,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        165,
+                        173,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        174,
+                        176,
+                        "SL",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 197,
+                "sent": "The broken line between the G\u2013G base pair in the ADE SL indicates a putative non-Watson\u2013Crick pairing.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        49,
+                        52,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        53,
+                        55,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        77,
+                        101,
+                        "non-Watson\u2013Crick pairing",
+                        "bond_interaction"
+                    ]
+                ]
+            },
+            {
+                "sid": 198,
+                "sent": "The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        8,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        9,
+                        12,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        18,
+                        20,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        29,
+                        32,
+                        "Tnf",
+                        "protein"
+                    ],
+                    [
+                        33,
+                        36,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        37,
+                        39,
+                        "SL",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 199,
+                "sent": "NMR analysis of the SL RNAs used in this study.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        20,
+                        22,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        23,
+                        27,
+                        "RNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 200,
+                "sent": "Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        40,
+                        46,
+                        "1H NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        47,
+                        54,
+                        "spectra",
+                        "evidence"
+                    ],
+                    [
+                        66,
+                        69,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        70,
+                        72,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        82,
+                        86,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        87,
+                        90,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        96,
+                        98,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        111,
+                        115,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        116,
+                        119,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        125,
+                        127,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        142,
+                        146,
+                        "free",
+                        "protein_state"
+                    ],
+                    [
+                        147,
+                        151,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        164,
+                        179,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        184,
+                        192,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        193,
+                        196,
+                        "ROQ",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 201,
+                "sent": "The respective SL RNAs and their base pairs are indicated.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        15,
+                        17,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        18,
+                        22,
+                        "RNAs",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 202,
+                "sent": "Red asterisks indicate NMR signals of the protein.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        23,
+                        26,
+                        "NMR",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 203,
+                "sent": "Green lines in the secondary structure schemes on the left refer to visible imino NMR signals and thus experimental confirmation of the base pairs indicated.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        82,
+                        85,
+                        "NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        86,
+                        93,
+                        "signals",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 204,
+                "sent": "The dotted green line between G6 and G15 in a highlights a G\u2013G base pair.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        30,
+                        32,
+                        "G6",
+                        "residue_name_number"
+                    ],
+                    [
+                        37,
+                        40,
+                        "G15",
+                        "residue_name_number"
+                    ],
+                    [
+                        59,
+                        60,
+                        "G",
+                        "residue_name"
+                    ],
+                    [
+                        61,
+                        62,
+                        "G",
+                        "residue_name"
+                    ]
+                ]
+            },
+            {
+                "sid": 205,
+                "sent": "Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        9,
+                        "Structure",
+                        "evidence"
+                    ],
+                    [
+                        17,
+                        25,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        26,
+                        29,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        37,
+                        45,
+                        "bound to",
+                        "protein_state"
+                    ],
+                    [
+                        46,
+                        50,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        51,
+                        54,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        60,
+                        63,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 206,
+                "sent": "(a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174\u2013325; blue) and the Ox40 ADE-like SL RNA (magenta).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        32,
+                        49,
+                        "crystal structure",
+                        "evidence"
+                    ],
+                    [
+                        57,
+                        60,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        78,
+                        85,
+                        "174\u2013325",
+                        "residue_range"
+                    ],
+                    [
+                        101,
+                        105,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        106,
+                        109,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        115,
+                        117,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        118,
+                        121,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 207,
+                "sent": "Selected RNA bases and protein secondary structure elements are labelled.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        9,
+                        12,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 208,
+                "sent": "(b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        25,
+                        29,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        30,
+                        33,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        39,
+                        41,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        56,
+                        59,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        60,
+                        68,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        123,
+                        132,
+                        "structure",
+                        "evidence"
+                    ],
+                    [
+                        140,
+                        151,
+                        "ROQ-Tnf CDE",
+                        "complex_assembly"
+                    ],
+                    [
+                        182,
+                        185,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 209,
+                "sent": "Only RNA-interacting residues that are different in both structures are shown.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        5,
+                        29,
+                        "RNA-interacting residues",
+                        "site"
+                    ],
+                    [
+                        57,
+                        67,
+                        "structures",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 210,
+                "sent": "Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        48,
+                        52,
+                        "RNAs",
+                        "chemical"
+                    ],
+                    [
+                        173,
+                        176,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        200,
+                        203,
+                        "U11",
+                        "residue_name_number"
+                    ],
+                    [
+                        208,
+                        211,
+                        "U13",
+                        "residue_name_number"
+                    ],
+                    [
+                        219,
+                        223,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        224,
+                        227,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        233,
+                        235,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        236,
+                        239,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 211,
+                "sent": "The nucleotides interact with the C-terminal end of helix \u03b14 (Tyr250 and Ser253) and the N-terminal part of strand \u03b23 (Phe255 and Val257).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        52,
+                        57,
+                        "helix",
+                        "structure_element"
+                    ],
+                    [
+                        58,
+                        60,
+                        "\u03b14",
+                        "structure_element"
+                    ],
+                    [
+                        62,
+                        68,
+                        "Tyr250",
+                        "residue_name_number"
+                    ],
+                    [
+                        73,
+                        79,
+                        "Ser253",
+                        "residue_name_number"
+                    ],
+                    [
+                        108,
+                        114,
+                        "strand",
+                        "structure_element"
+                    ],
+                    [
+                        115,
+                        117,
+                        "\u03b23",
+                        "structure_element"
+                    ],
+                    [
+                        119,
+                        125,
+                        "Phe255",
+                        "residue_name_number"
+                    ],
+                    [
+                        130,
+                        136,
+                        "Val257",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 212,
+                "sent": "The protein chain is shown in turquoise and the RNA is shown in grey.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        48,
+                        51,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 213,
+                "sent": "(e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        46,
+                        49,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        73,
+                        76,
+                        "U10",
+                        "residue_name_number"
+                    ],
+                    [
+                        78,
+                        81,
+                        "U11",
+                        "residue_name_number"
+                    ],
+                    [
+                        86,
+                        89,
+                        "U13",
+                        "residue_name_number"
+                    ],
+                    [
+                        97,
+                        100,
+                        "RNA",
+                        "chemical"
+                    ],
+                    [
+                        101,
+                        109,
+                        "hexaloop",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 214,
+                "sent": "U11 and U13 contact the C-terminal end of helix \u03b14: residues Tyr250 and Gln247.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "U11",
+                        "residue_name_number"
+                    ],
+                    [
+                        8,
+                        11,
+                        "U13",
+                        "residue_name_number"
+                    ],
+                    [
+                        42,
+                        47,
+                        "helix",
+                        "structure_element"
+                    ],
+                    [
+                        48,
+                        50,
+                        "\u03b14",
+                        "structure_element"
+                    ],
+                    [
+                        61,
+                        67,
+                        "Tyr250",
+                        "residue_name_number"
+                    ],
+                    [
+                        72,
+                        78,
+                        "Gln247",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 215,
+                "sent": "The side chain of Tyr250 makes hydrophobic interactions with the pyrimidine side chain of U10 on one side and U11 on the other side.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        18,
+                        24,
+                        "Tyr250",
+                        "residue_name_number"
+                    ],
+                    [
+                        31,
+                        55,
+                        "hydrophobic interactions",
+                        "bond_interaction"
+                    ],
+                    [
+                        90,
+                        93,
+                        "U10",
+                        "residue_name_number"
+                    ],
+                    [
+                        110,
+                        113,
+                        "U11",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 216,
+                "sent": "Lys259 interacts with the phosphate groups of U10 and U11.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        6,
+                        "Lys259",
+                        "residue_name_number"
+                    ],
+                    [
+                        46,
+                        49,
+                        "U10",
+                        "residue_name_number"
+                    ],
+                    [
+                        54,
+                        57,
+                        "U11",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 217,
+                "sent": "(f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        25,
+                        48,
+                        "hydrophobic interaction",
+                        "bond_interaction"
+                    ],
+                    [
+                        57,
+                        63,
+                        "Val257",
+                        "residue_name_number"
+                    ],
+                    [
+                        68,
+                        71,
+                        "U11",
+                        "residue_name_number"
+                    ],
+                    [
+                        95,
+                        108,
+                        "hydrogen bond",
+                        "bond_interaction"
+                    ],
+                    [
+                        112,
+                        118,
+                        "Lys259",
+                        "residue_name_number"
+                    ],
+                    [
+                        144,
+                        147,
+                        "U10",
+                        "residue_name_number"
+                    ],
+                    [
+                        152,
+                        155,
+                        "U11",
+                        "residue_name_number"
+                    ]
+                ]
+            },
+            {
+                "sid": 218,
+                "sent": "NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        3,
+                        "NMR",
+                        "experimental_method"
+                    ],
+                    [
+                        16,
+                        19,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        49,
+                        53,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        54,
+                        57,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        63,
+                        71,
+                        "hexaloop",
+                        "structure_element"
+                    ],
+                    [
+                        72,
+                        75,
+                        "RNA",
+                        "chemical"
+                    ]
+                ]
+            },
+            {
+                "sid": 219,
+                "sent": "(a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171\u2013326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "Overlay",
+                        "experimental_method"
+                    ],
+                    [
+                        15,
+                        26,
+                        "1H,15N HSQC",
+                        "experimental_method"
+                    ],
+                    [
+                        27,
+                        34,
+                        "spectra",
+                        "evidence"
+                    ],
+                    [
+                        49,
+                        53,
+                        "free",
+                        "protein_state"
+                    ],
+                    [
+                        54,
+                        57,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        66,
+                        73,
+                        "171\u2013326",
+                        "residue_range"
+                    ],
+                    [
+                        85,
+                        100,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        131,
+                        135,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        136,
+                        139,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        145,
+                        147,
+                        "SL",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 220,
+                "sent": "(b) Plot of chemical shift change versus residue number in the ROQ domain (residues 171\u2013326) from a. Grey negative bars indicate missing assignments in one of the spectra.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        12,
+                        33,
+                        "chemical shift change",
+                        "evidence"
+                    ],
+                    [
+                        63,
+                        66,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        84,
+                        91,
+                        "171\u2013326",
+                        "residue_range"
+                    ],
+                    [
+                        163,
+                        170,
+                        "spectra",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 221,
+                "sent": "Gaps indicate prolines.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        14,
+                        22,
+                        "prolines",
+                        "residue_name"
+                    ]
+                ]
+            },
+            {
+                "sid": 222,
+                "sent": "(c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        11,
+                        "Overlay",
+                        "experimental_method"
+                    ],
+                    [
+                        19,
+                        22,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        30,
+                        35,
+                        "alone",
+                        "protein_state"
+                    ],
+                    [
+                        47,
+                        62,
+                        "in complex with",
+                        "protein_state"
+                    ],
+                    [
+                        67,
+                        71,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        72,
+                        75,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        81,
+                        83,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        97,
+                        101,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        102,
+                        105,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        111,
+                        113,
+                        "SL",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 223,
+                "sent": "Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3\u2032-UTR.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        19,
+                        "Mutational analysis",
+                        "experimental_method"
+                    ],
+                    [
+                        23,
+                        31,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        50,
+                        54,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        55,
+                        58,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        64,
+                        66,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        71,
+                        75,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        76,
+                        82,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 224,
+                "sent": "(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        14,
+                        "EMSA assay",
+                        "experimental_method"
+                    ],
+                    [
+                        40,
+                        49,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        61,
+                        66,
+                        "Y250A",
+                        "mutant"
+                    ],
+                    [
+                        67,
+                        73,
+                        "mutant",
+                        "protein_state"
+                    ],
+                    [
+                        74,
+                        77,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        104,
+                        108,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        109,
+                        112,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        118,
+                        120,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        156,
+                        159,
+                        "Tnf",
+                        "protein"
+                    ],
+                    [
+                        160,
+                        163,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        164,
+                        166,
+                        "SL",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 225,
+                "sent": "A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (\u2212 tam).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        160,
+                        174,
+                        "Flow cytometry",
+                        "experimental_method"
+                    ],
+                    [
+                        178,
+                        182,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        187,
+                        191,
+                        "Icos",
+                        "protein"
+                    ],
+                    [
+                        234,
+                        239,
+                        "Rc3h1",
+                        "gene"
+                    ],
+                    [
+                        240,
+                        243,
+                        "2fl",
+                        "gene"
+                    ],
+                    [
+                        244,
+                        246,
+                        "fl",
+                        "gene"
+                    ],
+                    [
+                        267,
+                        271,
+                        "mice",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        285,
+                        294,
+                        "tamoxifen",
+                        "chemical"
+                    ],
+                    [
+                        312,
+                        317,
+                        "Rc3h1",
+                        "gene"
+                    ],
+                    [
+                        318,
+                        321,
+                        "2fl",
+                        "gene"
+                    ],
+                    [
+                        322,
+                        324,
+                        "fl",
+                        "gene"
+                    ],
+                    [
+                        325,
+                        333,
+                        "deletion",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 226,
+                "sent": "The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        74,
+                        84,
+                        "retrovirus",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        98,
+                        109,
+                        "doxycycline",
+                        "chemical"
+                    ],
+                    [
+                        141,
+                        149,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        150,
+                        152,
+                        "WT",
+                        "protein_state"
+                    ],
+                    [
+                        154,
+                        162,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        163,
+                        168,
+                        "Y250A",
+                        "mutant"
+                    ],
+                    [
+                        172,
+                        180,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        181,
+                        186,
+                        "K220A",
+                        "mutant"
+                    ],
+                    [
+                        188,
+                        193,
+                        "K239A",
+                        "mutant"
+                    ],
+                    [
+                        198,
+                        203,
+                        "R260A",
+                        "mutant"
+                    ],
+                    [
+                        204,
+                        211,
+                        "mutants",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 227,
+                "sent": "Functional importance of Roquin-1 target motifs in cells.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        25,
+                        33,
+                        "Roquin-1",
+                        "protein"
+                    ]
+                ]
+            },
+            {
+                "sid": 228,
+                "sent": "(a) Overview of the Ox40 3\u2032-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2\u2013440) and either the complete wild-type Ox40 3\u2032-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        20,
+                        24,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        25,
+                        31,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        36,
+                        45,
+                        "truncated",
+                        "protein_state"
+                    ],
+                    [
+                        46,
+                        53,
+                        "mutated",
+                        "protein_state"
+                    ],
+                    [
+                        83,
+                        87,
+                        "EMSA",
+                        "experimental_method"
+                    ],
+                    [
+                        134,
+                        138,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        155,
+                        159,
+                        "EMSA",
+                        "experimental_method"
+                    ],
+                    [
+                        208,
+                        216,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        245,
+                        250,
+                        "2\u2013440",
+                        "residue_range"
+                    ],
+                    [
+                        276,
+                        285,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        286,
+                        290,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        291,
+                        297,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        315,
+                        324,
+                        "mutations",
+                        "experimental_method"
+                    ],
+                    [
+                        332,
+                        335,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        341,
+                        343,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        349,
+                        352,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        358,
+                        360,
+                        "SL",
+                        "structure_element"
+                    ],
+                    [
+                        369,
+                        372,
+                        "SLs",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 229,
+                "sent": "It is noteworthy that the higher bands observed at large protein concentrations are probably additional nonspecific, lower-affinity interactions of Roquin-1 with the 3\u2032-UTR or protein aggregates.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        148,
+                        156,
+                        "Roquin-1",
+                        "protein"
+                    ],
+                    [
+                        166,
+                        172,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 230,
+                "sent": "(c) Relative Ox40 MFI normalized to expression levels from the Ox40 CDS construct.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        13,
+                        17,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        18,
+                        53,
+                        "MFI normalized to expression levels",
+                        "evidence"
+                    ],
+                    [
+                        63,
+                        67,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        68,
+                        71,
+                        "CDS",
+                        "structure_element"
+                    ]
+                ]
+            },
+            {
+                "sid": 231,
+                "sent": "Error bars show s.d. of seven (CDS, 1\u201340, 1\u201380, 1\u2013120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        31,
+                        34,
+                        "CDS",
+                        "structure_element"
+                    ],
+                    [
+                        36,
+                        40,
+                        "1\u201340",
+                        "residue_range"
+                    ],
+                    [
+                        42,
+                        46,
+                        "1\u201380",
+                        "residue_range"
+                    ],
+                    [
+                        48,
+                        53,
+                        "1\u2013120",
+                        "residue_range"
+                    ],
+                    [
+                        58,
+                        69,
+                        "full-length",
+                        "protein_state"
+                    ],
+                    [
+                        77,
+                        80,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        86,
+                        89,
+                        "mut",
+                        "protein_state"
+                    ],
+                    [
+                        94,
+                        97,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        98,
+                        101,
+                        "mut",
+                        "protein_state"
+                    ],
+                    [
+                        113,
+                        123,
+                        "double mut",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 232,
+                "sent": "Statistical significance was calculated by one-way analysis of variance (ANOVA) Kruskal\u2013Wallis test followed by Dunn\u2019s multiple comparison test (**P<0.01).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        43,
+                        71,
+                        "one-way analysis of variance",
+                        "experimental_method"
+                    ],
+                    [
+                        73,
+                        78,
+                        "ANOVA",
+                        "experimental_method"
+                    ],
+                    [
+                        80,
+                        99,
+                        "Kruskal\u2013Wallis test",
+                        "experimental_method"
+                    ],
+                    [
+                        112,
+                        143,
+                        "Dunn\u2019s multiple comparison test",
+                        "experimental_method"
+                    ]
+                ]
+            },
+            {
+                "sid": 233,
+                "sent": "(d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3\u2032-UTR (CDS, red line), Ox40 CDS with its wild-type 3\u2032-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line).",
+                "section": "FIG",
+                "ner": [
+                    [
+                        4,
+                        21,
+                        "mRNA decay curves",
+                        "evidence"
+                    ],
+                    [
+                        67,
+                        79,
+                        "retroviruses",
+                        "taxonomy_domain"
+                    ],
+                    [
+                        91,
+                        95,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        96,
+                        99,
+                        "CDS",
+                        "structure_element"
+                    ],
+                    [
+                        108,
+                        114,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        116,
+                        119,
+                        "CDS",
+                        "structure_element"
+                    ],
+                    [
+                        132,
+                        136,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        137,
+                        140,
+                        "CDS",
+                        "structure_element"
+                    ],
+                    [
+                        150,
+                        159,
+                        "wild-type",
+                        "protein_state"
+                    ],
+                    [
+                        160,
+                        166,
+                        "3\u2032-UTR",
+                        "structure_element"
+                    ],
+                    [
+                        168,
+                        179,
+                        "full length",
+                        "protein_state"
+                    ],
+                    [
+                        194,
+                        198,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        199,
+                        210,
+                        "full length",
+                        "protein_state"
+                    ],
+                    [
+                        216,
+                        223,
+                        "mutated",
+                        "protein_state"
+                    ],
+                    [
+                        224,
+                        227,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        240,
+                        243,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        249,
+                        252,
+                        "mut",
+                        "protein_state"
+                    ],
+                    [
+                        266,
+                        270,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        271,
+                        282,
+                        "full length",
+                        "protein_state"
+                    ],
+                    [
+                        288,
+                        295,
+                        "mutated",
+                        "protein_state"
+                    ],
+                    [
+                        296,
+                        299,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        312,
+                        315,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        321,
+                        324,
+                        "mut",
+                        "protein_state"
+                    ],
+                    [
+                        341,
+                        345,
+                        "Ox40",
+                        "protein"
+                    ],
+                    [
+                        346,
+                        357,
+                        "full length",
+                        "protein_state"
+                    ],
+                    [
+                        363,
+                        370,
+                        "mutated",
+                        "protein_state"
+                    ],
+                    [
+                        371,
+                        374,
+                        "ADE",
+                        "structure_element"
+                    ],
+                    [
+                        379,
+                        382,
+                        "CDE",
+                        "structure_element"
+                    ],
+                    [
+                        391,
+                        401,
+                        "Double mut",
+                        "protein_state"
+                    ]
+                ]
+            },
+            {
+                "sid": 234,
+                "sent": "mRNA half-life times were calculated with Graph Pad Prism.",
+                "section": "FIG",
+                "ner": [
+                    [
+                        0,
+                        20,
+                        "mRNA half-life times",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 235,
+                "sent": "Data collection and refinement statistics.",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        0,
+                        41,
+                        "Data collection and refinement statistics",
+                        "evidence"
+                    ]
+                ]
+            },
+            {
+                "sid": 236,
+                "sent": "\u00a0\tROQ-Ox40ADE-like SL\tROQ-ADE SL\t \tData collection\t \t\u2003space group\tP21212\tP212121\t \t\u00a0\t\u00a0\t\u00a0\t \t\u2003Cell dimensions\t \t\u2003a, b, c (\u00c5)\t89.66, 115.79, 42.61\t72.90, 89.30, 144.70\t \t\u2003\u03b1, \u03b2, \u03b3 (\u00b0)\t90, 90, 90\t90, 90, 90\t \t\u2003Resolution (\u00c5)\t50\u20132.23 (2.29\u20132.23)\t50\u20133.0 (3.08\u20133.00)\t \t\u2003Rmerge\t5.9 (68.3)\t14.8 (93.8)\t \t\u2003I/\u03c3I\t14.9 (2.1)\t16.7 (3.1)\t \t\u2003Completeness (%)\t98.7 (97.7)\t99.9 (99.9)\t \t\u2003Redundancy\t3.9 (3.7)\t13.2 (12.7)\t \t\u00a0\t\u00a0\t\u00a0\t \tRefinement\t \t\u2003Resolution (\u00c5)\t2.23\t3.00\t \t\u2003No. reflections\t21,018\t18,598\t \t\u2003Rwork/Rfree\t21.8/25.7\t18.6/23.4\t \t\u00a0\t\u00a0\t\u00a0\t \t\u2003No. atoms\t \t\u2003Protein\t2,404\t4,820\t \t\u2003Ligand/ion\t894\t1,708\t \t\u2003Water\t99\t49\t \t\u2003B-factor overall\t47.2\t60.4\t \t\u00a0\t\u00a0\t\u00a0\t \tRoot mean squared deviations\t \t\u2003Bond lengths (\u00c5)\t0.006\t0.014\t \t\u2003Bond angles (\u00b0)\t1.07\t1.77\t \t\u00a0\t\u00a0\t\u00a0\t \tRamachandran plot\t \t\u2003Most favoured (%)\t98.6\t99.8\t \t\u2003Additional allowed (%)\t1.4\t0.2\t \t",
+                "section": "TABLE",
+                "ner": [
+                    [
+                        2,
+                        5,
+                        "ROQ",
+                        "structure_element"
+                    ],
+                    [
+                        6,
+                        10,
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+                "sent": "For each data set, only one crystal has been used.",
+                "section": "TABLE",
+                "ner": [
+                    [
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+                    ]
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+                "sent": "KD for selected RNAs obtained from SPR measurements with immobilized ROQ domain of Roquin-1.",
+                "section": "TABLE",
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+                    ],
+                    [
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+                    [
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+                    ],
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+                    [
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+}
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+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection><source>PMC</source><date>20201216</date><key>pmc.key</key><document><id>4806292</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/srep23641</infon><infon key="article-id_pii">srep23641</infon><infon key="article-id_pmc">4806292</infon><infon key="article-id_pmid">27009356</infon><infon key="elocation-id">23641</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Klima;given-names:Martin</infon><infon key="name_1">surname:Tóth;given-names:Dániel J.</infon><infon key="name_10">surname:Humpolickova;given-names:Jana</infon><infon key="name_11">surname:Nencka;given-names:Radim</infon><infon key="name_12">surname:Veverka;given-names:Vaclav</infon><infon key="name_13">surname:Balla;given-names:Tamas</infon><infon key="name_14">surname:Boura;given-names:Evzen</infon><infon key="name_2">surname:Hexnerova;given-names:Rozalie</infon><infon key="name_3">surname:Baumlova;given-names:Adriana</infon><infon key="name_4">surname:Chalupska;given-names:Dominika</infon><infon key="name_5">surname:Tykvart;given-names:Jan</infon><infon key="name_6">surname:Rezabkova;given-names:Lenka</infon><infon key="name_7">surname:Sengupta;given-names:Nivedita</infon><infon key="name_8">surname:Man;given-names:Petr</infon><infon key="name_9">surname:Dubankova;given-names:Anna</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">6</infon><infon key="year">2016</infon><offset>0</offset><text>Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>121</offset><text>Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1035</offset><text>Phosphatidylinositol 4-kinase beta (PI4KB, also known as PI4K IIIβ) is a soluble cytosolic protein yet its function is to phosphorylate membrane lipids. It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P). PI4P is an essential lipid found in various membrane compartments including the Golgi and trans-Golgi network (TGN), the plasma membrane and the endocytic compartments. In these locations, PI4P plays an important role in cell signaling and lipid transport, and serves as a precursor for higher phosphoinositides or as a docking site for clathrin adaptor or lipid transfer proteins. A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories. These structures are essential for effective viral replication. Recently, highly specific PI4KB inhibitors were developed as potential antivirals.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2112</offset><text>PI4K kinases must be recruited to the correct membrane type to fulfill their enzymatic functions. Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins. In contrast, type III PI4Ks (PI4KA and PI4KB) are soluble cytosolic proteins that are recruited to appropriate membranes indirectly via protein-protein interactions. The recruitment of PI4KA to the plasma membrane by EFR3 and TTC7 is relatively well understood even at the structural level, but, the actual molecular mechanism of PI4KB recruitment to the Golgi is still poorly understood.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2697</offset><text>Acyl-coenzyme A binding domain containing protein 3 (ACBD3, also known as GCP60 and PAP7) is a Golgi resident protein. Its membrane localization is mediated by the interaction with the Golgi integral protein golgin B1/giantin. ACBD3 functions as an adaptor protein and signaling hub across cellular signaling pathways. ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2. ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3754</offset><text>We present a biochemical and structural characterization of the molecular complex composed of the ACBD3 protein and the PI4KB enzyme. We show that ACBD3 can recruit PI4KB to model membranes as well as redirect PI4KB to cellular membranes where it is not naturally found. Our data also show that ACBD3 regulates the enzymatic activity of PI4KB kinase through membrane recruitment rather than allostery.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4156</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4164</offset><text>ACBD3 and PI4KB interact with 1:1 stoichiometry with submicromolar affinity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4240</offset><text>In order to verify the interactions between ACBD3 and PI4KB we expressed and purified both proteins. To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423–522) was removed (Fig. 1A). This internal deletion does not significantly affect the kinase activity(SI Fig. 1A) or interaction with ACBD3 (SI Fig. 1B,C). In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction. Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain. We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5275</offset><text>Because it has been reported that ACBD3 can dimerize in a mammalian two-hybrid assay, we were interested in determining the stoichiometry of the ACBD3:PI4KB protein complex. The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively. This result suggests that both proteins are monomeric and the stoichiometry of the ACBD3: PI4KB protein complex is 1:1 (Fig. 1C, left panel). Similar results were obtained for the complex of the Q domain of ACBD3 and the N-terminal region of PI4KB (Fig. 1C, right panel). We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR). SPR measurements revealed a strong interaction with a Kd value of 320 +/−130 nM (Fig. 1D, SI Fig. 1D). We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>6446</offset><text>Structural analysis of the ACBD3:PI4KB complex</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6493</offset><text>Full length ACBD3 and PI4KB both contain large intrinsically disordered regions that impede crystallization. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of the complex to determine which parts of the complex are well folded (SI Fig. 2). However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6926</offset><text>For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization. As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules. Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously. Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44. Over 93% of non-exchangeable side-chain signals were assigned for the free ACBD3 Q domain. Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances. The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB). Assignments obtained for non-exchangeable side-chain signals were over 99% complete. The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation. Structural statistics for the final water-refined sets of structures are shown in SI Table 1.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8698</offset><text>This structure revealed that the Q domain forms a two helix hairpin. The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A). Preceding the kinase helix are three ordered residues (Val42, Ile43, and Asp44) that also contribute to the interaction (Fig. 2B). The remaining part of the PI4KB N-termini, however, is disordered (SI Fig. 5). Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9580</offset><text>To corroborate the structural data, we introduced a number of point mutations and validated their effect on complex formation using an in vitro pull-down assay (Fig. 2D). Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10144</offset><text>ACBD3 efficiently recruits the PI4KB enzyme to membranes</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10201</offset><text>We next sought to determine if the ACBD3:PI4KB interaction drives membrane localization of the PI4KB enzyme. To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate – the PI lipid. We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10759</offset><text>We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel). We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B). Given this result, overexpression of the Q domain should also interfere with the PI4KB dependent Golgi functions. Ceramide transport and accumulation in Golgi is a well-known PI4KB dependent process. We have used fluorescently labeled ceramide and analyzed its trafficking in non-transfected cells and cell overexpressing the Q domain. As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12033</offset><text>We further analyzed the function of ACBD3:PI4KB interaction in membrane recruitment of PI4KB in living cells using fluorescently tagged proteins. We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system. We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP. The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D). We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin. As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase. In every case the ACDB3 Q domain was rapidly (within 5 minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2). Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel). However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13272</offset><text>ACBD3 increases PI4KB enzymatic activity by recruiting PI4KB to close vicinity of its substrate</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13368</offset><text>To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate. We observed no effect on the kinase activity of PI4KB (Fig. 4A) suggesting that ACBD3 does not directly affect the enzyme (e.g. induction of a conformation change). However, in vivo ACBD3 is located at the Golgi membranes, whereas in this experiment, ACBD3 was located in the solution and PI is provided as micelles. We therefore designed a more physiologically relevant experiment. For this, we again turned to the GUV system with ACBD3 localized to the GUV membrane. The GUVs contained 10% PI to serve as a substrate for PI4KB kinase. The buffer also contained CFP-SidC, which binds to PI4P with nanomolar affinity. This enabled visualization of the kinase reaction using a confocal microscope. We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3. Reaction was also performed in the absence of ATP as a negative control (Fig. 4B). These experiments showed that PI4KB enzymatic activity increases when ACBD3 is membrane localized (Fig. 4C, SI Fig. 6). We conclude that enzyme activation proceeds through a membrane recruitment mechanism.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>14664</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>14675</offset><text>Membrane recruitment of PI4KB enzyme is crucial to ensure its proper function at the Golgi and TGN. However, the molecular mechanism and structural basis for PI4KB interaction with the membrane is poorly understood. In principle, any of the binding partners of PI4KB could play a role in membrane recruitment. To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>15258</offset><text>The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase. Although Rab11 does not appear to be required for recruitment of PI4KB to the Golgi, PI4KB is required for Golgi recruitment of Rab11. Arf1, the other small GTP binding protein, is known to influence the activity and localization of PI4KB, but it does not appear to interact directly with PI4KB (our unpublished data). The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear. NCS-1 is an N-terminally myristoylated protein that participates in exocytosis. It is expressed only in certain cell types, suggesting that if it contributes to PI4KB membrane recruitment, it does so in a tissues specific manner. The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation. However, 14-3-3 proteins do not appear to interfere with membrane recruitment of this kinase. ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase. For these reasons we focused on the interaction of the PI4KB enzyme with the Golgi resident ACBD3 protein in this study.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>16782</offset><text>Here we present the mechanism for membrane recruitment of PI4KB by the Golgi resident ACBD3 protein. We show that these proteins interact directly with a Kd value in the submicromolar range. The interaction is sufficient to recruit PI4KB to model membranes in vitro as well as to the mitochondria where PI4KB is never naturally found. To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain. The Q domain adopts a helical hairpin fold that is further stabilized upon binding the kinase helix (Fig. 2A). Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase. Based on our and previously published structures we built a pseudoatomic model of PI4KB multi-protein assembly on the membrane (Fig. 5) that illustrates how the enzyme is recruited and positioned towards its lipidic substrate and how it in turn recruits Rab11.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>18118</offset><text>+RNA viruses replicate at specific PI4P-enriched membranous compartments. These are called replication factories (because they enhance viral replication) or membranous webs (because of their appearance under the electron microscope). To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid. Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3. Our data suggest that they could do this via 3A:ACBD3:PI4KB complex formation. The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication. This could eventually have implications for therapeutic intervention to combat picornaviruses-mediated diseases ranging from polio to the common cold.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>19131</offset><text>Materials and Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>19153</offset><text>Plasmid construction, protein expression, and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>19212</offset><text>All proteins used in this study were recombinant and were expressed in E. coli using previously developed protocols. Briefly, full-length human ACBD3 (UniProtKB entry Q9H3P7) and PI4KB (UniProtKB entry Q9UBF8, isoform 1) lipid kinase and their deletion mutants were cloned into a previously modified pRSFD vector (Novagen) that already contained an N-terminal 6xHis tag followed by a GB1 solubility tag and TEV protease cleavage site. Mutations were generated using the Phusion Site-Directed Mutagenesis Kit (Thermo Scientific). The plasmids used are listed in the SI (SI Table 2). The proteins were expressed in E. coli BL21 Star cells as previously described. Upon overnight expression in autoinduction media bacterial cells were harvested and lysed in lysis buffer (50 mM Tris pH 8, 300 mM NaCl, 3 mM β-mercaptoethanol, 20 mM imidazole, 10% glycerol). The lysate was incubated with the Ni-NTA resin (Macherey-Nagel) and then extensively washed with the lysis buffer. The protein was eluted with the lysis buffer supplemented with 300 mM imidazole. When appropriate, tags were removed with TEV protease, and the protein was further purified using the size exclusion chromatography on Superdex 75 or Superdex 200 columns (GE Healthcare) in SEC buffer (10 mM Tris pH 8, 200 mM NaCl, 3 mM β-ME). Proteins were concentrated to 1–5 mg/ml (measured spectroscopically) and stored at −80 °C until needed.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>20638</offset><text>In vitro pull-downs</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20658</offset><text>Ni-NTA sepharose beads (Macherey-Nagel) were mixed with both binding partners (one of which was tagged with an N-terminal 6xHis tag) at a final concentration of 1 μM in a final volume of 200 μL binding buffer (30 mM Tris pH 8, 200 mM NaCl, 10 mM imidazole, and 1 mM TCEP). After 30 min incubation at 4 °C the beads were washed twice with 200 μL of the binding buffer, and total protein was directly eluted with the Laemmli sample buffer and analyzed by SDS-PAGE.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>21143</offset><text>SPR (Surface plasmon resonance) and AUC (Analytical ultracentrifugation)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21216</offset><text>PI4KB was chip-immobilized as detailed in the SI. Afterwards, the ACBD3 protein was injected in a series of concentrations for 3 min and then dissociation was monitored for another 5 min. The data were fit to a single-exponential model. Rate constants of association and dissociation were obtained by fitting the observed change in resonance signal using the following equations:</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21600</offset><text>where c is the protein concentration, t is time, kon is the association rate constant, koff is the dissociation rate constant, D1 and D2 are the linear drift terms, and Ras, Rdis, R0, R1, and Rmax are corresponding changes in the relative response signal.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21856</offset><text>AUC was used to perform sedimentation velocity experiments using a ProteomeLab XL-I Beckman Coulter analytical ultracentrifuge equipped with an AN50Ti rotor. All measurements were performed in 10 mM Tris pH 8, 200 mM NaCl, and 3 mM β-mercaptoethanol at 20 °C and 48000 rpm. All data were collected using an absorbance (230 nm and 280 nm) optical system. Data analysis was performed with the SEDFIT package and data were analyzed using a sedimentation coefficient distribution model c(s).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>22360</offset><text>In vitro kinase assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22382</offset><text>In vitro kinase activity was measured using a bioluminescent ADP-Glo assay (Promega) as described previously. Briefly, reactions were carried out in a total volume of 5 μL in 384-well plates by diluting the indicated amounts of the PI4KB enzyme and/or ACBD3 protein into the kinase buffer (20 mM Tris pH 7.5, 5 mM MgCl2, 0.2% Triton-X100, 0.1 mg/mL BSA, 2 mM DTT, 50 μM phosphatidylinositol). Reaction was initiated by adding ATP to a final concentration of 100 μM. Samples were incubated for 60 min at 25 °C and the amount of hydrolyzed ATP was measured according to the manufacturer’s protocol using a TECAN infinite M 1000 plate reader.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>23046</offset><text>NMR spectroscopy</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>23063</offset><text>NMR spectra were acquired at 25 °C on a 600 MHz and 850 MHz Bruker Avance spectrometers, both of which were equipped with a triple-resonance (15N/13C/1H) cryoprobe. The sample volume was 0.35 mL, with a 280 μM concentration for the free Q domain and a 470 μM concentration for the ACBD3:PI4KB complex in the NMR buffer (25 mM sodium phosphate pH 6.5, 100 mM NaCl, 1 mM TCEP, 0.01% NaN3), 5% D2O/95% H2O. A series of double- and triple-resonance spectra were recorded to determine essentially complete sequence-specific resonance backbone and side-chain assignments. Constraints for 1H-1H distance required to calculate the structure of free Q domain and ACBD3:PI4KB complex were derived from 3D 15N/1H NOESY-HSQC and 13C/1H NOESY-HMQC, which were acquired using a NOE mixing time of 100 ms.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>23877</offset><text>The families of converged structures for the ACBD3:PI4KB complex and free Q domain were calculated using standard software as detailed in the SI. The structures with the lowest total energy were selected and validated. The statistics for the resulting structures are summarized in SI Table 1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24170</offset><text>Protein labeling with fluorescent dyes</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24209</offset><text>PI4KB was labeled on native cysteine residues. Briefly, pure recombinant protein was incubated overnight at 4 °C with a 3x molar excess of Alexa 488 C5 maleimide (Life Technologies). The reaction was quenched by adding 10 mM β-mercaptoethanol (βME) and the protein was repurified by size exclusion chromatography.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24530</offset><text>Giant Unilamellar Vesicle Preparation and Imaging</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24580</offset><text>Giant Unilamellar Vesicles (GUVs) composed of POPC (54.9 mol %), POPS (10 mol %), cholesterol (20 mol %), PI (10 mol %), DGS-NTA(Ni) [1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) ] (5 mol %) (Avanti Polar lipids), and ATTO647N-DOPE (0.1 mol %) (ATTO-TEC GmbH) were prepared by electroformation as described previously, please see SI.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24979</offset><text>Live Cell Imaging</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24997</offset><text>COS-7 cells were plated onto 29-mm-diameter poly-L-Lysine coated glass bottom dishes (In Vitro Scientific) at 100,000 cells/well density and transfected using the Lipofectamine2000 reagent (Invitrogen) with plasmid DNAs (0.5–1 mg/well) according to manufacturer’s instructions. The plasmids are described in SI Table 2. 24 hr post transfection, COS-7 cells were washed with a modified Krebs-Ringer solution (10 mM Na-HEPES pH 7.4, 120 mM NaCl, 4.7 mM KCl, 2 mM CaCl2, 0.7 mM MgSO4, 10 mM glucose) in the same dish and were imaged using an LSM 710 confocal microscope (Carl Zeiss MicroImaging) with a 63 × 1.4-numerical-aperture planapochromatic objective. For ceramide uptake experiments, COS-7 cells were loaded with 0.05 μM BODIPY® FL C5-Ceramide (Molecular Probes, ThermoFisher Scientific) complexed with BSA in modified Krebs-Ringer solution at room temperature for 20 min. Cells were then washed three times and imaged using the above mentioned settings.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>25988</offset><text>Immunofluorescent imaging</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26014</offset><text>COS-7 cells were plated onto 25-mm-diameter poly-L-Lysine coated circular glass coverslips in six-well plates (100,000 cells/well), and transfected using the Lipofectamine2000 reagent (Invitrogen) with plasmid DNAs (0.5–1 mg/well) according to manufacturer’s instructions. Twenty four hours post transfection, cells were washed with PBS, fixed with 4% paraformaldehyde, stained with mouse anti-PI4KB primary antibody (BD Transduction Laboratories, 1:500 dilution) and then after washing with PBS stained with Alexa Fluor 647 conjugated donkey anti-mouse secondary antibody (Molecular Probes, ThermoFisher Scientific, 1:500 dilution). Cover slips were mounted and observed with the above mentioned microscopy settings.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>26738</offset><text>HD exchange</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26750</offset><text>Hydrogen/deuterium exchange was performed as previously described with the following modifications. The exchange was done in 10 mM Tris-HCl pD 8.0, 200 mM NaCl at 20 °C. Protein concentration during the exchange was 1 μM. Aliquots (50 μL) were removed after 10, 20, 60, 120, 600, 1800, and 3600 s and the exchange was quenched by the addition of 50 μL of 0.25 M glycine-HCl pH 2.3 and rapid freezing in liquid nitrogen.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27188</offset><text>Prior to the analysis each sample was quickly thawed and injected onto an immobilized rhizopuspepsin column (bed volume 66 μL). Digestion was driven by a flow of 0.4% formic acid in water at a flow rate of 100 μL/min (LC-20AD pump, Shimadzu). The resulting peptides were trapped and desalted online on a peptide microtrap (Optimize Technologies). After a desalting step (3 min), peptides were separated using a linear gradient of 10–25% buffer B for 2 min, followed by a quick jump to 99% buffer B (buffer A = 0.4% formic acid/2% acetonitrile in water; buffer B = 95% acetonitrile/0.4% formic acid in water). The outlet of the LC system was interfaced to an electrospray ionization source of a Fourier transform ion cyclotron resonance mass spectrometer (12 T SolariX XR, Bruker Daltonics). Exchange was followed on 32 peptides from PI4KB (N) and 26 peptides from ACBD3(Q), covering in both cases 100% of the protein sequence. Peptides were identified by LC-MS/MS and MASCOT search against a database containing the sequences of the studied proteins. Data from H/D exchange were analyzed by program DeutEx written in the laboratory (unpublished).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>28355</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28378</offset><text>Accession codes: The structures and assigned chemical shifts for the free Q domain and the ACBD3:PI4KB complex were deposited in PDB database under accession codes 2N72 and 2N73, and BMRB database under accession codes 25790 and 25791.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28614</offset><text>How to cite this article: Klima, M. et al. Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein. Sci. 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A.</infon><infon key="name_1">surname:Mezulis;given-names:S.</infon><infon key="name_2">surname:Yates;given-names:C. M.</infon><infon key="name_3">surname:Wass;given-names:M. N.</infon><infon key="name_4">surname:Sternberg;given-names:M. J.</infon><infon key="pub-id_doi">10.1038/nprot.2015.053</infon><infon key="pub-id_pmid">25950237</infon><infon key="section_type">REF</infon><infon key="source">Nat Protoc</infon><infon key="type">ref</infon><infon key="volume">10</infon><infon key="year">2015</infon><offset>33401</offset><text>The Phyre2 web portal for protein modeling, prediction and analysis</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>33469</offset><text>The authors declare no competing financial interests.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>33523</offset><text>Author Contributions M.K. and A.D. carried out DNA cloning, M.K., A.B., D.C. and E.B. carried out protein expression and purification, M.K. performed pull-down assays, L.R. carried out analytical ultracentrifugation, M.K. and J.T. performed S.P.R. experiments, R.H. and V.V. carried out NMR experiments, structure refinement, and deposition, A.B. and P.M. performed HDX/MS experiments, D.C. carried out in vitro kinase assay, E.B. performed protein labeling, E.B. and J.H. carried out GUV preparation and imaging, D.T. and N.S. performed some of the cloning and the cell-based experiments, E.B. supervised the project, E.B., M.K., M.N., V.V. and T.B. wrote the manuscript, all authors contributed to data analysis and commented on the manuscript.</text></passage><passage><infon key="file">srep23641-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>34270</offset><text>Biochemical characterization of the ACBD3:PI4KB complex.</text></passage><passage><infon key="file">srep23641-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>34327</offset><text>(A) Schematic representation of the ACBD3 and PI4KB constructs used for the experiments. ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD). PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes. (B) In vitro pull-down assay. Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3. The inputs and bound proteins were analyzed on SDS gels stained with Coomassie Blue. The asterisks mark the bands corresponding to specific interactions. Cropped gels ran the same experimental conditions are shown. Please, see SI Fig. 9 for original full-length gels. (C) Analytical Ultracentrifugation. AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance. SPR analysis of the PI4KB binding to immobilized ACBD3. Sensorgrams for four concentrations of PI4KB are shown.</text></passage><passage><infon key="file">srep23641-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>35540</offset><text>Structural analysis of the ACBD3:PI4KB complex.</text></passage><passage><infon key="file">srep23641-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>35588</offset><text>(A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region. Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex. The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. ACBD3 is shown in magenta and PI4KB in orange. (C) Top view of the kinase helix. The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta). Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein. Wild type proteins and selected point mutants of both PI4KB and ACBD3 were used. Inputs and bound proteins were analyzed on SDS gels and stained with Coomassie Blue. Cropped gels ran the same experimental conditions are shown. Please, see SI Fig. 9 for original full-length gels.</text></passage><passage><infon key="file">srep23641-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>36896</offset><text>ACBD3 is sufficient to recruit the PI4KB kinase to membranes.</text></passage><passage><infon key="file">srep23641-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>36958</offset><text>(A) GUVs recruitment assay. Top – Virtually no membrane bound kinase was observed when 600 nM PI4KB was added to the GUVs. Bottom – in the presence of 600 nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs. (B) Golgi displacement experiment. Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained. Middle panel: The same experiment performed with GFP alone. Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. Middle panel – The same experiment performed with mRFP-FKBP alone. Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment. – The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex. Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment. Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin. Right – The same experiment performed using the H264A Q domain mutant.</text></passage><passage><infon key="file">srep23641-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>38600</offset><text>ACBD3 indirectly increases the activity of PI4KB.</text></passage><passage><infon key="file">srep23641-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>38650</offset><text>(A) Micelles-based kinase assay – PI in TX100 micelles was used in a luminescent kinase assay and the production of PI4P was measured. Bar graph presents the mean values of PI4P generated in the presence of the proteins as indicated, normalized to the amount of PI4P generated by PI4KB alone. Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor. (C)–Quantification of the GUV phosphorylation assay – Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions. The mean membrane intensity value is relative to the background signal and the difference between the membrane and background signal in the reference system lacking ATP. The error bars stand for SEM based on three independent experiments (also SI Fig. 6).</text></passage><passage><infon key="file">srep23641-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>39617</offset><text>Pseudoatomic model of the PI4KB multiprotein complex assembly.</text></passage><passage><infon key="file">srep23641-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>39680</offset><text>PI4KB in orange, Rab11 in purple, ACBD3 in blue. The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server. The GOLD domain is tethered to the membrane by GolginB1 (also known as Giantin) which is not shown for clarity. Intrinsically disordered linkers are modeled in an arbitrary but physically plausible conformation.</text></passage></document></collection>
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To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Liberato;given-names:Marcelo V.</infon><infon key="name_1">surname:Silveira;given-names:Rodrigo L.</infon><infon key="name_10">surname:Polikarpov;given-names:Igor</infon><infon key="name_2">surname:Prates;given-names:Érica T.</infon><infon key="name_3">surname:de Araujo;given-names:Evandro A.</infon><infon key="name_4">surname:Pellegrini;given-names:Vanessa O. A.</infon><infon key="name_5">surname:Camilo;given-names:Cesar M.</infon><infon key="name_6">surname:Kadowaki;given-names:Marco A.</infon><infon key="name_7">surname:Neto;given-names:Mario de O.</infon><infon key="name_8">surname:Popov;given-names:Alexander</infon><infon key="name_9">surname:Skaf;given-names:Munir S.</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">6</infon><infon key="year">2016</infon><offset>0</offset><text>Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>105</offset><text>Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1053</offset><text>The production of biofuels from renewable sources is an important element of the global strategy for generating sustainable energy with reduced environmental impact. Current technologies for obtaining liquid biofuels and green chemicals rely on the enzymatic digestion of lignocellulosic biomass from a variety of feedstocks. Plant biomass-the most abundant source of carbohydrates on Earth-is primarily composed of cellulose microfibrils surrounded by a hydrated heteropolymeric matrix of hemicellulose and lignin. Plant biomass may be subjected to thermo-chemical pretreatments and enzymatic reactions to produce soluble fermentable sugars.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1696</offset><text>The canonical model of hydrolytic degradation of cellulose requires at least three classes of enzymes. Cellobiohydrolases (CBHs) processively cleave the glycosidic bonds at the reducing and non-reducing ends of cellulose chains in crystalline regions to produce cellobiose. Endoglucanases (EGs) introduce random cuts in the amorphous regions of cellulose and create new chain extremities for CBH attack; thus, these enzymes act synergistically. The released cellobiose molecules are then enzymatically converted into glucose by β-glucosidases.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2243</offset><text>The molecular architecture of glycoside hydrolases (GHs) frequently consists of a catalytic domain (CD), where hydrolysis occurs, and one or more ancillary modules (AMs), which are usually connected by less structured linkers. The most common type of AMs are carbohydrate-binding modules (CBMs), which are able to recognize and bind specific carbohydrate chains. Generally distinct and independent structural domains, the CBMs facilitate carbohydrate hydrolysis by increasing the local concentration of enzymes at the surface of insoluble substrates, thereby targeting the CD component to its cognate ligands. CBMs might also disrupt the crystalline structure of cellulose microfibrils, although the underlying mechanism remains poorly understood. Thus, CBMs enhance the accessibility of CDs to carbohydrate chains to improve enzymatic activity, making them important candidates for the development of effective biomass-degrading enzymes in industrial settings.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3205</offset><text>Although there are examples of active GHs that lack AMs, the majority of the enzymes depend on AMs for activity. In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B). A pioneer work of Sakon et al. revealed that rigid structural extension of the GH9 CD by a type C CBM3 imprints a processive mode of action to this endoglucanase. Further publications showed that CBM-based structural extensions of the active site are important for substrate engagement and recognition.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3974</offset><text>Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module. Removal of the CBM46 caused a ~60-fold reduction of the activity of the enzyme against β-glucans, but showed little or no effect against xyloglucan hydrolysis. Moreover, the CBM46 mediated a significant increase in the BhCel5B activity in plant cell wall settings. Modeling of cellotriose in the negative subsites of the active site of BhCel5B demonstrated the structural conservation of the -1 position, but provided little information about direct interactions between CBM46 and the substrate. It was speculated that β-1,3 kink of the β-glucan might allow the ligand to reach for the CBM46, whereas pure β-1,4 linkages in the backbone of xyloglucan chains would restrict binding to the CD, thus explaining the lack of influence of the CBM46 on the enzymatic activity of BhCel5B against xyloglucans in solution. It was also argued that the CBM46 could potentialize the activity by driving BhCel5B towards xyloglucan-rich regions in the context of the plant cell walls, but no large-scale conformational adjustments of the AMs have been shown to occur or suggested to take part in the enzymatic activity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5349</offset><text>The mechanisms of ligand binding mediated by large-scale conformational changes in proteins following the induced-fit or conformational selection models have recently attracted considerable attention. Although initially introduced as contradictory theories, these two limiting cases can be unified considering the flux description concept or the extended conformational selection model. While local ligand-induced conformational adjustments have been reported for carbohydrate-active enzymes, cognate ligands recognition and hydrolysis mediated by a large-scale conformational mobility of distinct domains in multidomain settings is uncommon for endoglucanases.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6011</offset><text>Here, we report the crystal structure of a full-length GH5_4 enzyme from Bacillus licheniformis (BlCel5B) that exhibits two AMs (Ig-like module and CBM46) appended to the CD. We structurally and functionally characterize the enzyme using a combination of protein crystallography, small-angle X-ray scattering (SAXS), molecular dynamics computer simulations and site-directed mutagenesis, and show that the AMs and their conformational mobility are essential for the enzymatic activity of BlCel5B. We find that the large-scale conformational adjustments of the distal CBM46 mediated by the Ig-like hinge domain are crucial in active-site assembly for optimal substrate binding and hydrolysis. We propose that the BlCel5B conformational selection/induced-fit mechanism of hydrolysis represents a novel paradigm that applies to several GH5_4 members and, possibly, to a number of other multidomain GHs.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>6911</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>6919</offset><text>BlCel5B Crystal Structure</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6945</offset><text>BlCel5B crystals in the substrate-free form and complexed with cellopentaose (C5) were obtained and diffracted to 1.7 Å and 1.75 Å resolutions, respectively (Supplementary Table 1). The substrate-free and complexed structures exhibited no substantial conformational differences (with the exception of the substrate). Because of minor variations in the loops located distal to the substrate-binding site, a root mean squared deviation (rmsd) of 0.33 Å between the complexed and substrate-free structures was observed. A single protein chain occupies the asymmetric unit, and most of the residues were built, with the exception of the first 17 residues and those in the loop between L398 and P405 due to weak electron density.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7679</offset><text>The BlCel5B structure comprises three distinct domains: an N-terminal CD (residues 18 to 330), an Ig-like module (residues 335 to 428) and a family 46 CBM (residues 432 to 533) (Fig. 1A,B). Similarly to other members of the GH5 family, the CD of BlCel5B has a typical TIM barrel fold with eight inner β-strands and eight outer α helices that are interconnected by loops and three short α helices. Very short linkers, D429-D430-P431 and V331-P332-N333-A334, connect the CBM46 to the Ig-like module and the Ig-like module to the CD, respectively. Both Ig-like module and CBM46 have a β-sandwich fold composed of two β-sheets of four and three antiparallel β-strands interconnected by loops and a short α helix between strands β3 and β4 (Fig. 1C). A structural comparison between the Ig-like module and the CBM46 using the Dali server yielded an rmsd of 2.3 Å and a Z-score of 10.2. However, despite their structural resemblance, these modules share only 17% sequence identity. A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd = 1.3 Å and Z-score = 15.3. The CBM46 from BhCel5B is the most structurally similar to BlCel5B CBM46, with rmsd = 1.6 Å and Z-score = 12.4. The sequence identity relative to BhCel5B, however, is low (28% for Ig-like and 25% for CBM46).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9161</offset><text>The Ig-like module, adjacent to the CD, contains only one tyrosine (Y367) exposed to solvent and no tryptophan residues. Because aromatic residues play a major role in glucose recognition, this observation suggests that substrate binding may not be the primary function of Ig-like module. In contrast, the CBM46 has three tryptophan residues, two of which face the CD substrate binding site (Fig. 1A), indicating that it may be actively engaged in the carbohydrate binding.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9635</offset><text>Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule. The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end. The ability of BlCel5B to cleave C5 into glucose and C4 molecules in solution was demonstrated by enzymatic product profile mass spectrometry analysis (Fig. 2A). The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D). These residues belong to the CD and are conserved in the GH5 family.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10667</offset><text>BlCel5B enzymatic activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10694</offset><text>BlCel5B exhibits optimum activity toward carboxymethylcellulose (CMC; 8.7 U/mg) at a pH of 4.0 and 55 °C and retains approximately half of its maximum activity at 80 °C, demonstrating considerable thermal stability (Fig. 2B,C). BlCel5B is also active on β-glucan (34 U/mg), lichenan (17.8 U/mg) and xyloglucan (15.7 U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-β-mannan or the insoluble substrate Azo-Avicel. Kinetic parameters were calculated assuming Michaelis-Menten behavior with CMC as substrate: KM = 1.78 g L−1 and Vmax = 1.41 × 10−4 g s−1 mg protein−1 (Fig. 2D). Although BlCel5B is not a highly active enzyme against one specific substrate as compared to others GH5_4, it has the advantage of being active against different substrates with β-1,3 and/or β-1,4 glycosidic linkages.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11580</offset><text>To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A. These mutants were expressed and purified as described for the wild-type enzyme. Strikingly, neither of the deletion variants exhibited detectable activity toward any of the substrates tested using full-length BlCel5B (Table 1), demonstrating that the Ig-like module and the CBM46 are essential for BlCel5B activity. Thermal shift assays were conducted to confirm structural stability of the mutants (Supplementary Fig. 1). All of the constructs showed similar melting temperatures: 62 °C for BlCel5B, 58 °C for BlCel5BΔCBM46, 56 °C for BlCel5BΔIg-CBM46, 65 °C for BlCel5BW479A and 59 °C for BlCel5BW479A, thus confirming their proper overall fold.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12539</offset><text>We also examined the function of the CBM46 inner surface residues W479 and W481 (Fig. 1A) in BlCel5B activity by performing enzymatic assays with W479A and W481A mutants. Both mutations reduced enzymatic activity toward all tested substrates (Table 1), with W481A having a stronger effect than W479A (~64% vs. 79% activity relative to wt BlCel5B using β-glucan and ~10% vs. 50% using CMC). This indicates that CBM46 must interact with the substrate via residues W479 and W481. However, since the BlCel5B crystal structure exhibits no close contact between these residues and the substrate, these results suggest the existence of large-amplitude interdomain motions that may enable direct interactions between CBM46 and the carbohydrate.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13279</offset><text>BlCelB5 dynamics and binding-site architecture</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13326</offset><text>Molecular dynamics (MD) simulations were performed to investigate the conformational mobility of BlCel5B. In the simulations of the crystal structure for BlCel5B bound to C4, the substrate dissociates from the protein within the first 100 ns of the simulation time (Supplementary Fig. 2A). This observation suggests that cellotetraose does not exhibit detectable affinity for this specific BlCel5B conformation in solution, as one might otherwise expect for a reaction product. No changes beyond local fluctuations were observed in any of the three BlCel5B domains within the time scale of these runs (400 ns; Supplementary Fig. 2B). However, the CBM46 and Ig-like domains did exhibit rigid body-like motions relative to the CD, with rmsd values around 2.3 Å and 1.8 Å, respectively, suggesting that BlCel5B may execute large-amplitude interdomain motions over longer time scales (Supplementary Fig. 2B,C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14243</offset><text>Accordingly, simulations were then performed using accelerated molecular dynamics (aMD) techniques to probe BlCel5B interdomain motions. aMD enhances conformational sampling by raising the basins of the dihedral potential energy surface without affecting the general form of the atomistic potential, thereby increasing transition rates between different local minima. aMD trajectories corresponding to more than 1.0 μs of conventional MD runs were generated. During these simulations, we observed occlusive conformations between CBM46 and CD that resulted in a rearrangement of the enzyme’s architecture around the active site (Video S1). Figure 3A shows BlCel5B in the crystallographic conformation (red) and in a selected configuration obtained with aMD (blue) in the absence of the substrate. Interdomain motions were gauged by the time evolution of the distance between the α carbons of residues I120 and E477 (represented as spheres in Fig. 3A), belonging to the CD and CBM46, respectively. Figure 3C shows that the I120-E477 distance (red curve) gradually decreases from ~35 Å to ~7 Å within the first half of the 1.0 μs aMD trajectory, indicating a transition between the semi-open (crystallographic) and occluded (aMD sampled) configurations. During the second half of the aMD simulation, the full-length enzyme remained in the closed conformation, with the CBM46 covering the carbohydrate-binding site. These results suggest that BlCel5B undergoes large-scale interdomain movements that enable interactions between CBM46 and the substrate bound to the CD.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15823</offset><text>To study the interactions of BlCel5B with a non-hydrolyzed glucan chain, we built a model structure with a cellooctaose (C8) chain spanning the entire positive (+1 to +4) and negative (−4 to −1) subsites of the enzyme. Starting from the crystallographic BlCel5B conformation, the C8 molecule deviated significantly from the active site and assumed a non-productive binding mode (Supplementary Fig. 2D). This observation suggests that the open conformation of BlCel5B is not able to hold the substrate in a position suitable for hydrolysis (Supplementary Fig. 2E). However, after subjecting the BlCel5B-C8 complex to a 0.5 μs aMD simulation with harmonic restraints on the C8 chain to prevent it from deviating from the productive binding mode, the CBM46 readily closed over the CD and trapped the C8 chain in position for hydrolysis (Fig. 3B). In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C). This BlCel5B-C8 configuration remains stable over an additional 500 ns of conventional MD simulation with no restraints (Fig. 3C cyan line, Supplementary Fig. 2E,F).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17220</offset><text>A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D. Together, these results indicate that CBM46 is a key component of the catalytic active complex, providing an explanation as to why CBM46 is essential for the enzymatic activity of BlCel5B.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17752</offset><text>To enable substantially longer time scales compared to atomistic simulations, we further explored the dynamics of BlCel5B using coarse-grained MD (CG-MD) simulations. We performed three independent ~120 μs CG-MD simulations, for a total of approximately 360 μs of sampling. The distance between the α carbons of two residues centrally positioned in the CD and CBM46 (Fig. 4A) was monitored, and the results shown in Fig. 4B indicate that the wide-amplitude events described above frequently appear in this time scale. The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18751</offset><text>BlCel5B conformers fit the SAXS envelope</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18792</offset><text>SAXS experiments were conducted to assess BlCel5B conformational states in solution, and the results revealed the enzyme in its monomeric form, with average values of Rg = 27.17 Å and Dmax = 87.59 Å (Supplementary Table 2). The ab initio dummy atom model (DAM) demonstrated that the SAXS-derived BlCel5B molecular envelope could not be single-handedly filled by any of the main conformational states encountered in the simulations (Fig. 4D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19248</offset><text>It is known that a Kratky plot exhibits a peak with an elevated baseline at high q for a monodisperse system composed of multi-domain particles with flexible extensions. Indeed, an elevation of the baseline toward a hyperbolic-like curve was observed for BlCel5B, indicating a considerable degree of molecular mobility in solution (Supplementary Fig. 3). Thus, the conformational heterogeneity of the enzyme can be decomposed in structural terms as a combination of conformational states identified in our crystallographic and MD studies. We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope. The resulting average scattering curve from this model fits the experimental protein scattering intensity, with χ = 1.89 (Supplementary Fig. 3).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>20397</offset><text>GH5_4 phylogenetic analysis</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20425</offset><text>To date, there are 427 sequences classified as subfamily 4 members in the CAZy database. After the exclusion of partial sequences and the suppression of highly identical members (higher than 90% identity), 144 sequences containing between 277 and 400 residues were aligned and used to construct a phylogenetic tree (Supplementary Fig. 4A). According to PFAM database conserved domain classification, 128 GH5 enzymes have an architecture consisting of an N-terminal catalytic module, a CBM_X2 module and an unknown module of approximately 100 residues at the C-terminus (Supplementary Fig. 4B). Of these, 12 enzymes have an additional CBM1, and 5 have a CBM2 at the N-terminal region. Based on this PFAM architecture and CAZy subfamily classification, all the 144 enzymes (including BlCel5B) belong to the GH5_4 subfamily and group together in the same branch of the phylogenetic tree, evidencing a common ancestor. These results support the hypothesis that the enzymes may employ the same mechanism by which ligand binding is mediated by an extensive conformational breathing of the enzyme that involves the large-scale movement of CBM46 around the Ig-like module (CBM_X2) as a structural hinge.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>21621</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>21632</offset><text>Growing interest in biotechnological applications of enzymes exhibiting activity toward lignocellulosic biomass has sparked efforts in the discovery and development of novel enzymes, as well as the search for a deeper understanding of their mechanisms of action. Here, we elucidate the trimodular molecular architecture of the full-length BlCel5B, a member of the GH5_4 subfamily, for which large-scale conformational dynamics appears to play a central role in its enzymatic activity. Full-length BlCel5B is active on both cellulosic and hemicellulosic substrates and auxiliary modules are crucial for its activity.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>22248</offset><text>Most carbohydrate-active enzymes are modular and consist of a catalytic domain appended to one or more separate AMs. AMs, such as CBMs, typically recognize carbohydrates and target their cognate catalytic domains toward the substrate. Because the structural analysis of the protein is challenging if the linkers connecting the structural subunits of the enzyme are long and flexible, the standard approach is to study the domains separately. In this work, a combination of protein crystallography, computational molecular dynamics, and SAXS analyses enabled the identification of a new conformational selection-based molecular mechanism that involves GH5 catalytic domain and two AMs in full-length BlCel5B. We observed that the BlCel5B distal CBM46 is directly involved in shaping the local architecture of the substrate-binding site. Although the CD alone appears unable to bind the substrate for catalysis, the AMs exhibit open-close motions that allow the substrate to be captured in a suitable position for hydrolysis. Here, we advocate that large-amplitude motions of AMs are crucial for assembling the enzyme into its active conformation, highlighting a new function of CBMs. This mechanism of substrate binding closely resembles the extended conformational selection model, with the induced-fit mechanism of reaction as its limiting case. To the best of our knowledge, this enzymatic mechanism has not been proposed previously for any GH.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>23695</offset><text>The CD binding site of BlCel5B is open and relatively flat and is thus barely able to properly hold the substrate in position for catalysis without assistance from the CBM46. In contrast, other GH5s belonging to subfamily 4 listed in the Protein Data Bank exhibit a deep binding cleft or tunnel that can effectively entrap the substrate for catalysis (Fig. 5). Due to the marked interdomain conformational rearrangement observed in our simulations, the CBM46 generates a confined binding site in BlCel5B that resembles the binding site architecture of the other GH5 enzymes that lack AMs. Thus, BlCel5B appears to have adopted a strategy of CBM46-mediated interactions for proper functioning. Although the homologous BhCel5B has the same domain architecture of BlCel5B and belongs to the same subfamily (a comparison of the sequence and structure of BlCel5B and BhCel5B is presented in Supplementary Fig. 5), its binding site exhibits important differences that may impact the catalytic mechanism. The BhCel5B binding site is V-shaped and deeper than the BlCel5B binding site (Figs 5 and 6). This is due to the loop between residues F177 and R185 from BhCel5B (absent in the BlCel5B), which contains residue W181 that forms part of the binding cleft (Fig. 6). Consistently, although BhCel5B CBM46 is important for β-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates. A closer inspection of results of the phylogenetic analysis, more specifically of the clade composed by GH5_4 enzymes with trimodular architecture (Supplementary Fig. 4C), reveals subclades whose main characteristic is the varying length of the loop located between residues 161 and 163 (BlCel5B residue numbering). Therefore, our results show that BlCel5B represents a smaller group of enzymes that are completely dependent on its AMs for hydrolysis of plant cell wall polysaccharides, and that the underlying mechanism may rely on large-scale interdomain motions.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>25816</offset><text>The amino acid sequence of the BlCel5B Ig-like module is recognized by BLASTP as belonging to CBM_X2, a poorly described group that has been compared with CBM-like accessory modules without a defined function. Despite the similarity of BlCel5B Ig-like module to CBMs, it lacks an identifiable aromatic residue-rich carbohydrate-binding site. Nonetheless, according to our results, the Ig-like module seems to play an important function as a structural hinge, dynamically holding the CBM46 and CD in positions that are appropriate for enzymatic activity.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>26370</offset><text>Based on the results of our crystallographic, computer simulation, and SAXS structural analyses, as well as site-directed mutagenesis and activity assays, we propose a molecular mechanism for BlCel5B substrate binding, which might apply to other GH5_4 subfamily enzymes that share this tri-modular architecture. BlCel5B can be found in several different conformational states ranging from CBM46/CD closed (or occluded) to extended conformations (Fig. 7). In extended configurations, the substrate may dock at the shallow substrate binding site of CD in one of the semi-closed conformations of the enzyme; however, its binding is properly stabilized for hydrolysis only with the aid of induced-fit repositioning mediated by CBM46. After cleavage, the intrinsic dynamics of BlCel5B would eventually allow the opening of the active site for product release. The proposed mechanism is consistent with our mutagenesis and enzymatic activity assays, which show that the Ig-like module and CBM46 are indispensable for BlCel5B catalytic activity and, together with the CD, form the unique catalytic domain of the enzyme. These experiments reveal a novel function for CBMs in which they are intimately involved in the assembly of the active site and catalytic process. Computer simulations suggest that large-scale motions of the CBM46 and Ig-like domains mediate conformational selection and final induced-fit adjustments to trap the substrate at the active site and promote hydrolysis. SAXS data support the modeling results, providing compelling evidence for highly mobile domains in solution.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>27958</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>27966</offset><text>Cloning, Expression and Purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28003</offset><text>The gene encoding BlCel5B (GenBank: AAU23417.1) was amplified from Bacillus licheniformis genomic DNA (ATCC 14580) without the predicted signal peptide sequence (nucleotides 1 to 81) using the primers Blcel5B_Fw and Blcel5B_Rv (Supplementary Table 3). The fragment was cloned into the expression vector pETTRXA-1a/LIC by ligation-independent cloning (LIC), as described elsewhere.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28384</offset><text>The same method was used for construction of domain deletions. For Ig-like + CBM46 deletion, Δ(Ig-CBM46), the fragment encoding the CD (nucleotides 82 to 1086) was amplified using the primers Blcel5B_Fw and Blcel5BΔ1087-1683_Rv. For CBM46 deletion, ΔCBM46, the fragment encoding the CD + Ig-like (nucleotides 82 to 1377) was amplified using the primers Blcel5B_Fw and Blcel5BΔ1378-1683_Rv (Supplementary Table 3). Both fragments were cloned into pETTRXA-1a/LIC.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28862</offset><text>The wt protein BlCel5B, mutated proteins and AM deletions were expressed in E. coli Rosetta2 (DE3) strain. The cells were grown at 37 °C and 150 RPM in Luria Bertani Broth medium supplemented with 50 μg/mL kanamycin to an A600 of 1.5–2.0, after which the temperature was reduced to 20 °C and protein expression was induced with 1 mM IPTG for 6 h.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>29224</offset><text>The extract was then loaded onto a NiNTA resin (Qiagen) equilibrated with a washing buffer (5 mM imidazole, 100 mM NaCl, 50 mM Tris-HCl, pH 8.0). Non-absorbed material was washed with ten times column volume with washing buffer and the purified protein was eluted with 200 mM imidazole, 100 mM NaCl, 50 mM Tris-HCl at pH = 7.0. His6 tag was removed by overnight digestion with TEV (Tobacco Etch Virus) at 4 °C, and untagged protein was purified by gel filtration through a HiLoad 16/60 Superdex 200 column in buffer containing 50 mM NaCl, 25 mM Tris-HCl at pH 7.0.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>29812</offset><text>Site-directed Mutagenesis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>29838</offset><text>The BlCel5B point mutations W479A and W481A were obtained by the inverse PCR method of site-directed mutagenesis. Phusion® “High-Fidelity” DNA polymerase (NEB, USA) was used for amplifications with the plasmid pETTRXA-1a/LIC-Blcel5B as a template. Mutagenic primers Blcel5BW479A_Fw/Rv and Blcel5BW481A_Fw/Rv (Supplementary Table 3) were generated by HTP-OligoDesigner tool (http://www.ifsc.usp.br/htpoligo/).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>30252</offset><text>Activity Assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>30268</offset><text>Enzymatic activity assays were performed by a colorimetric method using the 3,5-dinitrosalicylic acid (DNS), with glucose being a standard for the calibration curves. Assays of optimal temperature and pH were performed in triplicate with 1% medium-viscosity CMC as the substrate. For optimal temperature, the reaction mixture containing 10 μL of enzyme at 0.1 mg/mL, 50 μL of 1% (w/v) CMC and 40 μL of 50 mM sodium citrate buffer (pH 5.0) was incubated at 30 to 80 °C for 15 min and stopped by adding 100 μL of DNS solution. After this, the mixture was incubated again for 5 min at 100 °C and the absorbance was measured at 540 nm with a spectrophotometer. For optimal pH determination, the same amount of enzyme and substrate were diluted in 40 mM acetate/borate/phosphate buffer (ABF) with different pH values ranging from 2.0 to 10.0. The reactions were carried out under the predetermined optimal temperature.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31212</offset><text>The substrate specificity of the enzyme was determind using rye arabinan, xyloglucan, β-glucan, galactomannan, lichenan, β-mannan, Azo-Avicel and CMC as substrates. The substrates were diluted in water to 1% (w/v), and the reaction mixture was composed of 10 mL of purified enzyme at a concentration of 0.1 mg/mL, 0.4 mL of 50 mM sodium citrate buffer at pH 5.0, and 0.5 mL of 1% (w/v) substrate aqueous solution. The reaction was incubated at 50 °C for 15 min, followed by treatment with DNS as mentioned above. Enzyme unit was defined as the amount of enzyme that produces 1.0 μM of glucose in one minute for each substrate.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31859</offset><text>The kinetic parameters were determined by increasing concentrations of CMC. Reactions were performed in 50 mM sodium citrate buffer (pH = 4.0) at 50 °C, and measured by DNS method as well. Kinetic constants were determined by non-linear regression using OriginPro 8.0.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32137</offset><text>Thermal Shift Assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32158</offset><text>The thermal denaturation assays were performed using a Real Time PCR Machine (Stratagene Mx3005P) as described by Dupeux and co-workers. Briefly, the enzymes were diluted to 10 μM in 50 mM sodium citrate buffer (pH = 4.0) containing 1x SYPRO orange dye (Thermo Fisher Scientific). The fluorescence emission of the probe was monitored (excitation and emission at 492 and 516 nm, respectively) varying the temperature between 25 and 75 °C with the rate of 1 °C/min.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32639</offset><text>Cellopentaose Cleavage Experiment</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32673</offset><text>The full-length BlCel5B and AM deletion constructs were tested for product formation from cellopentaose. Cellopentaose (1.0 mM) was incubated with 25 μg of purified enzyme in 10 mM ammonium bicarbonate buffer (pH 7.0) in a total volume of 50 μL. The reaction was incubated for 90 min at 50 °C and then stopped by treatment at 100 °C for 5 min. After centrifugation for 10 min at 16,000 g the samples were subjected to MALDI/TOF-MS. Samples were supplemented with NaCl to a final concentration of 20 mM and 1 μL of the supernatant was co-crystallized with 1 μL 2,5-dihydroxybenzoic acid (10 mg/mL) in acetonitrile 30% and spotted on the target plate. The products were analyzed on Microflex LT MALDI-TOF (Bruker Daltonics) operating in positive ion mode. A single spectrum was obtained by averaging four independent spectra generated by 300 laser shots at 60% potency.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>33575</offset><text>Crystallization, Data Collection, and Structure Determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>33637</offset><text>After purification, BlCel5B was concentrated to 10 mg/mL for crystallization trials. Crystallization screens were set up using the sitting-drop vapor-diffusion method on a Cartesian PixSys 4200 (Genomic Solutions, United Kingdom) in a 96-well plate with drops formed by 100 nL protein solution plus 100 nL reservoir solution. The commercial kits Crystal Screen and Index (Hampton) were used as initial conditions. Crystals were grown at 18 °C between 3 and 7 days, and screened for diffraction.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34141</offset><text>Crystals were supplemented with cryoprotection solution, flash cooled in liquid nitrogen and diffraction data were collected at 100 K, at beamline ID23-1 (wavelength of 0.97 Å) from the European Synchrotron Radiation Facility (Grenoble, France). A crystal grown in condition containing 22.5% PEG 4000, 14% isopropanol and 0.1 M sodium citrate, pH 6.0, was selected to collect diffraction data to 1.7 Å resolution. The complex of the enzyme with substrate was obtained by crystal soaking with five times molar excess of cellopentaose for 24 hours. Diffraction data for the complexed enzyme were collected at 1.75 Å resolution.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34782</offset><text>Data were integrated with iMosflm and scaled with Aimless. The structure was solved by molecular replacement with Phaser using an endoglucanase from Clostridium cellulovoran (PDB code: 3NDY) as the search model. Coot was used for density fitting, and refinement was performed with PHENIX.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>35071</offset><text>Atomistic simulations</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35093</offset><text>We took the BlCel5B structure complexed with cellotetraose as the starting configuration for the MD simulations. The missing residues were taken from the apo BlCel5B structure after structural alignment using the LovoAlign server. Hydrogen atoms were then added according to the protonation states determined at the optimum pH of 4.0 using the H + + server. The following residues were considered protonated: H55, H77, D89, E96, E103, H114, E129, E159, E197, D198, E202, H205, E208, D211, H220, E245, E248, E260, H278, H292, D306, E312, E371, E375, E476, H416, E477, E489, D497, and E524. The remaining protonatable residues were considered in the standard protonation state. The BlCel5B-cellotetraose complex was then immersed in a rectangular simulation box of dimensions such that a solvent layer at least 16 Å thick surrounded the protein. The simulation box, built with Packmol, also contained 0.10 M NaCl aqueous solution with excess counter ions to keep the system electrically neutral. The final system comprised approximately 85500 atoms.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36152</offset><text>The simulations were performed using NAMD with the CHARMM force field and the TIP3P water model. Periodic boundary conditions were employed, using particle mesh Ewald to handle electrostatics and a 12-Å cutoff radius for truncating short-range potentials. Bonds involving hydrogen atoms were constrained at their equilibrium lengths and a time step of 2 fs was used to integrate the equations of motion. The simulations were carried out under constant pressure and temperature of 1 atm and 310 K, respectively, employing the Langevin barostat and thermostat.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>36714</offset><text>Accelerated Molecular Dynamics</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36745</offset><text>In accelerated molecular dynamics, the trajectory is propagated on a modified potential aimed to enhance conformational sampling. Whenever the potential energy drops below a given threshold E, a boost ΔV(r) is applied, so that the escaping rates of local minima increase. When the potential energy gets over the threshold E, the system evolves on the original energy surface. This method has the advantage of conserving the general shape of the potential energy surface and of requiring no prior definition of reaction coordinates, so the system is allowed to explore freely its conformational space. Here, we restricted the energy boost only to the dihedral potential energy, as changes in torsion angles are the main source of conformational changes in proteins. The energy boost assumes the form of equation (1) that depends on the energy threshold E and on the parameter α – which modulates the shape of the potential energy surface where the boost is applied. We set the parameters E and α according to previous studies, which recommend that E equals the average dihedral energy obtained from a conventional MD simulation plus 4 kcal/mol times the number of residues, and α equals 0.8 kcal/mol times the number of residues. The average dihedral energy was 2275.5 kcal/mol and the BlCel5B has 516 residues, so we set E = 2275.5 + 4 × 516 = 4339.5 kcal/mol and α = 0.8 × 516 = 418.8 kcal/mol.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>38188</offset><text>Simulation procedures</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38210</offset><text>BlCel5B-cellotetraose – Having built the system, we carried out the following steps for equilibration: (i) 1000 steps of energy minimization followed by 100 ps of MD simulation with all non-solvent heavy atoms fixed; (ii) same as (i), but with only the α carbons fixed; (iii) 5 ns of MD with all atoms free. After these preliminary steps, a trajectory lasting 400 ns was generated using conventional MD and then the aMD dihedral boost was applied for additional 1.0 μs. After 100 ns of conventional MD, the cellotetraose dissociated and the simulation began to represent the dynamics of unbound state of BlCel5B.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>38835</offset><text>BlCel5B-cellooctaose</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38856</offset><text>To build the cellooctaose chain, we extended the original cellotetraose chain in the crystal structure with 4 additional glucose residues spanning regions around the BlCel5B positive subsites. Then, we submitted the system to the following procedure: (i) 1000 steps of energy minimization followed by 1 ns of MD keeping all the non-solvent heavy atoms fixed, except the 4 modeled glucose residues of the cellooctaose chain; (ii) same as step (i), but with only the α carbons fixed; (iii) 1 ns of MD with only the non-modeled glucose residues fixed. We then performed a 200-ns-long MD with three harmonic potentials involving cellooctaose chain: first, between C3 atom (CHARMM atom names) of the second glucose residue from the cellooctaose non-reducing end and the CD2 atom of the W47 tryptophan residue; second, between the OH3 atom of the forth glucose residue from the cellooctaose non-reducing end and HE2 atom of the H113 histidine residue; and third, between the HE2 atom of the catalytic residue E159 and O4 glycosidic oxygen between the fourth and fifth glucose unit of the cellooctaose chain. After these preliminary relaxation steps, the harmonic potentials were removed and the trajectory was propagated by 400 ns using MD. To get a model of the BlCel5B-cellooctaose complex in the closed conformation, we took the configuration after 80 ns of the restrained 200-ns MD simulation as the starting point for a 500-ns-long restrained aMD simulation, in which the CBM46 moved towards the CD in the presence of the harmonically-restrained cellooctaose chain. After this procedure, we released the restraints and propagated the closed BlCel5B-cellooctaose complex for additional 500 ns of conventional, restraint-free MD simulation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>40605</offset><text>Coarse-grained MD simulations</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40635</offset><text>The coarse-grained model was constructed from the minimized all-atom protein. We have used the domELNEDIN CG model for the protein. In this representation, an elastic network is used within each domain as a structural scaffold in order to maintain the overall shape of the protein, and a slightly modified version of MARTINI CG model describes the interactions involving beads not connected by harmonic springs.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>41047</offset><text>The delimitation of each domain was quite clear considering the short linkers connecting them and the recognition of their structural patterns in databases. We assumed CD, Ig-like module, and CBM46 as consisted of residues 18–331, 332–430, and 431–533, respectively. Therefore, there were elastic network bonds only within these domains (domELNEDIN CG model in Supplementary Fig. 6A).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>41438</offset><text>The protonation state of each residue bead in the protein was the same adopted in the atomistic simulations. The system was then solvated by 10000 standard MARTINI CG water beads, including 10% of antifreeze particles. Also, 58 chloride and 48 sodium ions were added for charge neutrality. The size of final system was 109 Å × 109 Å × 109 Å.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>41799</offset><text>Preliminary simulations were performed to test the elastic network (EN) parameters. We have tested six different ENs in 100 ns of simulations, using combinations of cut-off distance (Rc) of 8 Å and 9 Å with spring force constant (ks) of 500, 800 and 1000 kJ mol−1 nm−2. The time evolution of root mean square deviation relative to the crystal structure as well as the mobility profile of the protein in these simulations were compared to the correspondent data from a 100 ns atomistic simulation. From this procedure, the parameters Rc = 9 Å and ks = 500 kJ mol−1 nm−2 resulted in the best match between atomistic and coarse-grained simulations (Supplementary Fig. 6B).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>42500</offset><text>The coarse-grained simulations were carried out using GROMACS. Periodic boundary conditions were employed. Van der Waals interactions were shifted to zero in the range 0.9–1.2 nm, and the electrostatic interactions, in the range 0.0–1.2 nm. The simulations were performed in the isothermal-isobaric ensemble (NpT), employing the Berendsen thermostat and barostat for temperature and pressure control, respectively, with time constants τT = 0.5 ps and τp = 1.2 ps.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>42982</offset><text>The CG simulations were carried out using the following protocol: the system was first minimized for 1000 steps using the steepest descent method. Then, it was submitted to a relaxation procedure comprising gradual increasing in time step or temperature. In the first stage of relaxation, the protein beads were restrained with a 1000 kJ mol−1 nm−2 force constant and a 50 ps simulation was carried out at 50 K, using the short time step of 1 fs. In the second stage, the time step was increased up to 5 ps lasting 1000 ps of simulation time. In the last stage of relaxation, all the system is released to move and it underwent a gradual increase in temperature, consisting on five segments of 100 ps at 50, 100, 150, 200 and 310 K. After achieving the desired temperature of 310 K, we performed three production simulations using 20-fs timestep. We have used a random number generator for assigning velocities to generate three independent simulations.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>43950</offset><text>In general, smoothing of the energy surface in CG model makes the time scales faster. A speed up factor of 4 is typically employed to rescale the time scale of MARTINI CG systems. Therefore, all CG simulations times described here and in the main text are effective times, i.e., 4× simulation time.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>44250</offset><text>Small Angle X-ray Scattering</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>44279</offset><text>SAXS data were collected at the SAXS2 beamline of the Brazilian Synchrotron Light Laboratory-LNLS (Campinas, Brazil) on a bi-dimensional position sensitive CCD detector (MarResearch, USA) using the radiation wavelength 1.54 Å. The sample-detector distance of 1000 mm allowed covering the momentum transfer range 0.01 Å−1 &lt; q &lt; 0.35 Å−1 (q = 4πsin θ/λ, where 2θ is the scattering angle).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>44697</offset><text>The protein samples were prepared in McIlvaine’s buffer at 50 mM, pH 5 and 20 °C. In each measurement, two successive frames of 300s were recorded for each sample at 1 and 2 mg/mL to monitor radiation damage. The patterns were integrated using the FIT2D program. The comparative analysis for each scattering curve at 1 and 2 mg/mL of BlCel5B (data not shown), as well as the radius of gyration values (Rg), indicated that concentration and aggregation effects did not exist. The linearity of the Guinier plot indicated that the preparation was monodisperse.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>45266</offset><text>The radius of gyration of the molecules (Rg) was estimated by two methods, using the Guinier equation-I(q) = I(0).exp[(−q2.Rg2)/3], q.Rg &lt; 1.3- and also with the inverse Fourier transform in GNOM. The same program was used to obtain the distance distribution function P(r) and the maximum diameter Dmax. Ten independent dummy atom models (DAMs) were restored by the ab initio proceeding implemented in DAMMIN package. The best model, selected using normalized spatial discrepancy parameter computed by DAMAVER program, was superimposed on the crystallographic model with the SUPCOMB.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>45860</offset><text>Then, based on the enzyme conformations reported by MD and protein crystallography, the computed X-ray scattering profile was fitted to a given experimental SAXS data by minimizing the χ function in the FOXS program.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>46079</offset><text>To assess the inter-domain information, the contribution of individual conformer and the flexibility of BlCel5B, we proceed in two approaches. First, the theoretical profiles and experimental data comparison was performed to infer the best-fit conformation of the ensemble-based analysis by the ensemble optimization method – EOM, which assumes coexistence of a range of conformations in solution for which an average scattering intensity fits the experimental SAXS data; all models were generated with the three individual domains (Ig-like, CBM46, and CD) free to randomly move in order to cover the entire conformational space. The second approach was based on a fractional volume calculation from three conformation members extracted from the MD simulations, each with a distinct scatter curve. OLIGOMER provided solution of a system of linear equations between the experimental and generated conformations by MD.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>46998</offset><text>The simulated scattering curves from the MD and crystallographic models were obtained using the CRYSOL.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>47102</offset><text>Phylogenetic assignment</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>47126</offset><text>Sequences for all GH5 members, in which only the catalytic domain were considered, were downloaded from PFAM database and their classification into subfamilies were obtained within the CAZy database. The sequences belonging to subfamily 4 were selected and those that had over 90% identity or represented partial coverage were rejected. Based on their multiple sequence alignment, the phylogenetic tree was constructed using the maximum likelihood method implemented in the MEGA program version 6.06. One hundred Bootstrap replications were performed to examine the reliability of the phylogenetic tree.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>47730</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>47753</offset><text>How to cite this article: Liberato, M. V. et al. Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism. Sci. 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Evol.</infon><infon key="type">ref</infon><infon key="volume">30</infon><infon key="year">2013</infon><offset>53178</offset><text>MEGA6: Molecular Evolutionary Genetics Analysis version 6.0</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>53238</offset><text>Author Contributions M.V.L. and I.P. designed the experiments; C.M.C. performed the gene mutations and cloning; M.V.L. and V.O.A.P. expressed and purified the enzymes and characterized the enzymatic activities; M.V.L. crystallized and determined the crystal structures, with data collection supervised by A.P.; M.A.K. performed mass spectrometry experiment; E.A.A. and M.O.N. collected and treated SAXS data. R.L.S., E.T.P. and M.S.S. designed the computer simulations; R.L.S. performed Accelerated Molecular Dynamics; E.T.P. performed Coarse-grained MD Simulations; M.V.L., R.L.S., M.S.S. and I.P. wrote the manuscript with the input from all the other authors; M.S.S. and I.P. supervised the project.</text></passage><passage><infon key="file">srep23473-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>53941</offset><text>Crystal models of BlCel5B.</text></passage><passage><infon key="file">srep23473-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>53968</offset><text>Complete structure is shown as a cartoon illustration in (a) and a van der Waals surface in (b). The CD module (red) has a typical TIM-barrel fold, and its substrate-binding site is adjacent to CBM46 (blue). Despite the proximity of the binding site in the crystallographic model, the CBM46 residues W479 and W481 are distant from the substrate cellotetraose (yellow). The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray.</text></passage><passage><infon key="file">srep23473-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>54730</offset><text>BlCel5B enzymatic activity characterization.</text></passage><passage><infon key="file">srep23473-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>54775</offset><text>(a) MALDI/TOF-MS spectra of the products released after incubation of BlCel5B and its two deletion constructs (ΔCBM46 and ΔIg-CBM46) with the substrate cellopentaose (C5). The first three spectra show the substrate, enzyme and buffer controls. The forth spectrum reveals that full length BlCel5B is capable of enzymatic hydrolysis of C5 into smaller oligosaccharides such as C4, C3 and C2. The last two spectra show that the C-terminal deletions eliminate the enzyme activity. BlCel5B activities on CMC as functions of pH and temperature are shown in (b) and (c), respectively. The enzyme exhibits optimal pH of 4.0 and optimal temperature of 55 °C, retaining about 50% of its activity at 80 °C. (d) Michaelis-Menten curve using CMC as a substrate.</text></passage><passage><infon key="file">srep23473-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>55532</offset><text>Open-close transitions of BlCel5B.</text></passage><passage><infon key="file">srep23473-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>55567</offset><text>(a) BlCel5B in the absence of substrate and (b) in the presence of cellooctaose, as observed in our aMD simulations. The distance between the α carbon of residues I120 (CD) and E477 (CBM46), illustrated as spheres in (a), is plotted in (c), revealing a transition by the decrease in the distance from 40 Å to 7 Å (substrate-free) or 20 Å (in presence of cellooctaose). For the substrate-free enzyme, the red line refers to a 1 μs-long aMD; for the BlCel5B-cellooctaose complex, the first 500 ns refers to aMD (in blue) and the second 500 ns to conventional MD (in turquoise). (d) A snapshot of the BlCel5B-cellooctaose complex, highlighting the tryptophan residues that interact with the glucan chain in subsites −4 to +4. Residues W479 and W481 belong to CBM46 and only become available for substrate interactions in the closed configuration of BlCel5B.</text></passage><passage><infon key="file">srep23473-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>56441</offset><text>Large-scale movements of BlCel5B modules and superposition of their representative conformations with the SAXS envelope.</text></passage><passage><infon key="file">srep23473-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>56562</offset><text>(a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations. Different colors separated by vertical lines correspond to independent simulations of approximately 120 μs. (c) The distance distribution indicates three major peaks: closed or occluded CBM46/CD conformations (I); semi-open (II), which is similar to the crystallographic structure; and extended conformers (III). (d) Superimposition of the three representative molecular conformations of BlCel5B with the SAXS model. (e) Average structures obtained from the simulation segments corresponding to population groups I-III, which are individually superposed on the SAXS envelope.</text></passage><passage><infon key="file">srep23473-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>57440</offset><text>Comparison of the binding site shape of GH5_4 enzymes available on the Protein Data Bank.</text></passage><passage><infon key="file">srep23473-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>57530</offset><text>(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).</text></passage><passage><infon key="file">srep23473-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>58176</offset><text>Comparison of the binding cleft of the BlCel5B and BhCel5B.</text></passage><passage><infon key="file">srep23473-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>58236</offset><text>The main difference between BlCel5B and BhCel5B is that the latter exhibits a deeper cleft due to the presence of residue W181 in the loop between F177 and R185. We conjecture that this difference in the binding site architecture relates to the importance that the CBM46 plays in the BlCel5B enzymatic mechanism.</text></passage><passage><infon key="file">srep23473-f7.jpg</infon><infon key="id">f7</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>58549</offset><text>Proposed molecular mechanism of BlCel5B conformational selection.</text></passage><passage><infon key="file">srep23473-f7.jpg</infon><infon key="id">f7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>58615</offset><text>As suggested by the simulations and SAXS data, BlCel5B spans multiple conformations ranging from closed to extended CBM46/CD states. In a given open state, the substrate may reach the active site and become entrapped by the capping of CBM46 onto CD and induced-fit conformational adjustments. After hydrolysis, the reaction product is released to yield apo-BlCel5B, which becomes ready for a new cycle.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>59018</offset><text>Activity of BlCel5B constructs against tested substrates.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot; border=&quot;1&quot;&gt;&lt;colgroup&gt;&lt;col align=&quot;left&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;/colgroup&gt;&lt;thead valign=&quot;bottom&quot;&gt;&lt;tr&gt;&lt;th rowspan=&quot;2&quot; align=&quot;left&quot; valign=&quot;bottom&quot; charoff=&quot;50&quot;&gt;Substrate (1%)&lt;/th&gt;&lt;th colspan=&quot;5&quot; align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Relative Activity (%)&lt;/th&gt;&lt;/tr&gt;&lt;tr&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;WT&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn1&quot;&gt;*&lt;/xref&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;W479A&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;W481A&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ΔCBM46&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ΔIg-CBM46&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;β-glucan&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;100&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;79.1&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;63.6&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;CMC&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;25.5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;12.2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2.4&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Lichenan&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;52.4&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;41&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;28.6&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Xyloglucan&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;45.2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;41.2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;30.8&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Azo-Avicel&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn2&quot;&gt;**&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Arabinoxylan&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Galactomannan&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1,4-β-mannan&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;nd&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>59076</offset><text>Substrate (1%)	Relative Activity (%)	 	WT*	W479A	W481A	ΔCBM46	ΔIg-CBM46	 	β-glucan	100	79.1	63.6	nd	nd	 	CMC	25.5	12.2	2.4	nd	nd	 	Lichenan	52.4	41	28.6	nd	nd	 	Xyloglucan	45.2	41.2	30.8	nd	nd	 	Azo-Avicel	nd**	nd	nd	nd	nd	 	Arabinoxylan	nd	nd	nd	nd	nd	 	Galactomannan	nd	nd	nd	nd	nd	 	1,4-β-mannan	nd	nd	nd	nd	nd	 	</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>59407</offset><text>*WT = wild type.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>59428</offset><text>**nd = not detected.</text></passage></document></collection>
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+<collection><source>PMC</source><date>20230107</date><key>pmc.key</key><document><id>4980666</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/srep31500</infon><infon key="article-id_pii">srep31500</infon><infon key="article-id_pmc">4980666</infon><infon key="article-id_pmid">27510745</infon><infon key="elocation-id">31500</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Carcelli;given-names:Mauro</infon><infon key="name_1">surname:Rogolino;given-names:Dominga</infon><infon key="name_2">surname:Gatti;given-names:Anna</infon><infon key="name_3">surname:De Luca;given-names:Laura</infon><infon key="name_4">surname:Sechi;given-names:Mario</infon><infon key="name_5">surname:Kumar;given-names:Gyanendra</infon><infon key="name_6">surname:White;given-names:Stephen W.</infon><infon key="name_7">surname:Stevaert;given-names:Annelies</infon><infon key="name_8">surname:Naesens;given-names:Lieve</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">6</infon><infon key="year">2016</infon><offset>0</offset><text>N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>97</offset><text>Influenza virus PA endonuclease has recently emerged as an attractive target for the development of novel antiviral therapeutics. This is an enzyme with divalent metal ion(s) (Mg2+ or Mn2+) in its catalytic site: chelation of these metal cofactors is an attractive strategy to inhibit enzymatic activity. Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays. Several N-acylhydrazones were found to have promising anti-influenza activity in the low micromolar concentration range and good selectivity. Computational docking studies are carried on to investigate the key features that determine inhibition of the endonuclease enzyme by N-acylhydrazones. Moreover, we here describe the crystal structure of PA-Nter in complex with one of the most active inhibitors, revealing its interactions within the protein’s active site.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1057</offset><text>Influenza virus is an enveloped virus with a segmented negative-oriented single-stranded RNA genome, belonging to the Orthomyxoviridae. Seasonal influenza A and B viruses affect each year approximately 5–10% of the adult and 20–30% of the paediatric population, and there is a permanent risk of sudden influenza pandemics, such as the notorious ‘Spanish flu’ in 1918 and the swine-origin H1N1 pandemic in 2009. Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir). The M2 inhibitors have limited clinical utility due to their central nervous system side effects and widespread resistance, as in the case of the 2009 pandemic H1N1 virus; resistance is also a growing concern for oseltamivir. Therefore, there is an urgent need for new antiviral drugs with an entirely different mode of action.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1984</offset><text>The influenza virus polymerase complex is composed of three subunits: PB1, PB2 and PA. The PA subunit performs the ‘cap-snatching’ endonuclease reaction, the PB2 subunit is responsible for initial binding of the capped RNAs, while the actual RNA synthesis is performed by the PB1 protein.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2277</offset><text>Given its crucial role in the viral life cycle, the influenza virus polymerase is widely recognized as a superior target for antiviral drug development and, in particular, inhibition of the PA endonuclease has deserved much attention in recent years.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2528</offset><text>The endonuclease catalytic site resides in the N-terminal domain of PA (PA-Nter; residues 1~195). It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions. The two-metal-ion model is consistent with numerous biochemical findings. Since the intracellular concentration of Mg2+ is at least 1000-fold higher than that of Mn2+, magnesium may be more biologically relevant. A controversy about number and type of metal ions exists also for the active site of HIV-1 integrase. HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3693</offset><text>In recent years, we focused our research on chemical scaffolds that are able to chelate metal ions of PA-Nter, resulting in inhibition of influenza virus replication. N-acylhydrazones represent an appealing class of chelating ligands with a broad spectrum of biological activities, such as activity against HIV, hepatitis A, vaccinia and influenza virus. In the present work, we report the biological activity of a series of N-acylhydrazones (Fig. 2), as determined in an enzymatic assay with PA-Nter endonuclease as well as in cell-based influenza viral ribonucleoprotein (vRNP) reconstitution and virus yield assays. Several N-acylhydrazones were found to have promising anti-influenza activity with 50% effective concentration values (EC50) in the range of 3–20 μM and good selectivity (Table 1 and Fig. 3). Computational docking studies of two candidate ligands in the PA-Nter active site gave information about the features that could determine inhibition of endonuclease activity. Moreover, we describe the X-ray crystal structure of PA-Nter in complex with one of the most active inhibitors.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4797</offset><text>Results and Discussion</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4820</offset><text>Chemistry</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4830</offset><text>N-acylhydrazones 1–27 (Fig. 2) were prepared in high yields by following literature methods (Fig. 2A); they were characterized by spectroscopic tools, mass spectrometry and elemental analysis. Even if isomerism around the C = N bond is possible, 1–27 are present in the E form in solution, as evidenced by the chemical shift values of the HC = N and NH protons in the 1H-NMR spectrum. Exceptions are represented by the alkyl-derivatives 3 and 4 (2:1 and 5:3 E:Z ratio, respectively).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5326</offset><text>If R’ (Fig. 2A) is a 2-hydroxy substituted phenyl ring, the corresponding acylhydrazones can coordinate one or, depending on denticity, two metal centers (modes A and B in Fig. 4). Starting from N’-(2,3-dihydroxybenzylidene)-semicarbazide (1) and its methoxy-analogue (2), we modified the acylhydrazonic substituent R” (3–8, 18, 19, Fig. 2A). In 18 and 19, also the gallic moiety can be involved in the chelation of the metal cofactors (mode C, Fig. 4). In order to investigate the role of hydroxyl substituents 9–11, 13–17, 20–23 and 27 were also synthesized. Compound 12 was synthesized in order to confirm the crucial influence of the gallic moiety. Finally, 26 was here considered, because it is an inhibitor of HIV RNase H, another enzyme with two magnesium ions in its active site.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6128</offset><text>Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+. Different reaction conditions were used (1:1 and 1:2 metal to ligand ratio, up to 4 equivalents of triethylamine), but in any case the same chemical species Mg(HL)2∙4H2O was recovered and conveniently characterized. The use of a coordinating solvent as d6-DMSO causes partial decoordination of the ligand, but the 1H-NMR spectrum in MeOD, instead, shows only the signals attributable to the complex. In the 13C-NMR spectrum, the signal of the C = O quaternary carbon is practically unaffected by complexation, suggesting that the C = O group is weakly involved in the coordination to the metal ion. This is confirmed, in the IR spectrum, by the shift of about 20 cm−1 of the C = O absorption, while a shift of 30–50 cm−1 is expected when the carbonylic oxygen is tightly bound to the metal ion. ESI-mass spectra and elemental analysis confirmed the formula Mg(HL)2∙4H2O.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7274</offset><text>The interaction between the N-acylhydrazone ligands and the magnesium cation was investigated also by means of UV-visible spectroscopy (UV-visible titrations of 23 and 19 with increasing amount of Mg(CH3COO)2 are shown in Figure S1). The spectrum of 19 includes a band at 313 nm assignable to n-π* transitions of the C = N and C = O groups. By adding increasing equivalents of Mg(CH3COO)2, the absorption around 400 nm increases, and a new band appears with a maximum at 397 nm. The opposite trend is observed in the range 300–350 nm, where an isosbestic point is present close to 335 nm. When the same experiment was performed with 23, a different behavior was observed. Increasing concentration of Mg2+, in fact, caused a diminution in the maximum absorption, an isosbestic point is visible at about 345 nm, but a new band at 400 nm does not appear. Ligands 19 and 23 coordinate the Mg2+ ions in different ways: 19 chelates the metal ion by using the deprotonated salicyl oxygen and the iminic nitrogen, while for 23, the gallic moiety is supposed to be involved (Fig. 4A,B versus C), leading to different, less extensive, modifications of the UV spectrum. These results will be revisited during the discussion of the biological activity.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8540</offset><text>Inhibition of the PA-Nter enzyme</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8573</offset><text>All the compounds were tested for their ability to inhibit the influenza endonuclease in an enzymatic plasmid-based assay with recombinant PA-Nter, as well as in cell-based influenza methods (i.e. virus yield and vRNP reconstitution assays). The results are shown in Table 1 and summarized in Fig. 3 to visualize the structure-activity relationships; Figure S2 shows the dose-response curves for three representative compounds (i.e. 10, 13 and 23) in either the PA-enzyme or vRNP reconstitution assay.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9075</offset><text>The moderate activity (IC50 = 24 μM) of N’-2,3-dihydroxybenzylidene semicarbazide (1) was completely lost when the NH2 moiety was replaced by a hydrophobic heptyl chain (3), but it is less affected when a phenyl or a 2-hydroxyphenyl is present (5 and 7, IC50 = 84 and 54 μM, respectively). When the hydroxyl in position 3 on R1 (2,3-dihydroxybenzylidene) was replaced by a methoxy group (2-hydroxy-3-methoxybenzylidene), the activity disappeared (compounds 2, 4, 6 and 8). The activity is unaffected (IC50 values ranging from 45 to 75 μM) when going from two hydroxyls in R1 (7) to compounds with three hydroxyls (i.e. 9, 10 and 11). Similarly, 11 (R1 = 3,4,5-trihydroxyphenyl, R2 = 2-hydroxyphenyl) had comparable activity as 27 (R1 = 3,4,5-trihydroxyphenyl, R2 = NH2). Within the series carrying a 2-hydroxyphenyl R2 group, the activity of 11 is particularly intriguing. 11 does not have the possibility to chelate in a tridentate ONO fashion (mode A in Fig. 4), but it can coordinate two cations by means of its three OH groups in R1 (mode C, Fig. 4). Note that a similar chelating mode was observed in a crystal structure, solved by Cusack and coworkers, of PA-Nter endonuclease in complex with the inhibitor EGCG.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10329</offset><text>The PA-Nter inhibitory activity strongly depends on the number and position of hydroxyl substituents in R1 and R2: this is clearly highlighted by the data obtained with compounds 13–23, in which R2 is a 3,4,5-trihydroxyphenyl (gallic) group, the most active scaffold in our series. The analogue carrying an unsubstituted aromatic ring as R1 (compound 13) had moderate activity (IC50 = 69 μM). When one OH was added at position 2 of the R1 ring (14), the activity was lost. Adding a second OH substituent at position 5 resulted in strong activity (compound 15, IC50 = 9 μM); medium activity for a 3-OH (18; IC50 = 83 μM), and marginal activity when the second OH is at position 4 (17, IC50 ≥ 370 μM). The addition of a 3-methoxy group (19) abolished all inhibitory activity. This cannot be related to variations in the chelating features displayed by the R1 moiety, since compounds 14–19 all have, in theory, the capacity to chelate one metal ion through the ortho-OH and iminic nitrogen (mode A in Fig. 4). Moreover, compound 18 can, in principle, chelate the two M2+ ions in the active site according to mode B (Fig. 4), yet it (IC50 = 83 μM) has nine-fold lower activity than 15, that does not possess this two-metal chelating feature. Therefore, we hypothesized that the inhibitory activity of the series containing the gallic moiety is determined by: (i) the capacity of the moiety R2 to chelate two metal ions in the active site of the enzyme, according to mode C (Fig. 4); and (ii) the presence and position of one or more hydroxyl substituents in R1, which may possibly result in ligand-protein interactions (e.g. through hydrogen bonds). This assumption was supported by molecular docking calculations and X-ray analysis of inhibitor 23 in complex with PA-Nter (vide infra). At this point, change of the substituents in R1 represents the next logical step. Substitution of the 5-hydroxyl in 15 by a methoxy group (16) causes a dramatic drop in activity (IC50 = 9 and 454 μM for 15 and 16, respectively). When two or three OH groups are present in R1, their spatial disposition greatly affects the activity. In particular, all the compounds with a trihydroxylated phenyl group as R1 (i.e. 20, 21, 22 and 23) were able to inhibit PA-Nter quite potently. The lowest IC50 values were obtained for 21 and 23 (IC50 = 13 and 7 μM, respectively), which both have one of their three hydroxyl groups at position 5. The most active compound in this series was 23, which lacks the hydroxyl group at position 2 of R1, further confirming that this function is undesirable or even detrimental for inhibitory activity against PA-Nter, as already noticed above for 14.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13044</offset><text>Consistent with a crucial role of the R2 gallic moiety in metal chelation, the strong activity of 15 was completely lost in its 3,4,5-trimethoxy analogue 12. On the other hand, the R2 gallic containing compounds displayed moderate activity (IC50 values around 40 μM) when R1 was absent (i.e. the 3,4,5-trihydroxybenzohydrazide 28, Fig. 2), or composed of an extended ring system (26) or a pyrrole ring (25). Still lower activity was seen with the pyridine analogue 24. Evidently, the 3,4,5-trihydroxybenzyl moiety at R2 is fundamental but not sufficient to ensure potent PA-Nter endonuclease inhibition, since the interactions of R1 with the amino acid side chains of the protein appear crucial in modulating activity.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13766</offset><text>Inhibition of vRNP activity or virus replication in cells</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13824</offset><text>To determine the anti-influenza virus activity of compounds 1–28 in cell culture, we performed an influenza vRNP reconstitution assay in human embryonic kidney 293 T (HEK293T) cells, then subjected the active compounds (i.e. EC50 &lt; 100 μM) to a virus yield assay in influenza virus-infected Madin-Darby canine kidney (MDCK) cells (Table 1 and Fig. 3). For some N-acylhydrazone compounds, we observed quite potent and selective activity in the vRNP reconstitution assay. This indicates that they are able to inhibit viral RNA synthesis and suggests that they could be classified as original PA inhibitors. Values for EC50 (vRNP) or EC90 (virus yield) in the range of 0.4–18 μM were obtained for compounds 15 and 20–23, which all carry a 3,4,5-trihydroxyphenyl as R2, and possess either two (15) or three (20–23) hydroxyl substituents in the R1 moiety. As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14990</offset><text>The hydrazide 28 displayed weak (virus yield) to moderate (vRNP reconstitution) activity, albeit less than the most active molecules in the 3,4,5-trihydroxyphenyl series (i.e. 18 and 21–23). Even if there are no data indicating that the compounds reported in the paper are subject to hydrolysis, the activity of 28 could raise the concern that for some N-acylhydrazones the antiviral activity in cell culture may be related to their intracellular hydrolysis. However, this is unlikely, since the antiviral potency showed large differences (i.e. EC50 values between 0.42 and 29 μM) for compounds with the same R2 but different R1 groups, meaning that R1 does play a role in modulating the antiviral effect.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15701</offset><text>Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8. Two notable exceptions are 18 and 19 (containing a 2,3-dihydroxybenzylidene or 2-hydroxy-3-methoxybenzylidene R1, respectively) which were not cytotoxic at 200 μM and displayed favorable antiviral selectivity.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16192</offset><text>Some N-acylhydrazone compounds were devoid of activity in the enzymatic assay, yet showed good to moderate efficacy in cell culture (e.g. 14 and 19, having EC50 values of 2.2 and 7.1 μM, respectively). For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay. On the other hand, this difference was 8-fold or more for 7, 10, 14, 22, 25 and 28. Some N-acylhydrazone compounds showed good to moderate efficacy in the vRNP assay (e.g. 14 and 19, having EC50 values of 2.3 and 5.7 μM, respectively), yet were devoid of activity in the enzymatic assay. This observation suggests that they may inhibit the viral polymerase in an endonuclease-independent manner. To achieve a clear insight into the antiviral profile of the N-acylhydrazones, specific mechanistic experiments are currently ongoing in our laboratory, in which we are analyzing in full depth their effects on virus entry, polymerase-dependent RNA synthesis or the late stage (maturation and release) of the virus replication cycle.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>17388</offset><text>Docking studies</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17404</offset><text>In order to explore the possible binding mode of the synthesized compounds, docking simulations by GOLD program were performed by using the structural coordinates (PDB code 4AWM) for the PA-Nter endonuclease in complex with EGCG. Considering that the position of the side-chains of some residues changes depending on which pocket the ligand is occupying, we superimposed some X-ray structures of complexes between PA-Nter endonuclease and known active ligands. It was observed that the side-chain of amino acid Tyr24 shows greater movement than the other residues and for this reason we considered it as a flexible residue during the docking procedure.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18057</offset><text>First, test docking calculations, using EGCG, L-742,001 and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one (Fig. 1), were carried out to compare experimental and predicted binding modes and validate docking procedure. Their best docking poses agreed well with the experimental binding modes (rmsd values of 0.8, 1.2 and 0.7, respectively).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18410</offset><text>Next, docking of several N-acylhydrazones was performed and this generated a number of possible binding conformations, highlighting that the active site cavity of the PA endonuclease is quite spacious, as already demonstrated by crystallographic studies, and confirming the ability of this scaffold to chelate the two M2+ ions in different ways (Mode A-C in Fig. 4).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18777</offset><text>Figure 5 displays the first (panel A) and second (panel B) GOLD cluster docked solutions for compound 23. These two complex structures represent the largest clusters with similar fitness values (59.20 and 58.65, respectively). In both cases, 23 appears able to coordinate the two M2+ ions in the active site through the three contiguous OH groups (Fig. 5). In addition, 23 was predicted to form two hydrogen bonding interactions, i.e. with the catalytic Lys134 on the one side and Glu26 on the other side. Furthermore, in these two different binding modes, 23 forms π–π interactions with the aromatic ring of Tyr24, in a fashion similar to that described for other endonuclease inhibitors, i.e. EGCG and L-742,001.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19496</offset><text>The best docked conformation for compound 15 (Fig. 6, fitness value 68.56), which has an activity slightly lower than 23, reveals a different role for the gallic moiety. The ligand seems to form two hydrogen bonding interactions with Tyr130 as well as a cation–π interaction with Lys134. Tyr130 lies in a pocket that also contains Arg124, a residue that was proposed to have a crucial role in binding of the RNA substrate. Compound 15 appears further stabilized by hydrogen bonding interactions between two hydroxyl groups and Arg82 and Asp108. In this case, chelation of the two M2+ ions is carried out by involving the imine group (mode A in Fig. 4).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20152</offset><text>It is important to highlight that compounds 23 and 15, although in different ways, both are able to chelate the metal cofactors and to establish interactions with highly conserved aminoacids (Tyr24, Glu26, Arg124, Tyr130 and Lys134) that are very important for both endonuclease activity and transcription in vitro. The crucial role of such interactions is underlined by the differences in activity between 15 (IC50 = 9.0 μM) and 19 (&gt;500 μM): their coordinating features are similar, since both coordinate to the divalent metal ion through the phenolic oxygen, the iminic nitrogen and the carbonylic oxygen (mode A in Fig. 4), but the biological activity could be related to their different ability to engage interactions with the protein environment.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>20915</offset><text>Crystallographic Studies</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20940</offset><text>Attempts were made to co-crystallize PA-Nter with 15, 20, 21 and 23 in one to four molar excess. While crystals appeared and diffracted well, upon data processing, no or very little electron density for the inhibitors was observed. Attempts to soak apo crystals in crystallization solution containing 5 mM inhibitor overnight also did not result in substantial electron density for the inhibitor. As a last resort, dry powder of the inhibitor was sprinkled over the crystallization drop containing apo crystals and left over night. This experiment was successful for compound 23, the crystals diffracted to 2.15 Å and diffraction data were collected (PDB ID 5EGA). The refined structure shows unambiguous electron density for the inhibitor (Table S1 and Fig. 7). The complex structure confirms one of the two binding modes predicted by the docking simulations (Fig. 5, panel B). The galloyl moiety chelates the manganese ions, while the trihydroxyphenyl group stacks against the Tyr24 side chain. It is interesting to note that two of these hydroxyl groups are in position to form hydrogen bonds with the side chain of Glu26 and Lys34 (Fig. 7). These interactions suggest that other functional groups, e.g. halogens, could be used in place of the hydroxyl groups for better interactions with Glu26 and Lys34 side chains, and the inhibitory potency of these compounds could be further improved.</text></passage><passage><infon key="section_type">CONCL</infon><infon key="type">title_1</infon><offset>22335</offset><text>Conclusions</text></passage><passage><infon key="section_type">CONCL</infon><infon key="type">paragraph</infon><offset>22347</offset><text>The development of new agents for the treatment of influenza infection that exert their action by inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is a strategy that recently is gaining a lot of interest. The results here presented add the N-acylhydrazone scaffold to the library of the chelating molecules with potent antiviral activity (EC90 &lt; 5 μM, virus yield assay in influenza virus-infected MDCK cells). The structure of the N-acylhydrazone 23 co-crystallized with PA-Nter is important not only because confirms that the polyhydroxypheyl group efficiently coordinates two metal ions in the active site of the enzyme, but also because highlights the importance of the (flexible) inhibitor backbone in order to engage effective interactions with crucial aminoacids of the protein. Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase could represent another example, after carbonic anhydrase, histone deacetylase, and HIV-1 integrase, of metal binding as a successful strategy in drug design.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>23412</offset><text>Experimental Section</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>23433</offset><text>Materials and methods. Chemistry</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>23466</offset><text>All reagents of commercial quality were purchased from Sigma-Aldrich and used without further purification. The purity of the compounds was determined by elemental analysis and verified to be ≥95% for all synthesized molecules. NMR spectra were recorded at 25 °C on a Bruker Avance 400 FT spectrophotometer. The attenuate total reflectance IR spectra were recorded by means of a Nicolet-Nexus (Thermo Fisher) spectrophotometer by using a diamond crystal plate in the range of 4000–400 cm−1. Elemental analyses were performed by using a FlashEA 1112 series CHNS/O analyzer (Thermo Fisher) with gas-chromatographic separation. Electrospray mass spectral analyses (ESI-MS) were performed with an electrospray ionization (ESI) time-of-flight Micromass 4LCZ spectrometer. MS spectra were acquired in positive EI mode by means of a direct exposure probe mounting on the tip of a Re-filament with a DSQII Thermo Fisher apparatus, equipped with a single quadrupole analyzer. UV–Vis spectra were recorded on an Evolution 260 Bio Thermo spectrophotometer by using cells of 1 cm path length. UV-vis absorption spectra of 19 and 23 were registered using a ca. 10−5 M solution in methanol. Each metal/ligand system was studied by titrating a 2.8 ml sample of the ligand solution with a methanolic solution of Mg(CH3COO)2; 8–12 spectra of samples with M:L molar ratio ranging from 0 to 6 were measured.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24870</offset><text>Synthesis of the ligands (general procedure)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24915</offset><text>All the N-acylhydrazones were prepared in a manner similar to reported procedures. Briefly, to a solution of the aldehyde in absolute ethanol or toluene, an equimolar amount of the hydrazide dissolved in the same solvent was added. The mixture was refluxed for 6 hours, cooled at room temperature and concentrated in vacuum. The resulting precipitate was filtered off, washed with cold ethanol and dried in vacuum.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>25330</offset><text>3,4,5-trihydroxybenzohydrazide (28) and 3,4,5-trimethoxybenzohydrazide (29) were obtained by reaction of the corresponding methyl esters with hydrazine monohydrate. Hydrazine was added to an ethanol suspension of the ester and stirred at room temperature until the solute completely dissolved. Reaction mixture was then refluxed overnight. On concentrating the solution, a precipitate was observed, which was filtered and washed with cold ethanol. Chemical characterization of 1–29 and of Mg(HL)2 4H2O is collected in the Supplementary Information.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>25881</offset><text>Computational Studies</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>25903</offset><text>The crystal structure of PA-Nter endonuclease in complex with EGCG was retrieved from the RCSB Protein Data Bank (entry code 4AWM). The ligand and water molecules were discarded and the hydrogens were added to the protein by Discovery Studio 2.5. The charge on the metal ions was set as +2. EGCG, L-742,001, and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one structures were extracted from their X-ray complexes (PDB IDs 4AWM, 4W9S and 4E5H respectively). The other ligand structures were constructed using Discovery Studio 2.5.5 (Accelrys, Discovery Studio) and energy minimized using the Smart Minimizer protocol (1000 steps) which combines the Steepest Descent and the Conjugate Gradient methods.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26618</offset><text>The minimized ligands were docked in their corresponding proteins by means of GOLD Suite 5.0.1. The region of interest used by the GOLD program was defined in order to contain the residues within 15 Å from the original position of the ligand in the X-ray structure. The side-chain of residue Tyr24 was allowed to rotate according to the internal rotamer libraries in GOLD Suite 5.0.1. GoldScore was chosen as fitness function. The standard default settings were used in all calculations and the ligands were submitted to 100 genetic algorithm runs. The “allow early termination” command was deactivated. Results differing by less than 0.75 Å in ligand-all atom rmsd, were clustered together. The best GOLD-calculated conformation was used both for analysis and representation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>27401</offset><text>Plasmid-based endonuclease assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27434</offset><text>This enzymatic assay was performed according to a previously published method. One microgram of recombinant PA-Nter (residues 1–217 from the PA protein of influenza virus strain A/X-31) was incubated with 1 μg (16.7 nM) of single-stranded circular DNA plasmid M13mp18 (Bayou Biolabs, Metairie, Louisiana) in the presence of the test compounds and at a final volume of 25 μL. The assay buffer contained 50 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM β-mercaptoethanol and 1 mM MnCl2. The reaction was incubated at 37 °C for 2 h and then stopped by heat inactivation (80 °C, 20 min), followed by visualization of the endonucleolytic digestion of the plasmid by gel electrophoresis on a 1% agarose gel with ethidium bromide staining. The amount of remaining intact plasmid was quantified by ImageQuant TL software (GE Healthcare, Diegem, Belgium). The percentage inhibition of PA endonuclease activity was plotted against the compound concentration on a semi-logarithmic plot, using GraphPad Prism software (GraphPad Software, La Jolla, CA). The 50% inhibitory concentrations (IC50) were obtained by nonlinear least-squares regression analysis of the results from three independent experiments. 2,4-Dioxo-4-phenylbutanoic acid (DPBA; Interchim, Montluçon, France) was included as the reference compound.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>28740</offset><text>Cells and media</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28756</offset><text>MDCK cells (a kind gift from M. Matrosovich, Marburg, Germany) and HEK293T cells (purchased from Thermo Fisher Scientific, Waltham, MA) were cultivated in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, and 0.075% sodium bicarbonate. During virus experiments, the MDCK cells were maintained in MDCK infection medium, consisting of Ultra MDCK medium (Lonza, Basel, Switzerland) supplemented with 0.0225% sodium bicarbonate, 2 mM L-glutamine, and 2 μg/ml tosyl phenylalanyl chloromethyl ketone-treated trypsin (Sigma-Aldrich, St. Louis, MO). The cells were incubated in a humidified atmosphere containing 5% CO2.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>29417</offset><text>vRNP reconstitution assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>29443</offset><text>The procedure to determine the inhibitory effect of the compounds on influenza virus vRNPs reconstituted in HEK293T cells, is described in full detail elsewhere. Briefly, the four relevant plasmids (i.e. the expression plasmids for PB1, PB2, PA and NP) were combined with the fluc reporter plasmid, and co-transfected into HEK293T cells using Lipofectamin 2000 (Invitrogen, Life Technologies, Gent, Belgium). After incubation at 37 °C for 24 h in the presence of serial dilutions of the test compounds, the ONE-Glo luciferase assay system (Promega, Madison, WI) was used to determine luciferase activity. EC50 was defined as the compound concentration causing 50% reduction in the vRNP-driven firefly luciferase signal, as compared to cells receiving medium instead of compound. These EC50 values were calculated by interpolation assuming a semi-log dose-response effect using GraphPad Prism software. In parallel, compound cytotoxic activity was determined in untransfected HEK293T cells which had been incubated with serial dilutions of the compounds for 24 h, using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay; Promega). These spectrophotometric data were used to calculate the 50% cytotoxic concentration (CC50), i.e. the concentration reducing cell viability by 50%, as compared to wells receiving medium instead of compound. Ribavirin (Virazole; ICN Pharmaceuticals, Costa Mesa, CA) was included as the reference compound.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>31011</offset><text>Virus yield assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31029</offset><text>We previously published in full detail the virus yield assay to determine the anti-influenza virus activity in MDCK cell cultures. Briefly, one day prior to infection, MDCK cells were seeded into 96-well plates at 25,000 cells per well. At day 0, serial dilutions of the test compounds were added, immediately followed by infection with influenza A/PR/8/34 virus. After 24 h incubation at 35 °C, the virus amount in the supernatants was estimated by determining the viral genome copy number in a one-step quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay (CellsDirect One-Step qRT-PCR kit; Invitrogen), with influenza virus M1-specific primers and probe. The compound concentration values causing a 2-log10 (EC99) and a 1-log10 (EC90) reduction in viral RNA (vRNA) copy number at 24 h p.i., as compared to the virus control receiving no compound, were calculated by interpolation from data of at least three experiments. In parallel, the CC50 values after 24 h incubation with compounds were determined in uninfected MDCK cells, using the spectrophotometric MTS cell viability assay described above, respectively. Ribavirin was included as the reference compound.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32233</offset><text>Crystallographic analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32259</offset><text>A PAN construct (PANΔLoop) with a loop (residues 51–72) deleted and replaced with GGS from A/California/04/2009 H1N1 strain was used for the crystallographic studies. The details of cloning, over-expression and purification are described elsewhere. Briefly, the gene was cloned into pET52b vector and transformed into BL21 (DE3) cells, and the protein was expressed in LB medium overnight at 18 °C after induction at an OD600 ~0.8 with 0.2 mM isopropyl-β-thiogalactopyranoside (IPTG). The protein was purified from cell lysates by HisTrap affinity chromatography and the 10xHis tag was removed by digestion with thrombin. The protein was further purified by gel filtration using a Superdex 75 size-exclusion chromatography column in 20 mM Tris pH 8.0, 150 mM NaCl and 1 mM TCEP. The protein was concentrated to 10–12 mg/ml for crystallization. Crystals were grown in 0.2 M MgCl2, 2 mM MnCl2, 0.1 M Tris pH 8.5, 30% (w/v) PEG 4000 using the hanging drop method. For determination of the protein-inhibitor complex structure, the powder of the inhibitor was sprinkled on a 2 μl drop of a 1:1 ratio mixture of protein solution and well solution, on a cover slide hanging over 500 μl well solution, and left overnight. Next day, the crystals were cryo-protected using well solution supplemented with 25% ethylene glycol and flash frozen in liquid nitrogen. The data were collected at the 22-ID beam line maintained by Southeast Regional Collaborative Access Team (SERCAT) at the Advanced Photon Source, Argonne National Laboratory. The data were indexed, integrated and scaled using the HKL2000 suite of programs. Phase determination, structure refinement and model building were completed using Phaser, Refmac and Coot (part of the CCP4 package). The apo structure of PANΔLoop (PDB ID: 5DES) was used as starting model for molecular replacement. The details of the data collection and refinement statistics are given in Table S1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>34198</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34221</offset><text>How to cite this article: Carcelli, M. et al. N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes. Sci. Rep. 6, 31500; doi: 10.1038/srep31500 (2016).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>34416</offset><text>Supplementary Material</text></passage><passage><infon key="fpage">1487</infon><infon key="lpage">1531</infon><infon key="name_0">surname:Lamb;given-names:R. A.</infon><infon key="name_1">surname:Krug;given-names:R. M.</infon><infon key="name_2">surname:Knipe;given-names:D. M.</infon><infon key="name_3">surname:Howley;given-names:P. M.</infon><infon key="name_4">surname:Griffin;given-names:D. E.</infon><infon key="name_5">surname:Lamb;given-names:R. A.</infon><infon key="name_6">surname:Martin;given-names:M. A.</infon><infon key="name_7">surname:Roizman;given-names:B.</infon><infon key="name_8">surname:Straus;given-names:S. 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M.</infon><infon key="section_type">REF</infon><infon key="source">In Methods in Enzymology, Volume 276: Macromolecular Crystallography, part A</infon><infon key="type">ref</infon><infon key="year">1997</infon><offset>39752</offset><text>Processing of X-ray diffraction data collected in oscillation mode</text></passage><passage><infon key="fpage">235</infon><infon key="lpage">42</infon><infon key="name_0">surname:Winn;given-names:M. D.</infon><infon key="pub-id_pmid">21460441</infon><infon key="section_type">REF</infon><infon key="source">Acta Crystallogr. D Biol Crystallogr</infon><infon key="type">ref</infon><infon key="volume">67</infon><infon key="year">2011</infon><offset>39819</offset><text>Overview of the CCP4 suite and current developments</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>39871</offset><text>Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. M.C., D.R., A.G. and M.S. drug design and chemical synthesis; L.D.L. docking studies; G.K. and S.W.W. crystallographic studies; A.S. and L.N. biological studies.</text></passage><passage><infon key="file">srep31500-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>40191</offset><text>Chemical structures of some prototype inhibitors of influenza virus endonuclease.</text></passage><passage><infon key="file">srep31500-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>40273</offset><text>Inhibitor activity in enzymatic assays (IC50, μM) as reported in: aref., bref., cref., dref..</text></passage><passage><infon key="file">srep31500-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>40370</offset><text>General synthesis for N-acylhydrazones 1–27 and hydrazides 28 and 29 (A). Chemical structures of compounds 1–27 (B).</text></passage><passage><infon key="file">srep31500-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>40491</offset><text>Overview of the structure-activity relationship for compounds 1–27.</text></passage><passage><infon key="file">srep31500-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>40561</offset><text>Scheme of possible binding modes of the studied N-acylhydrazones.</text></passage><passage><infon key="file">srep31500-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>40627</offset><text>First (A) and second (B) GOLD cluster docked solutions of compound 23 (orange and cyan, respectively) in complex with PA endonuclease. Key residues of the pocket are presented using PyMOL [ http://www.pymol.org] and LIGPLUS [Laskowski, R. A.; Swindells, M. B. Journal of chemical information and modeling
+2011,
+51, 2778]. Hydrogen bonds are illustrated by dotted lines, while the divalent metal ions are shown as purple spheres. Schematic drawings of the interactions of the first (C) and second (D) GOLD cluster docked solutions generated using LIGPLUS. Dashed lines are hydrogen bonds and ‘eyelashes’ show residues involved in hydrophobic interactions.</text></passage><passage><infon key="file">srep31500-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>41286</offset><text>(A) Binding mode of compound 15 (orange) in complex with PA endonuclease. Key residues of the pocket are presented using PyMOL [ http://www.pymol.org] and LIGPLUS [Laskowski, R. A.; Swindells, M. B. Journal of chemical information and modeling
+2011,
+51, 2778]. Hydrogen bonds are illustrated by dotted lines while the divalent metal ions are shown as purple spheres. (B) Schematic drawing of the interactions of compound 15 generated using LIGPLUS. Dashed lines are hydrogen bonds and ‘eyelashes’ show residues involved in hydrophobic interactions.</text></passage><passage><infon key="file">srep31500-f7.jpg</infon><infon key="id">f7</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>41839</offset><text>Crystal structure of PANΔLoop in complex with compound 23.</text></passage><passage><infon key="file">srep31500-f7.jpg</infon><infon key="id">f7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>41902</offset><text>Active site residues are shown in sticks with green carbons, manganese atoms are shown as purple spheres and water molecules as red spheres. Compound 23 is shown in sticks with yellow carbons. Protein secondary structure is shown as ribbons in salmon color. 2Fo-Fc electron density map contoured at 1σ is shown as blue mesh. Hydrogen bonds and metal coordination are shown with dotted lines. The H-bond distances from the side chain carboxyl group of Glu26 to p-OH and m-OH of the trihydroxyphenyl group of the inhibitor are 2.7 Å and 3.0 Å, respectively. The H-bond distance from the side chain of Lys34 to p-OH of the trihydroxyphenyl group is 3.6 Å. The H-bond distance to the water molecule from m-OH of the galloyl moiety is 3.0 Å, which in turn is H-bonded to the side chain of Tyr130 with a distance of 2.7 Å. Crystal structure has been deposited in the RCSB Protein Data Bank with PDB ID: 5EGA.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>42811</offset><text>Inhibitory activity of the N-acylhydrazones 1–27 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot; border=&quot;1&quot;&gt;&lt;colgroup&gt;&lt;col align=&quot;left&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;/colgroup&gt;&lt;thead valign=&quot;bottom&quot;&gt;&lt;tr&gt;&lt;th rowspan=&quot;3&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Compound&lt;/th&gt;&lt;th rowspan=&quot;2&quot; align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Enzyme assay with PA-Nter&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn1&quot;&gt;a&lt;/xref&gt;&lt;/th&gt;&lt;th colspan=&quot;4&quot; align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Virus yield assay in influenza virus-infected MDCK cells&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn2&quot;&gt;b&lt;/xref&gt;&lt;/th&gt;&lt;th colspan=&quot;2&quot; align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;vRNP reconstitution assay in HEK293T cells&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn3&quot;&gt;c&lt;/xref&gt;&lt;/th&gt;&lt;/tr&gt;&lt;tr&gt;&lt;th colspan=&quot;2&quot; align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Antiviral activity&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Cytotoxicity&lt;/th&gt;&lt;th rowspan=&quot;2&quot; align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;SI&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn4&quot;&gt;d&lt;/xref&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Activity&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Cytotoxicity&lt;/th&gt;&lt;/tr&gt;&lt;tr&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;IC&lt;sub&gt;50&lt;/sub&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;EC&lt;sub&gt;99&lt;/sub&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;EC&lt;sub&gt;90&lt;/sub&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;CC&lt;sub&gt;50&lt;/sub&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;EC&lt;sub&gt;50&lt;/sub&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;CC&lt;sub&gt;50&lt;/sub&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(1)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;24&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn6&quot;&gt;f&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;107&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(2)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;500&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;100&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(3)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;500&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;5.9&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;48&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(4)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;500&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;6.3&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;33&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(5)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;67&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;25&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;25&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;≥146&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2.6&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;10&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(6)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;500&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;50&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;50&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;15&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;14&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(7)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;54&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;172&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;100&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;2.0&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3.2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;8.9&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(8)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;500&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;12.5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;12.5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.9&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;15&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(9)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;34&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;16&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;5.3&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;38&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;5.5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(10)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;68&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;14&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;8.5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;111&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;13&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.40&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;132&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(11)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;45&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;30&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;12&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;17&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;5.6&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(12)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;500&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;12.5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;12.5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;20&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;39&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(13)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;69&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;71&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;34&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;5.9&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;6.3&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(14)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;500&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;63&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;37&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;5.4&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2.3&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(15)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;8.9&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;18&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;7.5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;≥172&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;≥23&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;14&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(16)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;454&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;67&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;28&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;7.1&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;5.2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(17)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;482&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;21&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;8.1&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;25&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;7.1&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(18)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;83&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;6.2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2.2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;91&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3.3&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(19)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;500&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;53&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;26&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;7.7&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;5.7&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(20)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;18&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;35&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;11&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;18&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2.2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(21)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;13&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;8.3&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3.6&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;56&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2.5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(22)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;75&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;7.4&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3.4&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;59&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.42&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(23)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;8.7&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;11&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3.5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;57&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3.1&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(24)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;131&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;58&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;26&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;7.7&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;25&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(25)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;40&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;132&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;70&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;2.9&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;4.1&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(26)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;30&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;36&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;13&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;15&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;5.5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(27)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;36&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;21&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;(28)&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;40&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;158&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;85&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;2.4&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;7.2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;DPBA&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn5&quot;&gt;e&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;5.3&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Ribavirin&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;13&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;8.5&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;24&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;9.4&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&amp;gt;200&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>42984</offset><text>Compound	Enzyme assay with PA-Ntera	Virus yield assay in influenza virus-infected MDCK cellsb	vRNP reconstitution assay in HEK293T cellsc	 	Antiviral activity	Cytotoxicity	SId	Activity	Cytotoxicity	 	IC50	EC99	EC90	CC50	EC50	CC50	 	(1)	24	NDf	ND	ND	 	107	&gt;200	 	(2)	&gt;500	ND	ND	ND	 	&gt;100	&gt;200	 	(3)	&gt;500	ND	ND	&gt;200	 	5.9	48	 	(4)	&gt;500	ND	ND	&gt;200	 	6.3	33	 	(5)	67	&gt;25	&gt;25	≥146	 	2.6	10	 	(6)	&gt;500	&gt;50	&gt;50	&gt;200	 	15	14	 	(7)	54	172	100	&gt;200	&gt;2.0	3.2	8.9	 	(8)	&gt;500	&gt;12.5	&gt;12.5	&gt;200	 	1.9	15	 	(9)	34	16	5.3	&gt;200	&gt;38	5.5	&gt;200	 	(10)	68	14	8.5	111	&gt;13	0.40	132	 	(11)	45	30	12	&gt;200	&gt;17	5.6	&gt;200	 	(12)	&gt;500	&gt;12.5	&gt;12.5	&gt;200	 	20	39	 	(13)	69	71	34	&gt;200	&gt;5.9	6.3	&gt;200	 	(14)	&gt;500	63	37	&gt;200	&gt;5.4	2.3	&gt;200	 	(15)	8.9	18	7.5	≥172	≥23	14	&gt;200	 	(16)	454	67	28	&gt;200	&gt;7.1	5.2	&gt;200	 	(17)	482	21	8.1	&gt;200	&gt;25	7.1	&gt;200	 	(18)	83	6.2	2.2	&gt;200	&gt;91	3.3	&gt;200	 	(19)	&gt;500	53	26	&gt;200	&gt;7.7	5.7	&gt;200	 	(20)	18	35	11	&gt;200	&gt;18	2.2	&gt;200	 	(21)	13	8.3	3.6	&gt;200	&gt;56	2.5	&gt;200	 	(22)	75	7.4	3.4	&gt;200	&gt;59	0.42	&gt;200	 	(23)	8.7	11	3.5	&gt;200	&gt;57	3.1	&gt;200	 	(24)	131	58	26	&gt;200	&gt;7.7	25	&gt;200	 	(25)	40	132	70	&gt;200	&gt;2.9	4.1	&gt;200	 	(26)	30	36	13	&gt;200	&gt;15	5.5	&gt;200	 	(27)	36	ND	ND	ND	 	21	&gt;200	 	(28)	40	158	85	&gt;200	&gt;2.4	7.2	&gt;200	 	DPBAe	5.3	ND	ND	ND	 	ND	ND	 	Ribavirin	ND	13	8.5	&gt;200	&gt;24	9.4	&gt;200	 	</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>44265</offset><text>aRecombinant PA-Nter was incubated with the ssDNA plasmid substrate, a Mn2+-containing buffer and test compounds. Cleavage of the substrate was assessed after 2 hr incubation. The IC50 represents the compound concentration (in μM) required to obtain 50% inhibition of cleavage, calculated by nonlinear least-squares regression analysis (using GraphPad Prism software) of the results from 2–4 independent experiments.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>44687</offset><text>bMDCK cells were infected with influenza A virus (strain A/PR/8/34) and incubated with the compounds during 24 h. The virus yield in the supernatant was assessed by real-time qPCR. The EC99 and EC90 values represent the compound concentrations (in μM) producing a 2-log10 or 1-log10 reduction in virus titer, respectively, determined in 2–3 independent experiments. The cytotoxicity, assessed in uninfected MDCK cells, was expressed as the CC50 value (50% cytotoxic concentration, determined with the MTS cell viability assay, in μM).</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>45226</offset><text>cHEK293T cells were co-transfected with the four vRNP-reconstituting plasmids and the luciferase reporter plasmid in the presence of the test compounds. The EC50 represents the compound concentration (in μM) producing 50% reduction in vRNP-driven firefly reporter signal, estimated at 24 h after transfection. The EC50 value was derived from data from 2–4 independent experiments, by nonlinear least-squares regression analysis (using GraphPad Prism software). The CC50 (in μM), i.e. the 50% cytotoxic concentration, was determined in untransfected HEK293T cells by MTS cell viability assay.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>45824</offset><text>dSI, selectivity index, defined as the ratio between the CC50 and EC90.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>45896</offset><text>eDPBA, 2,4-dioxo-4-phenylbutanoic acid.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>45936</offset><text>fND, not determined.</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201217</date><key>pmc.key</key><document><id>4981400</id><infon key="license">CC BY</infon><passage><infon key="alt-title">Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA</infon><infon key="article-id_doi">10.1371/journal.pone.0160694</infon><infon key="article-id_pmc">4981400</infon><infon key="article-id_pmid">27513867</infon><infon key="article-id_publisher-id">PONE-D-16-20928</infon><infon key="elocation-id">e0160694</infon><infon key="issue">8</infon><infon key="license">This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</infon><infon key="name_0">surname:Zhang;given-names:Yinglu</infon><infon key="name_1">surname:Rataj;given-names:Katarzyna</infon><infon key="name_2">surname:Simpson;given-names:Gordon G.</infon><infon key="name_3">surname:Tong;given-names:Liang</infon><infon key="name_4">surname:Candela;given-names:Hector</infon><infon key="name_5">surname:Tong;given-names:Liang</infon><infon key="name_6">surname:Tong;given-names:Liang</infon><infon key="name_7">surname:Simpson;given-names:Gordon G.</infon><infon key="name_8">surname:Simpson;given-names:Gordon G.</infon><infon key="notes">All relevant data are within the paper and its Supporting Information files.</infon><infon key="section_type">TITLE</infon><infon key="title">Data Availability</infon><infon key="type">front</infon><infon key="volume">11</infon><infon key="year">2016</infon><offset>0</offset><text>Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>80</offset><text>The Arabidopsis protein FPA controls flowering time by regulating the alternative 3′-end processing of the FLOWERING LOCUS (FLC) antisense RNA. FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain. The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower. We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution. The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them. Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs. Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1277</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1290</offset><text>Eukaryotic messenger RNAs (mRNAs) are made as precursors through transcription by RNA polymerase II (Pol II), and these primary transcripts undergo extensive processing, including 3′-end cleavage and polyadenylation. In addition, alternative 3′-end cleavage and polyadenylation is an essential and ubiquitous process in eukaryotes. Misregulation of (alternative) 3′-end processing can lead to various genetic defects, cancer and other diseases. There is currently great interest in understanding the molecular mechanisms and functional impacts of alternative 3′-end processing.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1876</offset><text>Recently, the split ends (SPEN) family of proteins was identified as RNA binding proteins that regulate alternative 3′-end cleavage and polyadenylation. They are characterized by possessing N-terminal RNA recognition motifs (RRMs) and a conserved SPEN paralog and ortholog C-terminal (SPOC) domain (Fig 1A). The SPOC domain is believed to mediate protein-protein interactions and has diverse functions among SPEN family proteins, but the molecular mechanism of these functions is not well understood.</text></passage><passage><infon key="file">pone.0160694.g001.jpg</infon><infon key="id">pone.0160694.g001</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>2379</offset><text>Sequence conservation of SPOC domains.</text></passage><passage><infon key="file">pone.0160694.g001.jpg</infon><infon key="id">pone.0160694.g001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>2418</offset><text>(A). Domain organization of A. thaliana FPA. (B). Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP. Residues in surface patch 1 are indicated with the orange dots, and those in surface patch 2 with the green dots. The secondary structure elements in the structure of FPA SPOC are labeled. Residues that are strictly conserved among the five proteins are shown in white with a red background, and those that are mostly conserved in red.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2931</offset><text>FPA, a SPEN family protein in Arabidopsis thaliana and other plants, was found to regulate the 3′-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene. FPA promotes the 3′-end processing of class I FLC antisense RNAs, which includes the proximal polyadenylation site. This is associated with histone demethylase activity and down-regulation of FLC transcription. However, the functional mechanism of this complex is still not clear.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3435</offset><text>Although a SPOC domain is found in all the SPEN family proteins, its sequence conservation is rather low. For example, the sequence identity between the SPOC domains of A. thaliana FPA and human SMRT/HDAC1 Associated Repressor Protein (SHARP) is only 19% (Fig 1B). Currently, the SHARP SPOC domain is the only one with structural information.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3778</offset><text>As a first step toward understanding the molecular basis for the regulation of alternative 3′-end processing and flowering by FPA, we have determined the crystal structure of the SPOC domain of A. thaliana FPA at 2.7 Å resolution. The overall structure is similar to that of the SHARP SPOC domain, although there are also substantial conformational differences between them. The structure reveals a surface patch that is conserved among FPA homologs.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4232</offset><text>Results and Discussion</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4255</offset><text>Structure of FPA SPOC domain</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4284</offset><text>The crystal structure of the SPOC domain of A. thaliana FPA has been determined at 2.7 Å resolution using the selenomethionyl single-wavelength anomalous dispersion method. The expression construct contained residues 433–565 of FPA, but only residues 439–460 and 465–565 are ordered in the crystal. The atomic model has good agreement with the X-ray diffraction data and the expected bond lengths, bond angles and other geometric parameters (Table 1). All the residues are located in the favored regions of the Ramachandran plot (data not shown). The structure has been deposited in the Protein Data Bank, with accession code 5KXF.</text></passage><passage><infon key="file">pone.0160694.t001.xml</infon><infon key="id">pone.0160694.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>4923</offset><text>Summary of crystallographic information.</text></passage><passage><infon key="file">pone.0160694.t001.xml</infon><infon key="id">pone.0160694.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;colgroup span=&quot;1&quot;&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;col align=&quot;left&quot; valign=&quot;middle&quot; span=&quot;1&quot;/&gt;&lt;/colgroup&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution range (Å)&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;t001fn001&quot;&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50–2.7 (2.8–2.7)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Number of observations&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;78,008&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;merge&lt;/sub&gt; (%)&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;10.5 (45.3)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;I/σI&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;24.1 (6.3)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Redundancy&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Completeness (%)&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100 (100)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;R&lt;/italic&gt; factor (%)&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;19.2 (25.0)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Free &lt;italic&gt;R&lt;/italic&gt; factor (%)&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;25.4 (35.4)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Rms deviation in bond lengths (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.017&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Rms deviation in bond angles (°)&lt;/td&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.9&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>4964</offset><text>Resolution range (Å)1	50–2.7 (2.8–2.7)	 	Number of observations	78,008	 	Rmerge (%)	10.5 (45.3)	 	I/σI	24.1 (6.3)	 	Redundancy		 	Completeness (%)	100 (100)	 	R factor (%)	19.2 (25.0)	 	Free R factor (%)	25.4 (35.4)	 	Rms deviation in bond lengths (Å)	0.017	 	Rms deviation in bond angles (°)	1.9	 	</text></passage><passage><infon key="file">pone.0160694.t001.xml</infon><infon key="id">pone.0160694.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>5272</offset><text>1The numbers in parentheses are for the highest resolution shell.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5338</offset><text>The crystal structure of the FPA SPOC domain contains a seven-stranded, mostly anti-parallel β-barrel (β1-β7) and three helices (αA-αC) (Fig 2A). Only two of the neighboring strands, β1 and β3, are parallel to each other. Helix αB covers one end of the barrel, while helices αA and αC are located next to each other at one side of the barrel (Fig 2B). The other end of the β-barrel is covered by the loop connecting strands β2 and β3, which contains the disordered 461–464 segment. The center of the barrel is filled with hydrophobic side chains and is not accessible to the solvent.</text></passage><passage><infon key="file">pone.0160694.g002.jpg</infon><infon key="id">pone.0160694.g002</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>5966</offset><text>Crystal structure of the SPOC domain of A. thaliana FPA.</text></passage><passage><infon key="file">pone.0160694.g002.jpg</infon><infon key="id">pone.0160694.g002</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>6023</offset><text>(A). Schematic drawing of the structure of FPA SPOC domain, colored from blue at the N terminus to red at the C terminus. The view is from the side of the β-barrel. The disordered segment (residues 460–465) is indicated with the dotted line. (B). Structure of the FPA SPOC domain, viewed from the end of the β-barrel, after 90° rotation around the horizontal axis from panel A. All structure figures were produced with PyMOL (www.pymol.org).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>6471</offset><text>Comparisons to structural homologs of the SPOC domain</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6525</offset><text>Only five structural homologs of the FPA SPOC domain were found in the Protein Data Bank with the DaliLite server, suggesting that the SPOC domain structure is relatively unique. The top hit is the SPOC domain of human SHARP (Fig 3A), with a Z score of 12.3. The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D). The next structural homolog has a Z score of 3.0.</text></passage><passage><infon key="file">pone.0160694.g003.jpg</infon><infon key="id">pone.0160694.g003</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>7135</offset><text>Structural homologs of the FPA SPOC domain.</text></passage><passage><infon key="file">pone.0160694.g003.jpg</infon><infon key="id">pone.0160694.g003</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>7179</offset><text>(A). Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray). The bound position of a doubly-phosphorylated peptide from SMRT is shown in magenta. (B). Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 β-barrel domain (gray). Ku80 contains a homologous domain (green), which forms a hetero-dimer with that in Ku70. The two domains, and inserted segments on them, mediate the binding of dsDNA (orange). The red rectangle highlights the region of contact between the two β-barrel domains. (C). Overlay of the structures of the FPA SPOC domain (cyan) and the homologous domain in Chp1 (gray). The binding partner of Chp1, Tas3, is shown in green. The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (D). Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8127</offset><text>SHARP is a transcriptional co-repressor in the nuclear receptor and Notch/RBP-Jκ signaling pathways. The SPOC domain of SHARP interacts directly with silencing mediator for retinoid and thyroid receptor (SMRT), nuclear receptor co-repressor (N-CoR), HDAC, and other components to represses transcription. While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the β-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A). In addition, the SHARP SPOC domain has three extra helices. One of them covers the other end of the β-barrel, and the other two shield an additional surface of the side of the β-barrel from solvent. A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands β1 and β3 (Fig 3A). Such a binding mode probably would not be possible in FPA, as the peptide would clash with the β1-β2 loop.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9139</offset><text>The Ku70-Ku80 hetero-dimer is involved in DNA double-strand break repair and the β-barrel domain contributes to DNA binding. In fact, the β-barrel domains of Ku70 and Ku80 form a hetero-dimer, primarily through interactions between the loops connecting the third and fourth strands of the barrel (Fig 3B). The open ends of the two β-barrels face the DNA binding sites, and contact the phosphodiester backbone of the dsDNA. In addition, a long insert connecting strands β2 and β3 in the two domains form an arch-like structure, encircling the dsDNA.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9703</offset><text>Chp1 is a subunit of the RNA-induced initiation of transcriptional gene silencing (RITS) complex. The partner of Chp1, Tas3, is bound between the barrel domain and the second domain of Chp1, and the linker between the two domains is also crucial for this interaction (Fig 3C). It is probably unlikely that the β-barrel itself is sufficient to bind Tas3. Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10263</offset><text>Mediator is a coactivator complex that promotes transcription by Pol II. The Med25 subunit ACID is the target of the potent activator VP16 of the herpes simplex virus. The structure of ACID contains a helix at the C-terminus as well as an extended β1-β2 loop. Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands β1 and β3, that is used by the SHARP and Tas3 SPOC domains for binding their partners.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10725</offset><text>A conserved surface patch in the FPA SPOC domain</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10774</offset><text>An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A). This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B). The first surface patch is electropositive in nature (Fig 4C), and residues Arg477 and Tyr515 are also conserved in the SHARP SPOC domain (Fig 1B). In fact, one of the phosphorylated residues of the SMRT peptide interacts with this surface patch (Fig 3A), suggesting that the FPA SPOC domain might also interact with a phosphorylated segment here. In comparison, the second surface patch is more hydrophobic in nature (Fig 4C).</text></passage><passage><infon key="file">pone.0160694.g004.jpg</infon><infon key="id">pone.0160694.g004</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>11638</offset><text>A conserved surface patch of FPA SPOC domain.</text></passage><passage><infon key="file">pone.0160694.g004.jpg</infon><infon key="id">pone.0160694.g004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11684</offset><text>(A). Two views of the molecular surface of FPA SPOC domain colored based on sequence conservation among plant FPA homologs. Purple: most conserved; cyan: least conserved. (B). Residues in the conserved surface patch of FPA SPOC domain. The side chains of the residues are shown in stick models, colored orange in the first sub-patch and green in the second. (C). Molecular surface of FPA SPOC domain colored based on electrostatic potential. Blue: positively charged; red: negatively charged.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>12177</offset><text>Testing the requirement of specific conserved amino acids for FPA functions</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12253</offset><text>We next examined the potential impact of the conserved surface patch on FPA function in vivo. We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain (Fig 1B) and were found to be functionally important. The mutations were introduced into a transgene designed to express FPA from its native control elements (promoter, introns and 3′ UTR). The resulting transgenes were then stably transformed into an fpa-8 mutant background so that the impact of the mutations on FPA function could be assessed. Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig). We examined independent transgenic lines expressing each R477A and Y515A mutation. In each case, we confirmed that detectable levels of FPA protein expression were restored close to wild-type levels in protein blot analyses using antibodies that specifically recognize FPA (S2 Fig).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13409</offset><text>We then examined the impact of the surface patch mutations on FPA’s function in controlling RNA 3′-end formation by determining whether the mutant proteins functioned in FPA autoregulation and the repression of FLC expression. FPA autoregulates its expression by promoting cleavage and polyadenylation within intron 1 of its own pre-mRNA, resulting in a truncated transcript that does not encode functional protein. We used RNA gel blot analyses to reveal that in each of three independent transgenic lines for each single mutant, rescue of proximally polyadenylated FPA pre-mRNA can be detected (Fig 5A and 5B). We therefore conclude that neither of these mutations disrupted the ability of FPA to promote RNA 3′-end formation in its own transcript.</text></passage><passage><infon key="file">pone.0160694.g005.jpg</infon><infon key="id">pone.0160694.g005</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>14166</offset><text>Impact of individual FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA.</text></passage><passage><infon key="file">pone.0160694.g005.jpg</infon><infon key="id">pone.0160694.g005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14261</offset><text>RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A
+(A), or FPA::FPA Y515A
+(B) using poly(A)+ purified mRNAs. A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs. RNA size (kb) marker (Ambion). TUBULIN was detected as an internal control. Proximally and distally polyadenylated FPA transcripts are marked with arrows. The ratio of distal:proximal polyadenylated forms is given under each lane. (C,D) Impact of individual FPA SPOC domain mutations on FLC transcript levels. qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A
+(C), FPA::FPA Y515A
+(D) plants. Transcript levels were normalized to the control UBC. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15153</offset><text>We next examined whether the corresponding mutations disrupted the ability of FPA to control FLC expression. We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15573</offset><text>Since each surface patch mutation appeared to be insufficient to disrupt FPA functions on its own, we combined both mutations into the same transgene. We could again confirm that near wild-type levels of FPA protein were expressed from three independent transgenic lines expressing the FPA R477A;Y515A doubly mutated protein in an fpa-8 mutant background (S3 Fig). We found that FPA R477A;Y515A protein functioned like wild-type FPA to restore FPA pre-mRNA proximal polyadenylation (Fig 6A) and FLC expression to wild-type levels (Fig 6B).</text></passage><passage><infon key="file">pone.0160694.g006.jpg</infon><infon key="id">pone.0160694.g006</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>16113</offset><text>Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and FLC expression.</text></passage><passage><infon key="file">pone.0160694.g006.jpg</infon><infon key="id">pone.0160694.g006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16223</offset><text>(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs. Black arrows indicate the proximally and distally polyadenylated FPA mRNAs. A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs. RNA size (kb) marker (Ambion). TUBULIN was detected as an internal control. The ratio of distal:proximal polyadenylated forms is given under each lane. (B). qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, and FPA::FPA R477A;Y515A plants. Transcript levels were normalized to the control UBC. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16983</offset><text>Together our findings suggest that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation, or that this combination of mutations is not sufficient to critically disrupt the function of the FPA SPOC domain. Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>17516</offset><text>Materials and Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>17538</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17574</offset><text>The SPOC domain (residue 433–565) of A. thaliana FPA was sub-cloned into the pET28a vector (Novagen). The recombinant protein, with an N-terminal hexa-histidine tag, was over-expressed in E. coli BL21 Star (DE3) cells (Novagen), which were induced with 0.4 mM IPTG and allowed to grow at 20°C for 14–18 h. The soluble protein was purified by nickel-charged immobilized-metal affinity chromatography and gel filtration chromatography. The purified protein was concentrated and stored at –80°C in a buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 10 mM DTT and 5% (v/v) glycerol. The His-tag was not removed for crystallization.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>18213</offset><text>The selenomethionine labeled SPOC domain was expressed in E. coli B834(DE3) strain using LeMaster media and purified with the same protocol as the native protein.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>18376</offset><text>Protein crystallization</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>18400</offset><text>Crystals of the native SPOC domain of FPA were grown at 20°C with the sitting-drop vapor diffusion method. The protein solution was at 30 mg/ml concentration, and the reservoir solution contained 0.2 M MgSO4, and 20% (v/v) PEG 3350. Fully-grown crystals were obtained two days after set-up. Crystals of the selenomethionine labeled SPOC domain were grown using the same condition as the native protein. The crystals were cryo-protected in the crystallization solution supplemented with 20% (v/v) glycerol and flash-frozen in liquid nitrogen for data collection at 100K.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>18971</offset><text>Data collection and processing</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>19002</offset><text>A single-wavelength anomalous dispersion (SAD) X-ray diffraction data set on a selenomethionine labeled SPOC domain crystal was collected at the National Synchrotron Light Source (NSLS) beamline X29A using an ADSC Q315r CCD. The diffraction images were processed and scaled with the HKL package. The crystal belongs to space group P65, with unit cell parameters of a = b = 108.2 Å, and c = 34.2 Å.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>19402</offset><text>Structure determination and refinement</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>19441</offset><text>The structure of the SPOC domain was solved by the selenomethionyl SAD method with the program SHELX. The phases were used by program PHENIX for automatic model building. Manual model rebuilding was carried out with Coot. The structure refinement was performed with the program PHENIX, with translation, libration, and screw-rotation (TLS) parameters. The data processing and refinement statistics are summarized in Table 1. The Ramachandran plot showed that 95.8% of the residues are located in the most favored regions, and 4.2% are in additional allowed regions.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>20007</offset><text>Generation of constructs with mutated genomic FPA sequence</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20066</offset><text>A series of constructs containing a mutated FPA genomic sequence was prepared based on pGreen I 0029 vector. pGreen I 0029 vector with inserted FPA genomic sequence was prepared. In this vector FPA genomic sequence is flanked by 2620bp of the native sequence upstream to the start codon and 1178bp downstream to the stop codon. The vector contains kanamycin resistance genes for both the bacteria and plant hosts. In order to obtain a series of constructs with mutated FPA genomic sequence, FPA sequence in this construct was modified using site-directed mutagenesis. Primers used to prepare required constructs are listed in S1 Table. After the mutagenesis reaction the presence of only the desired mutations was confirmed by sequencing of the whole FPA genomic sequence and flanking regions.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>20860</offset><text>Generation of Arabidopsis thaliana transgenic plants</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20913</offset><text>All transgenic plants were prepared in fpa-8 mutant background, which is in Col-0 accession. The prepared vectors for Arabidopsis transformations were introduced into electro-competent Agrobacterium tumefaciens cells (C58 CV3101 strain harbouring pSoup vector). The floral dip method was used for plant transformation. Transgenic plants were selected using kanamycin as a selection marker. Presence of the desired mutations in plants was confirmed with specific dCaps markers.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>21390</offset><text>Plant growth conditions</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21414</offset><text>Wild type Col-0 plants used in this study were obtained from the Nottingham Arabidopsis Stock Centre. Seed of fpa-8 and 35S::FPA:YFP were obtained from Professor Caroline Dean. Plants were grown in pots containing Universal Extra general purpose soil. The glasshouse temperature was maintained at 20°C and the 16 hour daylight was provided by high pressure sodium vapour lamps (Philips Powertone SON-T AGRO 400). In order to grow plants in sterile conditions, seeds were first surface sterilized by a 5 min treatment with sterilizing solution (3% v/v sodium hypochlorite, 0.02% v/v Triton X-100), followed by three washes with 0.02% v/v Triton X-100 and one wash with sterile water. The sterile seeds were sown on MS10 media supplemented with 0.8% w/v agar. MS10 medium was also supplemented with specific antibiotics if required. After sowing, the seeds were stratified at 4°C for two days in order to synchronize their germination. Plants were grown in the tissue culture room at the following conditions: temperature 22°C, 16 hours daylight provided by the Master TL-D 36W/840 (Philips) lamps.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>22514</offset><text>Plant protein analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22537</offset><text>Total protein samples were prepared using extraction buffer containing: 40 mM Tris-HCl, pH 6.8; 0.1 mM EDTA, pH 8.0; 8 M urea; 1.43 M β-mercaptoethanol, 7% v/v Complete Protease Inhibitors (Roche) and 5 mM PMSF. Equal volumes of samples were separated on 8% SDS-PAGE. Proteins were transferred onto Protran nitrocellulose transfer membrane (Whatman) using wet Criterion blotter system (BioRad). The transfer was performed at room temperature for two hours at a stable voltage of 70 V. Membrane was blocked in 3% (w/v) Milk in TBS for 1h at room temperature followed by overnight incubation with anti-FPA antibody (dilution 1:100 in 3% (w/v) Milk in TBS). After washes the membrane was incubated for 75 min with goat anti-rabbit antibody (Thermo Scientific) (1:3000 dilution in 3% (w/v) Milk in TBS). Protein was detected using SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific). Blots were re-probed following treatment with low pH solution (25mM glycine-HCl, pH 2, 1% (w/v) SDS) followed by blocking for 1h at room temperature in 3% (w/v) Milk in TBS. The membrane was incubated overnight with anti-TUBB2A, tubulin, beta 2A antibody (ARP40177_P050 Aviva systems biology; (dilution 1:1000 in 3% (w/v) Milk in TBS). After washes the membrane was incubated for 75 min with goat anti-rabbit antibody (Thermo Scientific) [1:3000 dilution in 3% (w/v) Milk in TBS]. Signal was detected using SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24024</offset><text>RNA gel blot analysis and RT-qPCR</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24058</offset><text>RNA gel blot analysis and RT-qPCR method performed as previously described.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>24134</offset><text>Supporting Information</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>24157</offset><text>References</text></passage><passage><infon key="fpage">688</infon><infon key="lpage">700</infon><infon key="name_0">surname:Moore;given-names:MJ</infon><infon key="name_1">surname:Proudfoot;given-names:NJ</infon><infon 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+<!DOCTYPE collection SYSTEM "BioC.dtd">
+<collection><source>PMC</source><date>20201219</date><key>pmc.key</key><document><id>4993997</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/srep31425</infon><infon key="article-id_pii">srep31425</infon><infon key="article-id_pmc">4993997</infon><infon key="article-id_pmid">27550639</infon><infon key="elocation-id">31425</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Chen;given-names:Ji-Yun</infon><infon key="name_1">surname:Liu;given-names:Liang</infon><infon key="name_2">surname:Cao;given-names:Chun-Ling</infon><infon key="name_3">surname:Li;given-names:Mei-Jun</infon><infon key="name_4">surname:Tan;given-names:Kemin</infon><infon key="name_5">surname:Yang;given-names:Xiaohan</infon><infon key="name_6">surname:Yun;given-names:Cai-Hong</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">6</infon><infon key="year">2016</infon><offset>0</offset><text>Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>96</offset><text>N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1386</offset><text>Acetylation is one of the most ubiquitous modifications that plays a vital role in many biological processes, such as transcriptional regulation, protein-protein interaction, enzyme activity, protein stability, antibiotic resistance, biological rhythm and so on. Protein acetylation can be grouped into lysine Nε-acetylation and peptide N-terminal acetylation (Nt-acetylation). Generally, Nε-acetylation refers to the transfer of an acetyl group from an acetyl coenzyme A (Ac-CoA) to the ε-amino group of lysine. This kind of modification is catalyzed by lysine acetyltransferases (KATs), some of which are named histone acetyltransferases (HATs) because early studies focused mostly on the post-transcriptional acetylation of histones.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2141</offset><text>Despite the prominent accomplishments in the field regarding Nε-acetylation by KATs for over 50 years, the significance of the more evolutionarily conserved Nt-acetylation is still inconclusive. Nt-acetylation is an abundant and evolutionarily conserved modification occurring in bacteria, archaea and eukaryotes. It is estimated that about 80–90% of soluble human proteins and 50–70% of yeast proteins are subjected to Nt-acetylation, where an acetyl moiety is transferred from Ac-CoA to the α-amino group of the first residue. Recently Nt-acetylome expands the Nt-acetylation to transmembrane proteins. Unlike Nε-acetylation that can be eliminated by deacetylases, Nt-acetylation is considered irreversible since no corresponding deacetylase is found to date. Although Nt-acetylation has been regarded as a co-translational modification traditionally, there is evidence that post-translational Nt-acetylation exists. During the past decades, a large number of Nt-acetylome researches have shed light on the functional roles of Nt-acetylation, including protein degradation, subcellular localization, protein-protein interaction, protein-membrane interaction, plant development, stress-response and protein stability.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3376</offset><text>The Nt-acetylation is carried out by N-terminal acetyltransferases (NATs) that belong to the GNAT superfamily. To date, six NATs (NatA/B/C/D/E/F) have been identified in eukaryotes. About 40 percent of Nt-acetylation of soluble proteins in cells is catalyzed by NatA complex which is composed of the catalytic subunit Naa10p and the auxiliary subunit Naa15p. NatE was found to physically interact with the NatA complex without any observation of impact on NatA-activity. Two other multimeric complexes of NATs are NatB and NatC which contain the catalytic subunits Naa20 and Naa30 and the auxiliary subunits Naa25 and Naa35/Naa38, respectively. Furthermore, only the catalytic subunits Naa40 and Naa60 were found for NatD and NatF, respectively. Besides Nt-acetylation, accumulating reports have proposed Nε-acetylation carried out by NATs.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4223</offset><text>There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family. Unlike other NATs that are highly conserved among lower and higher eukaryotes, NatF only exists in higher eukaryotes. Subsequent researches indicated that NatF displays its catalytic ability with both Nt-acetylation and lysine Nε-acetylation. As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91. Another important feature of NatF is that this protein is anchored on the Golgi apparatus through its C-terminal membrane-integrating region and takes part in the maintaining of Golgi integrity. With its unique intracellular organellar localization and substrate selectivity, NatF appears to provide more evolutionary information among the NAT family members.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5697</offset><text>It was recently found that NatF facilitates nucleosomes assembly and that NAA60 knockdown in MCF7-cell inhibits cell proliferation, sensitizes cells to DNA damage and induces cell apoptosis. In Drosophila cells, NAA60 knockdown induces chromosomal segregation defects during anaphase including lagging chromosomes and chromosomal bridges. Much recent attention has also been focused on the requirement of NatF for regulation of organellar structure. In HeLa cells, NAA60 knockdown causes Golgi apparatus fragmentation which can be rescued by overexpression Naa60. The systematic investigation of publicly available microarray data showed that NATs share distinct tissue-specific expression patterns in Drosophila and NatF shows a higher expression level in central nervous system of Drosophila.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6492</offset><text>In this study, we solved the structures of human Naa60 (NatF) in complex with coenzyme. The hNaa60 protein contains a unique amphipathic α-helix (α5) following its GNAT domain that might account for the Golgi localization of this protein. Crystal structures showed that the β7-β8 hairpin rotated about 50 degrees upon removing the C-terminal region of the protein and this movement substantially changed the geometry of the substrate-binding pocket. Remarkably, we find that Phe 34 may participate in the proper positioning of the coenzyme for the transfer reaction to occur. Further structure comparison and biochemical studies also identified other key structural elements essential for the enzyme activity of Naa60.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>7225</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>7233</offset><text>Overall structure of hNaa60</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7261</offset><text>In the effort to prepare the protein for structural studies, we tried a large number of hNaa60 constructs but all failed due to heavy precipitation or aggregation. Sequence alignment of Naa60 from different species revealed a Glu-Glu-Arg (EER) versus Val-Val-Pro (VVP) sequence difference near the N-terminus of the protein in Xenopus Laevis versus Homo sapiens (Fig. 1A). Considering that terminal residues may lack higher-order structure and hydrophobic residues in this region may expose to solvent and hence cause protein aggregation, we mutated residues 4–6 from VVP to EER for the purpose of improving solubility of this protein. According to previous studies, this N-terminal region should not interfere with hNaa60’s Golgi localization. We tried many hNaa60 constructs with the three-residues mutation but only the truncated variant 1-199 and the full-length protein behaved well. We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C). Hereafter, all deletions or point mutants of hNaa60 we describe here are with the EER mutation. The crystal structures of hNaa60(1-242)/Ac-CoA and hNaa60(1-199)/CoA were determined by molecular replacement and refined to 1.38 Å and 1.60 Å resolution, respectively (Table 1). The electron density maps were of sufficient quality to trace residues 1-211 of hNaa60(1-242) and residues 5-199 of hNaa60(1-199).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8783</offset><text>The structure of hNaa60 protein contains a central domain exhibiting a classic GCN5-related N-acetyltransferase (GNAT) folding, along with the extended N- and C-terminal regions (Fig. 1B,C). The central domain includes nine β strands (β1-β9) and four α-helixes (α1-α4) and is highly similar to the known hNaa50p and other reported NATs (Fig. 1D). However, in hNaa60, there is an extra 20-residue loop between β3 and β4 that forms a small subdomain with well-defined 3D structure (Fig. 1B–D). Furthermore, the β7-β8 strands form an approximately antiparallel β-hairpin structure remarkably different from that in hNaa50p (Fig. 1D). The N- and C-terminal regions form helical structures (α0 and α5) stretching out from the central GCN5-domain (Fig. 1C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9579</offset><text>Interestingly, we found that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) (Figure S1), indicating that residues 200–242 may have some auto-inhibitory effect on the activity of the enzyme. However, since this region was not visible in the hNaa60(1-242) crystal structure, we do not yet understand how this happens. Another possibility is that since hNaa60 is localized on Golgi apparatus, the observed low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies. For the convenience of studying the kinetics of mutants, the mutagenesis studies described hereafter were all based on hNaa60 (1-199).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10278</offset><text>An amphipathic α-helix in the C-terminal region may contribute to Golgi localization of hNaa60</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10377</offset><text>There is one hNaa60 molecule in the asymmetric unit in the hNaa60(1-242)/Ac-CoA structure. The C-terminal region extended from the GCN5-domain forms an amphipathic helix (α5) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between α5-helix and a hydrophobic groove between the N-terminal β1 and β3 strands of the neighbor molecule (Fig. 2A). The C-terminal extension following α5-helix forms a β-turn that wraps around and interacts with the neighbor protein molecule through hydrophobic interactions, too. In the hNaa60(1-199)/CoA structure, a part of the α5-helix is deleted due to truncation of the C-terminal region (Fig. 1B). Interestingly, the remaining residues in α5-helix still form an amphipathic helix although the hydrophobic interaction with the N-terminal hydrophobic groove of a neighbor molecule is abolished and the helix is largely exposed in solvent due to different crystal packing (Fig. 2B).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11364</offset><text>A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>11975</offset><text>The β7-β8 hairpin showed alternative conformations in the hNaa60 crystal structures</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12065</offset><text>Superposition of hNaa60(1-242)/Ac-CoA, hNaa60(1-199)/CoA and hNaa50/CoA/peptide (PDB 3TFY) revealed considerable difference in the β7-β8 hairpin region despite the overall stability and similarity of the GNAT domain (Fig. 1D). In hNaa60(1-242), the β7-β8 hairpin is located in close proximity to the α1-α2 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (β6-β7 loop). Upon removing the C-terminal region of hNaa60, we observed that hNaa60 (1-199) molecules pack in a different way involving the β7-β8 hairpin in the crystal, leading to about 50 degree rotation of the hairpin which moves away from the α1-α2 loop (Figs 1D and 2C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12821</offset><text>This conformational change substantially altered the geometry of the substrate binding site, which could potentially change the way in which the substrate accesses the active site of the enzyme. In hNaa60(1-242), the β7-β8 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only. KAT activity of hNaa60 toward histone H4 has been noted in previous study, and our enzyme kinetic data also indicated that hNaa60 can acetylate H3-H4 tetramer in vitro (Figure S3). Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4). We also conducted liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis on a synthetic peptide (NH2-MKGKEEKEGGAR-COOH) after treatment with hNaa60(1-199), and the data confirmed that both the N-terminal α-amine and lysine side-chain ε-amine were robustly acetylated after the treatment (Table S1). Despite these observations, the mechanism for this alternative activity remains unknown. Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site. Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D). Interestingly, in the hNaa60(1-199) crystal structure, the displaced β7-β8 hairpin opened a second way for the substrate to access the active center that would readily accommodate the binding of the H4 peptide (Fig. 2E), thus implied a potential explanation for KAT activity of this enzyme from a structural biological view. However, since hNaa60(1-242) and hNaa60(1-199) were crystallized in different crystal forms, the observed conformational change of the β7-β8 hairpin may simply be an artifact related to the different crystal packing. Whether the KAT substrates bind to the β7-β8 hairpin displaced conformation of the enzyme needs to be verified by further structural and functional studies.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>15485</offset><text>Phe 34 facilitates proper positioning of the cofactor for acetyl-transfer</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15559</offset><text>The electron density of Phe 34 side-chain is well defined in the hNaa60(1-242)/Ac-CoA structure, but becomes invisible in the hNaa60(1-199)/CoA structure, indicating displacement of the Phe 34 side-chain in the latter (Fig. 3A,B). A solvent-derived malonate molecule is found beside Phe 34 and the ethanethioate moiety of Ac-CoA in the high-resolution hNaa60(1-242)/Ac-CoA structure (Fig. 3A). Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A). Interestingly, in the structure of hNaa60(1-199)/CoA, the terminal thiol of CoA adopts alternative conformations. One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the α1-α2 loop and away from the substrate amine (Fig. 3B). To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17026</offset><text>Phe 27 in hNaa50p (equivalent to Phe 34 in hNaa60) has been implicated to facilitate the binding of N-terminal methionine of the substrate peptide through hydrophobic interaction. However, in the hNaa60/Ac-CoA structure, a hydrophilic malonate molecule is found at the same location where the N-terminal methionine should bind as is indicated by the superposition (Fig. 3A), suggesting that Phe 34 may accommodate binding of hydrophilic substrate, too. Moreover, orientation of Phe 34 side-chain seems to be co-related to positioning of the terminus of the co-enzyme and important for placing it at a location in close proximity to the substrate amine. We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme. Indeed, our enzyme kinetic data showed that hNaa60(1-199) F34A mutant showed no detectable activity (Fig. 5A). In order to rule out the possibility that the observed loss of activity may be related to bad folding of the mutant protein, we studied the circular dichroism (CD) spectrum of the protein (Fig. 5B) and determined its crystal structure (Fig. 3C). Both studies proved that the F34A mutant protein is well-folded. Many studies have addressed the crucial effect of α1-α2 loop on catalysis, showing that some residues located in this area are involved in the binding of substrates. We propose that Phe 34 may play a dual role both in interacting with the peptide substrate (recognition) and in positioning of the ethanethioate moiety of Ac-CoA to the right location to facilitate acetyl-transfer.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18850</offset><text>Structural basis for hNaa60 substrate binding</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18896</offset><text>Several studies have demonstrated that the substrate specificities of hNaa60 and hNaa50 are highly overlapped. The structure of hNaa50p/CoA/peptide provides detailed information about the position of substrate N-terminal residues in the active site of hNaa50. Comparing the active site of hNaa60(1-242)/Ac-CoA with hNaa50p/CoA/peptide revealed that key catalytic and substrate binding residues are highly conserved in both proteins (Fig. 4A). With respect to catalysis, hNaa50p has been shown to employ residues Tyr 73 and His 112 to abstract proton from the α-amino group from the substrate’s first residue through a well-ordered water. A well-ordered water was also found between Tyr 97 and His 138 in hNaa60 (1-199)/CoA and hNaa60 (1-242)/Ac-CoA (Fig. 4B). To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B). Purity of all proteins were also analyzed by SDS-PAGE (Figure S5). As show in Fig. 5A, the mutants Y97A, Y97F, H138A and H138F abolished the activity of hNaa60. In contrast, to mutate the nearby solvent exposed residue Glu 37 to Ala (E37A) has little impact on the activity of hNaa60 (Figs 4B and 5A). In conclusion, the structural and functional studies indicate that hNaa60 applies the same two base mechanism through Tyr 97, His 138 and a well-ordered water as was described for hNaa50.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20394</offset><text>The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A). Residues Tyr 38, Asn 143 and Tyr 165 are located around the malonate and interact with it through direct hydrogen bonds or water bridge (Fig. 4C). Although malonate is negatively charged, which is different from that of lysine ε-amine or peptide N-terminal amine, similar hydrophilic interactions may take place when substrate amine presents in the same position, since Tyr 38, Asn 143 and Tyr 165 are not positively or negatively charged. In agreement with this hypothesis, it was found that the Y38A, N143A and Y165A mutants all showed remarkably reduced activities as compared to WT, implying that these residues may be critical for substrate binding (Figs 4C and 5A).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>21378</offset><text>The β3-β4 loop participates in the regulation of hNaa60-activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21449</offset><text>Residues between β3 and β4 of hNaa60 form a unique 20-residue long loop (residues 73–92) that is a short turn in many other NAT members (Fig. 1D). Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain. We therefore used mass spectrometry to analyze if Lys 79 was acetylated in our bacterially purified proteins, and observed no modification on this residue (Figure S6). To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively. Interestingly, both K79R and K79Q mutants led to an increase in the catalytic activity of hNaa60, while K79A mutant led to modest decrease of the activity (Fig. 5A). These data indicate that the acetylation of Lys 79 is not required for optimal catalytic activity of hNaa60 in vitro.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22485</offset><text>It is noted that the β3-β4 loop of hNaa60 acts like a door leaf to partly cover the substrate-binding pathway. We hence hypothesize that the β3-β4 loop may interfere with the access of the peptide substrates and that the solvent-exposing Lys 79 may play a potential role to remove the door leaf when it hovers in solvent (Fig. 4D). Acidic residues Glu 80, Asp 81 and Asp 83 interact with His 138, His 159 and His 158 to maintain the conformation of the β3-β4 loop, thus contribute to control the substrate binding (Fig. 4D). To verify this hypothesis, we mutated Glu 80, Asp 81 and Asp 83 to Ala respectively. In line with our hypothesis, E80A, D81A and D83A mutants exhibit at least 2-fold increase in hNaa60-activity (Fig. 5A). Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>23536</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>23547</offset><text>Nt-acetylation, which is carried out by the NAT family acetyltransferases, is an ancient and essential modification of proteins. Although many NATs are highly conserved from lower to higher eukaryotes and the substrate bias of them appears to be partially overlapped, there is a significant increase in the overall level of N-terminal acetylation from lower to higher eukaryotes. In this study we provide structural insights into Naa60 found only in multicellular eukaryotes.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>24023</offset><text>The N-terminus of hNaa60 harbors three hydrophobic residues (VVP) that makes it very difficult to express and purify the protein. This problem was solved by replacing residues 4–6 from VVP to EER that are found in Naa60 from Xenopus Laevis. Since Naa60 from human and from Xenopus Laevis are highly homologous (Fig. 1A), we speculate that these two proteins should have the same biological function. Therefore it is deduced that the VVP to EER replacement on the N-terminus of hNaa60 may not interfere with its function. However, in the hNaa60(1-242) structure the N-terminus adopts an α-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing. Hence it is not clear if the N-terminal end of wild-type hNaa60 is an α-helix, and what roles the hydrophobic residues 4–6 play in structure and function of wild-type hNaa60. In addition to the three-residue mutation (VVP to EER), we also tried many other hNaa60 constructs, but only the full-length protein and the truncated variant 1-199 behaved well. The finding that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) is intriguing. We speculate that low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies or there remains some undiscovered auto-inhibitory regulation in the full-length protein.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>25544</offset><text>The hNaa60 protein was proven to be localized on Golgi apparatus. Aksnes and colleagues predicted putative transmembrane domains and two putative sites of S-palmitoylation, by bioinformatics means, to account for Golgi localization of the protein. They then mutated all five cysteine residues of hNaa60’s to serine, including the two putative S-palmitoylation sites. However, these mutations did not abolish Naa60 membrane localization, indicating that S-palmitoylation is unlikely to (solely) account for targeting hNaa60 on Golgi. Furthermore, adding residues 217–242 of hNaa60 (containing residues 217–236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182–216 are important for Golgi localization of hNaa60. We found that residues 190–202 formed an amphipathic helix with an array of hydrophobic residues located on one side. This observation is reminiscent of the protein/membrane interaction through amphipathic helices in the cases of KalSec14, Atg3, PB1-F2 etc. In this model an amphipathic helix can immerse its hydrophobic side into the lipid bilayer through hydrophobic interactions. Therefore we propose that the amphipathic helix α5 may contribute to Golgi localization of hNaa60. This model, though may need further studies, is supported by the Aksnes studies.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>26986</offset><text>Previous studies indicated that members of NAT family are bi-functional NAT and KAT enzymes. However, known structures of NATs do not well support this hypothesis, since the β6-β7 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the α1-α2 loop, which would be good for the NAT but not KAT activity of the enzyme. Kinetic studies have been conducted to compare the NAT and KAT activity of hNaa50 in vitro, and indicate that the NAT activity of Naa50 is much higher than KAT activity. However, the substrate used in this study for assessing KAT activity was a small peptide which could not really mimic the 3D structure of a folded protein substrate in vivo. Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro. Conformational change of the β7-β8 hairpin (corresponding to the β6-β7 loop of other NATs) is noted in our structures (Figs 1D and 2C), which might provide an explanation to the NAT/KAT dual-activity in a structural biological view, but we were unable to rule out the possibility that the observed conformational change of this hairpin might be an artifact related to crystal packing or truncation of the C-terminal end of the protein. Further studies are therefore needed to reveal the mechanism for the KAT activity of this enzyme.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28518</offset><text>The relationship between enzyme, co-enzyme and substrates has been documented for several years. In early years, researchers found adjustment of GCN5 histone acetyltransferase structure when it binds CoA molecule. The complexed form of NatA is more suitable for catalytic activation, since the α1-α2 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA). In the structure of hNaa50/CoA/peptide, Phe 27 in the α1-α2 loop appears to make hydrophobic interaction with the N-terminal Met of substrate peptide. However, the hNaa60(1-242)/Ac-CoA crystal structure indicated that its counterpart in hNaa60, Phe 34, could also accommodate the binding of a hydrophilic malonate that occupied the substrate binding site although it maintained the same conformation as that observed in hNaa50. Interestingly, the terminal thiol of CoA adopted alternative conformations in the structure of hNaa60(1-199)/CoA. One was to approach the substrate amine; the other was to approach the α1-α2 loop and away from the substrate amine. Same alternative conformations of CoA were observed in the hNaa60(1-199)(F34A) crystal structure, and our kinetic data showed that the F34A mutation abolished the activity of the enzyme. Taken together, our data indicated that Phe 34 in hNaa60 may play a role in placing co-enzyme at the right location to facilitate the acetyl-transfer. However, these data did not rule out that possibility that Phe 34 may coordinate the binding of the N-terminal Met through hydrophobic interaction as was proposed by previous studies.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30205</offset><text>Furthermore, we showed that hNaa60 adopts the classical two base mechanism to catalyze acetyl-transfer. Although sequence identity between hNaa60 and hNaa50 is low, key residues in the active site of both enzymes are highly conserved. This can reasonably explain the high overlapping substrates specificities between hNaa60 and hNaa50. Another structural feature of hNaa60 that distinguishes it from other NATs is the β3-β4 long loop which appears to inhibit the catalytic activity of hNaa60. However, this loop also seems to stabilize the whole hNaa60 structure, because deletion mutations of this region led to protein precipitation and aggregation (Figure S7). A previous study suggested that the auto-acetylation of Lys 79 was important for hNaa60-activity, whereas the point mutation K79R did not decrease the activity of hNaa60 in our study. Meanwhile, no electron density of acetyl group was found on Lys 79 in our structures and mass spectrometry analysis. Hence, it appears that the auto-acetylation of hNaa60 is not an essential modification for its activity for the protein we used here. As for the reason why K79R in Yang’s previous studies reduced the activity of the enzyme, but in our studies it didn’t, we suspect that the stability of this mutant may play some role. K79R is less stable than the wild-type enzyme as was judged by its poorer gel-filtration behavior and tendency to precipitate. In our studies we have paid special attention and carefully handled this protein to ensure that we did get enough of the protein in good condition for kinetic assays. The intracellular environment is more complicated than our in vitro assay and the substrate specificity of hNaa60 most focuses on transmembrane proteins. The interaction between hNaa60 and its substrates may involve the protein-membrane interaction which would further increase the complexity. It is not clear if the structure of hNaa60 is different in vivo or if other potential partner proteins may help to regulate its activity. Nevertheless, our study may be an inspiration for further studies on the functions and regulation of this youngest member of the NAT family.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>32362</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32370</offset><text>Cloning, expression and purification of Homo sapiens Naa60 (hNaa60)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32438</offset><text>The cDNA encoding hNaa60 residues 1–242 (full-length) or residues 1–199 were amplified by PCR and inserted into the pET23a vector, which had been modified to provide an N-terminal 6xHis-tag followed by a tobacco etch virus (TEV) protease cleavage site. The VVP to EER (residues 4–6) mutation and other mutations for functional studies were introduced using the quick change method. The protein was expressed in Escherichia coli BL21 (DE3) or Escherichia coli BL21 (DE3) pLysS at 16 °C for 15 h in the presence of 0.1 mM IPTG. Cells were harvested at 4 °C by centrifugation (4,000 g for 10 min) and resuspended in buffer A containing 20 mM Tris, pH 8.0, 500 mM NaCl, 50 mM imidazole, 10% glycerol, 1 mM protease inhibitor PMSF (Phenylmethylsulfonyl fluoride) and 1 mM Tris (2-carboxyethyl)phosphine (TCEP) hydrochloride. Cells were lysed by sonication and the lysate was cleared by centrifugation (18,000 g at 4 °C for 20 min). Then the supernatant was loaded onto a 5-mL Chelating Sepharose column (GE Healthcare) charged with Ni2+ and washed with buffer B (20 mM Tris, pH 8.0, 500 mM NaCl, 50 mM imidazole, 1% glycerol and 1 mM TCEP). The protein was eluted with buffer C (20 mM Tris, pH 8.0, 500 mM NaCl, 300 mM imidazole, 1% glycerol and 1 mM TCEP). The eluent was digested by His-tagged TEV protease and concentrated by ultrafiltration at the same time. After 3 hours, the concentrated eluent was diluted 10 times with buffer D (20 mM Tris, pH 8.0, 500 mM NaCl, 1% glycerol and 1 mM TCEP) and the diluent was passed through the nickel column once again to remove the His-tagged TEV protease and the un-cleaved His-hNaa60 protein. The flow-through was concentrated to 500 μl and loaded onto a Superose 6 or Superdex 200 10/300 gel-filtration column (GE Healthcare) equilibrated with buffer E (20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP). Fractions containing the protein were collected and concentrated to a final concentration of 10 mg/ml for crystallization or acetyltransferases assays.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>34517</offset><text>Circular Dichroism (CD) Spectroscopy</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34554</offset><text>CD spectra of the proteins were obtained using a Jasco J-810 circular dichroism spectropolarimeter scanning from 190 to 250 nm with a 1 mm quartz cuvette. The wild-type and mutant proteins were examined at 4.5 μM concentration in 20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP at room temperature. All samples were centrifuged at 10,000 g for 5 min before analysis.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>34949</offset><text>Crystallization, data collection and structure determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35010</offset><text>The purified hNaa60(1-242), hNaa60(1-199) or F34A(1-199) protein was mixed with acetyl coenzyme A (Ac-CoA) or coenzyme A (CoA) (Sigma), respectively, at a 1:5 molar ratio before crystallization. All crystals were made by the hanging-drop vapor diffusion method. The crystallization reservoir solution for hNaa60(1-242) was 10 mM Tris pH 8.0, 75 mM NaCl, 0.5% glycerol, 3% v/v Tacsimate pH 4.0 (Hampton Research) and 7.5% w/v polyethylene glycol 3350 (PEG 3350), and for hNaa60(1-199) was 0.2 M L-Proline, 0.1 M HEPES pH 7.5, 10% w/v PEG 3350. Crystals of F34A mutation were obtained in 0.2 M Lithium Sulfate monohydrate, 0.1 M Tris pH 8.5, 20% w/v PEG 3350. The crystals were flash-frozen in liquid nitrogen in a cryo-protectant made of the reservoir solution supplemented with 25% glycerol.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35814</offset><text>The diffraction data were collected at the Shanghai SSRF BL18U1 beamline or at the Argonne National Laboratory APS ID19 beamline at 100 K. The data were processed with HKL3000. The hNaa60(1-199) structure was determined by molecular replacement with Phaser using a previously reported GNAT family acetyltransferase structure (PDB 2AE6) as the search model. The hNaa60(1-242) structure was solved by molecular replacement using hNaa60(1-199) structure as the search model. To improve the model quality, the programs ARP/wARP in CCP4 or simulated-annealing in CNS were used. Iterative cycles of manual refitting and crystallographic refinement were performed using COOT and Phenix. Ac-CoA/CoA and malonate were modeled into the closely fitting positive Fo-Fc electron density and then included in following refinement cycles. Topology and parameter files for Ac-CoA/CoA and malonate were generated using PRODRG. All figures for the molecular models were prepared using the PyMOL program. Statistics of diffraction data processing and structure refinement are shown in Table 1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>36891</offset><text>Acetyltransferase assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36915</offset><text>Acetyltransferase assay of hNaa60 was conducted as described previously. Briefly, a reaction cocktail containing 100 mM Tris-HCl buffer, pH 8.5, 0.07% alkylated BSA, 0.01% NP-40, 1 mM EDTA, 150 μM Ac-CoA (Sigma) was prepared and varied concentrations of the substrate peptide (0–400 μM) (NH2-MKGKEEKEGGAR-COOH) was added in a 1.5-mL microfuge tube, and then the respective enzyme was added to initiate the reaction with a final assay volume of 100 μL. The reaction was carried out for 20 minutes at 37 °C. Aliquots (40 μL) of the reaction were then removed and quenched with 40 μL of ice-cold isopropanol in individual wells of a 96-well black microplate (Corning), and then mixed with 80 μl of 25 μM 7-diethylamino-3-(49 maleimidylphenyl)-4-methylcoumarin (CPM) (Sigma) in 100 mM Tris-HCl (pH 8.5) and 1% Triton X-100 and allowed to react in darkness for 10 minutes prior to reading. The fluorescence signal was monitored using a Varioskan Flash plate reader (Thermo Scientific) at Exmax = 385 nm and Emmax = 465 nm. Substrate inhibition appeared at high concentrations of substrate peptide prevented our kinetics assays from reaching saturation of the enzyme. Therefore, we determined the value of kcat/Km by fitting our data to the equation: v = (kcat/Km)[ET][S] when the substrate concentration was far less than Km. The assays were done in triplicate. The slope of the line indicates the kcat/Km value of the enzyme (Figure S1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>38401</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38424</offset><text>How to cite this article: Chen, J.-Y. et al. Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase. Sci. 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J.-Y.C. and C.-H.Y. wrote the manuscript.</text></passage><passage><infon key="file">srep31425-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>42521</offset><text>Overall structure of Naa60.</text></passage><passage><infon key="file">srep31425-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>42549</offset><text>(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus). Alignment was generated using NPS@ and ESPript.3.0 (http://espript.ibcp.fr/ESPript/ESPript/). Residues 4–6 are highlighted in red box. (B) The structure of hNaa60(1-199)/CoA complex is shown as a yellow cartoon model. The CoA molecule is shown as sticks. (C) The structure of hNaa60(1-242)/Ac-CoA complex is presented as a cartoon model in cyan. The Ac-CoA and malonate molecules are shown as cyan and purple sticks, respectively. The secondary structures are labeled starting with α0. (D) Superposition of hNaa60(1-242) (cyan), hNaa60(1-199) (yellow) and hNaa50 (pink, PDB 3TFY). The Ac-CoA of hNaa60(1-242)/Ac-CoA complex is represented as cyan sticks.</text></passage><passage><infon key="file">srep31425-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>43389</offset><text>Amphipathicity of the α5 helix and alternative conformations of the β7-β8 hairpin.</text></passage><passage><infon key="file">srep31425-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>43482</offset><text>(A) The α5 helix of hNaa60(1-242) in one asymmetric unit (slate) interacts with another hNaa60 molecule in a neighboring asymmetric unit (cyan). A close view of the interaction is shown in red box. Side-chains of hydrophobic residues on α5 helix and the neighboring molecule participating in the interaction are shown as yellow and green sticks, respectively. (B) The α5 helix of hNaa60(1-199) in one asymmetric unit (yellow) interacts with another hNaa60 molecule in the neighboring asymmetric units (green). A close view of the interaction is shown in the red box. Side-chains of hydrophobic residues on α5 helix and the neighboring molecule (green) participating in the interaction are shown as yellow and green sticks, respectively. The third molecule (pink) does not directly interact with the α5 helix. (C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E). The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D). The α-amine of the NAT substrate and ε-amine of the KAT substrate (along with the lysine side-chain) subject to acetylation are shown as sticks.</text></passage><passage><infon key="file">srep31425-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>44987</offset><text>Electron density map of the active site.</text></passage><passage><infon key="file">srep31425-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>45028</offset><text>The 2Fo-Fc maps contoured at 1.0σ are shown for hNaa60(1-242)/Ac-CoA (A), hNaa60(1-199)/CoA (B) and hNaa60(1-199) F34A/CoA (C). The putative substrate peptide binding site is indicated by the peptide (shown as pink sticks) from the hNaa50/CoA/peptide complex structure after superimposing hNaa50 on the hNaa60 structures determined in this study. The black arrow indicates the α-amine of the first Met (M1) (all panels). The purple arrow indicates the acetyl moiety of Ac-CoA (A). The red arrow indicates the alternative conformation of the thiol moiety of the co-enzyme when Phe 34 side-chain is displaced (B) or mutated to Ala (C).</text></passage><passage><infon key="file">srep31425-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>45670</offset><text>Structural basis for hNaa60 catalytic activity.</text></passage><passage><infon key="file">srep31425-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>45718</offset><text>(A) Superposition of hNaa60 active site (cyan) on that of hNaa50 (pink, PDB 3TFY). Side-chains of key catalytic and substrate-binding residues are highlighted as sticks. The malonate molecule in the hNaa60(1-242)/Ac-CoA structure and the peptide in the hNaa50/CoA/peptide structure are shown as purple and yellow sticks respectively. (B) A close view of the active site of hNaa60. Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks. The water molecules participating in catalysis in the hNaa60 and hNaa50 structures are showed as green and red spheres, separately. (C) The interaction between the malonate molecule and surrounding residues observed in the hNaa60(1-242)/Ac-CoA structure. The yellow dotted lines indicate the hydrogen bonds. (D) A zoomed view of β3-β4 loop of hNaa60. Key residues discussed in the text (cyan), the malonate (purple) and Ac-CoA (gray) are shown as sticks. The yellow dotted lines indicate the salt bridges.</text></passage><passage><infon key="file">srep31425-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>46876</offset><text>Catalytic activity of hNaa60 and mutant proteins.</text></passage><passage><infon key="file">srep31425-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>46926</offset><text>(A) Catalytic efficiency (shown as kcat/Km values) of hNaa60 (1-199) WT and mutants. Error bars indicate the Standard Error (SE). (B) CD spectra of wild-type and mutant proteins from 250 nm to 190 nm. The sample concentration was 4.5 μM in 20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP at room temperature.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>47259</offset><text>Data collection and refinement statistics.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot; border=&quot;1&quot;&gt;&lt;colgroup&gt;&lt;col align=&quot;left&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;/colgroup&gt;&lt;thead valign=&quot;bottom&quot;&gt;&lt;tr&gt;&lt;th align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Structure and PDB ID&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;hNaa60(1-242)/Ac-CoA 5HGZ&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;hNaa60(1-199)/CoA 5HH0&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;hNaa60(1-199)F34A/CoA 5HH1&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td colspan=&quot;4&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Data collection&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn1&quot;&gt;*&lt;/xref&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Space group&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;P2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;P2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;P2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;sub&gt;&lt;italic&gt;1&lt;/italic&gt;&lt;/sub&gt;&lt;italic&gt;2&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;4&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Cell dimensions&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;italic&gt;a, b, c&lt;/italic&gt; (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;53.3, 57.4, 68.8&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;67.8, 73.8, 43.2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;66.7, 74.0, 43.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; α,β,γ (°)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90.0, 90.0, 90.0&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90.0, 90.0, 90.0&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90.0, 90.0, 90.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Resolution (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;50–1.38 (1.42–1.38)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;50–1.60 (1.66–1.60)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;50–1.80 (1.86–1.80)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;p.i.m.&lt;/sub&gt;(%)&lt;xref ref-type=&quot;fn&quot; rid=&quot;t1-fn2&quot;&gt;**&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3.0 (34.4)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2.1 (32.5)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2.6 (47.8)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;I&lt;/italic&gt;/&lt;italic&gt;σ&lt;/italic&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;21.5 (2.0)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;31.8 (2.0)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;28.0 (2.4)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Completeness (%)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.8 (99.1)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.6 (98.5)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.9 (99.7)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Redundancy&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;6.9 (5.0)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;6.9 (6.2)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;6.3 (5.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;4&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Refinement&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Resolution (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;25.81–1.38&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;33.55–1.60&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;43.52–1.80&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; No. reflections&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;43660&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;28588&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;20490&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt; R&lt;/italic&gt;&lt;sub&gt;work&lt;/sub&gt;/&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.182/0.192&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.181/0.184&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.189/0.209&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;4&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;No. atoms&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Protein&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1717&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1576&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1566&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Ligand/ion&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;116&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;96&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;96&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Water&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;289&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;258&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;168&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;4&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;B&lt;/italic&gt;-factors&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Protein&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;23.8&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;32.0&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;37.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Ligand/ion&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;22.2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;34.6&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;43.7&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Water&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;35.1&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;46.4&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;49.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;4&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;R.m.s. deviations&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Bond lengths (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.018&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.017&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.015&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Bond angles (°)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.529&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.651&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.581&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;4&quot; align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Ramachandran Plot&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Favoured region&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;98.98%&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;98.93%&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;98.96%&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Allowed region&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.02%&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.07%&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.04%&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Outliers&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.00%&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.00%&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.00%&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>47302</offset><text>Structure and PDB ID	hNaa60(1-242)/Ac-CoA 5HGZ	hNaa60(1-199)/CoA 5HH0	hNaa60(1-199)F34A/CoA 5HH1	 	Data collection*	 	 Space group	P212121	P21212	P21212	 	Cell dimensions	 	 a, b, c (Å)	53.3, 57.4, 68.8	67.8, 73.8, 43.2	66.7, 74.0, 43.5	 	 α,β,γ (°)	90.0, 90.0, 90.0	90.0, 90.0, 90.0	90.0, 90.0, 90.0	 	Resolution (Å)	50–1.38 (1.42–1.38)	50–1.60 (1.66–1.60)	50–1.80 (1.86–1.80)	 	Rp.i.m.(%)**	3.0 (34.4)	2.1 (32.5)	2.6 (47.8)	 	I/σ	21.5 (2.0)	31.8 (2.0)	28.0 (2.4)	 	Completeness (%)	99.8 (99.1)	99.6 (98.5)	99.9 (99.7)	 	Redundancy	6.9 (5.0)	6.9 (6.2)	6.3 (5.9)	 	Refinement	 	 Resolution (Å)	25.81–1.38	33.55–1.60	43.52–1.80	 	 No. reflections	43660	28588	20490	 	 Rwork/Rfree	0.182/0.192	0.181/0.184	0.189/0.209	 	No. atoms	 	 Protein	1717	1576	1566	 	 Ligand/ion	116	96	96	 	 Water	289	258	168	 	B-factors	 	 Protein	23.8	32.0	37.4	 	 Ligand/ion	22.2	34.6	43.7	 	 Water	35.1	46.4	49.1	 	R.m.s. deviations	 	 Bond lengths (Å)	0.018	0.017	0.015	 	 Bond angles (°)	1.529	1.651	1.581	 	Ramachandran Plot	 	 Favoured region	98.98%	98.93%	98.96%	 	 Allowed region	1.02%	1.07%	1.04%	 	 Outliers	0.00%	0.00%	0.00%	 	</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>48477</offset><text>*Values in parentheses are for highest-resolution shell. One crystal was used for each data set.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>48574</offset><text>**Rp.i.m., a redundancy-independent R factor was used to evaluate the diffraction data quality as was proposed by Evans.</text></passage></document></collection>
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+<collection><source>PMC</source><date>20230811</date><key>pmc.key</key><document><id>5012862</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.7554/eLife.18972</infon><infon key="article-id_pmc">5012862</infon><infon key="article-id_pmid">27529188</infon><infon key="article-id_publisher-id">18972</infon><infon key="elocation-id">e18972</infon><infon key="kwd">encapsulin encapsulated ferritin ferritin Rhodospirillum rubrum Other</infon><infon key="license">This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.</infon><infon key="name_0">surname:He;given-names:Didi</infon><infon key="name_1">surname:Hughes;given-names:Sam</infon><infon key="name_10">surname:Losick;given-names:Richard</infon><infon key="name_11">surname:He;given-names:Didi</infon><infon key="name_12">surname:Hughes;given-names:Sam</infon><infon key="name_13">surname:Altenbach;given-names:Kirsten</infon><infon key="name_14">surname:Clarke;given-names:David J</infon><infon key="name_15">surname:Tarrant;given-names:Emma</infon><infon key="name_16">surname:Waldron;given-names:Kevin J</infon><infon key="name_17">surname:Clarke;given-names:David J</infon><infon key="name_18">surname:Marles-Wright;given-names:Jon</infon><infon key="name_19">surname:Marles-Wright;given-names:Jon</infon><infon key="name_2">surname:Vanden-Hehir;given-names:Sally</infon><infon key="name_3">surname:Georgiev;given-names:Atanas</infon><infon key="name_4">surname:Altenbach;given-names:Kirsten</infon><infon key="name_5">surname:Tarrant;given-names:Emma</infon><infon key="name_6">surname:Mackay;given-names:C Logan</infon><infon key="name_7">surname:Waldron;given-names:Kevin J</infon><infon key="name_8">surname:Clarke;given-names:David J</infon><infon key="name_9">surname:Marles-Wright;given-names:Jon</infon><infon key="section_type">TITLE</infon><infon key="title">Author Keywords Research Organism</infon><infon key="type">front</infon><infon key="volume">5</infon><infon key="year">2016</infon><offset>0</offset><text>Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>118</offset><text>Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage. We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid. We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface. EncFtn adopts an open decameric structure that is topologically distinct from other ferritins. While EncFtn acts as a ferroxidase, it cannot mineralize iron. Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage. This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1107</offset><text>DOI: http://dx.doi.org/10.7554/eLife.18972.001</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>1154</offset><text>eLife digest</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1167</offset><text>Iron is essential for life as it is a key component of many different enzymes that participate in processes such as energy production and metabolism. However, iron can also be highly toxic to cells because it readily reacts with oxygen. This reaction can damage DNA, proteins and the membranes that surround cells.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1482</offset><text>To balance the cell’s need for iron against its potential damaging effects, organisms have evolved iron storage proteins known as ferritins that form cage-like structures. The ferritins convert iron into a less reactive form that is mineralised and safely stored in the central cavity of the ferritin cage and is available for cells when they need it.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1836</offset><text>Recently, a new family of ferritins known as encapsulated ferritins have been found in some microorganisms. These ferritins are found in bacterial genomes with a gene that codes for a protein cage called an encapsulin. Although the structure of the encapsulin cage is known to look like the shell of a virus, the structure that the encapsulated ferritin itself forms is not known. It is also not clear how encapsulin and the encapsulated ferritin work together to store iron.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>2312</offset><text>He et al. have now used the techniques of X-ray crystallography and mass spectrometry to determine the structure of the encapsulated ferritin found in some bacteria. The encapsulated ferritin forms a ring-shaped doughnut in which ten subunits of ferritin are arranged in a ring; this is totally different from the enclosed cages that other ferritins form.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>2668</offset><text>Biochemical studies revealed that the encapsulated ferritin is able to convert iron into a less reactive form, but it cannot store iron on its own since it does not form a cage. Thus, the encapsulated ferritin needs to be housed within the encapsulin cage to store iron.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>2939</offset><text>Further work is needed to investigate how iron moves into the encapsulin cage to reach the ferritin proteins. Some organisms have both standard ferritin cages and encapsulated ferritins; why this is the case also remains to be discovered.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>3178</offset><text>DOI: http://dx.doi.org/10.7554/eLife.18972.002</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>3225</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3238</offset><text>Encapsulin nanocompartments are a family of proteinaceous metabolic compartments that are widely distributed in bacteria and archaea. They share a common architecture, comprising an icosahedral shell formed by the oligomeric assembly of a protein, encapsulin, that is structurally related to the HK97 bacteriophage capsid protein gp5. Gp5 is known to assemble as a 66 nm diameter icosahedral shell of 420 subunits. In contrast, both the Pyrococcus furiosus and Myxococcus xanthus encapsulin shell-proteins form 32 nm icosahedra with 180 subunits; while the Thermotoga maritima encapsulin is smaller still with a 25 nm, 60-subunit icosahedron. The high structural similarity of the encapsulin shell-proteins to gp5 suggests a common evolutionary origin for these proteins.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4011</offset><text>The genes encoding encapsulin proteins are found downstream of genes for dye-dependent peroxidase (DyP) family enzymes, or encapsulin-associated ferritins (EncFtn). Enzymes in the DyP family are active against polyphenolic compounds such as azo dyes and lignin breakdown products; although their physiological function and natural substrates are not known. Ferritin family proteins are found in all kingdoms and have a wide range of activities, including ribonucleotide reductase, protecting DNA from oxidative damage, and iron storage. The classical iron storage ferritin nanocages are found in all kingdoms and are essential in eukaryotes; they play a central role in iron homeostasis, where they protect the cell from toxic free Fe2+ by oxidizing it and storing the resulting Fe3+ as ferrihydrite minerals within their central cavity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4849</offset><text>The encapsulin-associated enzymes are sequestered within the icosahedral shell through interactions between the shell’s inner surface and a short localization sequence (Gly-Ser-Leu-Lys) appended to their C-termini. This motif is well-conserved, and the addition of this sequence to heterologous proteins is sufficient to direct them to the interior of encapsulins.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5217</offset><text>A recent study of the Myxococcus xanthus encapsulin showed that it sequesters a number of different EncFtn proteins and acts as an ‘iron-megastore’ to protect these bacteria from oxidative stress. At 32 nm in diameter, it is much larger than other members of the ferritin superfamily, such as the 12 nm 24-subunit classical ferritin nanocage and the 8 nm 12-subunit Dps (DNA-binding protein from starved cells) complex; and is thus capable of sequestering up to ten times more iron than these ferritins. The primary sequences of EncFtn proteins have Glu-X-X-His metal coordination sites, which are shared features of the ferritin family proteins. Secondary structure prediction identifies two major α-helical regions in these proteins; this is in contrast to other members of the ferritin superfamily, which have four major α-helices (Supplementary file 1). The ‘half-ferritin’ primary sequence of the EncFtn family and their association with encapsulin nanocompartments suggests a distinct biochemical and structural organization to other ferritin family proteins. The Rhodospirillum rubrum EncFtn protein (Rru_A0973) shares 33% protein sequence identity with the M. xanthus (MXAN_4464), 53% with the T. maritima (Tmari_0787), and 29% with the P. furiosus (PF1192) homologues. The GXXH motifs are strictly conserved in each of these species (Supplementary file 1).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6594</offset><text>Here we investigate the structure and biochemistry of EncFtn in order to understand iron storage within the encapsulin nanocompartment. We have produced recombinant encapsulin (Enc) and EncFtn from the aquatic purple-sulfur bacterium R. rubrum, which serves as a model organism for the study of the control of the bacterial nitrogen fixation machinery, in Escherichia coli. Analysis by transmission electron microscopy (TEM) indicates that their co-expression leads to the production of an icosahedral nanocompartment with encapsulated EncFtn. The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins. We identify a symmetrical iron bound ferroxidase center (FOC) formed between subunits in the decamer and additional metal-binding sites close to the center of the ring and on the outer surface. We also demonstrate the metal-dependent assembly of EncFtn decamers using native PAGE, analytical gel-filtration, and native mass spectrometry. Biochemical assays show that EncFtn is active as a ferroxidase enzyme. Through site-directed mutagenesis we show that the conserved glutamic acid and histidine residues in the FOC influence protein assembly and activity. We use our combined structural and biochemical data to propose a model for the EncFtn-catalyzed sequestration of iron within the encapsulin shell.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>8136</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8144</offset><text>Assembly of R. rubrum EncFtn encapsulin nanocompartments in E. coli</text></passage><passage><infon key="file">elife-18972-fig1-figsupp1.jpg</infon><infon key="id">fig1s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>8212</offset><text>Full-frame transmission electron micrographs of R. rubrum nanocompartments.</text></passage><passage><infon key="file">elife-18972-fig1-figsupp1.jpg</infon><infon key="id">fig1s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>8288</offset><text>(A/B) Negative stain TEM image of recombinant R. rubrum encapsulin and EncFtn-Enc nanocompartments. All samples were imaged at 143,000 x magnification; the scale bar length corresponds to 50 nm. (C) Histogram showing the distribution of nanocompartment diameters. A model Gaussian nonlinear least square function was fitted to the data to obtain a mean diameter of 24.6 nm with a standard deviation of 2.0 nm for encapsulin (grey) and a mean value of 23.9 nm with a standard deviation of 2.2 nm for co-expressed EncFtn and encapsulin (EncFtn-Enc, black).</text></passage><passage><infon key="file">elife-18972-fig1-figsupp1.jpg</infon><infon key="id">fig1s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>8845</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.004</text></passage><passage><infon key="file">elife-18972-fig1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>8892</offset><text>Purification of recombinant R. rubrum encapsulin nanocompartments.</text></passage><passage><infon key="file">elife-18972-fig1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>8959</offset><text>(A) Recombinantly expressed encapsulin (Enc) and co-expressed EncFtn-Enc were purified by sucrose gradient ultracentrifugation from E. coli B834(DE3) grown in SeMet medium. Samples were resolved by 18% acrylamide SDS-PAGE; the position of the proteins found in the complexes as resolved on the gel are shown with arrows. (B/C) Negative stain TEM image of recombinant encapsulin and EncFtn-Enc nanocompartments. Samples were imaged at 143,000 x magnification, with scale bar shown as 25 nm. Representative encapsulin and EncFtn-Enc complexes are indicated with red arrows.</text></passage><passage><infon key="file">elife-18972-fig1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>9531</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.003</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9578</offset><text>We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A). TEM imaging of uranyl acetate-stained samples revealed that, when expressed in isolation, the encapsulin protein forms empty compartments with an average diameter of 24 nm (Figure 1B and Figure 1—figure supplement 1A/C), consistent with the appearance and size of the T. maritima encapsulin. We were not able to resolve any higher-order structures of EncFtn by TEM. Protein purified from co-expression of the encapsulin and EncFtn resulted in 24 nm compartments with regions in the center that exclude stain, consistent with the presence of the EncFtn within the encapsulin shell (Figure 1C and Figure 1—figure supplement 1B/C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10437</offset><text>R. rubrum EncFtn forms a metal-ion stabilized decamer in solution</text></passage><passage><infon key="file">elife-18972-fig2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>10504</offset><text>Purification of recombinant R. rubrum EncFtnsH.</text></passage><passage><infon key="file">elife-18972-fig2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>10552</offset><text>(A) Recombinant SeMet-labeled EncFtnsH produced with 1 mM Fe(NH4)2(SO4)2 in the growth medium was purified by nickel affinity chromatography and size-exclusion chromatography using a Superdex 200 16/60 column (GE Healthcare). Chromatogram traces measured at 280 nm and 315 nm are shown with the results from ICP-MS analysis of the iron content of the fractions collected during the experiment. The peak around 73 ml corresponds to a molecular weight of around 130 kDa when compared to calibration standards; this is consistent with a decamer of EncFtnsH. The small peak at 85 ml corresponds to the 13 kDa monomer compared to the standards. Only the decamer peak contains significant amounts of iron as indicated by the ICP-MS analysis. (B) Peak fractions from the gel filtration run were resolved by 15% acrylamide SDS-PAGE and stained with Coomassie blue stain. The bands around 13 kDa and 26 kDa correspond to EncFtnsH, as identified by MALDI peptide mass fingerprinting. The band at 13 kDa is consistent with the monomer mass, while the band at 26 kDa is consistent with a dimer of EncFtnsH. The dimer species only appears in the decamer fractions. (C) SEC-MALLS analysis of EncFtnsH from decamer fractions and monomer fractions allows assignment of an average mass of 132 kDa to decamer fractions and 13 kDa to monomer fractions, consistent with decamer and monomer species (Table 2).</text></passage><passage><infon key="file">elife-18972-fig2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11944</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.005</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>11991</offset><text>Determination of the Fe/EncFtnsH protein ratio by ICP-MS. EncFtnsH was purified as a SeMet derivative from E. coli B834(DE3) cells grown in SeMet medium with 1 mM Fe(NH4)2(SO4)2. Fractions from SEC were collected, acidified and analysed by ICP-MS. EncFtnsH concentration was calculated based on the presence of two SeMet per mature monomer. Samples where the element was undetectable are labelled with n.d. These data were collected from EncFtnsH fractions from a single gel-filtration run.</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>12482</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.006</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead&gt;&lt;tr&gt;&lt;th rowspan=&quot;2&quot; colspan=&quot;1&quot;&gt;Peak&lt;/th&gt;&lt;th rowspan=&quot;2&quot; colspan=&quot;1&quot;&gt;EncFtn&lt;sub&gt;sH&lt;/sub&gt;
+&lt;break/&gt;retention volume (ml)&lt;/th&gt;&lt;th colspan=&quot;4&quot; rowspan=&quot;1&quot;&gt;Element concentration (µM)&lt;/th&gt;&lt;th rowspan=&quot;2&quot; colspan=&quot;1&quot;&gt;Derived EncFtn&lt;sub&gt;sH&lt;/sub&gt;concentration (µM)&lt;/th&gt;&lt;th rowspan=&quot;2&quot; colspan=&quot;1&quot;&gt;Derived Fe/ &lt;break/&gt;EncFtn&lt;sub&gt;sH&lt;/sub&gt; monomer&lt;/th&gt;&lt;/tr&gt;&lt;tr&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Ca&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Fe&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Zn&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Se&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td rowspan=&quot;8&quot; colspan=&quot;1&quot;&gt;Decamer&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;66.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;24.6&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;12.3&lt;/p&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;0.5&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;68.3&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;28.4&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;124.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;62.3&lt;/p&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;0.5&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;70.1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;93.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.4&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;301.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;150.9&lt;/p&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;0.6&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;71.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;120.6&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;379.8&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;189.9&lt;/p&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;0.6&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;73.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;64.4&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.8&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;240.6&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;120.3&lt;/p&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;0.5&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;75.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;21.1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;101.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;50.8&lt;/p&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;0.4&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;77.3&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;42.6&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;21.3&lt;/p&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;0.3&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;79.1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.4&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;26.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;13.3&lt;/p&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;0.2&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;80.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.0&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;22.3&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;11.2&lt;/p&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;0.1&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;82.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;29.2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;14.6&lt;/p&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;n.d&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;5&quot; colspan=&quot;1&quot;&gt;Monomer&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;84.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;34.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;17.5&lt;/p&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;n.d&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;86.3&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;28.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;14.4&lt;/p&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;n.d&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;88.1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;17.4&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;8.7&lt;/p&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;n.d.&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;89.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;2.8&lt;/p&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;n.d.&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;91.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;n.d.&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;0.07&lt;/p&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;p&gt;0.2&lt;/p&gt;&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>12529</offset><text>Peak	EncFtnsHretention volume (ml)	Element concentration (µM)	Derived EncFtnsHconcentration (µM)	Derived Fe/ EncFtnsH monomer	 	Ca	Fe	Zn	Se	 	Decamer	66.5	n.d.	6.7	n.d.	24.6	12.3	0.5	 	68.3	n.d.	28.4	n.d	124.5	62.3	0.5	 	70.1	2.9	93.7	2.4	301.7	150.9	0.6	 	71.9	6.9	120.6	3.7	379.8	189.9	0.6	 	73.7	1.9	64.4	0.8	240.6	120.3	0.5	 	75.5	0.9	21.1	n.d.	101.7	50.8	0.4	 	77.3	n.d.	6.2	n.d.	42.6	21.3	0.3	 	79.1	0.1	2.4	n.d.	26.5	13.3	0.2	 		80.9	1.0	1.5	n.d.	22.3	11.2	0.1	 		82.7	n.d.	0.2	n.d.	29.2	14.6	n.d	 	Monomer	84.5	n.d.	0.1	n.d.	34.9	17.5	n.d	 	86.3	n.d.	n.d	n.d.	28.9	14.4	n.d	 	88.1	n.d.	n.d.	n.d.	17.4	8.7	n.d.	 	89.9	n.d.	n.d.	n.d.	5.5	2.8	n.d.	 	91.7	n.d.	n.d.	n.d.	0.1	0.07	0.2	 	</text></passage><passage><infon key="file">tbl2.xml</infon><infon key="id">tbl2</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>13228</offset><text>Estimates of EncFtnsH molecular weight from SEC-MALLS analysis. EncFtnsH was purified from E. coli BL21(DE3) grown in minimal medium (MM) by nickel affinity chromatography and size-exclusion chromatography. Fractions from two peaks (decamer and monomer) were pooled separately (Figure 1C) and analysed by SEC-MALLS using a Superdex 200 10/300 GL column (GE Healthcare) and Viscotek SEC-MALLS instruments (Malvern Instruments) (Figure 2C). The decamer and monomer peaks were both symmetric and monodisperse, allowing the estimation of the molecular weight of the species in these fractions. The molecular weights are quoted to the nearest kDa due to the resolution limit of the instrument. The proteins analyzed by SEC-MALLS came from single protein preparation.</text></passage><passage><infon key="file">tbl2.xml</infon><infon key="id">tbl2</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>13997</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.007</text></passage><passage><infon key="file">tbl2.xml</infon><infon key="id">tbl2</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead&gt;&lt;tr&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Molecular Weight (kDa)&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Decamer peak&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Monomer peak&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Theoretical&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;133&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;13&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EncFtn&lt;sub&gt;sH&lt;/sub&gt;-decamer fractions&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;132&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;15&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EncFtn&lt;sub&gt;sH&lt;/sub&gt;-monomer fractions&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;126&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;13&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>14044</offset><text>Molecular Weight (kDa)	Decamer peak	Monomer peak	 	Theoretical	133	13	 	EncFtnsH-decamer fractions	132	15	 	EncFtnsH-monomer fractions	126	13	 	</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14189</offset><text>We purified recombinant R. rubrum EncFtn as both the full-length sequence (140 amino acids) and a truncated C-terminal hexahistidine-tagged variant (amino acids 1–96 plus the tag; herein EncFtnsH). In both cases the elution profile from size-exclusion chromatography (SEC) displayed two peaks (Figure 2A). SDS-PAGE analysis of fractions from these peaks showed that the high molecular weight peak was partially resistant to SDS and heat-induced denaturation; in contrast, the low molecular weight peak was consistent with monomeric mass of 13 kDa (Figure 2B). MALDI peptide mass fingerprinting of these bands confirmed the identity of both as EncFtn. Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the SEC fractions showed 100 times more iron in the oligomeric fraction than the monomer (Figure 2A, blue scatter points; Table 1), suggesting that EncFtn oligomerization is associated with iron binding. In order to determine the iron-loading stoichiometry in the EncFtn complex, further ICP-MS experiments were performed using selenomethionine (SeMet)-labelled protein EncFtn (Table 1). In these experiments, we observed sub-stoichiometric metal binding, which is in contrast to the classical ferritins. Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) analysis of samples taken from each peak gave calculated molecular weights consistent with a decamer for the high molecular weight peak and a monomer for the low molecular weight peak (Figure 2C, Table 2).</text></passage><passage><infon key="file">elife-18972-fig3-figsupp1.jpg</infon><infon key="id">fig3s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>15702</offset><text>Effect of metal ions on the oligomeric state of EncFtnsH in solution.</text></passage><passage><infon key="file">elife-18972-fig3-figsupp1.jpg</infon><infon key="id">fig3s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>15773</offset><text>(A/B) EncFtnsH-monomer was incubated with one mole equivalent of various metal salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column. Co2+ and Zn2+ induced the formation of the decameric form of EncFtnsH; while Mn2+, Mg2+ and Fe3+ did not significantly alter the oligomeric state of EncFtnsH.</text></passage><passage><infon key="file">elife-18972-fig3-figsupp1.jpg</infon><infon key="id">fig3s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16105</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.009</text></passage><passage><infon key="file">elife-18972-fig3-figsupp2.jpg</infon><infon key="id">fig3s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>16152</offset><text>PAGE analysis of the effect of metal ions on the oligomeric state of EncFtnsH.</text></passage><passage><infon key="file">elife-18972-fig3-figsupp2.jpg</infon><infon key="id">fig3s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16231</offset><text>50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE. (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.</text></passage><passage><infon key="file">elife-18972-fig3-figsupp2.jpg</infon><infon key="id">fig3s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16802</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.010</text></passage><passage><infon key="file">elife-18972-fig3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>16849</offset><text>Effect of Fe2+ and protein concentration on the oligomeric state of EncFtnsH in solution.</text></passage><passage><infon key="file">elife-18972-fig3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16939</offset><text>(A) Recombinant EncFtnsH was purified by Gel filtration Superdex 200 chromatography from E. coli BL21(DE3) grown in MM or in MM supplemented with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+). A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH
+in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column (GE Healthcare). Both Fe2+ salts tested induced the formation of decamer indicated by the peak between 1.2 and 1.6 ml. Monomeric and decameric samples of EncFtnsH are shown as controls. Peaks around 0.8 ml were seen as protein aggregation. (C) Analytical gel filtration of EncFtn monomer at different concentrations to illustrate the effect of protein concentration on multimerization. The major peak shows a shift towards a dimer species at high concentration of protein, but the ratio of this peak (1.5–1.8 ml) to the decamer peak (1.2–1.5 ml) does not change when compared to the low concentration sample.</text></passage><passage><infon key="file">elife-18972-fig3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>18097</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.008</text></passage><passage><infon key="file">tbl3.xml</infon><infon key="id">tbl3</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>18144</offset><text>Gel-filtration peak area ratios for EncFtnsH decamer and monomer on addition of different metal ions. EncFtnsH was produced in E. coli BL21(DE3) cultured in MM and MM with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+) and purified by gel-filtration chromatography using an Superdex 200 16/60 column (GE Healthcare). Monomer fractions of EncFtnsH purified from MM were pooled and run in subsequent analytical gel-filtration runs over the course of three days. Samples of EncFtnsH monomer were incubated with one molar equivalent of metal ion salts at room temperature for two hours before analysis by analytical gel filtration chromatography (AGF) using a Superdex 200 10/300 GL column. The area for resulting protein peaks were calculated using the Unicorn software (GE Healthcare); peak ratios were calculated to quantify the propensity of EncFtnsH to multimerize in the presence of the different metal ions. The change in the ratios of monomer to decamer over the three days of experiments may be a consequence of experimental variability, or the propensity of this protein to equilibrate towards decamer over time. The increased decamer: monomer ratio seen in the presence of Fe2+, Co2+, and Zn2+ indicates that these metal ions facilitate multimerization of the EncFtnsH protein, while the other metal ions tested do not appear to induce multimerization. The analytical gel filtration experiment was repeated twice using two independent preparations of protein, of which values calculated from one sample are presented here.</text></passage><passage><infon key="file">tbl3.xml</infon><infon key="id">tbl3</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>19662</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.011</text></passage><passage><infon key="file">tbl3.xml</infon><infon key="id">tbl3</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead&gt;&lt;tr&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Method&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Sample&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Monomer area&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Decamer area&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Decamer/Monomer&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td rowspan=&quot;2&quot; colspan=&quot;1&quot;&gt;Gel filtration Superdex 200 chromatography&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EncFtn&lt;sub&gt;sH&lt;/sub&gt;-MM&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;64.3&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;583.6&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EncFtn&lt;sub&gt;sH&lt;/sub&gt;-MM+Fe&lt;sup&gt;2+&lt;/sup&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1938.4&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;426.4&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;4&quot; colspan=&quot;1&quot;&gt;Analytical Gel filtration Day1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EncFtn&lt;sub&gt;sH&lt;/sub&gt;-decamer fractions&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;20.2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.8&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;11.2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EncFtn&lt;sub&gt;sH&lt;/sub&gt;-monomer fractions&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;21.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Fe(NH&lt;sub&gt;4)2&lt;/sub&gt;(SO&lt;sub&gt;4)2&lt;/sub&gt;/EncFtn&lt;sub&gt;sH&lt;/sub&gt;-monomer&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;11.0&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;13.0&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.8&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;FeSO&lt;sub&gt;4&lt;/sub&gt;-HCl/EncFtn&lt;sub&gt;sH&lt;/sub&gt;-monomer&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;11.3&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;11.4&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;5&quot; colspan=&quot;1&quot;&gt;Analytical Gel filtration Day2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EncFtn&lt;sub&gt;sH&lt;/sub&gt;-monomer fractions&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8.3&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;22.8&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;CoCl&lt;sub&gt;2&lt;/sub&gt;/EncFtn&lt;sub&gt;sH&lt;/sub&gt;-monomer&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;17.7&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;14.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;MnCl&lt;sub&gt;2&lt;/sub&gt;/EncFtn&lt;sub&gt;sH&lt;/sub&gt;-monomer&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;30.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ZnSO&lt;sub&gt;4&lt;/sub&gt;/EncFtn&lt;sub&gt;sH&lt;/sub&gt;-monomer&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;20.4&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;9.0&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;FeCl&lt;sub&gt;3&lt;/sub&gt;/EncFtn&lt;sub&gt;sH&lt;/sub&gt;-monomer&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;28.6&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;3&quot; colspan=&quot;1&quot;&gt;Analytical Gel filtration Day3&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;EncFtn&lt;sub&gt;sH&lt;/sub&gt;-monomer fractions&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.3&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;23.4&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;MgSO&lt;sub&gt;4&lt;/sub&gt;/EncFtn&lt;sub&gt;sH&lt;/sub&gt;-monomer&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5.8&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;30.2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Ca acetate/EncFtn&lt;sub&gt;sH&lt;/sub&gt;-monomer&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5.6&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;25.2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.2&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>19709</offset><text>Method	Sample	Monomer area	Decamer area	Decamer/Monomer	 	Gel filtration Superdex 200 chromatography	EncFtnsH-MM	64.3	583.6	0.1	 	EncFtnsH-MM+Fe2+	1938.4	426.4	4.5	 	Analytical Gel filtration Day1	EncFtnsH-decamer fractions	20.2	1.8	11.2	 	EncFtnsH-monomer fractions	2.9	21.9	0.1	 	Fe(NH4)2(SO4)2/EncFtnsH-monomer	11.0	13.0	0.8	 	FeSO4-HCl/EncFtnsH-monomer	11.3	11.4	1.0	 	Analytical Gel filtration Day2	EncFtnsH-monomer fractions	8.3	22.8	0.4	 	CoCl2/EncFtnsH-monomer	17.7	14.5	1.2	 	MnCl2/EncFtnsH-monomer	3.1	30.5	0.1	 	ZnSO4/EncFtnsH-monomer	20.4	9.0	2.3	 	FeCl3/EncFtnsH-monomer	3.9	28.6	0.1	 	Analytical Gel filtration Day3	EncFtnsH-monomer fractions	6.3	23.4	0.3	 	MgSO4/EncFtnsH-monomer	5.8	30.2	0.2	 	Ca acetate/EncFtnsH-monomer	5.6	25.2	0.2	 	</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20464</offset><text>We purified EncFtnsH from E. coli grown in MM with or without the addition of 1 mM Fe(NH4)2(SO4)2. The decamer to monomer ratio in the sample purified from cells grown in iron-supplemented media was 4.5, while that from the iron-free media was 0.11, suggesting that iron induces the oligomerization of EncFtnsH in vivo (Figure 3A, Table 3). To test the metal-dependent oligomerization of EncFtnsH in vitro, we incubated the protein with various metal cations and subjected samples to analytical SEC and non-denaturing PAGE. Of the metals tested, only Fe2+, Zn2+ and Co2+ induced the formation of significant amounts of the decamer (Figure 3B, Figure 3—figure supplement 1/2). While Fe2+ induces the multimerization of EncFtnsH, Fe3+ in the form of FeCl3 does not have this effect on the protein, highlighting the apparent preference this protein has for the ferrous form of iron. To determine if the oligomerization of EncFtnsH was concentration dependent we performed analytical SEC at 90 and 700 µM protein concentration (Figure 3C). At the higher concentration, no increase in the decameric form of EncFtn was observed; however, the shift in the major peak from the position of the monomer species indicated a tendency to dimerize at high concentration.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>21725</offset><text>Crystal structure of EncFtnsH</text></passage><passage><infon key="file">elife-18972-fig4-figsupp1.jpg</infon><infon key="id">fig4s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>21755</offset><text>Electrostatic surface of EncFtnsH.</text></passage><passage><infon key="file">elife-18972-fig4-figsupp1.jpg</infon><infon key="id">fig4s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>21790</offset><text>The solvent accessible surface of EncFtnsH is shown, colored by electrostatic potential as calculated using the APBS plugin in PyMOL. Negatively charged regions are colored red and positive regions in blue, neutral regions in grey. (A) View of the surface of the EncFtnsH decamer looking down the central axis. (B) Orthogonal view of (A). (C) Cutaway view of (B) showing the charge distribution within the central cavity.</text></passage><passage><infon key="file">elife-18972-fig4-figsupp1.jpg</infon><infon key="id">fig4s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>22212</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.013</text></passage><passage><infon key="file">elife-18972-fig4.jpg</infon><infon key="id">fig4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>22259</offset><text>Crystal structure of EncFtnsH.</text></passage><passage><infon key="file">elife-18972-fig4.jpg</infon><infon key="id">fig4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>22290</offset><text>(A) Overall architecture of EncFtnsH. Transparent solvent accessible surface view with α-helices shown as tubes and bound metal ions as spheres. Alternating subunits are colored blue and green for clarity. The doughnut-like decamer is 7 nm in diameter and 4.5 nm thick. (B) Monomer of EncFtnsH shown as a secondary structure cartoon. (C/D) Dimer interfaces formed in the decameric ring of EncFtnsH. Subunits are shown as secondary structure cartoons and colored blue and green for clarity. Bound metal ions are shown as orange spheres for Fe3+ and grey and white spheres for Ca2+.</text></passage><passage><infon key="file">elife-18972-fig4.jpg</infon><infon key="id">fig4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>22875</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.012</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22922</offset><text>We determined the crystal structure of EncFtnsH by molecular replacement to 2.0 Å resolution (see Table 1 for X-ray data collection and refinement statistics). The crystallographic asymmetric unit contained thirty monomers of EncFtn with visible electron density for residues 7 – 96 in each chain. The protein chains were arranged as three identical annular decamers, each with D5 symmetry. The decamer has a diameter of 7 nm and thickness of 4 nm (Figure 4A). The monomer of EncFtn has an N-terminal 310-helix that precedes two 4 nm long antiparallel α-helices arranged with their long axes at 25° to each other; these helices are followed by a shorter 1.4 nm helix projecting at 70° from α2 (Figure 4B). The C-terminal region of the crystallized construct extends from the outer circumference of the ring, indicating that the encapsulin localization sequence in the full-length protein is on the exterior of the ring and is thus free to interact with its binding site on the encapsulin shell protein.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23933</offset><text>The monomer of EncFtnsH forms two distinct dimer interfaces within the decamer (Figure 4 C/D). The first dimer is formed from two monomers arranged antiparallel to each other, with α1 from each monomer interacting along their lengths and α3 interdigitating with α2 and α3 of the partner chain. This interface buries one third of the surface area from each partner and is stabilized by thirty hydrogen bonds and fourteen salt bridges (Figure 4C). The second dimer interface forms an antiparallel four-helix bundle between helices 1 and 2 from each monomer (Figure 4D). This interface is less extensive than the first and is stabilized by twenty-one hydrogen bonds, six salt bridges, and a number of metal ions.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24659</offset><text>The arrangement of ten monomers in alternating orientation forms the decamer of EncFtn, which assembles as a pentamer of dimers (Figure 4A). Each monomer lies at 45° relative to the vertical central-axis of the ring, with the N-termini of alternating subunits capping the center of the ring at each end, while the C-termini are arranged around the circumference. The central hole in the ring is 2.5 nm at its widest in the center of the complex, and 1.5 nm at its narrowest point near the outer surface, although it should be noted that a number of residues at the N-terminus are not visible in the crystallographic electron density and these may occupy the central channel. The surface of the decamer has distinct negatively charged patches, both within the central hole and on the outer circumference, which form spokes through the radius of the complex (Figure 4—figure supplement 1).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>25550</offset><text>EncFtn ferroxidase center</text></passage><passage><infon key="file">elife-18972-fig5-figsupp1.jpg</infon><infon key="id">fig5s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>25576</offset><text>Putative ligand-binding site in EncFtnsH.</text></passage><passage><infon key="file">elife-18972-fig5-figsupp1.jpg</infon><infon key="id">fig5s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>25618</offset><text>(A) Wall-eyed stereo view of the dimer interface of EncFtn. Protein chains are shown as sticks, with 2mFo-DFc electron density shown in blue mesh and contoured at 1.5 σ and mFo-DFc shown in green mesh and contoured at 3 σ. (B) Wall-eyed stereo view of putative metal binding site at the external surface of EncFtnsH. Protein chains and electron density maps are shown as in (A).</text></passage><passage><infon key="file">elife-18972-fig5-figsupp1.jpg</infon><infon key="id">fig5s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26005</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.015</text></passage><passage><infon key="file">elife-18972-fig5.jpg</infon><infon key="id">fig5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>26052</offset><text>EncFtnsH metal binding sites.</text></passage><passage><infon key="file">elife-18972-fig5.jpg</infon><infon key="id">fig5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26082</offset><text>(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH. Protein residues are shown as sticks with blue and green carbons for the different subunits, iron ions are shown as orange spheres and calcium as grey spheres, and the glycolic acid ligand is shown with yellow carbon atoms coordinated above the di-iron center. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ and the NCS-averaged anomalous difference map is shown as an orange mesh and contoured at 10 σ. (B) Iron coordination within the FOC including residues Glu32, Glu62, His65 and Tyr39 from two chains. Protein and metal ions are shown as in A. Coordination between the protein and iron ions is shown as yellow dashed lines with distances indicated. (C) Coordination of calcium within the dimer interface by four glutamic acid residues (E31 and E34 from two chains). The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.</text></passage><passage><infon key="file">elife-18972-fig5.jpg</infon><infon key="id">fig5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27371</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.014</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27418</offset><text>The electron density maps of the initial EncFtnsH model displayed significant positive peaks in the mFo-DFc map at the center of the 4-helix bundle dimer (Figure 5—figure supplement 1). Informed by the ICP-MS data indicating the presence of iron in the protein we collected diffraction data at the experimentally determined iron absorption edge (1.74 Å) and calculated an anomalous difference Fourier map using this data. Inspection of this map showed two 10-sigma peaks between residues Glu32, Glu62 and His65 of two adjacent chains, and a statistically smaller 5-sigma peak between residues Glu31 and Glu34 of the two chains. Modeling metal ions into these peaks and refinement of the anomalous scattering parameters allowed us to identify these as two iron ions and a calcium ion respectively (Figure 5A). An additional region of asymmetric electron density near the di-iron binding site in the mFo-DFc map was modeled as glycolic acid, presumably a breakdown product of the PEG 3350 used for crystallization. This di-iron center has an Fe-Fe distance of 3.5 Å, Fe-Glu-O distances between 2.3 and 2.5 Å, and Fe-His-N distances of 2.5 Å (Figure 5B). This coordination geometry is consistent with the di-nuclear ferroxidase center (FOC) found in ferritin. It is interesting to note that although we did not add any additional iron to the crystallization trials, the FOC was fully occupied with iron in the final structure, implying that this site has a very high affinity for iron.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>28907</offset><text>The calcium ion coordinated by Glu31 and Glu34 adopts heptacoordinate geometry, with coordination distances of 2.5 Å between the metal ion and carboxylate oxygens of Glu31 and Glu34 (E31/34-site). A number of ordered solvent molecules are also coordinated to this metal ion at a distance of 2.5 Å. This heptacoordinate geometry is common in crystal structures with calcium ions (Figure 5C). While ICP-MS indicated that there were negligible amounts of calcium in the purified protein, the presence of 140 mM calcium acetate in the crystallization mother liquor favors the coordination of calcium at this site. The fact that the protein does not multimerize in solution in the presence of Fe3+ may indicate that these metal binding sites have a lower affinity for the ferric form of iron, which is the product of the ferroxidase reaction. A number of additional metal-ions were present at the outer circumference of at least one decamer in the asymmetric unit (Figure 5D). These ions are coordinated by His57, Glu61 and Glu64 from both chains in the FOC dimer and are 4.5 Å apart; Fe-Glu-O distances are between 2.5 and 3.5 Å and the Fe-His-N distances are 4 and 4.5 Å.</text></passage><passage><infon key="file">elife-18972-fig6-figsupp1.jpg</infon><infon key="id">fig6s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>30081</offset><text>Comparison of quaternary structure of EncFtnsH and ferritin.</text></passage><passage><infon key="file">elife-18972-fig6-figsupp1.jpg</infon><infon key="id">fig6s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>30142</offset><text>(A) Aligned FOC of EncFtnsH and Pseudo-nitzschia multiseries ferritin (PmFtn). The metal binding site residues from two EncFtnsH chains are shown in green and blue, while the PmFtn is shown in orange. Fe2+ in the FOC is shown as orange spheres and Ca2+ in EncFtnsH is shown as a grey sphere. The two-fold symmetry axis of the EncFtn FOC is shown with a grey arrow (B) Cross-section surface view of quaternary structure of EncFtnsH and PmFtn as aligned in (A) (dashed black box). The central channel of EncFtnsH is spatially equivalent to the outer surface of ferritin and its outer surface corresponds to the mineralization surface within ferritin.</text></passage><passage><infon key="file">elife-18972-fig6-figsupp1.jpg</infon><infon key="id">fig6s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>30791</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.017</text></passage><passage><infon key="file">elife-18972-fig6.jpg</infon><infon key="id">fig6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>30838</offset><text>Comparison of the symmetric metal ion binding site of EncFtnsH and the ferritin FOC.</text></passage><passage><infon key="file">elife-18972-fig6.jpg</infon><infon key="id">fig6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>30923</offset><text>(A) Structural alignment of the FOC residues in a dimer of EncFtnsH (green/blue) with a monomer of Pseudo-nitzschia multiseries ferritin (PmFtn) (PDBID: 4ITW) (orange). Iron ions are shown as orange spheres and a single calcium ion as a grey sphere. Residues within the FOC are conserved between EncFtn and ferritin PmFtn, with the exception of residues in the position equivalent to H65’ in the second subunit in the dimer (blue). The site in EncFtn with bound calcium is not present in other family members. (B) Secondary structure of aligned dimeric EncFtnsH and monomeric ferritin highlighting the conserved four-helix bundle. EncFtnsH monomers are shown in green and blue and aligned PmFtn monomer in orange as in A. (C) Cartoon of secondary structure elements in EncFtn dimer and ferritin. In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between α2 and α3 in ferritin PmFtn.</text></passage><passage><infon key="file">elife-18972-fig6.jpg</infon><infon key="id">fig6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>31941</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.016</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>31988</offset><text>Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins  (Figure 6A). The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65’) (Figure 6A). Structural superimposition of the FOCs of ferritin and EncFtn brings the four-helix bundle of the ferritin fold into close alignment with the EncFtn dimer, showing that the two families of proteins have essentially the same architecture around the di-iron center (Figure 6B). The linker connecting helices 2 and 3 of ferritin is congruent with the start of the C-terminal helix of one EncFtn monomer and the N-terminal 310 helix of the second monomer (Figure 6C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>33004</offset><text>Mass spectrometry of the EncFtn assembly</text></passage><passage><infon key="file">elife-18972-fig7-figsupp1.jpg</infon><infon key="id">fig7s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>33045</offset><text>Native IM-MS analysis of the apo-EncFtnsH monomer.</text></passage><passage><infon key="file">elife-18972-fig7-figsupp1.jpg</infon><infon key="id">fig7s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>33096</offset><text>(A) Mass spectrum of apo-EncFtnsH acquired from 100 mM ammonium acetate pH 8.0 under native MS conditions. The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1. The +8 to +15 protein charge states have observed CCS between 20–26 nm2, which is significantly higher than the calculated CCS for an EncFtnsH monomer taken from the decameric assembly crystal structure (15.8 nm2). The mobility of the +7 charge state displays broad drift-time distribution with maxima consistent with CCS of 15.9 and 17.9 nm2. Finally, the 6+ charge state of EncFtnsH has mobility consistent with a CCS of 12.3 nm2, indicating a more compact/collapsed structure. It is clear from this data that apo-EncFtnsH exists in several gas phase conformations. The range of charge states occupied by the protein (6+ to 15+) and the range of CCS in which the protein is observed (12.3 nm2 – 26 nm2) are both large. In addition, many of the charge states observed have higher charge than the theoretical maximal charge on spherical globular protein, as determined by the De La Mora relationship (ZR = 0.0778m; for the EncFtnsH monomer ZR = 8.9) Fernandez. As described by Beveridge et al., all these factors are indicative of a disordered protein.</text></passage><passage><infon key="file">elife-18972-fig7-figsupp1.jpg</infon><infon key="id">fig7s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>35036</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.019</text></passage><passage><infon key="file">elife-18972-fig7-figsupp2.jpg</infon><infon key="id">fig7s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>35083</offset><text>Gas-phase disassembly of the holo-EncFtnsH decameric assembly.</text></passage><passage><infon key="file">elife-18972-fig7-figsupp2.jpg</infon><infon key="id">fig7s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>35146</offset><text>The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles). The mass of the ejected-monomer is consistent with apo- EncFtnsH (13.2 kDa), suggesting unfolding of the monomer (and loss of Fe) occurs during ejection from the complex. This observation of asymmetric charge partitioning of the sub-complexes with respect to the mass of the complex is consistent with the 'typical' pathway of dissociation of protein assemblies by CID, as described by. In addition, a third, lower abundance, charge state distribution is observed which overlaps the EncFtn ejected monomer charge state distribution; this region of the spectrum is highlighted in (B). This distribution is consistent with an ejected EncFtnsH dimer (orange circles). Interestingly, closer analysis of the individual charge state of this dimeric CID product shows that this sub-complex exists in three forms – displaying mass consistent with an EncFtnsH dimer binding 0, 1, and 2 Fe ions. This is highlighted in (C), where the 15+ charge state of the EncFtnsH dimer is shown; 3 peaks are observed with m/z 1760.5, 1763.8, and 1767.0 Th – the lowest peak corresponds to neutral masses of 26392.5 Da [predicted EncFtnsH dimer, (C572H884N172O185S2)2; 26388.6 Da]. The two further peaks have a delta-mass of ~+50 Da, consistent with Fe binding. We interpret these observations as partial ‘atypical’ CID fragmentation of the decameric complex – i.e. fragmentation of the initial complex with retention of subunit and ligand interactions. A schematic summary of these results is displayed in (D). We postulate the high stability of this iron-bound dimer sub-complex is due to the metal coordination at the dimer interface, increasing the strength of the dimer interface. Taken together, these observations support our findings that the topology of the decameric EncFtnsH assembly is arranged as a pentamer of dimers, with two Fe ions at each dimer interface.</text></passage><passage><infon key="file">elife-18972-fig7-figsupp2.jpg</infon><infon key="id">fig7s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>37473</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.020</text></passage><passage><infon key="file">elife-18972-fig7.jpg</infon><infon key="id">fig7</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>37520</offset><text>Native mass spectrometry and ion mobility analysis of iron loading in EncFtnsH.</text></passage><passage><infon key="file">elife-18972-fig7.jpg</infon><infon key="id">fig7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>37600</offset><text>All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Native nanoelectrospray ionization (nESI) mass spectrometry of EncFtnsH at varying iron concentrations. A1, nESI spectrum of iron-free EncFtnsH displays a charge state distribution consistent with EncFtnsH monomer (blue circles, 13,194 Da). Addition of 100 µM (A2) and 300 µM (A3) Fe2+ results in the appearance of a second higher molecular weight charge state distribution consistent with a decameric assembly of EncFtnsH (green circles, 132.6 kDa). (B) Ion mobility (IM)-MS of the iron-bound holo-EncFtnsH decamer. Top, Peaks corresponding to the 22+ to 26+ charge states of a homo-decameric assembly of EncFtnsH are observed (132.6 kDa). Top Insert, Analysis of the 24+ charge state of the assembly at m/z 5528.2 Th. The theoretical average m/z of the 24+ charge state with no additional metals bound is marked by a red line (5498.7 Th); the observed m/z of the 24+ charge state indicates that the EncFtnsH assembly binds between 10 (green line, 5521.1 Th) and 15 Fe ions (blue line, 5532.4 Th) per decamer. Bottom, The arrival time distributions (ion mobility data) of all ions in the EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). Bottom right, The arrival time distribution of the 24+ charge state (dashed blue box) has been extracted and plotted. The drift time for this ion is shown (ms), along with the calibrated collision cross section (CCS), Ω (nm2).</text></passage><passage><infon key="file">elife-18972-fig7.jpg</infon><infon key="id">fig7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>39130</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.018</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>39177</offset><text>In order to confirm the assignment of the oligomeric state of EncFtnsH and investigate further the Fe2+-dependent assembly, we used native nano-electrospray ionization (nESI) and ion-mobility mass spectrometry (IM-MS). As described above, by recombinant production of EncFtnsH in minimal media we were able to limit the bioavailability of iron. Native MS analysis of EncFtnsH produced in this way displayed a charge state distribution consistent with an EncFtnsH monomer (blue circles, Figure 7A1) with an average neutral mass of 13,194 Da, in agreement with the predicted mass of the EncFtnsH protein (13,194.53 Da). Under these conditions, no significant higher order assembly was observed and the protein did not have any coordinated metal ions. Titration with Fe2+ directly before native MS analysis resulted in the appearance of a new charge state distribution, consistent with an EncFtnsH decameric assembly (+22 to +26; 132.65 kDa) (Figure 7A2/3). After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa). MS analysis of EncFtnsH after addition of further Fe2+ did not result in iron loading above this stoichiometry. Therefore, the extent of iron binding seen is limited to the FOC and Glu31/34 secondary metal binding site. These data suggest that the decameric assembly of EncFtnsH does not accrue iron in the same manner as classical ferritin, which is able to sequester around 4500 iron ions within its nanocage. Ion mobility analysis of the EncFtnsH decameric assembly, collected with minimal collisional activation, suggested that it consists of a single conformation with a collision cross section (CCS) of 58.2 nm2 (Figure 7B). This observation is in agreement with the calculated CCS of 58.7 nm2derived from our crystal structure of the EncFtnsH decamer. By contrast, IM-MS measurements of the monomeric EncFtnsH at pH 8.0 under the same instrumental conditions revealed that the metal-free protein monomer exists in a wide range of charge states (+6 to +16) and adopts many conformations in the gas phase with collision cross sections ranging from 12 nm2 to 26 nm2 (Figure 7—figure supplement 1). These observations are indicative of an unstructured protein with little secondary or tertiary structure. Thus, IM-MS studies highlight that higher order structure in EncFtnsH is mediated/stabilized by metal binding, an observation that is in agreement with our solution studies. Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>42254</offset><text>We subsequently performed gas phase disassembly of the decameric EncFtnsH using collision-induced dissociation (CID) tandem mass spectrometry. Under the correct CID conditions, protein assemblies can dissociate with retention of subunit and ligand interactions, and thus provide structurally-informative evidence as to the topology of the original assembly; this has been termed ‘atypical’ dissociation. For EncFtnsH, this atypical dissociation pathway was clearly evident; CID of the EncFtnsH decamer resulted in the appearance of a dimeric EncFtnsH subcomplex containing 0, 1, or 2 iron ions (Figure 7—figure supplement 2). In light of the crystal structure, this observation can be rationalized as dissociation of the EncFtnsH decamer by disruption of the non-FOC interface with at least partial retention of the FOC interface and the FOC-Fe. Thus, this observation supports our crystallographic assignment of the overall topology of the EncFtnsH assembly as a pentameric assembly of dimers with two iron ions located at the FOC dimer interface. In addition, this analysis provides evidence that the overall architecture of the complex is consistent in the crystal, solution and gas phases.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>43454</offset><text>Ferroxidase activity</text></passage><passage><infon key="file">elife-18972-fig8-figsupp1.jpg</infon><infon key="id">fig8s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>43475</offset><text>TEM visualization of iron-loaded bacterial nanocompartments and ferritin.</text></passage><passage><infon key="file">elife-18972-fig8-figsupp1.jpg</infon><infon key="id">fig8s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>43549</offset><text>Decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin, at 8.5 µM, were mixed with 147 µM, 1 mM, 1 mM and 215 µM acidic Fe(NH4)2(SO4)2, respectively. Protein mixtures were incubated at room temperature for 1 hr prior to TEM analysis with or without uranyl acetate stain. (A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.</text></passage><passage><infon key="file">elife-18972-fig8-figsupp1.jpg</infon><infon key="id">fig8s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>44180</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.022</text></passage><passage><infon key="file">elife-18972-fig8.jpg</infon><infon key="id">fig8</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>44227</offset><text>Spectroscopic evidence for the ferroxidase activity and comparison of iron loading capacity of apoferritin, EncFtnsH, encapsulin, and EncFtn-Enc.</text></passage><passage><infon key="file">elife-18972-fig8.jpg</infon><infon key="id">fig8</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>44373</offset><text>(A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer. The background oxidation of iron at 20 and 100 μM in enzyme-free controls are shown for reference. (B) Encapsulin and EncFtn-Enc complexes at 10 μM asymmetric unit concentration were incubated with Fe2+ at 20 and 100 μM and progress curves for iron oxidation at A315 were measured in a UV/visible spectrophotometer. Enzyme free controls for background oxidation of Fe2+ are shown for reference. (C) Histogram of the iron loading capacity per biological assembly of EncFtnsH, encapsulin, EncFtn-Enc and apoferritin. The results shown are for three technical replicates and represent the optimal iron loading by the complexes after three hours when incubated with Fe2+.</text></passage><passage><infon key="file">elife-18972-fig8.jpg</infon><infon key="id">fig8</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>45367</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.021</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>45414</offset><text>In light of the identification of an iron-loaded FOC in the crystal structure of EncFtn and our native mass spectrometry data, we performed ferroxidase and peroxidase assays to demonstrate the catalytic activity of this protein. In addition, we also assayed equine apoferritin, an example of a classical ferritin enzyme, as a positive control. Unlike the Dps family of ferritin-like proteins, EncFtn showed no peroxidase activity when assayed with the substrate ortho-phenylenediamine. The ferroxidase activity of EncFtnsH was measured by recording the progress curve of Fe2+ oxidation to Fe3+ at 315 nm after addition of 20 and 100 µM Fe2+ (2 and 10 times molar ratio Fe2+/FOC). In both experiments the rate of oxidation was faster than background oxidation of Fe2+ by molecular oxygen, and was highest for 100 µM Fe2+ (Figure 8A). These data show that recombinant EncFtnsH acts as an active ferroxidase enzyme. When compared to apoferritin, EncFtnsH oxidized Fe2+ at a slower rate and the reaction did not run to completion over the 1800 s of the experiment. Addition of higher quantities of iron resulted in the formation of a yellow/red precipitate at the end of the reaction. We also performed these assays on purified recombinant encapsulin; which, when assayed alone, did not display ferroxidase activity above background Fe2+ oxidation (Figure 8B). In contrast, complexes of the full EncFtn encapsulin nanocompartment (i.e. the EncFtn-Enc protein complex) displayed ferroxidase activity comparable to apoferritin without the formation of precipitates (Figure 8B).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>46994</offset><text>We attributed the precipitates observed in the EncFtnsH ferroxidase assay to the production of insoluble Fe3+ complexes, which led us to propose that EncFtn does not directly store Fe3+ in a mineral form. This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>47549</offset><text>To analyze the products of these reactions and determine whether the EncFtn and encapsulin were able to store iron in a mineral form, we performed TEM on the reaction mixtures from the ferroxidase assay. The EncFtnsH reaction mixture showed the formation of large, irregular electron-dense precipitates (Figure 8—figure supplement 1A). A similar distribution of particles was observed after addition of Fe2+ to the encapsulin protein (Figure 8—figure supplement 1B). In contrast, addition of Fe2+ to the EncFtn-Enc nanocompartment resulted in small, highly regular, electron dense particles of approximately 5 nm in diameter (Figure 8—figure supplement 1C); we interpret these observations as controlled mineralization of iron within the nanocompartment. Addition of Fe2+ to apoferritin resulted in a mixture of large particles and small (~2 nm) particles consistent with partial mineralization by the ferritin and some background oxidation of the iron (Figure 8—figure supplement 1D). Negative stain TEM of these samples revealed that upon addition of iron, the EncFtnsH protein showed significant aggregation (Figure 8—figure supplement 1F); while the encapsulin, EncFtn-Enc system, and apoferritin are present as distinct nanocompartments without significant protein aggregation (Figure 8—figure supplement 1G–I).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>48882</offset><text>Iron storage in encapsulin nanocompartments</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>48926</offset><text>The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage. To investigate this further, we added Fe2+ at various concentrations to samples of apo-ferritin, EncFtn, isolated encapsulin, and the EncFtn-Enc protein complex, and subjected these samples to a ferrozine assay to quantify the amount of iron associated with the proteins after three hours of incubation. The maximum iron loading capacity of these systems was calculated as the quantity of iron per biological assembly (Figure 8C). In this assay, the EncFtnsH decamer binds a maximum of around 48 iron ions before excess iron induces protein precipitation. The encapsulin shell protein can sequester about 2200 iron ions before significant protein loss occurs, and the reconstituted EncFtn-Enc nanocompartment sequestered about 4150 iron ions. This latter result is significantly more than the apoferritin used in our assay, which sequesters approximately 570 iron ions in this assay (Figure 8C, Table 5).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>50186</offset><text>Consideration of the functional oligomeric states of these proteins, where EncFtn is a decamer and encapsulin forms an icosahedral cage, and estimation of the iron loading capacity of these complexes gives insight into the role of the two proteins in iron storage and mineralization. EncFtn decamers bind up to 48 iron ions (Figure 8C), which is significantly higher than the stoichiometry of fifteen metal ions visible in the FOC and E31/34-site of the crystal structure of the EncFtnsH decamer and our MS analysis. The discrepancy between these solution measurements and our MS analysis may indicate that there are additional metal-binding sites on the interior channel and exterior faces of the protein; this is consistent with our identification of a number of weak metal-binding sites at the surface of the protein in the crystal structure (Figure 5D). These observations are consistent with hydrated Fe2+ ions being channeled to the active site from the E31/34-site and the subsequent exit of Fe3+ products on the outer surface, as is seen in other ferritin family proteins. While the isolated encapsulin shell does not display any ferroxidase activity, it binds around 2200 iron ions in our assay (Table 5). This implies that the shell can bind a significant amount of iron on its outer and inner surfaces. While the maximum reported loading capacity of classical ferritins is approximately 4500 iron ions, in our assay system we were only able to load apoferritin with around 570 iron ions. However, the recombinant EncFtn-Enc nanocompartment was able to bind over 4100 iron ions in the same time period, over seven times the amount seen for the apoferritin. We note we do not reach the experimental maximum iron loading for apoferritin and therefore the total iron-loading capacity of our system may be significantly higher than in this experimental system.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>52055</offset><text>Taken together, our data show that EncFtn can catalytically oxidize Fe2+ to Fe3+; however, iron binding in EncFtn is limited to the FOC and several surface metal binding sites. In contrast, the encapsulin protein displays no catalytic activity, but has the ability to bind a considerable amount of iron. Finally, the EncFtn-Enc nanocompartment complex retains the catalytic activity of EncFtn, and sequesters iron within the encapsulin shell at a higher level than the isolated components of the system, and at a significantly higher level than the classical ferritins. Furthermore, our recombinant nanocompartments may not have the physiological subunit stoichiometry, and the iron-loading capacity of native nanocompartments is potentially much higher than the level we have observed.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>52843</offset><text>Mutagenesis of the EncFtnsHferroxidase center</text></passage><passage><infon key="file">elife-18972-fig9.jpg</infon><infon key="id">fig9</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>52889</offset><text>Purification of recombinant R. rubrum EncFtnsH FOC mutants.</text></passage><passage><infon key="file">elife-18972-fig9.jpg</infon><infon key="id">fig9</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>52949</offset><text>Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex 200 size-exclusion chromatography. (A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.</text></passage><passage><infon key="file">elife-18972-fig9.jpg</infon><infon key="id">fig9</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>53626</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.023</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>53673</offset><text>To investigate the structural and biochemical role played by the metal binding residues in the di-iron FOC of EncFtnsH we produced alanine mutations in each of these residues: Glu32, Glu62, and His65. These EncFtnsH mutants were produced in E. coli cells grown in MM, both in the absence and presence of additional iron. The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9). For all of the mutants studied, no change in oligomerization state was apparent upon addition of Fe2+ in vitro.</text></passage><passage><infon key="file">elife-18972-fig10.jpg</infon><infon key="id">fig10</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>54339</offset><text>Native mass spectrometry of EncFtnsH mutants.</text></passage><passage><infon key="file">elife-18972-fig10.jpg</infon><infon key="id">fig10</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>54385</offset><text>All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles). (C) E62A EncFtnsH displays charge states consistent with a decamer (green circles). (D) H65A EncFtnsH displays charge states consistent with both monomer (blue circles) and dimer (purple circles).</text></passage><passage><infon key="file">elife-18972-fig10.jpg</infon><infon key="id">fig10</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>54994</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.024</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>55041</offset><text>In addition to SEC studies, native mass spectrometry of the apo-EncFtnsH mutants was performed and compared with the wild-type apo-EncFtnsH protein (Figure 10). As described above, the apo-EncFtnsH has a charge state distribution consistent with an unstructured monomer, and decamer formation is only initiated upon addition of ferrous iron. Both the E32A mutant and E62A mutant displayed charge state distributions consistent with decamers, even in the absence of Fe2+. This gas-phase observation is consistent with SEC measurements, which indicate both of these variants were also decamers in solution. Thus it seems that these mutations allow the decamer to form in the absence of iron in the FOC. In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>55950</offset><text>We propose that the observed differences in the oligomerization state of the E32A and E62A mutants compared to wild-type are due to the changes in the electrostatic environment within the FOC. At neutral pH the glutamic acid residues are negatively charged, while the histidine residues are predominantly in their uncharged state. In the wild-type (WT) EncFtnsH this leads to electrostatic repulsion between subunits in the absence of iron. Coordination of Fe2+ in this site stabilizes the dimer and reconstitutes the active FOC. The geometric arrangement of Glu32 and Glu62 in the FOC explains their behavior in solution and the gas phase, where they both favor the formation of decamers due to the loss of a repulsive negative charge. The FOC in the H65A mutant is destabilized through the loss of this metal coordinating residue and potential positive charge carrier, thus favoring the monomer in solution and the gas phase.</text></passage><passage><infon key="file">tbl4.xml</infon><infon key="id">tbl4</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>56879</offset><text>Data collection and refinement statistics. Statistics for the highest-resolution shell are shown in parentheses. Friedel mates were averaged when calculating reflection numbers and statistics.</text></passage><passage><infon key="file">tbl4.xml</infon><infon key="id">tbl4</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>57072</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.025</text></passage><passage><infon key="file">tbl4.xml</infon><infon key="id">tbl4</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead&gt;&lt;tr&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;styled-content style=&quot;color: #000000;&quot;&gt;WT&lt;/styled-content&gt;&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;styled-content style=&quot;color: #000000;&quot;&gt;E32A&lt;/styled-content&gt;&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;styled-content style=&quot;color: #000000;&quot;&gt;E62A&lt;/styled-content&gt;&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;styled-content style=&quot;color: #000000;&quot;&gt;H65A&lt;/styled-content&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Data collection&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Wavelength (Å)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.74&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.73&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.73&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.74&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution range (Å)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;49.63 - 2.06 &lt;break/&gt;(2.10 - 2.06)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;48.84 - 2.59 &lt;break/&gt;(2.683 - 2.59)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;48.87 - 2.21 &lt;break/&gt;(2.29 - 2.21)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;48.86 - 2.97 &lt;break/&gt;(3.08 - 2.97)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Space group&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;P&lt;/italic&gt; 1 2&lt;sub&gt;1&lt;/sub&gt; 1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;P&lt;/italic&gt; 1 2&lt;sub&gt;1&lt;/sub&gt; 1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;P&lt;/italic&gt; 1 2&lt;sub&gt;1&lt;/sub&gt; 1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;P&lt;/italic&gt; 1 2&lt;sub&gt;1&lt;/sub&gt; 1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Unit cell (Å) &lt;italic&gt;a&lt;/italic&gt;
+&lt;break/&gt; b &lt;break/&gt;
+&lt;italic&gt; c&lt;/italic&gt;
+&lt;break/&gt; β (°)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.18 &lt;break/&gt;120.53 &lt;break/&gt;140.30 &lt;break/&gt;95.36&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;97.78 &lt;break/&gt;120.28 &lt;break/&gt;140.53 &lt;break/&gt;95.41&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.09 &lt;break/&gt;120.23 &lt;break/&gt;140.36 &lt;break/&gt;95.50&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.03 &lt;break/&gt;120.29 &lt;break/&gt;140.43 &lt;break/&gt;95.39&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Total reflections&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1,264,922 &lt;break/&gt;(41,360)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;405,488 &lt;break/&gt;(36,186)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1,069,345 &lt;break/&gt;(95,716)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;323,853 &lt;break/&gt;(32,120)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Unique reflections&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;197,873 &lt;break/&gt;(8,766)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100,067 &lt;break/&gt;(9,735)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;162,379 &lt;break/&gt;(15,817)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;66,658 &lt;break/&gt;(6,553)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Multiplicity&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.4 (4.7)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4.1 (3.7)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.6 (6.1)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4.9 (4.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Anomalous multiplicity&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.2 (2.6)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;N/A&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;N/A&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;N/A&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Completeness (%)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.2 (88.6)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.0 (97.0)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100 (97.0)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100 (99.0)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Anomalous completeness (%)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;96.7 (77.2)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;N/A&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;N/A&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;N/A&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Mean I/sigma(I)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;10.6 (1.60)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8.46 (1.79)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;13.74 (1.80)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8.09 (1.74)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Wilson B-factor&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;26.98&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;40.10&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;33.97&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;52.20&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;R&lt;sub&gt;merge&lt;/sub&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.123 (0.790)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.171 (0.792)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.0979 (1.009)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.177 (0.863)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;R&lt;sub&gt;meas&lt;/sub&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.147 (0.973)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.196 (0.923)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.1064 (1.107)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.199 (0.966)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;CC1/2&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.995 (0.469)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.985 (0.557)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.998 (0.642)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.989 (0.627)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;CC*&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.999 (0.846)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.996 (0.846)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.999 (0.884)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.997 (0.878)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Image DOI&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;10.7488/ds/1342&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;10.7488/ds/1419&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;10.7488/ds/1420&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;10.7488/ds/1421&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Refinement&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;R&lt;sub&gt;work&lt;/sub&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.171 (0.318)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.183 (0.288)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.165 (0.299)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.186 (0.273)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;R&lt;sub&gt;free&lt;/sub&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.206 (0.345)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.225 (0351)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.216 (0.364)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.237 (0.325)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Number of non-hydrogen atoms&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;23,222&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;22,366&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;22,691&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;22,145&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;macromolecules&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;22,276&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;22,019&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;21,965&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;22,066&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ligands&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;138&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;24&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;74&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;water&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;808&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;339&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;702&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Protein residues&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2,703&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2,686&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2,675&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2,700&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RMS(bonds) (Å)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.012&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.005&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.011&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.002&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RMS(angles) (°)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.26&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.58&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.02&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.40&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Ramachandran favored (%)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;100&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Ramachandran allowed (%)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Ramachandran outliers (%)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Clash score&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.42&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.42&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.79&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.97&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Average B-factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;33.90&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;42.31&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;41.34&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;47.68&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;macromolecules&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;33.80&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;42.35&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;41.31&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;47.60&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ligands&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;40.40&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;72.80&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;65.55&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;72.34&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;solvent&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;36.20&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;38.95&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;41.46&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;33.85&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;PDB ID&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5DA5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5L89&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5L8B&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5L8G&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>57119</offset><text>	WT	E32A	E62A	H65A	 	Data collection					 	Wavelength (Å)	1.74	1.73	1.73	1.74	 	Resolution range (Å)	49.63 - 2.06 (2.10 - 2.06)	48.84 - 2.59 (2.683 - 2.59)	48.87 - 2.21 (2.29 - 2.21)	48.86 - 2.97 (3.08 - 2.97)	 	Space group	P 1 21 1	P 1 21 1	P 1 21 1	P 1 21 1	 	Unit cell (Å) a b  c β (°)	98.18 120.53 140.30 95.36	97.78 120.28 140.53 95.41	98.09 120.23 140.36 95.50	98.03 120.29 140.43 95.39	 	Total reflections	1,264,922 (41,360)	405,488 (36,186)	1,069,345 (95,716)	323,853 (32,120)	 	Unique reflections	197,873 (8,766)	100,067 (9,735)	162,379 (15,817)	66,658 (6,553)	 	Multiplicity	6.4 (4.7)	4.1 (3.7)	6.6 (6.1)	4.9 (4.9)	 	Anomalous multiplicity	3.2 (2.6)	N/A	N/A	N/A	 	Completeness (%)	99.2 (88.6)	99.0 (97.0)	100 (97.0)	100 (99.0)	 	Anomalous completeness (%)	96.7 (77.2)	N/A	N/A	N/A	 	Mean I/sigma(I)	10.6 (1.60)	8.46 (1.79)	13.74 (1.80)	8.09 (1.74)	 	Wilson B-factor	26.98	40.10	33.97	52.20	 	Rmerge	0.123 (0.790)	0.171 (0.792)	0.0979 (1.009)	0.177 (0.863)	 	Rmeas	0.147 (0.973)	0.196 (0.923)	0.1064 (1.107)	0.199 (0.966)	 	CC1/2	0.995 (0.469)	0.985 (0.557)	0.998 (0.642)	0.989 (0.627)	 	CC*	0.999 (0.846)	0.996 (0.846)	0.999 (0.884)	0.997 (0.878)	 	Image DOI	10.7488/ds/1342	10.7488/ds/1419	10.7488/ds/1420	10.7488/ds/1421	 	Refinement					 	Rwork	0.171 (0.318)	0.183 (0.288)	0.165 (0.299)	0.186 (0.273)	 	Rfree	0.206 (0.345)	0.225 (0351)	0.216 (0.364)	0.237 (0.325)	 	Number of non-hydrogen atoms	23,222	22,366	22,691	22,145	 	macromolecules	22,276	22,019	21,965	22,066	 	ligands	138	8	24	74	 	water	808	339	702	5	 	Protein residues	2,703	2,686	2,675	2,700	 	RMS(bonds) (Å)	0.012	0.005	0.011	0.002	 	RMS(angles) (°)	1.26	0.58	1.02	0.40	 	Ramachandran favored (%)	100	99	100	99	 	Ramachandran allowed (%)	0	1	0	1	 	Ramachandran outliers (%)	0	0	0	0	 	Clash score	1.42	1.42	1.79	0.97	 	Average B-factor (Å2)	33.90	42.31	41.34	47.68	 	macromolecules	33.80	42.35	41.31	47.60	 	ligands	40.40	72.80	65.55	72.34	 	solvent	36.20	38.95	41.46	33.85	 	PDB ID	5DA5	5L89	5L8B	5L8G	 	</text></passage><passage><infon key="file">tbl5.xml</infon><infon key="id">tbl5</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>59111</offset><text>Iron loading capacity of EncFtn, encapsulin and ferritin. Protein samples (at 8.5 µM) including decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were mixed with Fe(NH4)2(SO4) (in 0.1% (v/v) HCl) of different concentrations in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl buffer at room temperature for 3 hrs in the air. Protein-Fe mixtures were centrifuged at 13,000 x g to remove precipitated material and desalted prior to the Fe and protein content analysis by ferrozine assay and BCA microplate assay, respectively. Fe to protein ratio was calculated to indicate the Fe binding capacity of the protein. Protein stability was compromised at high iron concentrations; therefore, the highest iron loading with the least protein precipitation was used to derive the maximum iron loading capacity per biological assembly (underlined and highlighted in bold). The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin. Errors are quoted as the standard deviation of three technical repeats in both the ferrozine and BCA microplate assays. The proteins used in Fe loading experiment came from a single preparation.</text></passage><passage><infon key="file">tbl5.xml</infon><infon key="id">tbl5</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>60373</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.026</text></passage><passage><infon key="file">tbl5.xml</infon><infon key="id">tbl5</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead&gt;&lt;tr&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Protein sample&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Fe(NH&lt;sub&gt;4)2&lt;/sub&gt;(SO&lt;sub&gt;4)2&lt;/sub&gt; loading (µM)&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Fe detected by ferrozine assay (µM)&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Protein detected by BCA microplate assay (µM)&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Fe / monomeric protein&lt;/th&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Maximum Fe loading per biological assembly unit&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td rowspan=&quot;6&quot; colspan=&quot;1&quot;&gt;8.46 µM EncFtn&lt;sub&gt;sH&lt;/sub&gt;-10mer&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4.73 ± 2.32&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5.26 ± 0.64&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.90 ± 0.44&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;39.9&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;9.93 ± 1.20&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5.36 ± 0.69&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.85 ± 0.22&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;84&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;17.99 ± 2.01&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4.96 ± 0.04&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.63 ± 0.41&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;147&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;21.09 ± 1.94&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4.44 ± 0.21&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4.75 ± 0.44&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;48 ± 4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;224&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;28.68 ± 0.30&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.73 ± 0.53&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7.68 ± 0.08&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;301&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;11.27 ± 1.10&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.50 ± 0.05&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4.51 ± 0.44&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;8&quot; colspan=&quot;1&quot;&gt;8.50 µM Encapsulin&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;-1.02 ± 0.54&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8.63 ± 0.17&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;-0.12 ± 0.06&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;224&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;62.24 ± 2.49&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;10.01 ± 0.58&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.22 ± 0.35&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;301&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;67.94 ± 3.15&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8.69 ± 0.42&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7.81 ± 0.36&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;450&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;107.96 ± 8.88&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8.50 ± 0.69&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;12.71 ± 1.05&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;700&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;97.51 ± 3.19&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7.26 ± 0.20&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;13.44 ± 0.44&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1000&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;308.63 ± 2.06&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8.42 ± 0.34&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;36.66 ± 0.24&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2199 ± 15&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1500&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;57.09 ± 0.90&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.44 ± 0.21&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;39.77 ± 0.62&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2000&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;9.2 ± 1.16&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.21 ± 0.14&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;44.73 ± 5.63&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;8&quot; colspan=&quot;1&quot;&gt;8.70 µM EncFtn-Enc&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.31 ± 1.57&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.85 ± 0.07&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.48 ± 0.23&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;224&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;116.27 ± 3.74&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7.63 ± 0.12&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;15.25 ± 0.49&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;301&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;132.86 ± 4.03&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.66 ± 0.31&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;19.96 ± 0.61&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;450&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;220.57 ± 27.33&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.12 ± 1.07&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;36.06 ± 4.47&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;700&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;344.03 ± 40.38&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.94 ± 0.17&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;49.58 ± 5.82&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1000&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;496.00 ± 38.48&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7.19 ± 0.08&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;68.94 ± 5.35&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4137 ± 321&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1500&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;569.98 ± 73.63&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5.73 ± 0.03&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.44 ± 12.84&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2000&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;584.30 ± 28.33&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;4.88 ± 0.22&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;119.62 ± 5.80&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;6&quot; colspan=&quot;1&quot;&gt;8.50 µM Apoferritin&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.95 ± 2.26&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;9.37 ± 0.24&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.42 ± 0.25&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;42.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;10.27 ± 1.12&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8.27 ± 0.30&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.24 ± 0.18&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;212.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;44.48 ± 2.76&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7.85 ± 0.77&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5.67 ± 0.83&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;637.5&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;160.93 ± 4.27&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.76 ± 0.81&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;23.79 ± 3.12&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;571 ± 75&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1275&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;114.92 ± 3.17&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.84 ± 0.30&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;29.91 ± 2.95&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1700&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;91.40 ± 3.37&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.14 ± 0.35&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;29.13 ± 3.86&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>60420</offset><text>Protein sample	Fe(NH4)2(SO4)2 loading (µM)	Fe detected by ferrozine assay (µM)	Protein detected by BCA microplate assay (µM)	Fe / monomeric protein	Maximum Fe loading per biological assembly unit	 	8.46 µM EncFtnsH-10mer	0	4.73 ± 2.32	5.26 ± 0.64	0.90 ± 0.44		 	39.9	9.93 ± 1.20	5.36 ± 0.69	1.85 ± 0.22		 	84	17.99 ± 2.01	4.96 ± 0.04	3.63 ± 0.41		 	147	21.09 ± 1.94	4.44 ± 0.21	4.75 ± 0.44	48 ± 4	 	224	28.68 ± 0.30	3.73 ± 0.53	7.68 ± 0.08		 	301	11.27 ± 1.10	2.50 ± 0.05	4.51 ± 0.44		 	8.50 µM Encapsulin	0	-1.02 ± 0.54	8.63 ± 0.17	-0.12 ± 0.06		 	224	62.24 ± 2.49	10.01 ± 0.58	6.22 ± 0.35		 	301	67.94 ± 3.15	8.69 ± 0.42	7.81 ± 0.36		 	450	107.96 ± 8.88	8.50 ± 0.69	12.71 ± 1.05		 	700	97.51 ± 3.19	7.26 ± 0.20	13.44 ± 0.44		 	1000	308.63 ± 2.06	8.42 ± 0.34	36.66 ± 0.24	2199 ± 15	 	1500	57.09 ± 0.90	1.44 ± 0.21	39.77 ± 0.62		 	2000	9.2 ± 1.16	0.21 ± 0.14	44.73 ± 5.63		 	8.70 µM EncFtn-Enc	0	3.31 ± 1.57	6.85 ± 0.07	0.48 ± 0.23		 	224	116.27 ± 3.74	7.63 ± 0.12	15.25 ± 0.49		 	301	132.86 ± 4.03	6.66 ± 0.31	19.96 ± 0.61		 	450	220.57 ± 27.33	6.12 ± 1.07	36.06 ± 4.47		 	700	344.03 ± 40.38	6.94 ± 0.17	49.58 ± 5.82		 	1000	496.00 ± 38.48	7.19 ± 0.08	68.94 ± 5.35	4137 ± 321	 	1500	569.98 ± 73.63	5.73 ± 0.03	99.44 ± 12.84		 	2000	584.30 ± 28.33	4.88 ± 0.22	119.62 ± 5.80		 	8.50 µM Apoferritin	0	3.95 ± 2.26	9.37 ± 0.24	0.42 ± 0.25		 	42.5	10.27 ± 1.12	8.27 ± 0.30	1.24 ± 0.18		 	212.5	44.48 ± 2.76	7.85 ± 0.77	5.67 ± 0.83		 	637.5	160.93 ± 4.27	6.76 ± 0.81	23.79 ± 3.12	571 ± 75	 	1275	114.92 ± 3.17	3.84 ± 0.30	29.91 ± 2.95		 	1700	91.40 ± 3.37	3.14 ± 0.35	29.13 ± 3.86		 	</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>62115</offset><text>To understand the impact of the mutants on the organization and metal binding of the FOC, we determined the X-ray crystal structures of each of the EncFtnsH mutants (See Table 4 for data collection and refinement statistics). The crystal packing of all of the mutants in this study is essentially isomorphous to the EncFtnsH structure. All of the mutants display the same decameric arrangement in the crystals as the EncFtnsH structure, and the monomers superimpose with an average RMSDCα of less than 0.2 Å.</text></passage><passage><infon key="file">elife-18972-fig11-figsupp1.jpg</infon><infon key="id">fig11s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>62628</offset><text>FOC dimer interface of EncFtnsH-E32A mutant.</text></passage><passage><infon key="file">elife-18972-fig11-figsupp1.jpg</infon><infon key="id">fig11s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>62673</offset><text>(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-E32A. Protein residues are shown as sticks with blue and green carbons for the different subunits. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ. (B) Views of the FOC of the EncFtnsH-E32Amutant. Protein atoms shown as in (A).</text></passage><passage><infon key="file">elife-18972-fig11-figsupp1.jpg</infon><infon key="id">fig11s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>63016</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.028</text></passage><passage><infon key="file">elife-18972-fig11-figsupp2.jpg</infon><infon key="id">fig11s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>63063</offset><text>FOC dimer interface of EncFtnsH-E62A mutant.</text></passage><passage><infon key="file">elife-18972-fig11-figsupp2.jpg</infon><infon key="id">fig11s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>63108</offset><text>(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-E62A. Protein residues are shown as sticks with blue and green carbons for the different subunits. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ. The single coordinated calcium ion is shown as a grey sphere. (B) Views of the FOC of the EncFtnsH-E62A mutant. Protein atoms shown as in (A).</text></passage><passage><infon key="file">elife-18972-fig11-figsupp2.jpg</infon><infon key="id">fig11s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>63514</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.029</text></passage><passage><infon key="file">elife-18972-fig11-figsupp3.jpg</infon><infon key="id">fig11s3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>63561</offset><text>FOC dimer interface of EncFtnsH-H65A mutant.</text></passage><passage><infon key="file">elife-18972-fig11-figsupp3.jpg</infon><infon key="id">fig11s3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>63606</offset><text>(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-H65A. Protein residues are shown as sticks with blue and green carbons for the different subunits. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ. The coordinated calcium ions are shown as a grey spheres with coordination distances in the FOC highlighted with yellow dashed lines. (B) Views of the FOC of the EncFtnsH-H65A mutant. Protein atoms and metal ions shown as in (A).</text></passage><passage><infon key="file">elife-18972-fig11-figsupp3.jpg</infon><infon key="id">fig11s3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>64099</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.030</text></passage><passage><infon key="file">elife-18972-fig11.jpg</infon><infon key="id">fig11</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>64146</offset><text>Comparison of the EncFtnsH FOC mutants vs wild type.</text></passage><passage><infon key="file">elife-18972-fig11.jpg</infon><infon key="id">fig11</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>64199</offset><text>The structures of the three EncFtnsH mutants were all determined by X-ray crystallography. The E32A, E62A and H65A mutants were crystallized in identical conditions to the wild type. EncFtnsH structure and were essentially isomorphous in terms of their unit cell dimensions. The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue. Of the mutants, only H65A has any coordinated metal ions, which appear to be calcium ions from the crystallization condition. The overall organization of FOC residues is retained in the mutants, with almost no backbone movements. Significant differences center around Tyr39, which moves to coordinate the bound calcium ions in the H65A mutant; and Glu32, which moves away from the metal ions in this structure.</text></passage><passage><infon key="file">elife-18972-fig11.jpg</infon><infon key="id">fig11</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>65108</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.027</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>65155</offset><text>Close inspection of the region of the protein around the FOC in each of the mutants highlights their effect on metal binding (Figure 11 and Figure 11—figure supplement 1–3). In the E32A mutant the position of the side chains of the remaining iron coordinating residues in the FOC is essentially unchanged, but the absence of the axial-metal coordinating ligand provided by the Glu32 side chain abrogates metal binding in this site. The Glu31/34-site also lacks metal, with the side chain of Glu31 rotated by 180° at the Cβ in the absence of metal (Figure 11—figure supplement 1). The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11—figure supplement 2). The H65A mutant diverges significantly from the wild type in the position of the residues Glu32 and Tyr39 in the FOC. E32 appears in either the original orientation as the wild type and coordinates Ca2+ in this position, or it is flipped by 180° at the Cβ, moving away from the coordinated calcium ion in the FOC. Tyr39 moves closer to Ca2+ compared to the wild-type and coordinates the calcium ion (Figure 11—figure supplement 3). A single calcium ion is present in the entry site of this mutant; however, Glu31 of one chain is rotated away from the metal ion and is not involved in coordination.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>66541</offset><text>Taken together the results of our data show that these changes to the FOC of EncFtn still permit the formation of the decameric form of the protein. While the proteins all appear decameric in crystals, their solution and gas-phase behavior differs considerably and the mutants no longer show metal-dependent oligomerization. These results highlight the importance of metal coordination in the FOC for the stability and assembly of the EncFtn protein.</text></passage><passage><infon key="file">elife-18972-fig12-figsupp1.jpg</infon><infon key="id">fig12s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>66992</offset><text>Progress curves recording ferroxidase activity of EncFtnsH mutants. 20 µM wild-type EncFtnsH, E32A, E62A and H65A mutants were mixed with 20 µM or 100 µM acidic Fe(NH4)2(SO4)2, respectively.</text></passage><passage><infon key="file">elife-18972-fig12-figsupp1.jpg</infon><infon key="id">fig12s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>67195</offset><text>Absorbance at 315 nm was recorded for 1800 s at 25°C as an indication of Fe3+ formation. Protein free samples (dashed and dotted lines) were measured for Fe2+ background oxidation as controls. Assays were performed with three technical repeats. Error bars were showed in shadows behind each curves.</text></passage><passage><infon key="file">elife-18972-fig12-figsupp1.jpg</infon><infon key="id">fig12s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>67495</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.032</text></passage><passage><infon key="file">elife-18972-fig12.jpg</infon><infon key="id">fig12</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>67542</offset><text>Relative ferroxidase activity of EncFtnsH mutants.</text></passage><passage><infon key="file">elife-18972-fig12.jpg</infon><infon key="id">fig12</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>67593</offset><text>EncFtnsH, and the mutant forms E32A, E62A and H65A, each at 20 µM, were mixed with 100 µM acidic Fe(NH4)2(SO4)2. Ferroxidase activity of the mutant forms is determined by measuring the absorbance at 315 nm for 1800 s at 25 °C as an indication of Fe3+ formation. The relative ferroxidase activity of mutants is plotted as a proportion of the activity of the wild-type protein using the endpoint measurement of A315. Three technical repeats were performed and the plotted error bars represent the calculated standard deviations. The FOC mutants showed reduced ferroxidase activity to varied extents, among which E62A significantly abrogated the ferroxidase activity.</text></passage><passage><infon key="file">elife-18972-fig12.jpg</infon><infon key="id">fig12</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>68266</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.031</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>68313</offset><text>To address the question of how mutagenesis of the iron coordinating residues affects the enzymatic activity of the EncFtnsH protein we recorded progress curves for the oxidation of Fe2+ to Fe3+ by the different mutants as before. Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%; the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12). Collectively, the effect of mutating these residues in the FOC confirms the importance of the iron coordinating residues for the ferroxidase activity of the EncFtnsH protein.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>68975</offset><text>Discussion</text></passage><passage><infon key="file">elife-18972-fig13.jpg</infon><infon key="id">fig13</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>68986</offset><text>Phylogenetic tree of ferritin family proteins.</text></passage><passage><infon key="file">elife-18972-fig13.jpg</infon><infon key="id">fig13</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>69033</offset><text>The tree was built using the Neighbor-Joining method based on step-wise amino acid sequence alignment of the four-helical bundle portions of ferritin family proteins (Supplementary file 1). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree; the likely root of the tree is indicated by a red arrow. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 2.5). The analysis involved 104 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 262 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 </text></passage><passage><infon key="file">elife-18972-fig13.jpg</infon><infon key="id">fig13</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>69870</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.033</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>69917</offset><text>Our study reports on a new class of ferritin-like proteins (EncFtn), which are associated with bacterial encapsulin nanocompartments (Enc). By studying the EncFtn from R. rubrum we demonstrate that iron binding results in assembly of EncFtn decamers, which display a unique annular architecture. Despite a radically different quaternary structure to the classical ferritins, the four-helical bundle scaffold and FOC of EncFtnsH are strikingly similar to ferritin (Figure 6A). A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn). The analysis revealed that the EncFtn and Non-EncFtn proteins form groups distinct from the other clearly delineated groups of ferritins, and represent outliers in the tree (Figure 13). While it is difficult to infer ancestral lineages in protein families, the similarity seen in the active site scaffold of these proteins highlights a shared evolutionary relationship between EncFtn proteins and other members of the ferritin superfamily that has been noted in previous studies (; ). From this analysis, we propose that the four-helical fold of the classical ferritins may have arisen through gene duplication of an ancestor of EncFtn. This gene duplication would result in the C-terminal region of one EncFtn monomer being linked to the N-terminus of another and thus stabilizing the four-helix bundle fold within a single polypeptide chain (Figure 6B). Linking the protein together in this way relaxes the requirement for the maintenance of a symmetrical FOC and thus provides a path to the diversity in active-site residues seen across the ferritin family (Figure 6A, residues Glu95, Gln128 and Glu131 in PmFtn, Supplementary file 1).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_2</infon><offset>71858</offset><text>Relationship between ferritin structure and activity</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>71911</offset><text>The quaternary arrangement of classical ferritins into an octahedral nanocage and Dps into a dodecamer is absolutely required for their function as iron storage compartments. The oxidation and mineralization of iron must be spatially separated from the host cytosol to prevent the formation of damaging hydroxyl radicals in the Fenton and Haber-Weiss reactions. This is achieved in all ferritins by confining the oxidation of iron to the interior of the protein complex, thus achieving sequestration of the Fe3+ mineralization product. A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A). This overlay highlights the fact that the ferroxidase center of EncFtn faces in the opposite direction relative to the classical ferritins and is essentially inside out regarding iron storage space (Figure 6—figure supplement 1B, boxed region). Analysis of each of the single mutations (E32A, E62A and H65A) made in the FOC highlights the importance of the iron-coordinating residues in the catalytic activity of EncFtn. Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC. The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>73772</offset><text>The absolute requirement for the spatial separation of oxidation and mineralization in ferritins suggests that the EncFtn family proteins are not capable of storing iron minerals due to the absence of an enclosed compartment in their structure (Figure 6—figure supplement 1B). Our biochemical characterization of EncFtn supports this hypothesis, indicating that while this protein is capable of oxidizing iron, it does not accrue mineralized iron in an analogous manner to classical ferritins. While EncFtn does not store iron itself, its association with the encapsulin nanocage suggests that mineralization occurs within the cavity of the encapsulin shell. Our ferroxidase assay data on the recombinant EncFtn-Enc nanocompartments, which accrue over 4100 iron ions per complex and form regular nanoparticles, are consistent with the encapsulin protein acting as the store for iron oxidized by the EncFtn enzyme. TEM analysis of the reaction products shows the production of homogeneous iron nanoparticles only in the EncFtn-Enc nanocompartment (Figure 8—figure supplement 1).</text></passage><passage><infon key="file">elife-18972-fig14.jpg</infon><infon key="id">fig14</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>74854</offset><text>Model of iron oxidation in encapsulin nanocompartments.</text></passage><passage><infon key="file">elife-18972-fig14.jpg</infon><infon key="id">fig14</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>74910</offset><text>(A) Model of EncFtnsH docking to the encapsulin shell. A single pentamer of the icosahedral T. maritima encapsulin structure (PDBID: 3DKT) is shown as a blue surface with the encapsulin localization sequence of EncFtn shown as a purple surface. The C-terminal regions of the EncFtn subunits correspond to the position of the localization sequences seen in 3DKT. Alignment of EncFtnsH with 3DKT positions the central channel directly above the pore in the 3DKT pentamer axis (shown as a grey pentagon). (B) Surface view of EncFtn within the encapsulin nanocompartment (grey and blue respectively). The lumen of the encapsulin nanocompartment is considerably larger than the interior of ferritin (shown in orange behind the encapsulin for reference) and thus allows the storage of significantly more iron. The proposed pathway for iron movement through the encapsulin shell and EncFtn FOC is shown with arrows. (C) Model ofiron oxidation within an encapsulin nanocompartment. As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.</text></passage><passage><infon key="file">elife-18972-fig14.jpg</infon><infon key="id">fig14</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>76242</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.034</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>76289</offset><text>Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT)  shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A). Thus, it appears that the EncFtn decamer is the physiological state of this protein. This arrangement positions the central ring of EncFtn directly above the pore at the five-fold symmetry axis of the encapsulin shell and highlights a potential route for the entry of iron into the encapsulin and towards the active site of EncFtn. A comparison of the encapsulin nanocompartment and the ferritin nanocage highlights the size differential between the two complexes (Figure 14B) that allows the encapsulin to store significantly more iron. The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment. This arrangement of multiple reaction centers in a single protein assembly is reminiscent of classical ferritins, which has 24 FOCs distributed around the nanocage.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>77576</offset><text>Our structural data, coupled with biochemical and ICP-MS analysis, suggest a model for the activity of the encapsulin iron-megastore (Figure 14C). The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4—figure supplement 1). The five metal-binding sites on the interior of the ring (Glu31/34-sites) may select for the Fe2+ ion and direct it towards their cognate FOCs. We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by  in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>78575</offset><text>Here we describe for the first time the structure and biochemistry of a new class of encapsulin-associated ferritin-like protein and demonstrate that it has an absolute requirement for compartmentalization within an encapsulin nanocage to act as an iron store. Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>79099</offset><text>Materials and methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>79121</offset><text>Cloning</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>79129</offset><text>Genes of interest were amplified by PCR using R. rubrum ATCC 11,170 genomic DNA (DSMZ) as the template and KOD Hot Start DNA Polymerase (Novagen). Primers used in this study are listed in Supplementary file 2. PCR products were visualized in 0.8% agarose gel stained with SYBR Safe (Life Technologies, UK). Fragments of interest were purified by gel extraction (Qiagen, UK) before digestion by endonuclease restriction enzymes (Thermo Fisher Scientific, UK) at 37°C for 1 hr, followed by ligation with similarly digested vector pET-28a(+) or pACYCDuet-1 at room temperature for 1 hr. Ligation product was transformed into chemically competent Escherichia coli Top10 cells and screened against 50 ng/μl kanamycin for pET-28a(+) based constructs or 34 ng/μl chloramphenicol for pACYCDuet-1 based constructs. DNA insertion was confirmed through Sanger sequencing (Edinburgh Genomics, The University of Edinburgh, UK). Sequence verified constructs were transformed into E. coli BL21(DE3) or Tuner(DE3) for protein production. Alternatively, plasmids transformed into E. coli B834(DE3) cells were cultured in selenomethionine medium.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>80264</offset><text>Protein production and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>80300</offset><text>A single colony of E. coli BL21(DE3) or Tuner(DE3) cells, transformed with protein expression plasmid, was transferred into 10 ml LB medium, or M9 minimal medium (MM), supplemented with appropriate antibiotic, and incubated overnight at 37 °C with 200 rpm shaking. The overnight pre-culture was then inoculated into 1 liter of LB medium and incubated at 37 °C with 200 rpm shaking. Recombinant protein production was induced at OD600= 0.6 by the addition of 1 mM IPTG and the incubation temperature was reduced to 18°C for overnight incubation. Cells were pelleted by centrifugation at 4000 g for 20 min at 4 °C, and resuspended 10-fold (volume per gram of cell pellet) in PBS to wash cells before a second centrifugation step. Cells were resuspended in 10-times (v/w) of appropriate lysis buffer for the purification method used (see details of buffers below) and lysed by sonication on ice, with ten cycles of 30-second burst of sonication at 10 µm amplitude and 30 s of cooling. Cell lysate was clarified by centrifugation at 20,000 x g, 30 min, 4 °C; followed by filtration using a 0.22 µM syringe filter (Millipore, UK).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>81438</offset><text>Selenomethionine labelled protein was produced by growing a single colony of E. coli B834 (DE3) cells transformed with protein expression plasmids in 100 ml LB medium supplemented with appropriate antibiotic overnight at 37 °C with shaking at 200 rpm. The overnight pre-culture was pelleted by centrifugation 3,000 x g, 4 °C, 15 min and washed twice with M9 minimal medium. The washed cells were transferred to 1 liter of SeMet medium, which contains M9 minimal medium, 40 mg/L of each L-amino acid (without methionine), 40 mg/L selenomethionine, 2 mM MgSO4, 0.4% (w/v) glucose and 1 mM Fe(NH4)2(SO4)2. Cells were incubated at 37 °C with 200 rpm shaking and recombinant protein production was induced at OD600= 0.6 by the addition of 1 mM IPTG and the incubation temperature was reduced to 18 °C for overnight incubation. Cells were harvested and lysed as above.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>82309</offset><text>His-tagged protein purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>82341</offset><text>Clarified cell lysate was loaded onto a 5 ml HisTrap column (GE Healthcare, UK) pre-equilibrated with HisA buffer (50 mM Tris-HCl, 500 mM NaCl and 50 mM imidazole, pH 8.0). Unbound proteins were washed from the column with HisA buffer. His-tagged proteins were then eluted by a step gradient of 50% HisA buffer and 50% HisB buffer (50 mM Tris-HCl, 500 mM NaCl and 500 mM imidazole, pH 8.0). Fractions containing the protein of interest, as determined by 15% (w/v) acrylamide SDS-PAGE, were pooled before loading onto a gel-filtration column (HiLoad 16/600 Superdex 200, GE Healthcare) equilibrated with GF buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl). Fractions were subjected to 15% SDS-PAGE and those containing the protein of interest were pooled for further analysis.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>83115</offset><text>Sucrose gradient ultracentrifugation purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>83165</offset><text>Co-expressed encapsulin and EncFtn (EncFtn-Enc) and encapsulin protein were both purified according to the protocol used by M. Sutter. Briefly, EncFtn-Enc or encapsulin was expressed based on pACYCDuet-1 vector. The E. coli cells were grown, induced, harvested and sonicated in a similar way as described above. GF buffer used in this purification contains 50 mM Tris-HCl, pH 8.0, and 150 mM NaCl. To remove RNA contamination, the lysate was supplemented with 50 μg/ml RNase A and rotated at 10 rpm and room temperature for 2 hrs, followed by centrifugation at 34,000 x g and 4 °C for 20 min and filtering through 0.22 µM syringe filter. Proteins were pelleted through 38% (w/v) sucrose cushion by ultracentrifugation at 100,000 x g and 4 °C for 21 hrs. 10% - 50% (w/v) sucrose gradient ultracentrifugation was applied to further separate the proteins at 100,000 x g and 4 °C for 17 hrs. Protein was dialyzed against GF buffer to remove sucrose before being used in chemical assays or TEM.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>84163</offset><text>Transmission electron microscopy</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>84196</offset><text>TEM imaging was performed on purified encapsulin, EncFtn, and EncFtn-Enc and apoferritin. Purified protein at 0.1 mg/ml concentration was spotted on glow-discharged 300 mesh carbon-coated copper grids and excess liquid wicked off with filter paper (Whatman, UK). The grids were washed with distilled water and blotted with filter paper three times before staining with 0.2% uranyl acetate, blotting and air-drying. Grids were imaged using a JEM1400 transmission electron microscope and images were collected with a Gatan CCD camera. Images were analyzed using ImageJ (NIH, Bethesda, MD) and size-distribution histograms were plotted using Prism 6 (GraphPad software). To observe iron mineral formation by TEM, protein samples at 8.5 µM concentration including EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were supplemented with acidic Fe(NH4)2(SO4)2 at their maximum iron loading ratio in room temperature for 1 hr. The mixtures were subjected to TEM analysis with or without uranyl acetate staining. TEM experiments without Fe loading were repeated three times, a representative set of images are presented here. Proteins loaded with Fe and imaged by TEM were from single preparation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>85392</offset><text>Protein crystallization and X-ray data collection</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>85442</offset><text>EncFtnsH was purified by anion exchange and Superdex 200 size- exclusion chromatography and concentrated to 10 mg/ml (based on extinction coefficient calculation). Crystallization drops were set up using the hanging drop vapor diffusion method at 292 K. Glass coverslips were set up with 1–2 μl protein mixed with 1 μl well solution (0.14 M calcium acetate and 15% (w/v) PEG 3350) and sealed over 1 ml of well solution. Crystals appeared after 5 days and were harvested from the well using a LithoLoop (Molecular Dimensions Limited, UK), transferred briefly to a cryoprotection solution containing well solution supplemented with 1 mM FeSO4 (in 0.1% (v/v) HCl), 20% (v/v) PEG 200, and subsequently flash cooled in liquid nitrogen. Crystals of the EncFtnsHsingle mutations were produced in the same manner as for the EncFtnsH wild-type protein.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>86295</offset><text>All crystallographic datasets were collected on the macromolecular crystallography beamlines at Diamond Light Source (Didcot, UK) at 100 K using Pilatus 6M detectors. Diffraction data were integrated and scaled using XDS and symmetry related reflections were merged with Aimless . Data collection statistics are shown in Table 4. The resolution cut-off used for structure determination and refinement was determined based on the CC1/2 criterion proposed by.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>86754</offset><text>The structure of EncFtnsH was determined by molecular replacement using PDB ID: 3K6C as the search model, modified to match the sequence of the target protein using Chainsaw. A single solution comprising three decamers in the asymmetric unit was found by molecular replacement using Phaser. The initial model was rebuilt using Phenix.autobuild followed by cycles of refinement with Phenix.refine, with manual rebuilding and model inspection in Coot . The final model was refined with isotropic B-factors, torsional NCS restraints, and with anomalous group refinement. The model was validated using MolProbity. Structural superimpositions were calculated using Coot. Crystallographic figures were generated with PyMOL. Multiple sequence alignment of EncFtn and ferritin family proteins was performed using Clustal Omega Sievers and Higgins, 2014 and displayed with Espript 3.0. Model refinement statistics are shown in Table 4. The final models and experimental data are deposited in the PDB and diffraction image files are available at the Edinburgh DataShare repository.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>87828</offset><text>Horse spleen apoferritin preparation</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>87865</offset><text>Horse spleen apoferritin purchased from Sigma Aldrich (UK) was dissolved in deaerated MOPS buffer (100 mM MOPS, 100 mM NaCl, 3 g/100 ml Na2S2O4 and 0.5 M EDTA, pH 6.5). Protein was dialyzed against 1 liter MOPS buffer in room temperature for two days before buffer exchanging to GF buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl) in a vivaspin column with 5 kDa cut-off (Sartorius, UK) for several times. Fe content of apoferritin was detected using ferrozine assay. Protein concentration was determined using Pierce Microplate BCA Protein Assay Kit. Apoferritin containing less than 0.5 Fe per 24-mer was used in the ferroxidase assay. Apoferritin used in the Fe loading capacity experiment was prepared in the same way with 5–15 Fe per 24-mer.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>88612</offset><text>Ferroxidase assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>88630</offset><text>1 mM and 200 µM Fe(NH4)2(SO4)2 stock solutions were prepared in 0.1% (v/v) HCl anaerobically. Protein solutions with 20 µM FOC were diluted from ~10 mg/ml frozen stock in GF buffer (50 mM Tris-HCl, pH 8.0 and 150 mM NaCl) anaerobically. Ferroxidase activity was initiated by adding 450 μl protein to 50 μl of acidic Fe(NH4)2(SO4)2 at the final concentration of 100 µM and 20 µM in the air, respectively. The ferroxidase activity was measured by monitoring the Fe3+ formation which gives rise to the change of the absorbance at 315 nm. Absorbance at 315 nm was recorded every second over 1800 s using a quartz cuvette in a JASCO V-730 UV/VIS spectrophotometer (JASCO Inc., Easton, MD). In recombinantly coexpressed nanocompartments the ratio of EncFtn to Enc was assumed as 2 to 1, assuming each of the twelve pentameric vertices of the icosahedral encapsulin were occupied with decameric EncFtn. The data are presented as the mean of three technical replicates with error bars indicating one standard deviation from the mean. Proteins used here were from a single preparation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>89725</offset><text>Iron loading capacity of ferritins</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>89760</offset><text>In order to determine the maximum iron loading capacity, around 8.5 µM proteins including decameric EncFtnsH, Encapsulin, EncFtn-Enc and apoferritin were loaded with various amount of acidic Fe(NH4)2(SO4)2 ranging from 0 to 1700 µM. Protein mixtures were incubated in room temperature for 3 hrs before desalting in Zebra spin desalting columns (7 kDa cut-off, Thermo Fisher Scientific, UK) to remove free iron ions. The protein concentration was determined using PierceMicroplate BCA assay kit (Thermo Fisher Scientific). The protein standard curve was plotted according to the manufacturer. The Fe content in the samples was determined using modified ferrozine assay. Briefly speaking, 100 μl protein sample was mixed with 100 μl mixture of equal volume of 1.4 M HCl and 4.5% (w/v) KMnO4 and incubated at 60 °C for 2 hrs. 20 μl of the iron-detection reagent (6.5 mM ferrozine, 6.5 mM neocuproine, 2.5 M ammonium acetate, and 1 M ascorbic acid dissolved in H2O) was added to the cooled tubes. 30 min later, 200 μl of the solution was transferred into a well of 96-well plate and the absorbance at 562 nm was measured on the plate reader Spectramax M5 (Molecular Devices, UK). The standard curve was plotted using various concentrations of FeCl3 (in 10 mM HCl) diluted in the gel-filtration buffer. Three technical repeats were performed for both the ferrozine and microplate BCA assays. Samples analyzed by ICP-MS were prepared in the same way by mixing protein and ferrous ions and desalting. The proteins used in the Fe loading experiment came from a single preparation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>91345</offset><text>Peroxidase assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>91362</offset><text>The peroxidase activity of EncFtnsH was determined by measuring the oxidation of ortho-phenylenediamine (OP) by H2O2 . EncFtnsH decameric and monomeric fractions purified from MM were both used in the assay. Ortho-phenylenediamine was prepared as a 92.5 mM stock solution in 50 mM Tris-HCl (pH 8.0). 80, 70, 60, 50, 40, 30, 20 and 10 mM of OP were prepared by diluting the stock solution in the 50 mM Tris-HCl (pH 8.0). 100 μl of each diluted OP was added to a 96-well plate in 3 repeats. 1 μl of 32 µM protein was supplemented into each well to a final concentration of 160 nM, followed by the addition of 2 μl of 30% H2O2. After 15 min shaking in the dark, the reaction was stopped by adding 100 μl of 0.5 M H2SO4. The peroxidase activity was measured by monitoring the absorbance at 490 nm in the SpectraMax M5 Microplate Reader (Molecular Devices).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>92223</offset><text>ICP-MS analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>92239</offset><text>Protein samples were diluted 50-fold into a solution of 2.5% HNO3 (Suprapur, Merck, UK) containing 20 µg/L Pt as internal standard. Matrix-matched elemental standards (containing analyte metal concentrations 0 – 1000 µg/L) were prepared by serial dilution from individual metal standard stocks (VWR) with identical solution compositions, including the internal standard. All standards and samples were analyzed by ICP-MS using a Thermo x-series instrument (Thermo Fisher Scientific) operating in collision cell mode (using 3.0 ml min-1 flow of 8% H2 in He as the collision gas). Isotopes 44Ca, 56Fe, 66Zn, 78Se, and 195Pt were monitored using the peak-jump method (100 sweeps, 25–30 ms dwell time on 5 channels per isotope, separated by 0.02 atomic mass units) in triplicate. The protein samples used in ICP-MS came from a single protein preparation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>93098</offset><text>Mass spectrometry analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>93125</offset><text>For native MS analysis, all protein samples were buffer exchanged into 100 mM ammonium acetate (pH 8.0; adjusted with dropwise addition of 1% ammonia solution) using Micro Biospin Chromatography Columns (Bio-Rad, UK) prior to analysis and the resulting protein samples were analyzed at a final concentration of ~5 µM (oligomer concentration). In order to obtain Fe-bound EncFtn, 100 µM or 300 µM of freshly prepared FeCl2 was added to apo-EncFtnsH (monomer peak) immediately prior to buffer exchange into 100 mM ammonium acetate (pH 8.0). Samples were analyzed on a quadrupole ion-mobility time of flight instrument (Synapt G2, Waters Corp., Manchester, UK), equipped with a nanomate nanoelectrospray infusion robot (Advion Biosciences, Ithaca, NY). Instrument parameters were tuned to preserve non-covalent protein complexes. After optimization, typical parameters were: nanoelectrospray voltage 1.54 kV; sample cone 50 V; extractor cone 0 V; trap collision voltage 4 V; source temperature 80°C; and source backing pressure 5.5 mbar. For improved mass resolution the sample cone was raised to 155 V. Ion mobility mass spectrometry (IM-MS) was performed using the travelling-wave mobility cell in the Synapt G2, employing nitrogen as the drift gas. Typically, the IMS wave velocity was set to 300 m/s; wave height to 15 V; and the IMS pressure was 1.8 mbar. All native MS experiments were performed on samples from two independent protein preparations. For collision cross section determination, IM-MS data was calibrated using denatured equine myoglobin and data was analyzed using Driftscope v2.5 and MassLynx v4.1 (Waters Corp., UK). Theoretical collision cross sections (CCS) were calculated from pdb files using IMPACT software v. 0.9.1. In order to obtain information on the topology of the EncFtnsH assembly, gas-phase dissociation of the Fe-associated EncFtnsH complex was achieved by increasing the sample cone and/or trap collision voltage prior to MS analysis.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>95109</offset><text>SEC-MALLS</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>95119</offset><text>Size-exclusion chromatography (ÄKTA-Micro; GE Healthcare) coupled to UV, static light scattering and refractive index detection (Viscotec SEC-MALS 20 and Viscotec RI Detector:VE3580; Malvern Instruments, UK) were used to determine the molecular mass of fractions decamer and monomer of EncFtnsH in solution individually. Protein concentration was determined by measurement of absorbance at 280 nm and calculated using the extinction coefficient ε0.1%= 1.462 mg−1 ml-1 cm−1. 100 μl of 1.43 mgml-1 fractions of EncFtnsH decamer and 4.03 mg ml -1 fractions of EncFtnsH monomer were run individually on a Superdex 200 10/300 GL size-exclusion column pre-equilibrated in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl at 22°C with a flow rate of 0.5 ml/min. Light scattering, refractive index (RI) and A280nm were analyzed by a homo-polymer model (OmniSEC software, v 5.1; Malvern Instruments) using the following parameters for fractions of decamer and monomer: the extinction coefficient (dA/dc) at 280 nm was 1.46 AU mg ml−1 and specific refractive index increment (dn/dc) was 0.185 ml g−1. The proteins analyzed by SEC-MALLS came from single protein preparation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>96289</offset><text>Metal binding analysis by PAGE</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>96320</offset><text>Recombinant EncFtnsH fractions at 50 µM concentration were incubated with one molar equivalent of metal ions at room temperature for 2 hrs. Half of each sample was mixed with 5 x native loading buffer (65 mM Tris-HCl, pH 8.5, 20% glycerol and 0.01% bromophenol blue) and run on non-denaturing PAGE gels (10% acrylamide) and run in Tris/glycine buffer, 200 V, 4 °C for 50 min. The remaining samples were left for an additional three hours prior to SDS-PAGE (15% acrylamide) analysis. SDS-PAGE gels were run at room temperature at 200 V, room temperature for 50 min. Gels were stained with Coomassie Brilliant Blue R250 and scanned after de-staining in water. The proteins used in this experiment came from single protein preparation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>97057</offset><text>Analytical size-exclusion chromatography</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>97098</offset><text>For analysis of the multimeric state of EncFtn proteins by analytical size-exclusion gel-filtration chromatography (AGF) 25 μl of 90 µM protein was loaded into Superdex 200 PC 3.2/30 column (GE Healthcare) at 15 °C with GF buffer running at 0.05 ml/min and pressure limit 0.45 MPa. In order to use AGF to determine how metal ions influence the assembly of EncFtnsH, 90 µM EncFtnsH monomer fractions were mixed with equal molar concentrations of metal ion solutions including FeSO4 in 0.1% (v/v) HCl, Fe(NH4)2(SO4)2, FeCl3, CoCl2, calcium acetate (CaAc), ZnSO4 and MnCl2 at room temperature for 2 hrs prior to AGF analysis. Protein samples without metal titration were also analyzed as a control group. Both monomer and decamer fractions of EncFtnsH left at room temperature for 2 hrs, or overnight, were also analysed as controls to show the stability of the protein samples in the absence of additional metal ions. The AGF results have been repeated twice using two independent preparations of protein, of which only one representative trace is presented in the paper.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>98177</offset><text>Accession codes and datasets</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>98206</offset><text>Coordinates and structure factors for the structures presented in this paper have been deposited in the PDB under the following accession codes: EncFtnsH, 5DA5; EncFtnsH-E32A, 5L89; EncFtnsH-E62A, 5L8B; EncFtnsH-H65A, 5L8G (DOIs for X-ray diffraction image data are shown in Table 4). All MS datasets presented in this paper can be found, in the raw format at http://dx.doi.org/10.7488/ds/1449.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">title_1</infon><offset>98601</offset><text>Funding Information</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>98621</offset><text>This paper was supported by the following grants:</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>98671</offset><text> to Didi He.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>98684</offset><text> to Sam Hughes, Kirsten Altenbach, David J Clarke.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>98735</offset><text>  to Emma Tarrant, Kevin J Waldron.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>98771</offset><text>  to David J Clarke, Jon Marles-Wright.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>98811</offset><text>  to Jon Marles-Wright.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">title_1</infon><offset>98835</offset><text>Additional information</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">title_1</infon><offset>98858</offset><text>Competing interests</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">footnote</infon><offset>98878</offset><text>The authors declare that no competing interests exist.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title_1</infon><offset>98933</offset><text>Author contributions</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>98954</offset><text>DH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99073</offset><text>SH, Acquisition of data, Analysis and interpretation of data.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99135</offset><text>SV-H, Acquisition of data, Analysis and interpretation of data.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99199</offset><text>AG, Acquisition of data, Drafting or revising the article.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99258</offset><text>KA, Acquisition of data, Contributed unpublished essential data or reagents.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99335</offset><text>ET, Acquisition of data, Analysis and interpretation of data.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99397</offset><text>CLM, Acquisition of data, Contributed unpublished essential data or reagents.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99475</offset><text>KJW, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99572</offset><text>DJC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99692</offset><text>JM-W, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title_1</infon><offset>99813</offset><text>Additional files</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title_2</infon><offset>99830</offset><text>Major datasets</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">paragraph</infon><offset>99845</offset><text>The following datasets were generated:</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>99884</offset><text>References</text></passage><passage><infon key="fpage">276</infon><infon key="lpage">278</infon><infon key="name_0">surname:Aberg;given-names:A</infon><infon key="name_1">surname:Nordlund;given-names:P</infon><infon key="name_2">surname:Eklund;given-names:H</infon><infon key="pub-id_doi">10.1038/361276a0</infon><infon 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key="name_2">surname:Gutmann;given-names:S</infon><infon key="name_3">surname:Günther;given-names:S</infon><infon key="name_4">surname:Prangishvili;given-names:D</infon><infon key="name_5">surname:Loessner;given-names:MJ</infon><infon key="name_6">surname:Stetter;given-names:KO</infon><infon key="name_7">surname:Weber-Ban;given-names:E</infon><infon key="name_8">surname:Ban;given-names:N</infon><infon key="pub-id_doi">10.1038/nsmb.1473</infon><infon key="section_type">REF</infon><infon key="source">Nature Structural &amp; Molecular Biology</infon><infon key="type">ref</infon><infon key="volume">15</infon><infon key="year">2008</infon><offset>104104</offset><text>Structural basis of enzyme encapsulation into a bacterial nanocompartment</text></passage><passage><infon key="section_type">REF</infon><infon key="type">paragraph</infon><offset>104178</offset><text>10.7554/eLife.18972.048</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">title</infon><offset>104202</offset><text>Decision letter</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>104218</offset><text>Losick</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>104225</offset><text>Richard</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>104233</offset><text>In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>104517</offset><text>[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The previous decision letter after peer review is shown below.]</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>104711</offset><text>Thank you for submitting your work entitled &quot;Structural characterisation of an encapsulated ferritin provides insight into iron storage in bacterial nanocompartments&quot; for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Losick as the Senior Editor. Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>105289</offset><text>In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently, and thus represent an interesting frontier in microbial cell biology. While this study significantly advances our understanding of the structural and biochemical relationship between encapsulins and EncFer, it requires significant revision prior to publication. We do, however, encourage the authors to resubmit when and if they are address the issues raised below.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>105888</offset><text>Reviewer #1: </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>105902</offset><text>1) Methods: What procedures and analyses did the author use to assess whether the iron added to the various ferritin derivatives was protein coated or was simply balls of rust attached to protein fragments? If the latter, it could easily generate reactive oxygen species in air under physiological conditions.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>106212</offset><text>2) Results:</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>106224</offset><text>A) Critical data, such as the comparison of maximum amount of iron bound by a monomer in the dodocamer is in the Supplementary information.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>106364</offset><text>B) The data in Supplementary file 2, shows that the amount of iron bound by an ENCFTN decamer monomer is sub -stoichiometric, ranging from 0.18 to 0.64. In a bona fide ferritin, with ~ 2000 iron atoms/ protein cage (24 subunits), the same parameter is much, much higher.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>106635</offset><text>Even an experimental situation: 24 subunit (monomer) ferritin with a biomineral prepared experimentally from apoferritin and containing, on average, only 1000 iron atoms/24 subunit cage, the equivalent parameter appears to be 1000/24 = 42. This Fe/protein ratio is 66 times more iron than in the test system described. Moreover, in nature, some ferritin protein cages contain as much as 4500 Fe atoms, several hundred times higher than the test system. Thus the significance of the experimental results in the paper are unclear.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>107164</offset><text>3) Table 4: Data are shown for three proteins, Encapsulin, Enc-Ftn-10mer, and EncFTN-Enc. Missing are data for the starting material, 24 subunit ferritin or apoferritin (ferritin with the iron removed, by reduction and chelation, as a control.)</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>107409</offset><text>Reviewer #2: </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>107423</offset><text>In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently. While the structure of the encapsulin shell has been determined, that of its cargo, the ferritin-like protein (EncFer), has remained elusive. Here, the authors provide the structure of one such cargo and show that it assembles in a manner that is topologically distinct from ferritin. Additionally, the authors provide evidence that metal binding promotes the assembly of the EncFer and that it does act as a ferroxidase. Altogether, there is a substantial amount of work here that will likely be viewed as a major step forward in understanding these unique bacterial organelles. I have a few suggestions and questions that are listed below:</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>108298</offset><text>1) The authors grow E. coli in minimal media with and without added iron to show that assembly is iron dependent. The output of these experiments is the ratio of decamer vs. monomer. However, we don't have information on whether the growth conditions altered either the total amount of protein produced or the total amount soluble complex/monomer. Perhaps, lower protein concentrations lead to less efficient assembly (a critical concentration is needed).</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>108754</offset><text>2) There is no information regarding the reason for the use of R. rubrum encapsulins. As far as I can tell, these have not been a model for either in vivo or in vitro work. Is there even evidence that they are produced by R. rubrum? What is their size/appearance in that organism? Do they have a physiological role?</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>109070</offset><text>3) Also, how similar are the Enc and EncFer to those of M. xanthus? Are the putative iron-binding sites conserved?</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>109185</offset><text>4) I would have liked to see some mutagenesis experiments to test the models of assembly, iron binding and ferroxidase activity. These do not have to be in vivo and can be performed in vitro with the available system.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>109403</offset><text>5) I would like some more phylogenetic data for the model that ferritin evolved from EncFer. Perhaps, EncFer evolved from ferritin? Do any of the existing phylogenetic analyses support one model over another.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>109612</offset><text>10.7554/eLife.18972.049</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">title</infon><offset>109636</offset><text>Author response</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>109652</offset><text>[Editors’ note: the author responses to the first round of peer review follow.]</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>109734</offset><text>In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently, and thus represent an interesting frontier in microbial cell biology. While this study significantly advances our understanding of the structural and biochemical relationship between encapsulins and EncFer, it requires significant revision prior to publication. We do, however, encourage the authors to resubmit when and if they are address the issues raised below. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>110334</offset><text>Reviewer #1: </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>110348</offset><text>1) Methods: What procedures and analyses did the author use to assess whether the iron added to the various ferritin derivatives was protein coated or was simply balls of rust attached to protein fragments? If the latter, it could easily generate reactive oxygen species in air under physiological conditions. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>110659</offset><text>The reviewer makes an excellent point here. To ascertain whether the iron in the assays forms ‘balls of rust’ we performed transmission electron microscopy on the ferroxidase reaction mixtures after completion of the reaction to assess the formation of free, or encapsulated iron minerals. We provide an additional supplemental figure (Figure 8—figure supplement 1) and discuss the observation of iron mineral crystals and nanoparticles in the main text, subsection “Ferroxidase activity”, last paragraph. We also attempted to use a commercial luminescence-based ROS detection kit on the reactions to address the possibility that H2O2 is produced as a reaction intermediate by the EncFtn protein. We found that the results from this particular kit were inconsistent between repeats, but for the benefit of the reviewer we provide a graph of the results obtained (see Author response image 1). These results show the production of ROS by apoferritin, which is consistent with the published data on the reaction mechanism of certain ferritins; however, no significant ROS were detected for the EncFtn or encapsulin proteins.</text></passage><passage><infon key="file">elife-18972-resp-fig1.jpg</infon><infon key="id">fig15</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>111792</offset><text>DOI:
+http://dx.doi.org/10.7554/eLife.18972.037</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>111839</offset><text>We acknowledge that the reaction mechanism of the EncFtn merits further investigation in a follow up study.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>111947</offset><text>2) Results:</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>111959</offset><text>A) Critical data, such as the comparison of maximum amount of iron bound by a monomer in the dodocamer is in the Supplementary information. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>112100</offset><text>We acknowledge that the data for iron loading merits inclusion in the main text, we have now moved this data and other supplementary data tables to the main text.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>112263</offset><text>B) The data in Supplementary file 2, shows that the amount of iron bound by an ENCFTN decamer monomer is sub -stoichiometric, ranging from 0.18 to 0.64. In a bona fide ferritin, with ~ 2000 iron atoms/ protein cage (24 subunits), the same parameter is much, much higher. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>112535</offset><text>One of the central arguments of our paper is the fact that the EncFtnsH monomer must dimerize to produce a functional ferroxidase active site and that its iron binding properties are highly divergent from those of the classical ferritin nanocages. We have added additional text to the manuscript to highlight these differences (Introduction, last paragraph, and Mass spectrometry section) and discuss the functional consequences at length.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>112975</offset><text>Even an experimental situation: 24 subunit (monomer) ferritin with a biomineral prepared experimentally from apoferritin and containing, on average, only 1000 iron atoms/24 subunit cage, the equivalent parameter appears to be 1000/24 = 42. This Fe/protein ratio is 66 times more iron than in the test system described. Moreover, in nature, some ferritin protein cages contain as much as 4500 Fe atoms, several hundred times higher than the test system! Thus the significance of the experimental results in the paper are unclear. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>113505</offset><text>We have clarified this key difference in the discussion of the iron storage function of the encapsulin nanocompartment (subsection “Iron storage in encapsulin nanocompartments”, second paragraph). The key conclusion of the paper is that the iron storage and iron oxidation functions that are combined in classical ferritins are split between the encapsulin nanocompartment and the EncFtn protein.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>113906</offset><text>3) Table 4: Data are shown for three proteins, Encapsulin, Enc-Ftn-10mer, and EncFTN-Enc. Missing are data for the starting material, 24 subunit ferritin or apoferritin (ferritin with the iron removed, by reduction and chelation, as a control.) </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>114152</offset><text>The data for the starting material are shown in Table 5. Control data for apoferritin have been added to this table and are illustrated in Figure 8. We note that we do not reach the experimental maximum loading capacity for apoferritin; however, we also note that the EncFtn-encapsulin nanocompartment sequesters five times more iron than the ferritin under the same reaction conditions, supporting the published observations that these nanocompartments can store more iron than classical ferritin nanocages.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>114661</offset><text>Reviewer #2: </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>114675</offset><text>In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently. While the structure of the encapsulin shell has been determined, that of its cargo, the ferritin-like protein (EncFer), has remained elusive. Here, the authors provide the structure of one such cargo and show that it assembles in a manner that is topologically distinct from ferritin. Additionally, the authors provide evidence that metal binding promotes the assembly of the EncFer and that it does act as a ferroxidase. Altogether, there is a substantial amount of work here that will likely be viewed as a major step forward in understanding these unique bacterial organelles. I have a few suggestions and questions that are listed below: </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>115551</offset><text>1) The authors grow E. coli in minimal media with and without added iron to show that assembly is iron dependent. The output of these experiments is the ratio of decamer vs. monomer. However, we don't have information on whether the growth conditions altered either the total amount of protein produced or the total amount soluble complex/monomer. Perhaps, lower protein concentrations lead to less efficient assembly (a critical concentration is needed). </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>116008</offset><text>The reviewer makes an interesting point about growth conditions and we acknowledge that production of the protein in LB medium leads to varying protein yields and monomer/decamer proportions. We therefore adopted the use of M9 minimal medium throughout the study to give better reproducibility, which also enables better control of metal ion availability than the complex LB medium. Given the fact that the protein is produced recombinantly in E. coli it is not particularly instructive to prove the in vivoproduction of the EncFtn multimer in this host. We have added a panel to Figure 3 to show the effect of protein concentration on multimerization in vitro(Figure 3C). Our mass spectrometry results show that the protein spontaneously multimerized in the presence of iron in vitroto form decameric species and that this is metal ion concentration dependent (Figure 7).</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>116881</offset><text>2) There is no information regarding the reason for the use of R. rubrum encapsulins. As far as I can tell, these have not been a model for either in vivo or in vitro work. Is there even evidence that they are produced by R. rubrum? What is their size/appearance in that organism? Do they have a physiological role? </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>117198</offset><text>We have put a comment in the Introduction to introduce R. rubrum (last paragraph). A preliminary study in the laboratory identified encapsulins in a preparation of lipid vesicles from R. rubrum containing chromatophores. We chose to follow up on these structures in this study. We do not feel this particular information is key to the central argument of the paper.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>117564</offset><text>3) Also, how similar are the Enc and EncFer to those of M. xanthus? Are the putative iron-binding sites conserved? </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>117680</offset><text>We have noted this in the Introduction of the manuscript.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>117738</offset><text>4) I would have liked to see some mutagenesis experiments to test the models of assembly, iron binding and ferroxidase activity. These do not have to be in vivo and can be performed in vitro with the available system. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>117957</offset><text>To address this question we have produced three FOC mutants of the EncFtn protein and characterized these in solution, by mass spectrometry, and crystallographically (section: Mutagenesis of the EncFtnsH Ferroxidase center). We thank the reviewer for this suggestion as it highlighted the importance of the FOC residues for assembly and activity, and our new data has provided interesting insights into the EncFtn protein.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>118380</offset><text>5) I would like some more phylogenetic data for the model that ferritin evolved from EncFer. Perhaps, EncFer evolved from ferritin? Do any of the existing phylogenetic analyses support one model over another. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>118590</offset><text>We now include a phylogenetic tree (Figure 13) and consider the question of ferritin evolution in the Discussion (first paragraph). None of the authors of this study are evolutionary biologists but we appreciate the difficulty inherent in tracing the history of protein folds, especially in bacterial lineages. We refer to previous studies in this section and make a suggestion that can be followed up in subsequent studies.</text></passage></document></collection>
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+<collection><source>PMC</source><date>20201220</date><key>pmc.key</key><document><id>5014086</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1016/j.str.2016.06.020</infon><infon key="article-id_pmc">5014086</infon><infon key="article-id_pmid">27524201</infon><infon key="article-id_publisher-id">S0969-2126(16)30167-8</infon><infon key="fpage">1599</infon><infon key="issue">9</infon><infon key="license">This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</infon><infon key="lpage">1605</infon><infon key="name_0">surname:Zebisch;given-names:Matthias</infon><infon key="name_1">surname:Jackson;given-names:Verity A.</infon><infon key="name_2">surname:Zhao;given-names:Yuguang</infon><infon key="name_3">surname:Jones;given-names:E. Yvonne</infon><infon key="notes">Published: August 11, 2016</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">24</infon><infon key="year">2016</infon><offset>0</offset><text>Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>115</offset><text>Summary</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>123</offset><text>Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling. We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains. The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor. We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD. DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC. Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>1127</offset><text>Graphical Abstract</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>1146</offset><text>Highlights</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1157</offset><text>The structure of the KREMEN 1 ectodomain is solved from three crystal forms</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1233</offset><text>Kringle, WSC, and CUB subdomains interact tightly to form a single structural unit</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1316</offset><text>The interface to DKKs is formed from the Kringle and WSC domains</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1381</offset><text>The CUB domain is found to interact directly with LRP6PE1PE2</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1442</offset><text>Zebisch et al. describe the ectodomain structure of KREMEN 1, a receptor for Wnt antagonists of the DKK family. Apo structures and a complex with functional fragments of DKK1 and LRP6 shed light on the function of this dual-mode regulator of Wnt signaling.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1700</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1713</offset><text>Signaling by Wnt morphogens is renowned for its fundamental roles in embryonic development, tissue homeostasis, and stem cell maintenance. Due to these functions, generation, delivery, and interpretation of Wnt signals are all heavily regulated in the animal body. Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6. Kremen proteins (Krm1 and Krm2) have been identified as additional high-affinity transmembrane receptors for Dkk. Krm and Dkk synergize in Wnt inhibition during Xenopus embryogenesis to regulate anterior-posterior patterning. Mechanistically it is thought that, in the presence of Dkk, Krm forms a ternary complex with Lrp6, which is then rapidly endocytosed. This amplifies the intrinsic Wnt antagonistic activity of Dkk by efficiently depleting the cell surface of the Wnt co-receptor. In accordance with this, Krm1−/− and Krm2−/− double knockout mice show a high bone mass phenotype typical of increased Wnt signaling, as well as growth of ectopic forelimb digits. Growth of ectopic digits is further enhanced upon additional loss of dkk expression. The Wnt antagonistic activity of Krm1 is also linked to its importance for correct thymus epithelium formation in mice. The importance of intact KRM1 for normal human development and health is highlighted by the recent finding that a homozygous mutation in the ectodomain of KRM1 leads to severe ectodermal dysplasia including oligodontia. Interestingly, the Wnt antagonistic activity of Krm is context dependent, and Krm proteins are actually dual-mode Wnt regulators. In the absence of Dkk, Krm1 and 2 change their function from inhibition to enhancement of Lrp6-mediated signaling. By direct binding to Lrp6 via the ectodomains, Krm proteins promote Lrp6 cell-surface localization and hence increase receptor availability. Further increasing the complexity of Krm functionality, it was recently found that Krm1 (but not Krm2) can also act independently of LRP5/6 and Wnt as a dependence receptor, triggering apoptosis unless bound to Dkk.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3834</offset><text>Structurally, Krm1 and 2 are type I transmembrane proteins with a 40 kDa ectodomain and a flexible cytoplasmic tail consisting of 60–75 residues. The ectodomain consists of three similarly sized structural domains of around 10 kDa each: the N-terminal Kringle domain (KR) is followed by a WSC domain of unknown fold. The third structural domain is a CUB domain. An approximately 70-residue linker connects the CUB domain to the transmembrane span. An intact KR-WSC-CUB domain triplet and membrane attachment is required for Wnt antagonism. The transmembrane span and cytoplasmic tail can be replaced with a GPI linker without impact on Wnt antagonism.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4490</offset><text>We sought to provide structural insights into the multi-functionality of this cell-surface receptor. The structures presented here reveal the unknown fold of the WSC domain and the tight interactions of all three domains. We further succeeded in determination of a low-resolution LRP6PE3PE4-DKK1CRD2-KRM1ECD complex, defining the architecture of the Wnt inhibitory complex that leads to Lrp6 cell-surface depletion.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4907</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4915</offset><text>The recombinant production of the extracellular domain of Krm for structural studies proved challenging (see Experimental Procedures). We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown). Several crystal forms were obtained from these complexes, however, crystals always contained only KRM1 protein.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5366</offset><text>We solved the structure of KRM1ECD in three crystal forms at 1.9, 2.8, and 3.2 Å resolution (Table 1). The high-resolution structure is a near full-length model (Figure 1). The small, flexible, and charged 98AEHED102 loop could only be modeled in a slightly lower resolution structure and in crystal form III. The KR, WSC, and CUB are arranged in a roughly triangular fashion with tight interactions between all three domains. The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements. The structurally most similar Kringle domain is that of human plasminogen (PDB: 1PKR) with an root-mean-square deviation (RMSD) of 1.7 Å for 73 aligned Cα (Figure 1B). The KRM1 structure reveals the fold of the WSC domain for the first time. The structure is best described as a sandwich of a β1-β5-β3-β4-β2 antiparallel β sheet and a single α helix. The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198). Using the PDBeFold server, we detected a surprising yet significant homology to PAN module domains. The closest structural relative is hepatocyte growth factor (HGF, PDB: 1GP9), which superposes with an RMSD of 2.3 Å for 58 aligned Cα (Figure 1B). The CUB domain bears two glycosylation sites. Although present, the quality of the electron density around N217 did not allow modeling of the sugar moiety. In crystal form I, a calcium ion is present at the canonical position coordinated by the carboxylates of D263, D266 (bidentate), and D306, as well as the carbonyl of N309 and a water molecule. The coordination sphere deviates significantly from perfectly octahedral (not shown). This might result in the site having a low affinity and may explain why calcium is not present in the two low-resolution crystal forms. Loss of calcium has led to loop rearrangements and partial disorder in these crystal forms. The closest structural relative is the CUB_C domain of Tsg-6 (PDB: 2WNO), which superposes with KRMCUB with an RMSD of 1.6 Å for 104 Cα (Figure 1B).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7578</offset><text>A superposition of the three KRM1 structures reveals no major structural differences (Figure 1C) as anticipated from the plethora of interactions between the three domains. Minor differences are caused by the collapse of the Ca2+ binding site in crystal forms II and III and loop flexibility in the KR domain. The F207S mutation recently found to cause ectodermal dysplasia in Palestinian families maps to the hydrophobic core of the protein at the interface of the three subdomains (Figure 1A). Such a mutation is bound to severely destabilize the protein structure of KRM1, leading to disturbance of its Wnt antagonistic, Wnt stimulatory, and Wnt independent activity.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8251</offset><text>Low-Resolution Insight into Ternary Complex Formation</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8305</offset><text>Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands. One face of the rather flat DKK1CRD2 fragment binds to the third β propeller of LRP6. Mutational analyses further implied that the LRP6PE3-averted face of DKK1CRD2 bears the Krm binding site, hence suggesting how Dkk can recruit both receptors into a ternary complex.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8755</offset><text>To obtain direct insight into ternary complex formation by Lrp5/6, Dkk, and Krm, we subjected an LRP6PE3PE4-DKK1fl-KRM1ECD complex to crystallization trials. Diffraction data collected from the resulting crystals were highly anisotropic with diffraction extending in the best directions to 3.5 Å and 3.7 Å but only to 6.4 Å in the third direction. Despite the lack of high-resolution diffraction, the general architecture of the ternary complex is revealed (Figure 2A). DKK1CRD2 binds to the top face of the LRP6 PE3 β propeller as described earlier for the binary complex. KRM1ECD does indeed bind on the opposite side of DKK1CRD2 with only its KR and WSC domains engaged in binding (Figure 2A). Although present in the complex subjected to crystallization, we observe no density that could correspond to CRD1 or the domain linker (L). We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure 2B). A SUMO fusion of DKK1L-CRD2 displayed a similar (slightly higher) affinity. In contrast, a SUMO fusion of DKK1CRD1-L did not display binding for concentrations tested up to 325 μM (Figure 2B).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10014</offset><text>Overall, the DKK1-KRM1 interface is characterized by a large number of polar interactions but only few hydrophobic contacts (Figure 2C). The crystal structure gives an explanation for DKK1 loss-of-binding mutations identified previously: R191 of DKK1 forms a double salt bridge to D125 and E162 of KRM1 (Figure 2C). A charge reversal as in the mouse Dkk1 (mDkk1) R197E variant would severely disrupt the binding. Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90. Again, a charge reversal as shown before for mDkk1 K232E would be incompatible with binding. The side chain of DKK1 S192 was also predicted to be involved in Krm binding. Indeed, we found (Figure 2C) that the side chain of D201 of KRM1 forms two hydrogen bonds to the side-chain hydroxyl and the backbone amide of S192 (mouse, S198). Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively. The carbonyl of DKK1 R224 is hydrogen bonded to Y105 and W106 of KRM1. We suspect that the Dkk charge reversal mutations performed in the murine background and shown to diminish Krm binding K211E and R203E (mouse K217E and R209E) do so likely indirectly by disruption of the Dkk fold. We further validated the DKK1 binding site on KRM1 by introducing glycosylation sites at the KR (90DVS92→NVS) and WSC (189VCF191→NCS) domains pointing toward DKK (Figures 2A and 2D). Introduction of N-linked glycans in protein-protein-binding sites is an established way of disrupting protein-binding interfaces. Both ectodomain mutants were secreted comparably with the wild-type, indicating correct folding, but failed to achieve any detectable binding in SPR using full-length DKK1 as analyte. In contrast, a mutant carrying an additional N-glycan outside the interface at the CUB domain (309NQA311→NQS), was wild-type-like in DKK1 binding (Figure 2D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>12076</offset><text>Identification of a Direct LRP6-KRM1 Binding Site</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12126</offset><text>The LRP6PE3PE4-DKK1CRD2-KRM1ECD complex structure reveals no direct interactions between KRM1 and LRP6. We constructed in silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data. An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12642</offset><text>For this crude approximation of a true ternary complex, we noted very close proximity between the Ca2+-binding region of KRM1 and the top face of the PE2 β propeller of LRP6. The solvent-exposed residues R307, I308, and N309 of the central Ca2+-binding β connection loop of KRM1 would be almost ideally positioned for binding to this face, which is commonly used as a binding site on β propellers. Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure 3B). To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment. Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2 (Figure 3C). In contrast, no saturable binding was observed between KRM1 and LRP6PE3PE4. Introduction of an N-glycosylation site at N309 in KRM1ECD abolished LRP6PE1PE2 binding (Figure 3C), while binding to DKK1 was unaffected (Figure 2D). We conclude that the predicted binding site between KRM1CUB and LRP6PE2 is a strong candidate for mediating the direct Lrp6-Krm interaction, which is thought to increase Wnt responsiveness by stabilizing Lrp6 at the cell surface. Further experiments are required to pinpoint the exact binding site. Although LRP6PE1 appears somewhat out of reach in the modeled ternary complex, it cannot be excluded as the Krm binding site in the ternary complex and LRP6-Krm binary complex. The presence of DKK may govern which propeller (PE1 versus PE2) of LRP6 is available for Krm binding.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14374</offset><text>Apparent binding across the proposed KRM1CUB-LRP6PE2 interface is expected to be higher once Krm is also cross-linked to LRP6PE3 via DKK1CRD2 (Figure 3D). Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3. It is therefore possible that the interplay of Krm and Dkk binding can promote changes in LRP6 ectodomain conformation with functional consequences; however, such ideas await investigation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15012</offset><text>Taken together, the structural and biophysical studies we report here extend our mechanistic understanding of Wnt signal regulation. We describe the ectodomain structure of the dual Wnt regulator Krm1, providing an explanation for the detrimental effect on health and development of a homozygous KRM1 mutation. We also reveal the interaction mode of Krm-Dkk and the architecture of the ternary complex formed by Lrp5/6, Dkk, and Krm. Furthermore, the ternary crystal structure has guided in silico and biophysical analyses to suggest a direct LRP6-KRM1 interaction site. Our findings provide a solid foundation for additional studies to probe how ternary complex formation triggers internalization, whereas Krm binding in the absence of Dkk stabilizes the Wnt co-receptor at the cell surface.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>15806</offset><text>Experimental Procedures</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>15830</offset><text>Large-Scale Mammalian Expression and Protein Purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>15888</offset><text>KrmECD fragments were cloned into pHLsec or variants thereof. Full ectodomain variants (e.g., KRM1 isoform 3, P30-T377) were well secreted into the conditioned medium (CM) of HEK293T cells, but exhibited extensive O-glycosylation (as judged from smeary bands in western blot), which would be detrimental to crystallization. Fragments truncated to the KR-WSC-CUB core gave sharp bands but were barely secreted. We therefore engineered an A23-G373 (isoform 1 numbering used throughout the article) full ectodomain construct (KRM1ECD-TEV) with a C-terminal His10 tag that contained a TEV protease cleavage site after E324. The expected sequence of the secreted protein is ETG-23APSPGLGPGPE31 … 320AVKEE324-GSENLYFQGGS-325LPQ … VPG373-THHHHHHHHHH (the isoform-2-specific PG insertion and the TEV site are underlined). This construct was well secreted and could be processed using TEV protease. However, 80%–90% of the protein eluted as aggregates from a size-exclusion column even before TEV treatment. The same applied to analog constructs for Krm1 from zebrafish, frog, and mouse. No monomeric protein at all could be obtained for several Krm2 constructs from multiple species. A KRM1ECD-TEV expressing stable GntI-deficient HEK293S cell line was generated by excision of an EcoRI-XhoI fragment, sub-cloning into pNeo-Sec-1, and selection of neomycin-resistant cells. The stable cell line showed expression levels superior to transiently transfected cells (not shown).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17365</offset><text>Human LRP6PE1PE2, LRP6PE3PE4, and full-length DKK1 were produced in a similar way as described. Shorter constructs of DKK1 lacking the N-terminal flexible region and CRD1 were not secreted from HEK cells. However, using the approach of an N-terminal fusion to a modified SUMO protein as described earlier, we succeeded in secretory expression of a SUMO-DKK1Linker-CRD2 construct encompassing residues S141-H266. A variant of this containing a TEV cleavage site just before T181, SUMO-DKK1Linker-TEV-CRD2, was also well expressed and allowed removal of the flexible linker region.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17945</offset><text>To obtain complexes of KRM1ECD-TEV, we (co-)transfected the stable cell line with DKK and LRP6PE3PE4 constructs described earlier. Binary and ternary KRM1ECD-DKK1fl and KRM1ECD-DKK1fl-LRP6PE3PE4 complexes were stable in gel-filtration eluting as distinct monodisperse peaks.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>18220</offset><text>Crystallization and Data Collection</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>18256</offset><text>All samples subjected to crystallization were purified from CM by affinity and size-exclusion chromatography. After treatment with TEV protease and endoglycosidase F1 overnight using mass equivalents of 1%, samples were subjected to size-exclusion chromatography in 10 mM HEPES/NaOH (pH 7.5), 150 mM NaCl. The crystals giving rise to the 1.9 Å dataset for KRM1 in crystal form I were obtained from a KRM1ECD-DKK1Linker-CRD2 complex concentrated to 12 mg/mL. Out of this complex, KRM1ECD crystallized alone in 2.0 M ammonium sulfate, 5% (v/v) iso-propanol. For cryoprotection, crystals were transferred to mother liquor mixed 1:1 with 3.4 M sodium malonate (pH 7.0). The slightly less well-ordered crystal of crystal form I and crystals of form II were obtained from a KRM1ECD-DKK1CRD2 complex using the SUMO-DKK1Linker-TEV-CRD2 construct and releasing SUMO and the DKK linker region by TEV and 3C protease treatment. Crystals of form I (2.1 Å) appeared from protein at 12 mg/mL in 1.0 M (NH4)H2PO4, 0.100 M sodium citrate (pH 5.6) and were cryoprotected by transfer to 2.9 M sodium malonate (pH 5.0). Crystals of form II grew from protein concentrated to 17 mg/mL in 1.0 M MgSO4, 0.1 M trisodium citrate (final pH 5.6). For cryoprotection, crystals were transferred to mother liquor mixed 1:3 with 3.0 M ammonium sulfate, 18% glycerol. Crystal form III appeared after 11 months in a dried-out drop of condition H5 of the Morpheus screen. The protein concentration had been 9 mg/mL. For cryoprotection, fresh liquid from Morpheus/H5 was added. The ternary complex structure was obtained from an LRP6PE3PE4-DKK1fl-KRM1ECD complex at 9 mg/mL that grew in condition E10 of the PACTpremier screen (pH approximately 6.8) over the course of 2–11 months. For cryoprotection, 10% PEG200 was added. By mistake, the crystals were incubated for 1 hr with 1 mM platinum compound in this cryosolution before cryocooling.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>20188</offset><text>Structure Determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20212</offset><text>Diffraction data were collected at DIAMOND synchrotron light source at the beamlines detailed in Table 1. The structure was initially solved from crystal form III by molecular replacement (MR) with PHASER, placing models for the CUB domain (PDB: 2WNO, CUB_C domain of Tsg-6, 37% sequence identity), and the KR domain (PDB: 1PKR, Kringle 1 of plasminogen; 39% sequence identity). Traceable density for the WSC domain became immediately evident. The KRM1 structure was then built and refined by cycling between the various crystal forms.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20748</offset><text>For the ternary complex, we obtained only a low-resolution, highly anisotropic dataset extending to Bragg spacings of 3.5 Å, 6.4 Å, and 3.7 Å along the three principle directions (&lt;I/σI&gt; = 2). All data to 3.5 Å were used during structure determination by MR. LRP6PE3PE4 (PDB: 4A0P) and KRM1ECD (both stripped of glycosylation sites) could be placed independently by PHASER, giving Z scores of &gt;10 and log likelihood gains (LLG) of &gt;200. The combined LLG was 673, increasing to 901 after rigid-body refinement. Strong electron density became apparent at glycosylation sites and close to methionines (see platinum soak above), further supporting the MR solution. Additional strong density was evident between LRP6 and KRM1, suggesting the presence of DKK1. A model of the DKK1CRD2 (PDB: 3S2K and 3S8V) could then be placed with PHASER by testing all rotation function peaks. This increased the LLG from 901 to 973 indicating a correct solution. The individually placed LRP6 and DKK models were then replaced with chains B and C from the LRP6-DKK complex in PDB: 3S2K. The structure was subjected to rigid-body refinement using single structural domains as individually positioned bodies.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21945</offset><text>We then performed restrained refinement of the coordinates against the ellipsoidally truncated and anisotropically scaled diffraction data as obtained from the diffraction anisotropy server at UCLA. The resolution cutoffs were 3.5 Å, 6.4 Å, and 3.7 Å. Strong geometric restraints generated by PROSMART from the available high-resolution reference structures were used during refinement. No manual model building was attempted. Restrained refinement was followed by ten cycles of structure idealization. The final model had Rwork/Rfree errors of 32.5%/36.1% against the anisotropy-corrected data and 32.1%/35.5% against the unmodified but ellipsoidally truncated diffraction data.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>22631</offset><text>Surface Plasmon Resonance</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22657</offset><text>Equilibrium experiments were performed as described before with the addition of 2 mM CaCl2 for experiments investigating the direct LRP6PE1PE2-KRM1ECD interaction.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title_1</infon><offset>22822</offset><text>Author Contributions</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">paragraph</infon><offset>22843</offset><text>M.Z. and V.A.J. performed experiments with support from Y.Z., who generated the stable cell line. 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Cysteines as ball and sticks, glycosylation sites as sticks. The bound calcium is shown as a gray sphere. The site of the F207S mutation associated with ectodermal dysplasia in humans is shown as mesh.</text></passage><passage><infon key="file">gr1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27356</offset><text>(B) Superposition of the three KRM1ECD subdomains (solid) with their next structurally characterized homologs (half transparent).</text></passage><passage><infon key="file">gr1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27486</offset><text>(C) Superposition of KRM1ECD from the three crystal forms. Alignment scores for each pairing are indicated on the dashed triangle.</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27617</offset><text>Insight into Ternary Complex Formation</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27656</offset><text>(A) The structure of the ternary LRP6PE3PE4-DKK1CRD2-KRM1ECD complex. DKK1 (orange) is sandwiched between the PE3 module of LRP6 (blue) and the KR-WSC domain pair of KRM1 (green). Colored symbols indicate introduced N-glycan attachment sites (see D).</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27907</offset><text>(B) SPR data comparing binding of full-length DKK1 and SUMO fusions of DKK1 truncations for binding to immobilized wild-type KRM1ECD.</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28041</offset><text>(C) Close-up view of the DKK1CRD2-KRM1ECD interface. Residues involved in interface formation are shown as sticks; those mentioned in the text are labeled. Salt bridges are in pink and hydrogen bonds in black. Model bias cannot be excluded as single atoms and bonds are not resolved at 6.4–3.5 Å. See also Figure S1.</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28364</offset><text>(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange). See also symbols in (A).</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28614</offset><text>LRP6-KRM1 Direct Interaction and Summary</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28655</offset><text>(A) In a construction of a ternary complex with all four β propellers of LRP6 intact, the CUB domain points via its Ca2+-binding region toward the top face of the second β propeller.</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28844</offset><text>(B) Close-up view of the potential interaction site. In addition, LRP6PE2 has been superimposed with DKK1 (yellow) and SOST (pink) peptide complexes of LRP6PE1.</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>29005</offset><text>(C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309.</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>29131</offset><text>(D) Schematic representation of structural and biophysical findings and their implications for Wnt-dependent (left, middle) and independent (right) signaling. Conformational differences in the depictions of LRP6 are included purely for ease of representation.</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>29391</offset><text>Diffraction and Refinement Statistics</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table xmlns:xlink=&quot;http://www.w3.org/1999/xlink&quot; frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead&gt;&lt;tr&gt;&lt;th/&gt;&lt;th&gt;KRM1&lt;sub&gt;ECD&lt;/sub&gt;&lt;/th&gt;&lt;th&gt;KRM1&lt;sub&gt;ECD&lt;/sub&gt;&lt;/th&gt;&lt;th&gt;KRM1&lt;sub&gt;ECD&lt;/sub&gt;&lt;/th&gt;&lt;th&gt;KRM1&lt;sub&gt;ECD&lt;/sub&gt;&lt;/th&gt;&lt;th&gt;LRP6&lt;sub&gt;PE3PE4&lt;/sub&gt;-DKK&lt;sub&gt;CRD2&lt;/sub&gt;-KRM1&lt;sub&gt;ECD&lt;/sub&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td&gt;Crystal form&lt;/td&gt;&lt;td&gt;I&lt;/td&gt;&lt;td&gt;I&lt;/td&gt;&lt;td&gt;II&lt;/td&gt;&lt;td&gt;III&lt;/td&gt;&lt;td&gt;I&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;X-ray source&lt;/td&gt;&lt;td&gt;Diamond i04&lt;/td&gt;&lt;td&gt;Diamond i03&lt;/td&gt;&lt;td&gt;Diamond i03&lt;/td&gt;&lt;td&gt;Diamond i04&lt;/td&gt;&lt;td&gt;Diamond i04&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Wavelength (Å)&lt;/td&gt;&lt;td&gt;0.9793&lt;/td&gt;&lt;td&gt;0.9700&lt;/td&gt;&lt;td&gt;0.9700&lt;/td&gt;&lt;td&gt;0.9795&lt;/td&gt;&lt;td&gt;0.9795&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Space group&lt;/td&gt;&lt;td&gt;&lt;italic&gt;P&lt;/italic&gt;3&lt;sub&gt;1&lt;/sub&gt;21&lt;/td&gt;&lt;td&gt;&lt;italic&gt;P&lt;/italic&gt;3&lt;sub&gt;1&lt;/sub&gt;21&lt;/td&gt;&lt;td&gt;&lt;italic&gt;P&lt;/italic&gt;4&lt;sub&gt;3&lt;/sub&gt;&lt;/td&gt;&lt;td&gt;&lt;italic&gt;P&lt;/italic&gt;4&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;/td&gt;&lt;td&gt;&lt;italic&gt;C&lt;/italic&gt;222&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Unit cell a/α (Å/°)&lt;/td&gt;&lt;td&gt;50.9/90&lt;/td&gt;&lt;td&gt;50.5/90&lt;/td&gt;&lt;td&gt;65.8/90&lt;/td&gt;&lt;td&gt;67.8/90&lt;/td&gt;&lt;td&gt;86.9/90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;b/β (Å/°)&lt;/td&gt;&lt;td&gt;50.9/90&lt;/td&gt;&lt;td&gt;50.5/90&lt;/td&gt;&lt;td&gt;65.8/90&lt;/td&gt;&lt;td&gt;67.8/90&lt;/td&gt;&lt;td&gt;100.1/90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;c/γ (Å/°)&lt;/td&gt;&lt;td&gt;188.4/120&lt;/td&gt;&lt;td&gt;187.4/120&lt;/td&gt;&lt;td&gt;75.0/90&lt;/td&gt;&lt;td&gt;198.2/90&lt;/td&gt;&lt;td&gt;270.7/90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Wilson B factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/td&gt;&lt;td&gt;31&lt;/td&gt;&lt;td&gt;41&lt;/td&gt;&lt;td&gt;76&lt;/td&gt;&lt;td&gt;77&lt;/td&gt;&lt;td&gt;NA&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Resolution range (Å)&lt;/td&gt;&lt;td&gt;47.10–1.90 (1.95–1.90)&lt;/td&gt;&lt;td&gt;62.47–2.10 (2.16–2.10)&lt;/td&gt;&lt;td&gt;75.00–2.80 (2.99–2.80)&lt;/td&gt;&lt;td&gt;67.80–3.20 (3.42–3.20)&lt;/td&gt;&lt;td&gt;67.68–3.50 (7.16–6.40, 3.92–3.50)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Unique reflections&lt;/td&gt;&lt;td&gt;23,300 (1,524)&lt;/td&gt;&lt;td&gt;17,089 (1,428)&lt;/td&gt;&lt;td&gt;7,964 (1,448)&lt;/td&gt;&lt;td&gt;8,171 (1,343)&lt;/td&gt;&lt;td&gt;8,070 (723, 645)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Average multiplicity&lt;/td&gt;&lt;td&gt;9.1 (9.2)&lt;/td&gt;&lt;td&gt;5.2 (5.3)&lt;/td&gt;&lt;td&gt;3.7 (3.7)&lt;/td&gt;&lt;td&gt;22.7 (12.6)&lt;/td&gt;&lt;td&gt;3.8 (3.5, 4.4)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Completeness (%)&lt;/td&gt;&lt;td&gt;99.8 (98.5)&lt;/td&gt;&lt;td&gt;100 (100)&lt;/td&gt;&lt;td&gt;99.8 (100)&lt;/td&gt;&lt;td&gt;98.8 (93.4)&lt;/td&gt;&lt;td&gt;51.6 (98.5, 14.1)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&amp;lt;&lt;italic&gt;I&lt;/italic&gt;/&lt;italic&gt;σI&lt;/italic&gt;&amp;gt;&lt;/td&gt;&lt;td&gt;11.4 (1.7)&lt;/td&gt;&lt;td&gt;12.0 (1.7)&lt;/td&gt;&lt;td&gt;14.9 (1.5)&lt;/td&gt;&lt;td&gt;13.1 (1.9)&lt;/td&gt;&lt;td&gt;4.6 (4.1, 2.2)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;merge&lt;/sub&gt; (%)&lt;/td&gt;&lt;td&gt;14.8 (158.3)&lt;/td&gt;&lt;td&gt;9.3 (98.0)&lt;/td&gt;&lt;td&gt;6.2 (98.9)&lt;/td&gt;&lt;td&gt;29.8 (142.2)&lt;/td&gt;&lt;td&gt;44.9 (40.5, 114.2)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;pim&lt;/sub&gt; (%)&lt;/td&gt;&lt;td&gt;15.7 (55.3)&lt;/td&gt;&lt;td&gt;10.3 (109.0)&lt;/td&gt;&lt;td&gt;3.7 (53.8)&lt;/td&gt;&lt;td&gt;6.3 (40.0)&lt;/td&gt;&lt;td&gt;24.7 (23.9, 59.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;bold&gt;Refinement&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;work&lt;/sub&gt; (%)&lt;/td&gt;&lt;td&gt;17.9&lt;/td&gt;&lt;td&gt;18.4&lt;/td&gt;&lt;td&gt;21.6&lt;/td&gt;&lt;td&gt;20.2&lt;/td&gt;&lt;td&gt;32.1&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt; (%)&lt;/td&gt;&lt;td&gt;22.7&lt;/td&gt;&lt;td&gt;23.2&lt;/td&gt;&lt;td&gt;30.7&lt;/td&gt;&lt;td&gt;27.1&lt;/td&gt;&lt;td&gt;35.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;bold&gt;No. of Non-Hydrogen Atoms&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Protein&lt;/td&gt;&lt;td&gt;2,260&lt;/td&gt;&lt;td&gt;2,301&lt;/td&gt;&lt;td&gt;2,102&lt;/td&gt;&lt;td&gt;2,305&lt;/td&gt;&lt;td&gt;7,730&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;N-glycans&lt;/td&gt;&lt;td&gt;42&lt;/td&gt;&lt;td&gt;42&lt;/td&gt;&lt;td&gt;28&lt;/td&gt;&lt;td&gt;28&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Water&lt;/td&gt;&lt;td&gt;79&lt;/td&gt;&lt;td&gt;54&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;td&gt;2&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Ligands&lt;/td&gt;&lt;td&gt;6&lt;/td&gt;&lt;td&gt;6&lt;/td&gt;&lt;td&gt;2&lt;/td&gt;&lt;td&gt;5&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;bold&gt;Average B factor (Å&lt;sup&gt;2&lt;/sup&gt;)&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Protein&lt;/td&gt;&lt;td&gt;63&lt;/td&gt;&lt;td&gt;65&lt;/td&gt;&lt;td&gt;108&lt;/td&gt;&lt;td&gt;84&lt;/td&gt;&lt;td&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;N-glycans&lt;/td&gt;&lt;td&gt;35&lt;/td&gt;&lt;td&gt;46&lt;/td&gt;&lt;td&gt;102&lt;/td&gt;&lt;td&gt;18&lt;/td&gt;&lt;td&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Water&lt;/td&gt;&lt;td&gt;68&lt;/td&gt;&lt;td&gt;85&lt;/td&gt;&lt;td&gt;–&lt;/td&gt;&lt;td&gt;75&lt;/td&gt;&lt;td&gt;–&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Ligands&lt;/td&gt;&lt;td&gt;36&lt;/td&gt;&lt;td&gt;47&lt;/td&gt;&lt;td&gt;91&lt;/td&gt;&lt;td&gt;75&lt;/td&gt;&lt;td&gt;66&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;bold&gt;RMSD from Ideality&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Bond lengths (Å)&lt;/td&gt;&lt;td&gt;0.020&lt;/td&gt;&lt;td&gt;0.016&lt;/td&gt;&lt;td&gt;0.019&lt;/td&gt;&lt;td&gt;0.016&lt;/td&gt;&lt;td&gt;0.004&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Bond angles (°)&lt;/td&gt;&lt;td&gt;2.050&lt;/td&gt;&lt;td&gt;1.748&lt;/td&gt;&lt;td&gt;1.952&lt;/td&gt;&lt;td&gt;1.796&lt;/td&gt;&lt;td&gt;0.770&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;bold&gt;Ramachandran Plot&lt;/bold&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;6&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Favored (%)&lt;/td&gt;&lt;td&gt;96.8&lt;/td&gt;&lt;td&gt;95.5&lt;/td&gt;&lt;td&gt;96.9&lt;/td&gt;&lt;td&gt;94.9&lt;/td&gt;&lt;td&gt;92.3&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Allowed (%)&lt;/td&gt;&lt;td&gt;99.7&lt;/td&gt;&lt;td&gt;100.0&lt;/td&gt;&lt;td&gt;100.0&lt;/td&gt;&lt;td&gt;99.7&lt;/td&gt;&lt;td&gt;99.8&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;Number of outliers&lt;/td&gt;&lt;td&gt;1&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;td&gt;0&lt;/td&gt;&lt;td&gt;1&lt;/td&gt;&lt;td&gt;2&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td&gt;PDB code&lt;/td&gt;&lt;td&gt;&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;pdb:5FWS&quot; id=&quot;intref0085&quot;&gt;5FWS&lt;/ext-link&gt;&lt;/td&gt;&lt;td&gt;&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;pdb:5FWT&quot; id=&quot;intref0090&quot;&gt;5FWT&lt;/ext-link&gt;&lt;/td&gt;&lt;td&gt;&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;pdb:5FWU&quot; id=&quot;intref0095&quot;&gt;5FWU&lt;/ext-link&gt;&lt;/td&gt;&lt;td&gt;&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;pdb:5FWV&quot; id=&quot;intref0100&quot;&gt;5FWV&lt;/ext-link&gt;&lt;/td&gt;&lt;td&gt;&lt;ext-link ext-link-type=&quot;uri&quot; xlink:href=&quot;pdb:5FWW&quot; id=&quot;intref0105&quot;&gt;5FWW&lt;/ext-link&gt;&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>29429</offset><text>	KRM1ECD	KRM1ECD	KRM1ECD	KRM1ECD	LRP6PE3PE4-DKKCRD2-KRM1ECD	 	Crystal form	I	I	II	III	I	 	X-ray source	Diamond i04	Diamond i03	Diamond i03	Diamond i04	Diamond i04	 	Wavelength (Å)	0.9793	0.9700	0.9700	0.9795	0.9795	 	Space group	P3121	P3121	P43	P41212	C2221	 	Unit cell a/α (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	86.9/90	 	b/β (Å/°)	50.9/90	50.5/90	65.8/90	67.8/90	100.1/90	 	c/γ (Å/°)	188.4/120	187.4/120	75.0/90	198.2/90	270.7/90	 	Wilson B factor (Å2)	31	41	76	77	NA	 	Resolution range (Å)	47.10–1.90 (1.95–1.90)	62.47–2.10 (2.16–2.10)	75.00–2.80 (2.99–2.80)	67.80–3.20 (3.42–3.20)	67.68–3.50 (7.16–6.40, 3.92–3.50)	 	Unique reflections	23,300 (1,524)	17,089 (1,428)	7,964 (1,448)	8,171 (1,343)	8,070 (723, 645)	 	Average multiplicity	9.1 (9.2)	5.2 (5.3)	3.7 (3.7)	22.7 (12.6)	3.8 (3.5, 4.4)	 	Completeness (%)	99.8 (98.5)	100 (100)	99.8 (100)	98.8 (93.4)	51.6 (98.5, 14.1)	 	&lt;I/σI&gt;	11.4 (1.7)	12.0 (1.7)	14.9 (1.5)	13.1 (1.9)	4.6 (4.1, 2.2)	 	Rmerge (%)	14.8 (158.3)	9.3 (98.0)	6.2 (98.9)	29.8 (142.2)	44.9 (40.5, 114.2)	 	Rpim (%)	15.7 (55.3)	10.3 (109.0)	3.7 (53.8)	6.3 (40.0)	24.7 (23.9, 59.9)	 		 	Refinement	 		 	Rwork (%)	17.9	18.4	21.6	20.2	32.1	 	Rfree (%)	22.7	23.2	30.7	27.1	35.5	 		 	No. of Non-Hydrogen Atoms	 		 	Protein	2,260	2,301	2,102	2,305	7,730	 	N-glycans	42	42	28	28	0	 	Water	79	54	0	2	0	 	Ligands	6	6	2	5	0	 		 	Average B factor (Å2)	 		 	Protein	63	65	108	84	–	 	N-glycans	35	46	102	18	–	 	Water	68	85	–	75	–	 	Ligands	36	47	91	75	66	 		 	RMSD from Ideality	 		 	Bond lengths (Å)	0.020	0.016	0.019	0.016	0.004	 	Bond angles (°)	2.050	1.748	1.952	1.796	0.770	 		 	Ramachandran Plot	 		 	Favored (%)	96.8	95.5	96.9	94.9	92.3	 	Allowed (%)	99.7	100.0	100.0	99.7	99.8	 	Number of outliers	1	0	0	1	2	 	PDB code	5FWS	5FWT	5FWU	5FWV	5FWW	 	</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>31236</offset><text>Values in parentheses refer to the highest-resolution shell. An additional shell given for the ternary complex corresponds to the last shell with near-complete diffraction data. NA, not announced.</text></passage></document></collection>
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+<collection><source>PMC</source><date>20230813</date><key>pmc.key</key><document><id>5063996</id><infon key="license">CC BY</infon><passage><infon key="alt-title">Mechanism of Arabinoxylanase</infon><infon key="article-id_doi">10.1074/jbc.M116.743948</infon><infon key="article-id_pmc">5063996</infon><infon key="article-id_pmid">27531750</infon><infon key="article-id_publisher-id">M116.743948</infon><infon key="fpage">22149</infon><infon key="issue">42</infon><infon key="kwd">cellulosome crystallography enzyme kinetics enzyme mechanism glycoside hydrolase</infon><infon key="license">Author's Choice—Final version free via Creative Commons CC-BY license.</infon><infon key="lpage">22159</infon><infon key="name_0">surname:Labourel;given-names:Aurore</infon><infon key="name_1">surname:Crouch;given-names:Lucy I.</infon><infon key="name_10">surname:Najmudin;given-names:Shabir</infon><infon key="name_11">surname:Baslé;given-names:Arnaud</infon><infon key="name_12">surname:Cuskin;given-names:Fiona</infon><infon key="name_2">surname:Brás;given-names:Joana L. A.</infon><infon key="name_3">surname:Jackson;given-names:Adam</infon><infon key="name_4">surname:Rogowski;given-names:Artur</infon><infon key="name_5">surname:Gray;given-names:Joseph</infon><infon key="name_6">surname:Yadav;given-names:Madhav P.</infon><infon key="name_7">surname:Henrissat;given-names:Bernard</infon><infon key="name_8">surname:Fontes;given-names:Carlos M. G. A.</infon><infon key="name_9">surname:Gilbert;given-names:Harry J.</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">291</infon><infon key="year">2016</infon><offset>0</offset><text>The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans*</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>79</offset><text>The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance. Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains. A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans. The mechanism of substrate recognition displayed by the enzyme, however, remains unclear. Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands. The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain. The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility. The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center. The −2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants. Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity. Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed. This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1757</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1770</offset><text>The plant cell wall is an important biological substrate. This complex composite structure is depolymerized by microorganisms that occupy important highly competitive ecological niches, whereas the process makes an important contribution to the carbon cycle. Lignocellulosic degradation is also of continued interest to environmentally sensitive industries such as the biofuels and biorefinery sectors, where the use of sustainable or renewable substrates is of increasing importance. Given that the plant cell wall is the most abundant source of renewable organic carbon on the planet, this macromolecular substrate has substantial industrial potential.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2425</offset><text>An example of the chemical complexity of the plant cell wall is provided by xylan, which is the major hemicellulosic component. This polysaccharide comprises a backbone of β-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with α-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated. In addition, the Araf side chain decorations can also be esterified to ferulic acid that, in some species, provide a chemical link between hemicellulose and lignin. The precise structure of xylans varies between plant species, in particular in different tissues and during cellular differentiation. In specialized plant tissues, such as the outer layer of cereal grains, xylans are extremely complex, and side chains may comprise a range of other sugars including l- and d-galactose and β- and α-Xylp units. Indeed, in these cereal brans, xylans have very few backbone Xylp units that are undecorated, and the side chains can contain up to six sugars.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3517</offset><text>Reflecting the chemical and physical complexity of the plant cell wall, microorganisms that utilize these composite structures express a large number of polysaccharide-degrading enzymes, primarily glycoside hydrolases, but also polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. These carbohydrate active enzymes are grouped into sequence-based families in the CAZy database. With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8. The extensive decoration of the xylan backbone generally restricts the capacity of these enzymes to attack the polysaccharide prior to removal of the side chains by a range of α-glucuronidases, α-arabinofuranosidases, and esterases. Two xylanases, however, utilize the side chains as essential specificity determinants and thus target decorated forms of the hemicellulose. The GH30 glucuronoxylanases require the Xylp bound at the −2 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites −1 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively). The GH5 arabinoxylanase (CtXyl5A) derived from Clostridium thermocellum displays an absolute requirement for xylans that contain Araf side chains. In this enzyme, the key specificity determinant is the Araf appended to O3 of the Xylp bound in the active site (−1 subsite). The reaction products generated from arabinoxylans, however, suggest that Araf can be accommodated at subsites distal to the active site.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5280</offset><text>CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1. Previous studies of Fn3 domains have indicated that they might function as ligand-binding modules, as a compact form of peptide linkers or spacers between other domains, as cellulose-disrupting modules, or as proteins that help large enzyme complexes remain soluble. The dockerin domain recruits the enzyme into the cellulosome, a multienzyme plant cell wall degrading complex presented on the surface of C. thermocellum. CtCBM6 stabilizes CtGH5, and CtCBM62 binds to d-galactopyranose and l-arabinopyranose. The function of the CtCBM13 and Fn3 modules remains unclear. Similarly, the mechanism of substrate recognition and its impact on specificity are key unresolved issues. This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity. The data show that the plasticity in substrate recognition enables the enzyme to hydrolyze highly complex xylans that are not accessible to classical GH10 and GH11 endo-xylanases.</text></passage><passage><infon key="file">zbc0441653440001.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>6633</offset><text>Molecular architecture of GH5_34 enzymes. Modules prefaced by GH, CBM, or CE are modules in the indicated glycoside hydrolase, carbohydrate binding module, or carbohydrate esterase families, respectively. Laminin_3_G domain belongs to the concanavalin A lectin superfamily, and FN3 denotes a fibronectin type 3 domain. Segments labeled D are dockerin domains.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>6993</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>7001</offset><text>Substrate Specificity of CtXyl5A</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7034</offset><text>Previous studies showed that CtXyl5A is an arabinoxylan-specific xylanase that generates xylooligosaccharides with an arabinose linked O3 to the reducing end xylose. The enzyme is active against both wheat and rye arabinoxylans (abbreviated as WAX and RAX, respectively). It was proposed that arabinose decorations make productive interactions with a pocket (−2*) that is abutted onto the active site or −1 subsite. Arabinose side chains of the other backbone xylose units in the oligosaccharides generated by CtXyl5A were essentially random. These data suggest that O3, and possibly O2, on the xylose residues at subsites distal to the active site and −2* pocket are solvent-exposed, implying that the enzyme can access highly decorated xylans. To test this hypothesis, the activity of CtXyl5A against xylans from cereal brans was assessed. CtXyl5a was incubated with a range of xylans for 16 h at 60 °C, and the limit products were visualized by TLC. These xylans are highly decorated not only with Araf and GlcA units but also with l-Gal, d-Gal, and d-Xyl. Indeed, very few xylose units in the backbone of bran xylans lack side chains. The data presented in Table 1 showed that CtXyl5A was active against corn bran xylan (CX). In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues). The limit products generated by CtXyl5A from CX consisted of an extensive range of oligosaccharides. These data support the view that in subsites out with the active site the O2 and O3 groups of the bound xylose units are solvent-exposed and will thus tolerate decoration.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>8773</offset><text>Kinetics of GH5_34 arabinoxylanases</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>8809</offset><text>ND, not determined; NA, no activity.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead valign=&quot;bottom&quot;&gt;&lt;tr&gt;&lt;th align=&quot;center&quot; rowspan=&quot;2&quot; colspan=&quot;1&quot;&gt;Enzyme&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;2&quot; colspan=&quot;1&quot;&gt;Variant&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;3&quot;&gt;&lt;italic&gt;k&lt;/italic&gt;&lt;sub&gt;cat&lt;/sub&gt;/&lt;italic&gt;K&lt;sub&gt;m&lt;/sub&gt;&lt;/italic&gt;&lt;hr/&gt;&lt;/th&gt;&lt;/tr&gt;&lt;tr&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;WAX&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;RAX&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;CX&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;3&quot;&gt;&lt;italic&gt;min&lt;/italic&gt;&lt;sup&gt;−&lt;italic&gt;1&lt;/italic&gt;&lt;/sup&gt;
+&lt;italic&gt;mg&lt;/italic&gt;&lt;sup&gt;−&lt;italic&gt;1&lt;/italic&gt;&lt;/sup&gt;
+&lt;italic&gt;ml&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ct&lt;/italic&gt;Xyl5A&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ct&lt;/italic&gt;GH5-CBM6-CBM13-Fn3-CBM62&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;800&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;460&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ct&lt;/italic&gt;Xyl5A&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ct&lt;/italic&gt;GH5-CBM6-CBM13-Fn3&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1,232&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;659&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ct&lt;/italic&gt;Xyl5A&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ct&lt;/italic&gt;GH5-CBM6-CBM13&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1,307&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;620&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ct&lt;/italic&gt;Xyl5A&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ct&lt;/italic&gt;GH5-CBM6&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;488&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;102&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ct&lt;/italic&gt;Xyl5A&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ct&lt;/italic&gt;GH5-CBM6: E68A&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NA&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NA&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NA&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ct&lt;/italic&gt;Xyl5A&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ct&lt;/italic&gt;GH5-CBM6: Y92A&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NA&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NA&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NA&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ct&lt;/italic&gt;Xyl5A&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ct&lt;/italic&gt;GH5-CBM6: N135A&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;260&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ND&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ct&lt;/italic&gt;Xyl5A&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ct&lt;/italic&gt;GH5-CBM6: N139A&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NA&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NA&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;NA&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Ac&lt;/italic&gt;GH5&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Wild type&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;628&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1,641&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;289&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Gp&lt;/italic&gt;GH5&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Wild type&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2,600&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;9,986&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;314&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Vb&lt;/italic&gt;GH5&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Wild type&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ND&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ND&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;Vb&lt;/italic&gt;GH5&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;D45A&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;102&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;203&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;23&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>8846</offset><text>Enzyme	Variant	kcat/Km	 	WAX	RAX	CX	 			min−1mg−1ml	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3-CBM62	800	ND	460	 	CtXyl5A	CtGH5-CBM6-CBM13-Fn3	1,232	ND	659	 	CtXyl5A	CtGH5-CBM6-CBM13	1,307	ND	620	 	CtXyl5A	CtGH5-CBM6	488	ND	102	 	CtXyl5A	CtGH5-CBM6: E68A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: Y92A	NA	NA	NA	 	CtXyl5A	CtGH5-CBM6: N135A	260	ND	ND	 	CtXyl5A	CtGH5-CBM6: N139A	NA	NA	NA	 	AcGH5	Wild type	628	1,641	289	 	GpGH5	Wild type	2,600	9,986	314	 	VbGH5	Wild type	ND	ND	ND	 	VbGH5	D45A	102	203	23	 	</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9333</offset><text>To explore whether substrate bound only at −2* and −1 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study. HPAEC analysis of the smallest oligosaccharide fraction (pool 4) contained two species with retention times of 14.0 min (oligosaccharide 1) and 20.8 min (oligosaccharide 2) (Fig. 2). Positive mode electrospray mass spectrometry showed that pool 4 contained exclusively molecular ions with a m/z = 305 [M + Na]+, which corresponds to a pentose-pentose disaccharide (molecular mass = 282 Da) as a sodium ion adduct, whereas a dimer of the disaccharide with a sodium adduct (m/z = 587 [2M+Na]+) was also evident. The monosaccharide composition of pool 4 determined by TFA hydrolysis contained xylose and arabinose in a 3:1 ratio. This suggests that the two oligosaccharides consist of two disaccharides: one consisting of two xylose residues and the other consisting of an arabinose linked to a xylose. Treatment of pool 4 with the nonspecific arabinofuranosidase, CjAbf51A, resulted in the loss of oligosaccharide 2 and the production of both xylose and arabinose, indicative of a disaccharide of xylose and arabinose. Incubation of pool 4 with a β-1,3-xylosidase (XynB) converted oligosaccharide 1 into xylose, demonstrating that this molecule is the disaccharide β-1,3-xylobiose. This view is supported by the inability of a β-1,4-specific xylosidase to hydrolyze oligosaccharide 1 or oligosaccharide 2 (data not shown). The crucial importance of occupancy of the −2* pocket for catalytic competence is illustrated by the inability of the enzyme to hydrolyze linear β-1,4-xylooligosaccharides. The generation of Araf-Xylp and Xyl-β-1,3-Xyl as reaction products demonstrates that occupancy of the −2 subsite is not essential for catalytic activity, which is in contrast to all endo-acting xylanases where this subsite plays a critical role in enzyme activity. Indeed, the data demonstrate that −2* plays a more important role in productive substrate binding than the −2 subsite. Unfortunately, the inability to generate highly purified (Xyl-β-1,4)n-[β-1,3-Xyl/Ara]-Xyl oligosaccharides from arabinoxylans prevented the precise binding energies at the negative subsites to be determined.</text></passage><passage><infon key="file">zbc0441653440002.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11762</offset><text>Identification of the disaccharide reaction products generated from CX. The smallest reaction products were purified by size exclusion chromatography and analyzed by HPAEC (A) and positive mode ESI-MS (B), respectively. The samples were treated with a nonspecific arabinofuranosidase (CjAbf51A) and a GH3 xylosidase (XynB) that targeted β-1,3-xylosidic bonds. X, xylose; A, arabinose. The m/z = 305 species denotes a pentose disaccharide as a sodium adduct [M + Na]+, whereas the m/z = 587 signal corresponds to an ESI-MS dimer of the pentose disaccharide also as a sodium adduct [2M + Na]+.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>12357</offset><text>Crystal Structure of the Catalytic Module of CtXyl5A in Complex with Ligands</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12434</offset><text>To understand the structural basis for the biochemical properties of CtXyl5A, the crystal structure of the enzyme with ligands that occupy the substrate binding cleft and the critical −2* subsite were sought. The data presented in Fig. 3A show the structure of the CtXyl5A derivative CtGH5-CtCBM6 in complex with arabinose bound in the −2* pocket. Interestingly, the bound arabinose was in the pyranose conformation rather than in its furanose form found in arabinoxylans. O1 was facing toward the active site −1 subsite, indicative of the bound arabinose being in the right orientation to be linked to the xylan backbone via an α-1,3 linkage. As discussed on below, the axial O4 of the Arap did not interact with the −2* subsite, suggesting that the pocket might be capable of binding a xylose molecule. Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the −2* subsite in its pyranose conformation (Fig. 3B). These crystal structures support the biochemical data presented above showing that the enzyme generated β-1,3-xylobiose from CX, which would require the disaccharide to bind at the −1 and −2* subsites. A third product complex was generated by co-crystallizing the nucleophile inactive mutant CtGH5E279S-CtCBM6 with a WAX-derived oligosaccharide (Fig. 3C). The data revealed a pentasaccharide bound to the enzyme, comprising β-1,4-xylotetraose with an Araf linked α-1,3 to the reducing end xylose. The xylotetraose was positioned in subsites −1 to −4 and the Araf in the −2* pocket. Analysis of the three structures showed that O1, O2, O3, and the endocyclic oxygen occupied identical positions in the Arap, Araf, and Xylp ligands bound in the −2* subsite and thus made identical interactions with the pocket. O1 makes a polar contact with Nδ2 of Asn139, O2 is within hydrogen bonding distance with Oδ1 of Asn139 and the backbone N of Asn135, and O3 interacts with the N of Gly136 and Oϵ2 of Glu68. Although O4 of Arap does not make a direct interaction with the enzyme, O4 and O5 of Xylp and Araf, respectively, form hydrogen bonds with Oϵ1 of Glu68. Finally Tyr92 makes apolar parallel interactions with the pyranose or furanose rings of the three sugars.</text></passage><passage><infon key="file">zbc0441653440003.jpg</infon><infon key="id">F3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14671</offset><text>Representation of the residues involved in the ligands recognition at the −2* subsite. The protein backbone is represented as a cartoon in gray. Interacting residues are represented as stick in blue, and the catalytic residues and the mutated glutamate (into a serine) are in magenta. A, CtGH5-CBM6 in complex with an arabinopyranose. B, CtGH5-CBM6 in complex with a xylopyranose. C, CtGH5E279S-CBM6 in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose). The xylan backbone is shown transparently for more clarity. Densities shown in blue are RefMac maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ. The figure and all other structural figures were made with PyMOL unless otherwise stated.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15430</offset><text>The importance of the interactions between the ligands and the side chains of the residues in the −2* pocket were evaluated by alanine substitution of these amino acids. The mutants E68A, Y92A, and N139A were all inactive (Table 1), demonstrating the importance of the interactions of these residues with the substrate and reinforcing the critical role the −2* subsite plays in the activity of the enzyme. N135A retained wild type activity because the O2 of the sugars interacts with the backbone N of Asn135 and not with the side chain. Because the hydroxyls of Xylp or Araf in the −2* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration. This may explain why the kcat/Km for CtXyl5A against WAX was 2-fold higher than against CX (Table 1). WAX is likely to have a higher concentration of single Araf decorations compared with CX and thus contain more substrate available to the arabinoxylanase.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16441</offset><text>In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92. O3 (O1 of the Araf at the −2* subsite) makes a polar contact with Nδ2 of Asn139; the endocyclic oxygen hydrogens bonds with the OH of Tyr255. The Xylp in the active site makes strong parallel apolar interactions with Phe310. Substrate recognition in the active site is conserved between CtXyl5A and the closest GH5 structural homolog, the endoglucanase BaCel5A (PDB code 1qi2) as noted previously.</text></passage><passage><infon key="file">zbc0441653440004.jpg</infon><infon key="id">F4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>17270</offset><text>Comparison of the ligand recognition at the distal negative subsites between CtGH5E279S-CBM6, the cellulase BaCel5A, and the xylanase GH10.
+A–C show CtGH5E279S-CBM6 is in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose). A, Poseview representation highlighting the hydrogen bonding and the hydrophobic interactions that occur in the negative subsites. C, density of the ligand shown in blue is RefMac maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ. D and E display BaCel5A in complex with deoxy-2-fluoro-β-d-cellotrioside (PDB code 1qi2), and F and G show CmXyn10B in complex with a xylotriose (PDB code 1uqy). The ligand are represented as sticks. B, D, and F are surface representations (CtGH5E279S-CBM6 in gray, BaCel5A in cyan, and the xylanase GH10 in light brown). C, E, and G show the protein backbone as a cartoon representation with the interacting residues represented as sticks. The black dashes represent the hydrogen bonds.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18283</offset><text>The capacity of CtXyl5A to act on the highly decorated xylan CX indicates that O3 and possibly O2 of the backbone Xylp units are solvent-exposed. This is consistent with the interaction of the xylotetraose backbone with the enzyme distal to the active site. A surface representation of the enzyme (Fig. 4B) shows that O3 and O2 of xylose units at subsites −2 to −4 are solvent-exposed and are thus available for decoration. Indeed, these pyranose sugars make very weak apolar interactions with the arabinoxylanase. At −2, Xylp makes planar apolar interactions with the Araf bound to the −2* subsite (Fig. 4C). Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69. Comparison of the distal negative subsites of CtXyl5A with BaCel5A and a typical GH10 xylanase (CmXyn10B, PDB code 1uqy) highlights the paucity of interactions between the arabinoxylanase and its substrate out with the active site (Fig. 4). Thus, the cellulase contains three negative subsites and the sugars bound in the −2 and −3 subsites make a total of 9 polar interactions with the enzyme (Fig. 4, D and E). The GH10 xylanase also contains a −2 subsite that, similar to the cellulase, makes numerous interactions with the substrate (Fig. 4, F and G).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>19682</offset><text>The Influence of the Modular Architecture of CtXyl5A on Catalytic Activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19757</offset><text>CtXyl5A, in addition to its catalytic module, contains three CBMs (CtCBM6, CtCBM13, and CtCBM62) and a fibronectin domain (CtFn3). A previous study showed that although the CBM6 bound in an exo-mode to xylo- and cellulooligosaccharides, the primary role of this module was to stabilize the structure of the GH5 catalytic module. To explore the contribution of the other non-catalytic modules to CtXyl5A function, the activity of a series of truncated derivatives of the arabinoxylanase were assessed. The data in Table 1 show that removal of CtCBM62 caused a modest increase in activity against both WAX and CX, whereas deletion of the Fn3 domain had no further impact on catalytic performance. Truncation of CtCBM13, however, caused a 4–5-fold reduction in activity against both substrates. Members of CBM13 have been shown to bind to xylans, mannose, and galactose residues in complex glycans, hinting that the function of CtCBM13 is to increase the proximity of substrate to the catalytic module of CtXyl5A. Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown). It would appear, therefore, that CtCBM13 makes a structural contribution to the function of CtXyl5A.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>21061</offset><text>Crystal Structure of CtXyl5A-D</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21092</offset><text>To explore further the role of the non-catalytic modules in CtXyl5A the crystal structure of CtXyl5A extending from CtGH5 to CtCBM62 was sought. To obtain a construct that could potentially be crystallized, the protein was generated without the C-terminal dockerin domain because it is known to be unstable and prone to cleavage. Using this construct (CtXyl5A-D) the crystal structure of the arabinoxylanase was determined by molecular replacement to a resolution of 2.64 Å with Rwork and Rfree at 23.7% and 27.8%, respectively. The structure comprises a continuous polypeptide extending from Ala36 to Trp742 displaying four modules GH5-CBM6-CBM13-Fn3. Although there was some electron density for CtCBM62, it was not sufficient to confidently build the module (Fig. 5). Further investigation of the crystal packing revealed a large solvent channel adjacent to the area the CBM62 occupies. We postulate that the reason for the poor electron density is due to the CtCBM62 being mobile compared with the rest of the protein. The structures of CtGH5 and CtCBM6 have been described previously.</text></passage><passage><infon key="file">zbc0441653440005.jpg</infon><infon key="id">F5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>22184</offset><text>Surface representation of the tetra-modular arabinoxylanase and zoom view on the CtGH5 loop. The blue module is the CtGH5 catalytic domain, the green module corresponds to the CtCBM6, the yellow module is the CtCBM13, and the salmon module is the fibronectin domain. Surfaces are semitransparent with the protein backbone represented as a cartoon. The CtGH5 loop is stabilized between the CtCBM6 and the CtCBM13 modules. The black dashes represent the hydrogen bonds. The protein backbone is represented as cartoon, and interacting residues are shown as sticks.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22746</offset><text>CtCBM13 extends from Gly567 to Pro648. Typical of CBM13 proteins CtCBM13 displays a β-trefoil fold comprising the canonical pseudo 3-fold symmetry with a 3-fold repeating unit of 40–50 amino acid residues characteristic of the Ricin superfamily. Each repeat contains two pairs of antiparallel β-strands. A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23509</offset><text>The Fn3 module displays a typical β-sandwich fold with the two sheets comprising, primarily, three antiparallel strands in the order β1-β2-β5 in β-sheet 1 and β4-β3-β6 in β-sheet 2. Although β-sheet 2 presents a cleft-like topology, typical of endo-binding CBMs, the surface lacks aromatic residues that play a key role in ligand recognition, and in the context of the full-length enzyme, the cleft abuts into CtCBM13 and thus would not be able to accommodate an extended polysaccharide chain (see below).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24045</offset><text>In the structure of CtXyl5A-D, the four modules form a three-leaf clover-like structure (Fig. 5). Between the interfaces of CtGH5-CBM6-CBM13 there are a number of interactions that maintain the modules in a fixed position relative to each other. The interaction of CtGH5 and CtCBM6, which buries a substantial apolar solvent-exposed surface of the two modules, has been described previously. The polar interactions between these two modules comprise 14 hydrogen bonds and 5 salt bridges. The apolar and polar interactions between these two modules likely explaining why they do not fold independently compared with other glycoside hydrolases that contain CBMs. CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of ∼450, 350, and 500 Å2, respectively, to form a compact heterotetramer. With respect to the CtCBM6-CBM13 interface, the linker (SPISTGTIP) between the two modules, extending from Ser514 to Pro522, adopts a fixed conformation. Such sequences are normally extremely flexible; however, the two Ile residues make extensive apolar contacts within the linker and with the two CBMs, leading to conformational stabilization. The interactions between CtGH5 and the two CBMs, which are mediated by the tip of the loop between β-7 and α-7 (loop 7) of CtGH5, not only stabilize the trimodular clover-like structure but also make a contribution to catalytic function. Central to the interactions between the three modules is Trp285, which is intercalated between the two CBMs. The Nϵ of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5). Indeed, loop 7 is completely disordered in the truncated derivative of CtXyl5A comprising CtGH5 and CtCBM6, demonstrating that the interactions with CtCBM13 stabilize the conformation of this loop. Although the tip of loop 7 does not directly contribute to the topology of the active site, it is only ∼12 Å from the catalytic nucleophile Glu279. Thus, any perturbation of the loop (through the removal of CtCBM13) is likely to influence the electrostatic and apolar environment of the catalytic apparatus, which could explain the reduction in activity associated with the deletion of CtCBM13.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>26451</offset><text>Similar to the interactions between CtCBM6 and CtCBM13, there are extensive hydrophobic interactions between CtCBM13 and CtFn3, resulting in very little flexibility between these modules. As stated above, the absence of CtCBM62 in the structure suggests that the module can adopt multiple positions with respect to the rest of the protein. The CtCBM62, by binding to its ligands (d-Galp and l-Arap) in plant cell walls, may be able to recruit the enzyme onto its target substrate. Xylans are not generally thought to contain such sugars. d-Galp, however, has been detected in xylans in the outer layer of cereal grains and in eucalyptus trees, which are substrates used by CtXyl5A. Thus, CtCBM62 may direct the enzyme to particularly complex xylans containing d-Galp at the non-reducing termini of the side chains, consistent with the open substrate binding cleft of the arabinoxylanase that is optimized to bind highly decorated forms of the hemicellulose. In general CBMs have little influence on enzyme activity against soluble substrates but have a significant impact on glycans within plant cell walls. Thus, the role of CBM62 will likely only be evident against insoluble composite substrates.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>27651</offset><text>Exploring GH5 Subfamily 34</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27678</offset><text>CtXyl5A is a member of a seven-protein subfamily of GH5, GH5_34. Four of these proteins are distinct, whereas the other three members are essentially identical (derived from different strains of C. thermocellum). To investigate further the substrate specificity within this subfamily, recombinant forms of three members of GH5_34 that were distinct from CtXyl5A were generated. AcGH5 has a similar molecular architecture to CtXyl5A with the exception of an additional carbohydrate esterase family 6 module at the C terminus (Fig. 1). The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function. The Verrucomicobiae enzyme also has an N-terminal GH43 subfamily 10 (GH43_10) catalytic module. The fungal GH5_34, GpGH5, unlike the two bacterial homologs, comprises a single GH5 catalytic module lacking all of the other accessory modules (Fig. 1). GpGh5 is particularly interesting as Gonapodya prolifera is the only fungus of the several hundred fungal genomes that encodes a GH5_34 enzyme. In fact there are four potential GH5_34 sequences in the G. prolifera genome, all of which show high sequence homology to Clostridium GH5_34 sequences. G. prolifera and Clostridium occupy similar environments, suggesting that the GpGH5_34 gene was acquired from a Clostridium species, which was followed by duplication of the gene in the fungal genome. The sequence identity of the GH5_34 catalytic modules with CtXyl5A ranged from 55 to 80% (supplemental Fig. S1). All the GH5_34 enzymes were active on the arabinoxylans RAX, WAX, and CX but displayed no activity on BX (Table 1 and Fig. 6) and are thus defined as arabinoxylanases. The limit products generated by CtXyl5A, AcGH5, and GpGH5 comprised a range of oligosaccharides with some high molecular weight material. The oligosaccharides with low degrees of polymerization were absent in the VbGH5 reaction products. However, the enzyme generated a large amount of arabinose, which was not produced by the other arabinoxylanases. Given that GH43_10 is predominantly an arabinofuranosidase subfamily of GH43, the arabinose generated by VbGH5 is likely mediated by the N-terminal catalytic module (see below). Kinetic analysis showed that AcGH5 displayed similar activity to CtXyl5A against both WAX and RAX and was 2-fold less active against CX. When initially measuring the activity of wild type VbGH5 against the different substrates, no clear data could be obtained, regardless of the concentration of enzyme used the reaction appeared to cease after a few minutes. We hypothesized that the N-terminal GH43_10 rapidly removed single arabinose decorations from the arabinoxylans depleting the substrate available to the arabinoxylanase, explaining why this activity was short lived. To test this hypothesis, the conserved catalytic base (Asp45) of the GH43_10 module of VbGH5 was substituted with alanine, which is predicted to inactivate this catalytic module. The D45A mutant did not produce arabinose consistent with the arabinofuranosidase activity displayed by the GH43_10 module in the wild type enzyme (Fig. 6). The kinetics of the GH5_34 arabinoxylanase catalytic module was now measurable, and activities were determined to be between ∼6- and 10-fold lower than that of CtXyl5A. Interestingly, the fungal arabinoxylanase displays the highest activities against WAX and RAX, ∼4- and 6-fold higher, respectively, than CtXyl5A; however, there is very little difference in the activity between the eukaryotic and prokaryotic enzymes against CX. Attempts to express individual modules of a variety of truncations of AcGH5 and VbGH5 were unsuccessful. This may indicate that the individual modules can only fold correctly when incorporated into the full-length enzyme, demonstrating the importance of intermodule interactions to maintain the structural integrity of these enzymes.</text></passage><passage><infon key="file">zbc0441653440006.jpg</infon><infon key="id">F6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>31787</offset><text>Products profile generated of GH5_34 enzymes. The enzymes at 1 μm were incubated with the four different xylans at 1% in 50 mm sodium phosphate buffer for 16 h at 37 °C (GpGH5, VbGH5, and AcGH5) or 60 °C. The limit products were separated by TLC. The xylooligosaccharide standards (X) are indicated by their degrees of polymerization.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>32125</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>32136</offset><text>A characteristic feature of enzymes that attack the plant cell wall is their complex molecular architecture. The CBMs in these enzymes generally play a role in substrate targeting and are appended to the catalytic modules through flexible linker sequences. CtXyl5A provides a rare visualization of the structure of multiple modules within a single enzyme. The central feature of these data is the structural role played by two of the CBMs, CtCBM6 and CtCBM13, in maintaining the active conformation of the catalytic module, CtGH5. The crystallographic data described here are supported by biochemical data showing either that these two modules do not bind to glycans (CtCBM13) or that the recognition of the non-reducing end of xylan or cellulose chains (CtCBM6) is unlikely to be biologically significant. It should be emphasized, however, that glycan binding and substrate targeting may only be evident in the full-length enzyme acting on highly complex structures such as the plant cell wall, as observed recently by a CBM46 module in the Bacillus xyloglucanase/mixed linked glucanase BhCel5B.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>33233</offset><text>CtXyl5A is a member of GH5 that contains 6644 members. These proteins have been subdivided into 51 subfamilies based on sequence similarity. CtXyl5A is a member of subfamily GH5_34. Here we have explored the substrate specificity of the other members of this subfamily. Despite differences in sequence identity all of the homologs were shown to be arabinoxylanases. Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the −2* subsite, which are 1,3-linked to the xylose positioned in the active site. The presence of a CBM62 in CtXyl5A and AcGH5 suggests that these enzymes target highly complex xylans that contain d-galactose in their side chains. The absence of a “non-structural” CBM in GpGH5 may indicate that this arabinoxylanase is designed to target simpler arabinoxylans present in the endosperm of cereals. Although the characterization of all members of GH5_34 suggests that this subfamily is monospecific, differences in specificity are observed in other subfamilies of GHs including GH43 and GH5. Thus, as new members of GH5_34 are identified from genomic sequence data and subsequently characterized, the specificity of this family may require reinterpretation.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>34568</offset><text>An intriguing feature of VbGH5 is that the limited products generated by this enzymes are much larger than those produced by the other arabinoxylanases. This suggests that although arabinose decorations contribute to enzyme specificity (VbGH5 is not active on xylans lacking arabinose side chains), the enzyme requires other specificity determinants that occur less frequently in arabinoxylans. This has some resonance with a recently described GH98 xylanase that also exploits specificity determinants that occur infrequently and are only evident in highly complex xylans (e.g. CX).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>35152</offset><text>To conclude, this study provides the molecular basis for the specificity displayed by arabinoxylanases. Substrate specificity is dominated by the pocket that binds single arabinose or xylose side chains. The open xylan binding cleft explains why the enzyme is able to attack highly decorated forms of the hemicellulose. It is also evident that appending additional catalytic modules and CBMs onto the core components of these enzymes generates bespoke arabinoxylanases with activities optimized for specific functions. The specificities of the arabinoxylanases described here are distinct from the classical endo-xylanases and thus have the potential to contribute to the toolbox of biocatalysts required by industries that exploit the plant cell wall as a sustainable substrate.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>35932</offset><text>Experimental Procedures</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>35956</offset><text>Cloning, Expression, and Purification of Components of CtXyl5A</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36019</offset><text>All recombinant forms of CtXyl5A used in this study were expressed in the cytoplasm of Escherichia coli because they lacked a signal peptide. DNA encoding CtGH5-CtCBM6 and CtXyl5A-D (CtXyl5A lacking the C-terminal dockerin domain (CtGH5-CtCBM6-CtCBM13-Fn3-CtCBM62)) were described previously. DNA encoding CtGH5-CtCBM6-CtCBM13-Fn3 and CtGH5-CtCBM6-CtCBM13 and mature Acetivibrio cellulolyticus GH5 (AcGH5) were amplified by PCR using plasmid encoding the full-length C. thermocellum arabinoxylanase or A. cellulolyticus genomic DNA as the respective templates. DNA encoding the G. prolifera GH5 (GpGH5) and V. bacterium GH5 (VbGH5) were initially generated by GeneArt® gene synthesis (Thermo Fisher Scientific). DNA encoding VbGH5 lacking the C-terminal cell surface anchoring residues was also amplified by PCR using the synthesized nucleic acid as the template. All the primers used in the PCRs required restriction sites and plasmids used are listed inj supplemental Table S1. All constructs were cloned such that the encoded proteins contain a C-terminal His6 tag. Site-directed mutagenesis was carried out using the PCR-based QuikChange method (Stratagene) deploying the primers listed in supplemental Table S1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>37237</offset><text>To express the recombinant proteins, E. coli strain BL21(DE3), harboring appropriate recombinant plasmids, was cultured to mid-exponential phase in Luria broth at 37 °C. Isopropyl β-d-galactopyranoside at 1 mm was then added to induce recombinant gene expression, and the culture incubated for a further 18 h at 16 °C. The recombinant proteins were purified to &gt;90% electrophoretic purity by immobilized metal ion affinity chromatography using TalonTM (Clontech), cobalt-based matrix, and elution with 100 mm imidazole, as described previously. When preparing the selenomethionine derivative of CtXyl5A-D for crystallography, the proteins were expressed in E. coli B834 (DE3), a methionine auxotroph, cultured in medium comprising 1 liter of SelenoMet Medium BaseTM, 50 ml of SelenoMetTM nutrient mix (Molecular Dimensions), and 4 ml of a 10 mg/ml solution of l-selenomethionine. Recombinant gene expression and protein purification were as described above except that all purification buffers were supplemented with 10 mm β-mercaptoethanol.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>38285</offset><text>Enzyme Assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38299</offset><text>CtXyl5A-D and its derivatives were assayed for enzyme activity using the method of Miller to detect the release of reducing sugar. The standard assay was carried out in 50 mm sodium phosphate buffer, pH 7.0, containing 0.1 mg/ml BSA and at substrate concentrations ranging from 1 to 6 mg/ml. The pH and temperature optima were previously determined to be 7 and 60 °C, respectively, for the CtXyl5A-D and its derivatives. The optimum temperature for the other enzymes was found to be 37 °C, and pH optima of 5, 7, and 4 were determined for AcGH5, GpGH5 and VbGH5, respectively. All enzymes were assayed for activity at their individual temperature and pH optimum. A FLUOstar Omega microplate reader (BMG Labtech) was used to measure activity in 96-well plates. Overnight assays to assess end point products were carried out with 6 mg/ml substrate and 1 μm enzyme concentrations. The identification of potential reaction products was also assessed by HPAEC or TLC using methodology described previously.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>39304</offset><text>Oligosaccharide Analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>39329</offset><text>Approximately 5 g of CX or WAX were digested to completion (no further increase in reducing sugar and change in the HPAEC product profile) with 3 μm of CtXyl5A-D at 60 °C for 48 h. The oligosaccharide products were purified by size exclusion chromatography using a Bio-Gel P2 column as described previously. The structures of the oligosaccharides were analyzed by positive ion-mode infusion/offline electrospray ionization (ESI)-MS following either dilution with 30% acetonitrile or via desalting as described previously </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>39853</offset><text>Crystallography</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>39869</offset><text>Purified SeMet CtXyl5A-D was concentrated and stored in 5 mm DTT, 2 mm CaCl2. Crystals of seleno-l-methionine-containing protein were obtained by hanging drop vapor diffusion in 40% (v/v) 2-methyl-2,4-pentandiol. The data were collected on Beamlines ID14-1 and ID14-4 at the European Synchrotron Radiation Facility (Grenoble, France) to a resolution of 2.64 Å. The data were processed using the programs iMOSFLM and SCALA from the CCP4 suite (Collaborative Computational Project, Number 4, 1994). The crystal belongs to the orthorhombic space group (P21212). The structure was solved by molecular replacement using independently solved structures of some of the modules of the CtXyl5A: CtGH5-CBM6 (PDB code 2y8k), Fn3 (PDB code 3mpc), and CtCBM62 (PDB codes 2y8m, 2yfz, and 2y9s) using PHASER. The CtCBM13 domain was built de novo. BUCCANEER and PHENIX were initially used for auto building. The structure was completed by iterative cycles of manual rebuilding in COOT in tandem with refinement with RefMac5. The final values for Rwork and Rfree) were 23.73 and 27.80%) using TLS and restraining refinement to amino acid residues 36–373 representing the CtGH5 module, 374–516 for the CtCBM6, 517–652 for CtCBM13, and 653–742 for CtFn3. Stereochemistry was assessed with COOT and PDBSUM (with 677 residues (96%) in preferred, 22 in allowed regions (3%), and 6 outliers (1%) in the Ramachandran plot).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>41279</offset><text>To obtain structures of CtGH5-CBM6 in complex with ligand the protein was crystallized using the sitting drop vapor phase diffusion method with an equal volume (100 nl) of protein and reservoir solution (unless otherwise stated), using the robotic nanodrop dispensing systems (mosquitoR LCP; TTPLabTech). Crystals of the protein (10 mg/ml) co-crystallized with arabinose (300 mm) were obtained in 1 m ammonium sulfate, 0.1 m Bis-Tris, pH 5.5, and 1% PEG 3350. Crystals with xylose (300 mm) grew in 100 mm sodium/potassium phosphate, 100 mm MES, pH 6.5, and 2 m sodium chloride. To obtain crystals of the arabinoxylanase in complex with an oligosaccharide, the nucleophile mutant E279S was used and mixed with a range of arabinoxylooligosaccharides that was generated by digestion of WAX with CtGH5-CBM6 (see above) and thereafter by 100 nm of the Cellvibrio japonicus GH43 exo-1,4-β-xylosidase. Only the inclusion of the largest purified oligosaccharide generated crystals of the arabinoxylanase. Crystals of CtGH5E279S-CBM6 were obtained by mixing an equal volume (100 nl) of the protein (11 mg/ml)/oligosaccharide (10 mm) solution and mother liquor solution consisting of 100 mm Tris-Bicine, pH 8.5, 12.5% (w/v) polyethylene glycol with an average molecular mass of 1,000 Da, 12.5% (w/v) polyethylene glycol with an average molecular mass of 3,350 Da and 12.5% (R,S)-2-methyl-2,4-pentanediol (racemic). Crystallographic data were collected on Beamlines IO2, IO4-1, and I24 at the DIAMOND Light Source (Harwell, UK). The data were processed using XDS The crystal structures were solved by molecular replacement using MolRep with CtGH5-CtCBM6 (PDB code 5AK1) as the search model. The refinement was done in RefMac5, and COOT was used for model (re)building. The final model were validated using Molprobity. The data collection and refinement statistics are listed in Table 2.</text></passage><passage><infon key="file">T2.xml</infon><infon key="id">T2</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>43158</offset><text>Data collection and refinement statistics</text></passage><passage><infon key="file">T2.xml</infon><infon key="id">T2</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>43200</offset><text>The values in parentheses are for highest resolution shell.</text></passage><passage><infon key="file">T2.xml</infon><infon key="id">T2</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table xmlns:xlink=&quot;http://www.w3.org/1999/xlink&quot; frame=&quot;hsides&quot; rules=&quot;groups&quot;&gt;&lt;thead valign=&quot;bottom&quot;&gt;&lt;tr&gt;&lt;th rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;CtXyl5A&lt;sub&gt;-D&lt;/sub&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;GH5-CBM6-&lt;italic&gt;Arap&lt;/italic&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;GH5-CBM6-&lt;italic&gt;Xylp&lt;/italic&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;GH5-CBM6- (&lt;italic&gt;Araf&lt;/italic&gt;-Xyl&lt;italic&gt;p&lt;/italic&gt;&lt;sub&gt;4&lt;/sub&gt;)&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;Data collection&lt;/bold&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Source&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;ESRF-ID14-1&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Diamond I04–1&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Diamond I24&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Diamond I02&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Wavelength (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.9334&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.9173&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.9772&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.9791&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Space group&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;P2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Cell dimensions&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        &lt;italic&gt;a&lt;/italic&gt;, &lt;italic&gt;b&lt;/italic&gt;, &lt;italic&gt;c&lt;/italic&gt; (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;147.4, 191.7, 50.7&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;67.1, 72.4, 109.1&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;67.9, 72.5, 109.5&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;76.3, 123.2, 125.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        α, β, γ (°)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;90, 90, 90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    No. of measured reflections&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;244,475 (29,324)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;224,842 (11,281)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;152,004 (4,996)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;463,237 (23,068)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    No. of independent reflections&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;42246 (5,920)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;63,523 (3,175)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;42,716 (2,334)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;140,288 (6,879)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Resolution (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;50.70–2.64 (2.78–2.64)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;44.85–1.65 (1.68–1.65)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;45.16–1.90 (1.94–1.90)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;48.43–1.65 (1.68–1.65)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;merge&lt;/sub&gt; (%)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;16.5 (69.5)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;6.7 (65.1)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;2.8 (8.4)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5.7 (74.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    CC&lt;sub&gt;1/2&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.985 (0.478)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.998 (0.594)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.999 (0.982)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.998 (0.484)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    &lt;italic&gt;I&lt;/italic&gt;/σ&lt;italic&gt;I&lt;/italic&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;8.0 (2.0)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;13 (1.6)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;26.6 (8.0)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;11.2 (1.6)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Completeness (%)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.5 (96.4)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.5 (99.4)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.6 (85.0)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.8 (99.4)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Redundancy&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5.8 (5.0)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.5 (3.6)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.6 (2.1)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.3 (3.4)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;5&quot; rowspan=&quot;1&quot;&gt;&lt;hr/&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;Refinement&lt;/bold&gt;&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;work&lt;/sub&gt;/&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;23.7/27.8&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;12.2/17.0&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;12.9/16.1&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;14.5/19.9&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    No. atoms&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        Protein&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5446&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3790&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3729&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7333&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        Ligand&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;19&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;20&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;20&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;92&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        Water&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;227&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;579&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;601&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;923&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    B-factors&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        Protein&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;41.6&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;17.8&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;15.8&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;21.0&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        Ligand&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;65.0&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;19.4&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;24.2&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;39.5&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        Water&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;35.4&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;38.5&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;32.2&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;37.6&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    R.m.s deviations&lt;/td&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        Bond lengths (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.008&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.015&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.012&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.012&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;        Bond angles (°)&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.233&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.502&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.624&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.554&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;    Protein Data Bank code&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;ext-link ext-link-type=&quot;pdb&quot; xlink:href=&quot;5G56&quot;&gt;5G56&lt;/ext-link&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;ext-link ext-link-type=&quot;pdb&quot; xlink:href=&quot;5LA0&quot;&gt;5LA0&lt;/ext-link&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;ext-link ext-link-type=&quot;pdb&quot; xlink:href=&quot;5LA1&quot;&gt;5LA1&lt;/ext-link&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;ext-link ext-link-type=&quot;pdb&quot; xlink:href=&quot;2LA2&quot;&gt;2LA2&lt;/ext-link&gt;&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>43260</offset><text>	CtXyl5A-D	GH5-CBM6-Arap	GH5-CBM6-Xylp	GH5-CBM6- (Araf-Xylp4)	 	Data collection					 	    Source	ESRF-ID14-1	Diamond I04–1	Diamond I24	Diamond I02	 	    Wavelength (Å)	0.9334	0.9173	0.9772	0.9791	 	    Space group	P21212	P212121	P212121	P212121	 	    Cell dimensions					 	        a, b, c (Å)	147.4, 191.7, 50.7	67.1, 72.4, 109.1	67.9, 72.5, 109.5	76.3, 123.2, 125.4	 	        α, β, γ (°)	90, 90, 90	90, 90, 90	90, 90, 90	90, 90, 90	 	    No. of measured reflections	244,475 (29,324)	224,842 (11,281)	152,004 (4,996)	463,237 (23,068)	 	    No. of independent reflections	42246 (5,920)	63,523 (3,175)	42,716 (2,334)	140,288 (6,879)	 	    Resolution (Å)	50.70–2.64 (2.78–2.64)	44.85–1.65 (1.68–1.65)	45.16–1.90 (1.94–1.90)	48.43–1.65 (1.68–1.65)	 	    Rmerge (%)	16.5 (69.5)	6.7 (65.1)	2.8 (8.4)	5.7 (74.9)	 	    CC1/2	0.985 (0.478)	0.998 (0.594)	0.999 (0.982)	0.998 (0.484)	 	    I/σI	8.0 (2.0)	13 (1.6)	26.6 (8.0)	11.2 (1.6)	 	    Completeness (%)	98.5 (96.4)	98.5 (99.4)	98.6 (85.0)	98.8 (99.4)	 	    Redundancy	5.8 (5.0)	3.5 (3.6)	3.6 (2.1)	3.3 (3.4)	 		 	Refinement					 	    Rwork/Rfree	23.7/27.8	12.2/17.0	12.9/16.1	14.5/19.9	 	    No. atoms					 	        Protein	5446	3790	3729	7333	 	        Ligand	19	20	20	92	 	        Water	227	579	601	923	 	    B-factors					 	        Protein	41.6	17.8	15.8	21.0	 	        Ligand	65.0	19.4	24.2	39.5	 	        Water	35.4	38.5	32.2	37.6	 	    R.m.s deviations					 	        Bond lengths (Å)	0.008	0.015	0.012	0.012	 	        Bond angles (°)	1.233	1.502	1.624	1.554	 	    Protein Data Bank code	5G56	5LA0	5LA1	2LA2	 	</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title_1</infon><offset>45145</offset><text>Author Contributions</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">paragraph</infon><offset>45166</offset><text>A. L. obtained crystals of the GH5-CBM6 complex. L. I. C. analyzed the biochemistry of GH5 subfamilies. J. L. A. B. obtained crystals of CtXyl5A-D. A. J. analyzed the biochemistry of GH5-CBM6 and obtained crystals of GH5-CBM6. A. R. analyzed the biochemistry of GH5-CBM6 products. J. G. performed mass spectrometry. M. P. Y. provided the substrate. B. H. performed analysis of GH5 sequences. C. M. G. A. F. designed the experiments. H. J. G. designed the experiments, analyzed data, and contributed to writing the paper. S. N. solved the structure of CtXyl5A-D and contributed to writing the paper. A. B. used crystallography to solve GH5-CBM6 structures. F. C. analyzed the biochemistry of GH5-CBM6 mutants, obtained crystals of GH5-CBM6, and contributed to writing the paper.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>45944</offset><text>Supplementary Material</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>45967</offset><text>This work was supported in part by European Research Council Grant 322820 (to H. J. G. and B. H.), Biotechnology and Biological Research Council Grants BB/K020358/1 and BB/K001949/1 (to H. J. G.), Wellcome Trust Grant RES/0120/7613 (to H. J. G.), Agence Nationale de la Recherche Grant ANR 12-BIME-0006-01 (to B. H.), and Fundação para a Ciência e Tecnologia Grants PTDC/BIAPRO/103980/2008 and PTDC/BIAMIC/5947/2014 (to C. M. G. A. F.). The authors declare that they have no conflicts of interest with the contents of this article.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46502</offset><text>This article contains supplemental Table S1 and Fig. S1.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46559</offset><text>GH</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46562</offset><text>glycoside hydrolase</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46582</offset><text>CtXyl5A</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46590</offset><text>C. thermocellum arabinoxylanase</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46622</offset><text>CBM</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46626</offset><text>non-catalytic carbohydrate binding module</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46668</offset><text>Fn</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46671</offset><text>fibronectin</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46683</offset><text>WAX</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46687</offset><text>wheat arabinoxylan</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46706</offset><text>RAX</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46710</offset><text>rye arabinoxylan</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46727</offset><text>CX</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46730</offset><text>corn bran xylan</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46746</offset><text>HPAEC</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46752</offset><text>high performance anion exchange chromatography</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46799</offset><text>PDB</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46803</offset><text>Protein Data Bank</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46821</offset><text>BX</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46824</offset><text>birchwood xylan</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46840</offset><text>ESI</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46844</offset><text>electrospray ionization.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46869</offset><text>The abbreviations used are: </text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>46898</offset><text>References</text></passage><passage><infon key="fpage">444</infon><infon key="lpage">455</infon><infon key="name_0">surname:Gilbert;given-names:H. 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+<collection><source>PMC</source><date>20230815</date><key>pmc.key</key><document><id>5173035</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.18632/oncotarget.9692</infon><infon key="article-id_pmc">5173035</infon><infon key="article-id_pmid">27259995</infon><infon key="article-id_publisher-id">9692</infon><infon key="fpage">40965</infon><infon key="issue">27</infon><infon key="kwd">DNA N6-adenine methyltransferase M1.HpyAVI substrate recognition AdoMet-binding Helicobacter pylori Immunology and Microbiology Section Immune response Immunity</infon><infon key="license">This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</infon><infon key="lpage">40977</infon><infon key="name_0">surname:Ma;given-names:Bo</infon><infon key="name_1">surname:Ma;given-names:Ji</infon><infon key="name_2">surname:Liu;given-names:Dong</infon><infon key="name_3">surname:Guo;given-names:Ling</infon><infon key="name_4">surname:Chen;given-names:Huiling</infon><infon key="name_5">surname:Ding;given-names:Jingjin</infon><infon key="name_6">surname:Liu;given-names:Wei</infon><infon key="name_7">surname:Zhang;given-names:Hongquan</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">7</infon><infon key="year">2016</infon><offset>0</offset><text>Biochemical and structural characterization of a DNA N6-adenine methyltransferase from Helicobacter pylori </text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>108</offset><text>DNA N6-methyladenine modification plays an important role in regulating a variety of biological functions in bacteria. However, the mechanism of sequence-specific recognition in N6-methyladenine modification remains elusive. M1.HpyAVI, a DNA N6-adenine methyltransferase from Helicobacter pylori, shows more promiscuous substrate specificity than other enzymes. Here, we present the crystal structures of cofactor-free and AdoMet-bound structures of this enzyme, which were determined at resolutions of 3.0 Å and 3.1 Å, respectively. The core structure of M1.HpyAVI resembles the canonical AdoMet-dependent MTase fold, while the putative DNA binding regions considerably differ from those of the other MTases, which may account for the substrate promiscuity of this enzyme. Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity. Taken together, our data revealed the structural basis underlying DNA N6-adenine methyltransferase substrate promiscuity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1269</offset><text>INTRODUCTION</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1282</offset><text>DNA methylation is a common form of modification on nucleic acids occurring in both prokaryotes and eukaryotes. Such a modification creates a signature motif recognized by DNA-interacting proteins and functions as a mechanism to regulate gene expression. DNA methylation is mediated by DNA methyltransferases (MTases), which catalyze the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to a given position of a particular DNA base within a specific DNA sequence.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1763</offset><text>Three classes of DNA MTases have been identified to transfer a methyl group to different positions of DNA bases. C5-cytosine MTases, for example, methylate C5 of cytosine (m5C). In eukaryotes, m5C plays an important role in gene expression, chromatin organization, genome maintenance and parental imprinting, and is involved in a variety of human diseases including cancer. By contrast, the functions of the prokaryotic DNA cytosine MTase remain unknown. N4-cytosine MTases, which are frequently present in thermophilic or mesophilic bacteria, transfer a methyl group to the exocyclic amino group of cytosine (4mC). N4 methylation seems to be primarily a component of bacterial immune system against invasion by foreign DNA, such as conjugative plasmids and bacteriophages. The third group, N6-adenine MTases methylate the exocyclic amino groups of adenine (6mA), which exists in prokaryotes as a signal for genome defense, DNA replication and repair, regulation of gene expression, control of transposition and host-pathogen interactions. Recent studies utilizing new sequencing approaches have showed the existence of 6mA in several eukaryotic species. DNA 6mA modification is associated with important biological processes including nucleosome distribution close to the transcription start sites in Chlamydomonas, carrying heritable epigenetic information in C.elegans or controlling development of Drosophila.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3177</offset><text>All the three types of methylation exist in prokaryotes, and most DNA MTases are components of the restriction-modification (R-M) systems. The R-M systems are composed of two enzymes displaying opposing activities. “R” stands for a restriction endonuclease cleaving specific DNA sequences, while “M” symbolizes a modification methyltransferase rendering these sequences resistant to cleavage. The cooperation of these two enzymes provides a defensive mechanism to protect bacteria from infection by bacteriophages. The R-M systems are classified into three types based on specific structural features, position of DNA cleavage and cofactor requirements. In types I and III, the DNA adenine or cytosine methyltransferase is part of a multi-subunit enzyme that catalyzes both restriction and modification. By contrast, two separate enzymes exist in type II systems, where a restriction endonuclease and a DNA adenine or cytosine methyltransferase recognize the same targets.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4158</offset><text>To date, a number of bacterial DNA MTases have been structurally characterized, covering enzymes from all the three classes. All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base. Conserved amino acid motifs have been identified from reported structures, including ten motifs (I-X) in cytosine MTases and nine motifs (I-VIII and X) in adenine MTases, all of which are arranged in an almost constant order. According to the linear arrangement of three conserved domains, exocyclic amino MTases are subdivided into six groups (namely α, β, γ, ζ, δ and ε). N6-adenine and N4-cytosine MTases, in particular, are closely related by sharing common structural features. Despite the considerable similarity among bacterial MTases, some differences were observed among the enzymes from various species. For example, the structural regions of MTases beyond the catalytic domain are rather variable, such as the C-terminal domain of M.TaqI, the extended arm of M.MboIIA and M.RsrI, the helix bundle of EcoDam, and so on.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5553</offset><text>DNA methylation is thought to influence bacterial virulence. DNA adenine methyltransferase has been shown to play a crucial role in colonization of deep tissue sites in Salmonella typhimurium and Aeromonas hydrophila. Importantly, DNA adenine methylation is a global regulator of genes expressed during infection and inhibitors of DNA adenine methylation are likely to have a broad antimicrobial action. Dam was considered a promising target for antimicrobial drug development.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6031</offset><text>Helicobacter pylori is a Gram-negative bacterium that persistently colonizes in human stomach worldwide. It is a major pathogen of gastritis and peptic ulcer diseases as well as a cancer-causing factor for gastric cancer. H. pylori is involved in 90% of all gastric malignancies, infecting nearly 50% of the world's population and is the most crucial etiologic agent for gastric adenocarcinoma. H. pylori strains possess a few R-M systems like other bacteria to function as defensive systems. H. pylori 26695, for example, has 23 R-M systems. Methyltransferases were suggested to be involved in H. pylori pathogenicity. M1.HpyAVI is a DNA adenine MTase that belongs to the type II R-M system. This system contains two DNA MTases named M1.HpyAVI and M2.HpyAVI, and a putative restriction enzyme. M1.HpyAVI encoded by ORF hp0050 is an N6-adenine methyltransferase belonging to the β-class MTase. It has been reported recently that this enzyme recognizes the sequence of 5′-GAGG-3′, 5′-GGAG-3′ or 5′-GAAG-3′ and methylates adenines in these sequences. Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5′-GAAG-3′ seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5′-GAGG-3′. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.Here, we report the crystal structure of M1.HpyAVI from H. pylori 26695, which is the first determined N6-adenine MTase structure in H. pylori. The structure reveals a similar architecture as the canonical fold of homologous proteins, but displays several differences in the loop regions and TRD. Based on structural and biochemical analyses, we then identified two conserved amino acids, D29 at the catalytic site and E216 close to the C-terminus, as crucial residues for cofactor binding and methyltransferase activity of M1.HpyAVI. In addition, a non-conserved amino acid, P41, seems to play a key role in substrate recognition.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>8207</offset><text>RESULTS</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8215</offset><text>Overall structure</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8233</offset><text>Recombinant full-length M1.HpyAVI was produced as a soluble protein in Escherichia coli, but was quite unstable and tended to aggregate in low salt environment. The protein, however, remained fully soluble in a buffer containing higher concentration of sodium chloride (&gt;300 mM), which prompted that M1.HpyAVI is likely a halophilic protein.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8575</offset><text>The cofactor-free and AdoMet-bound proteins were crystallized at different conditions. Both structures were determined by means of molecular replacement, and refined to 3.0 Å and 3.1 Å, respectively. Statistics of X-ray data collection and structure refinement were summarized in Table 1.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>8866</offset><text>Data collection and structure refinement statistics of M1.HpyAVI</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;box&quot; rules=&quot;all&quot;&gt;&lt;thead&gt;&lt;tr&gt;&lt;th align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;th align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;M1.HpyAVI&lt;/th&gt;&lt;th align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;M1.HpyAVI-AdoMet complex&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;Data collection&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Wavelength (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.0000&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.97772&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Space group&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;P&lt;/italic&gt;4&lt;sub&gt;3&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;P&lt;/italic&gt;6&lt;sub&gt;5&lt;/sub&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Unit-cell parameters (Å, ˚)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;a&lt;/italic&gt; = &lt;italic&gt;b&lt;/italic&gt; = 69.73, &lt;italic&gt;c&lt;/italic&gt; = 532.75&lt;break/&gt;&lt;italic&gt;α = β = γ&lt;/italic&gt; = 90&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;a = b&lt;/italic&gt; = 135.60, &lt;italic&gt;c&lt;/italic&gt; = 265.15&lt;break/&gt;&lt;italic&gt;α = β&lt;/italic&gt; = 90, &lt;italic&gt;γ&lt;/italic&gt; = 120&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Resolution range (Å) &lt;xref ref-type=&quot;table-fn&quot; rid=&quot;tfn_001&quot;&gt;&lt;sup&gt;a&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;49.09-3.00 (3.09-3.00)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;48.91-3.10 (3.18-3.10)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Unique reflections &lt;xref ref-type=&quot;table-fn&quot; rid=&quot;tfn_001&quot;&gt;&lt;sup&gt;a&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;27243&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;49833&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Multiplicity &lt;xref ref-type=&quot;table-fn&quot; rid=&quot;tfn_001&quot;&gt;&lt;sup&gt;a&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;3.7 (3.8)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;5.6 (4.0)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Completeness (%)&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;tfn_001&quot;&gt;&lt;sup&gt;a&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;98.7 (98.9)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;99.7 (97.8)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Mean &lt;italic&gt;I/δ&lt;/italic&gt; (&lt;italic&gt;I&lt;/italic&gt;) &lt;xref ref-type=&quot;table-fn&quot; rid=&quot;tfn_001&quot;&gt;&lt;sup&gt;a&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;12.1 (3.4)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;14.0 (1.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Solvent content (%)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;58.67&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;61.96&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;merge&lt;/sub&gt;
+&lt;xref ref-type=&quot;table-fn&quot; rid=&quot;tfn_001&quot;&gt;&lt;sup&gt;a&lt;/sup&gt;&lt;/xref&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.073 (0.378)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.106 (0.769)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;Structure refinement&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;work&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.251&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.221&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.308&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.276&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;R.m.s.d., bond lengths (Å)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.007&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.007&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;R.m.s.d., bond angles (˚)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.408&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.651&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;&lt;bold&gt;Ramachandran plot&lt;/bold&gt;&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;/&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Favoured region (%)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;89.44&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;91.44&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Allowed region (%)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;9.58&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;7.11&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;Outliers (%)&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;0.99&lt;/td&gt;&lt;td align=&quot;left&quot; valign=&quot;middle&quot; rowspan=&quot;1&quot; colspan=&quot;1&quot;&gt;1.45&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>8931</offset><text>	M1.HpyAVI	M1.HpyAVI-AdoMet complex	 	Data collection			 	Wavelength (Å)	1.0000	0.97772	 	Space group	P43212	P65	 	Unit-cell parameters (Å, ˚)	a = b = 69.73, c = 532.75α = β = γ = 90	a = b = 135.60, c = 265.15α = β = 90, γ = 120	 	Resolution range (Å) a	49.09-3.00 (3.09-3.00)	48.91-3.10 (3.18-3.10)	 	Unique reflections a	27243	49833	 	Multiplicity a	3.7 (3.8)	5.6 (4.0)	 	Completeness (%)a	98.7 (98.9)	99.7 (97.8)	 	Mean I/δ (I) a	12.1 (3.4)	14.0 (1.9)	 	Solvent content (%)	58.67	61.96	 	Rmergea	0.073 (0.378)	0.106 (0.769)	 	Structure refinement			 	Rwork	0.251	0.221	 	Rfree	0.308	0.276	 	R.m.s.d., bond lengths (Å)	0.007	0.007	 	R.m.s.d., bond angles (˚)	1.408	1.651	 	Ramachandran plot			 	Favoured region (%)	89.44	91.44	 	Allowed region (%)	9.58	7.11	 	Outliers (%)	0.99	1.45	 	</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>9745</offset><text>Values in parentheses are statistics of the highest resolution shell.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9815</offset><text>Four and eight protein monomers resided in the asymmetric units of the two crystal structures. Some amino acids, particularly those within two loops (residues 32-61 and 152-172) in both structures, were poorly defined in electron density and had to be omitted from the refined models. Details of invisible amino acids are given in Table S1.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10156</offset><text>The two structures are very similar to each other (Figure 1) and could be well overlaid with an RMSD of 0.76 Å on 191 Cα atoms. The overall architecture of M1.HpyAVI revealed in these structures resembles the AdoMet-dependent MTase fold in which a twisted seven-stranded β-sheet flanked by six α-helices forms the structural core. Like the reported structures of the larger domain of MTases, three helices (αA, αB and αZ) are located at one face of the central β-sheet, while the other three αD, αE and αC sit at the other side. All these conserved structural motifs form a typical α/β Rossmann fold. The catalytic motif DPPY lies in a loop connecting αD and β4, and the cofactor AdoMet binds in a neighboring cavity. The loop (residues 136-166) located between β7 and αZ corresponds to a highly diverse region in other MTases that is involved in target DNA recognition. The hairpin loop (residues 101-133) bridging β6 and β7, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII. The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA. This loop, however, was well ordered in an M.TaqI-DNA complex structure and was shown to play a crucial role in DNA methylation by contacting the flipping adenine and recognizing specific DNA sequence.</text></passage><passage><infon key="file">oncotarget-07-40965-g001.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>11634</offset><text>Overall structure of M1.HpyAVI</text></passage><passage><infon key="file">oncotarget-07-40965-g001.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11665</offset><text>A. Free form B. AdoMet-bound form. Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY. Rainbow coloring from blue through green to red indicates the N- to C-terminal position of the residues in the model. The α-helices and β-strands are labelled and numbered according to the commonly numbering rule for the known MTases. The AdoMet molecule is shown in green.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>12286</offset><text>Dimeric state of M1.HpyAVI in crystal and solution</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12337</offset><text>Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state. Increasing number of dimeric DNA MTases, however, has been identified from later studies. For instance, M.DpnII, M.RsrI, M.KpnI, and M.MboIIA have been found as dimers in solution. In addition, several MTases including M.MboIIA, M.RsrI and TTH0409 form tightly associated dimers in crystal structures. Nonetheless, some DNA MTases such as M.CcrMI and the Bacillus amyloliquefaciens MTase dissociate from dimer into monomer upon DNA-binding.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13063</offset><text>According to the arrangement of the three conserved domains, M1.HpyAVI belongs to the β-subgroup, in which a conserved motif NXXTX9−11AXRXFSXXHX4WX6−9 YXFXLX3RX9−26NPX1−6NVWX29−34A has been identified at the dimerization interface in crystal structures. Most of conserved amino acids within that motif are present in the sequence of M1.HpyAVI (Figure 2A), implying dimerization of this protein. In agreement, a dimer of M1.HpyAVI was observed in our crystal structures with the two monomers related by a two-fold axis (Figure 2B and 2C). An area of ~1900 Å2 was buried at the dimeric interface, taking up ca 17% of the total area. The dimeric architecture was greatly stabilized by hydrogen bonds and salt bridges formed among residues R86, D93 and E96. In addition, comparison of the dimer structure of M1.HpyAVI with some other β-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3).</text></passage><passage><infon key="file">oncotarget-07-40965-g002.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>14040</offset><text>M1.HpyAVI exists as dimer in crystal and solution</text></passage><passage><infon key="file">oncotarget-07-40965-g002.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14090</offset><text>A. A conserved interface area of β-class MTases is defined in M1.HpyAVI. Residues that involved are signed in red color; Dimerization of free-form M1.HpyAVI B. and cofactor-bound M1.HpyAVI C. The two monomers are marked in green and blue, AdoMet molecules are marked in magenta. D. Gel-filtration analysis revealed that M1.HpyAVI exist as a dimer in solution. FPLC system coupled to a Superdex 75 10/300 column. Elution profiles at 280 nm (blue) and 260 nm (red) are: different concentration (0.05, 0.1, 0.2, 0.5 mg/ml) of M1.HpyAVI protein.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14635</offset><text>To probe the oligomeric form of M1.HpyAVI in solution, different concentrations of purified enzyme was loaded onto a Superdex 75 10/300 column. The protein was eluted at ~10 ml regardless of the protein concentrations, corresponding to a dimeric molecular mass of 54 kDa (Figure 2D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14919</offset><text>Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other β-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution. This variance might be caused by an addition of 100 mM arginine before cell lysis to keep protein solubility and also by later replacement of arginine with 30% glycerol by dialysis. These treatments probably changed protein conformation somehow and also the oligomeric state.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>15513</offset><text>Structure comparisons</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15535</offset><text>As a β-class N6 adenine MTase, the M1.HpyAVI structure displayed a good similarity with M.MboIIA (PDB ID 1G60) and M.RsrI (PDB ID 1NW7), which are falling into the same subgroup. Superimposition of M1.HpyAVI onto them gave RMSDs of 1.63 Å and 1.9 Å on 168 and 190 Cα atoms, respectively. The most striking structural difference was found to locate on the TRD region (residues 133-163 in M1.HpyAVI) (Figure 3A–3C), where the secondary structures vary among these structures. By comparison with the other two enzymes that possess protruding arms containing several α-helices and/or β-strands, the TRD of M1.HpyAVI is much shorter in length (Figure S1), wrapping more closely around the structural core and lacking apparent secondary structures. Given the proposed role of the TRD for DNA interaction at the major groove, some differences of DNA recognition mode can be expected. Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.</text></passage><passage><infon key="file">oncotarget-07-40965-g003.jpg</infon><infon key="id">F3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>16816</offset><text>Structural comparisons between M1.HpyAVI and other DNA MTases</text></passage><passage><infon key="file">oncotarget-07-40965-g003.jpg</infon><infon key="id">F3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16878</offset><text>A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17375</offset><text>N6-adenine and N4-cytosine MTases, in particular, are closely related by sharing common structural features. Structural comparison between M1.HpyAVI and a putative β-class N4 cytosine MTase named TTHA0409 (PDB ID 2ZIF) showed a good similarity as well, giving an RMSD of 1.73 Å on 164 Cα atoms (Figure 3D). Exactly like the above comparison, the most significant difference exists in the TRD, where the structures vary in terms of length and presence of α-helices (Figure S1).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17863</offset><text>M1.HpyAVI displayed a considerable structural dissimilarity in comparison with N6-adenine MTases from other subgroups including the α-class DpnM (PDB ID 2DPM) and the γ-class M.TaqI (PDB ID 2ADM). Both comparisons gave RMSDs above 3.0 Å (Figure 3E and 3F). These two enzymes lack a counterpart loop present in the TRD of M1.HpyAVI, but instead rely on an extra domain for DNA binding and sequence recognition.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18281</offset><text>Collectively, M1.HpyAVI possesses a long disordered TRD, which is in sharp contrast to the secondary structure-rich TRD in other β-class N6 adenine or N4 cytosine MTases or the extra DNA binding domain present in DNA MTases from other subgroups. This striking difference may be a significant determinant of the wider substrate spectrum of this H. pylori enzyme.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18646</offset><text>AdoMet-binding pocket</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18668</offset><text>The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases. A hydrogen bond between D29 in the catalytic motif DPPY and the amino group of bound AdoMet is preserved as other MTase structures. Residues D8 and A9 from hydrogen-bonds with N6 and N1 of the purine ring, respectively, and E216 also locates at hydrogen bonding distance with O2′ and O3′ of the ribose. In addition, H168, T200 and S198 contact the terminal carboxyl of AdoMet. Superposition of M1.HpyAVI with the five structures shown in Figure 3 reveals that the orientation of cofactor is rather conserved except for M.TaqI (Figure 4B). The different conformation of the bound cofactor observed in M.TaqI might be attributable to the absence of corresponding residues of the conserved AdoMet-binding motif FXGXG in that structure.</text></passage><passage><infon key="file">oncotarget-07-40965-g004.jpg</infon><infon key="id">F4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>19568</offset><text>Structural and biochemical analyses define two conserved residues D29 and E216 to be the key sites for AdoMet binding</text></passage><passage><infon key="file">oncotarget-07-40965-g004.jpg</infon><infon key="id">F4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>19686</offset><text>A. The cofactor-binding cavity of M1.HpyAVI. Residues (yellow) that form direct hydrogen bonds with AdoMet (green) are indicated, distance of the hydrogen bond is marked. B. Superposition of AdoMet in the structures of M1.HpyAVI (green), DpnM (yellow) and M.TaqI (orange). The AdoMet terminal carboxyl of M.TaqI reveals different orientations. C. Cofactor binding affinity of wt-/mutants M1.HpyAVI proteins analyzed by microscale thermophoresis (MST). The binding affinity was determined between fluorescently labelled M1.HpyAVI protein and unlabeled AdoMet. The bound fraction is shown on the y-axis against the protein concentration. AdoMet (15 nM to 1 mM) was titrated into a fixed concentration of M1.HpyAVI wt/mutant proteins (800 nM). The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated. Measurements were made with 40% LED and 40% laser power at 25°C. D. DNA methyltransferase activity of wide type protein and the mutants is quantified using radioactive assay. [3H]-methyl transferred to duplex DNA containing 5′-GAGG-3′ was quantified by Beckman LS6500 for 10 min, experiments were repeated for three times and data were corrected by subtraction of the background. E. Superposition of M1.HpyAVI (green) with M.MboIIA (cyan) and M.RsrI (magenta). Residues D29 and E216 are conserved through all the DNA MTases mentioned in Figure 3 (not shown in Figure 4).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21373</offset><text>To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay. As shown in Figure 4C, by contrast to the wild type enzyme, most mutants displayed variable reduction of KD value, among them the D29A and E216A mutants displayed no protein-AdoMet affinity at all.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21808</offset><text>The results suggested that the hydrogen bonds formed by D29 and E216 with AdoMet were most crucial interactions for cofactor binding. Mutation of the two residues may directly prevent the methyl transfer reaction of M1.HpyAVI. The importance of D29 is preserved because it belongs to the catalytic active site DPPY, but the residue E216 has not been fully investigated even being a conserved amino acid throughout MTases (Figure 4E). E216 is the last residue of β2, which contacts the two hydroxyls of the ribose of AdoMet. Replacement of this residue by alanine completely abolishes the key hydrogen bonds for AdoMet-binding, and very likely blocks the methyl transfer reaction. To confirm this notion, [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the mutants. As shown in Figure 4D, the result of radiological assay agreed well with the MST measurement. The D29A and E216A mutants showed little or no methyl transfer activity, while other mutants exhibited reduced methyltransferase activity.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22847</offset><text>As mentioned previously, FXGXG is a conserved AdoMet-binding motif of DNA MTases. We also made mutants of “FMGSG” to alanine for every amino acid, and found that the F195A mutant was insoluble probably due to decreasing the local hydrophobicity upon this mutation. We subsequently investigated the ligand binding affinity and methyl transfer reaction of the other mutants using MST and a radiological assay. We found that G197 played a crucial role in AdoMet-binding, while mutagenesis of M196 and G199 did not influence cofactor binding and catalytic activity (Figure S2A and B). G197 is a conserved residue throughout the DNA MTases, and replacing by alanine at this site likely change the local conformation of cofactor-binding pocket. Mutagenesis on this glycine residue in M.EcoKI or M.EcoP15I also abolished the AdoMet-binding activity. Although mutational study could not tell the role of F195 in ligand binding due to the insolubility of the F195A mutant, structural analysis suggested the importance of this residue in AdoMet-binding. The phenyl ring of F195 forms a perpendicular π-stacking interaction with the purine ring of AdoMet, which stabilizes the orientation of AdoMet bound in the pocket of M1.HpyAVI (Figure S2C). In a separate scenario, mutagenesis of this residue in M.EcoRV has been proven to play an important role in AdoMet binding.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>24211</offset><text>Potential DNA-binding sites</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24239</offset><text>The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58). The hairpin loop between β6 and β7 strands that carries a conserved HRRY sequence signature in the middle is proposed to insert into the minor groove of the bound DNA. As aforementioned, the TRD of M1.HpyAVI shows striking difference from the other DNA MTases, and the relaxed specificity of substrate recognition may be at least partially attributable to the disordered TRD.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24784</offset><text>In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either. This loop, however, was largely stabilized upon DNA binding, as observed in the protein-DNA complex structures of M.TaqI (PDB ID 2IBS), M.HhaI (PDB ID 1MHT) and M.HaeIII (PDB ID 1DCT). The well-ordered loop in those structures directly contacts the flipping adenine and forms hydrogen bond with neighboring bases. These observations implied that the corresponding loop in other MTases, e.g. M1.HpyAVI, is likely responsible for reducing sequence recognition specificity and thus plays crucial roles in catalysis.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>25548</offset><text>Key residue for wider spectrum of substrate recognition</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>25604</offset><text>Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment. Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A). Then, a [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the wide type protein and the mutants. As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.</text></passage><passage><infon key="file">oncotarget-07-40965-g005.jpg</infon><infon key="id">F5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>26782</offset><text>Sequence alignment, structural analysis and radioactive methyl transfer activity define the key residue for wider substrate specificity of M1.HpyAVI</text></passage><passage><infon key="file">oncotarget-07-40965-g005.jpg</infon><infon key="id">F5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26931</offset><text>A. Sequence alignment of M1.HpyAVI from 50 H. pylori strains including 26695 revealed several variant residues. Residues P41, N111, K165 and T166 of M1.HpyAVI from strain 26695 were chosen based on structural analysis and sequence alignment (shown in red arrow). Amino-acid conservation is depicted using WebLogo (Crooks et al, 2004). B., C., D. Methyl transfer reactions were performed using wt-M1.HpyAVI, M1.HpyAVI-P41S, M1.HpyAVI-N111T, and M1.HpyAVI-K165R T166V, respectively. Radioactivity incorporated into the duplex DNA containing 5′-GAGG-3′, 5′-GAAG-3′ or 5′-GGAG-3′ was quantified by Beckman LS6500 for 10 min. The experiments were repeated for three times and data were corrected by subtraction of the background.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27668</offset><text>Our experimental data identified P41 as a key residue determining the recognition of GGAG of M1.HpyAVI. This amino acid locates in the highly flexible loop between residues 33 and 58, which is involved in DNA binding and substrate recognition as shown above. Replacement by serine at this position definitely changes the local conformation and hydrophobicity, and probably some structural properties of the whole loop, which may in turn result in reduced specificity for sequence recognition of the enzyme from strain 26695.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>28193</offset><text>DISCUSSION</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28204</offset><text>Although the DNA-bound structure of previous investigation on a γ-class N6-adenine MTase revealed that the target adenine was rotated out of DNA helix, details of the methyl transfer process were still unclear. Additionally, recent studies reported the importance of N6-methyladenine in some eukaryotic species, but until now there has not been any N6-adenine MTases being identified in eukaryotes. Biochemical and structural characterization of M1.HpyAVI provides a new model for uncovering the methyl transfer mechanism and for investigating the N6-methyladenine in eukaryotes.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28788</offset><text>Oligomeric state of DNA MTases was long accepted as monomer, but our study indicated here that M1.HpyAVI exists as a dimer both in crystal and solution. Interestingly, some other β-class DNA exocyclic MTases showed similar oligomeric state in crystal and in solution, indicating that dimer may be the functional state shared by a subgroup of DNA MTases.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>29145</offset><text>The highly flexible region (residues 33-58) and TRD (residues 133-163) of M1.HpyAVI are supposed to interact with DNA at minor and major grooves, respectively. These two structural characteristics may account for the substrate promiscuity of this enzyme. And residue P41 might be a key residue partially determining the substrate spectrum of M1.HpyAVI.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>29498</offset><text>The missing loop between residues 33 and 58 may need DNA binding so as to form a stable conformation, which is similar to the condition of M.TaqI. Crystallization of M1.HpyAVI-DNA complex warrants future investigations, with the purpose of revealing the mechanism behind the wider substrate specificity of this enzyme.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>29817</offset><text>DNA methylation plays an important role in bacterial pathogenicity. DNA adenine methylation was known to regulate the expression of some virulence genes in bacteria including H.pylori. Inhibitors of DNA adenine methylation may have a broad antimicrobial action by targeting DNA adenine methyltransferase. As an important biological modification, DNA methylation directly influences bacterial survival. Knockout of M1.HpyAVI largely prevents the growth of H. pylori. Importantly, H. pylori is involved in 90% of all gastric malignancies. Appropriate antibiotic regimens could successfully cure gastric diseases caused by H.pylori infection. However, eradication of H. pylori infection remains a big challenge for the significantly increasing prevalence of its resistance to antibiotics. The development of new drugs targeting adenine MTases such as M1.HpyAVI offers a new opportunity for inhibition of H. pylori infection. Residues that play crucial roles for catalytic activity like D29 or E216 may influence the H.pylori survival. Small molecules targeting these highly conserved residues are likely to emerge less drug resistance.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30950</offset><text>In summary, the structure of M1.HpyAVI is featured with a disordered TRD and a key residue P41that located in the putative DNA binding region that may associate with the wider substrate specificity. Residues D29 and E216 were identified to play a crucial role in cofactor binding. As the first crystal structure of N6-adenine MTase in H.pylori, this model may shed light on design of new antibiotics to interfere the growth and pathogenesis of H.pylori in human.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>31413</offset><text>MATERIALS AND METHODS</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>31435</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31471</offset><text>The ORF encoding M1.HpyAVI was inserted into the expression plasmid pET22b (Novagen, Massachusetts, USA) to produce a recombinant protein containing a C-terminal His-tag. In order to produce soluble protein, a chaperone plasmid PG-KJE8 (TaKaRa, Dalian, China) was co-expressed with M1.HpyAVI. The recombinant protein was purified with a three-step chromatography protocol using a Ni-NTA affinity column, a HiLoad 16/60 Superdex 200 column and a mono-S HR 5/5 column (1ml) (GE Healthcare, Uppsala, Sweden). Mutants of M1.HpyAVI were generated using the Muta-direct Site-directed Mutagenesis kit (SBS Genetech, Beijing, China) and produced using the same protocol with wide type protein.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32157</offset><text>Crystallization and data collection</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32193</offset><text>Crystallization trials were carried out for both the AdoMet-free and AdoMet-bound proteins using the hanging drop vapor diffusion technique. Crystals used for diffraction data collection of the apoprotein were grown under the condition of 1.0 M Bis-Tris, pH 9.0, 1.4 M ammonium tartrate, and the optimal crystallization condition for AdoMet-bound protein was 1.0 M Bis-Tris, pH 6.0, 14% PEG2000, 0.2 M lithium sulfate. X-ray diffraction data were collected at 100 K on beamline BL17U1 at the Shanghai Synchrotron Radiation Facility (SSRF) using an ADSC Quantum 315r CCD detector. All data were indexed, integrated and scaled using the XDS program.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32841</offset><text>Structure determination and refinement</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32880</offset><text>The structure of ligand-free M1.HpyAVI was determined by means of molecular replacement using the M.MboIIA (PDB ID 1G60) as a search model. Automated structure determination using Phaser gave a solution showing four subunits sitting in the asymmetric unit. The model was refined using the COOT graphics package manually and phenix.refine. The AdoMet-bound structure was determined by means of molecular replacement using the refined model of the apoprotein, and refined in the same way. Statistics from the data collection and structure refinement are summarized in Table 1. All figures representing the M1.HpyAVI structures were generated using the molecular visualization program PyMol.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>33569</offset><text>Detection of protein dimerization</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>33603</offset><text>The interface information of M1.HpyAVI free form and AdoMet-bound form structures were analyzed using the PDBePISA (Proteins, Interface, Structures and Assemblies) web server.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>33779</offset><text>The protein molecular weight was determined by gel filtration using a FPLC system coupled to a Superdex 75 HR 10 / 30 column. The sizing standard was calibrated using the gel filtration calibration kit LMW (GE Healthcare, Uppsala, Sweden).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>34019</offset><text>Binding affinity quantification via microscale thermophoresis (MST)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34087</offset><text>Microscale thermophoresis was performed using the NT115 nanotemper technologies. M1.HpyAVI-wt and M1.HpyAVI-mutant proteins were fluorescently labeled using the protein label kit according to manufacturer's protocol. Affinity measurements were performed by using MST buffer (0.05% Tween-20 added as final concentration). A solution of unlabeled AdoMet was serially diluted from 1 mM to 15 nM. Equal volume of 0.8 μM labeled protein was mixed with the AdoMet and loaded into the silica capillaries. This binding curve can directly be fitted with the nonlinear solution of the law of mass action, with the dissociation constant (KD) as a result. Measurement was performed at 25°C using 40% LED power and 40%IR-laser power. The dissociation constant was calculated using the Nano-temper Analysis software.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>34892</offset><text>Radioactive methyltransferase analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34931</offset><text>Several different DNA duplexes containing single site of 5′- GAGG-3′, 5′- GAAG-3′ or 5′-GGAG-3′ were used as substrate for methyl transfer reaction (Table S2). 0.1 μM of enzyme and 2 μM of S-[methyl-3H] adenosly methionine (China Isotope and Radiation Corporation, Beijing, China) were incubated at 37°C for 5 min, and then 5 μM of DNA substrate was added to initiate the reaction. Aliquots (20 μl) were taken out at 4-min time intervals and quenched with 2 N HCl. Subsequently, DNA of the mixture was purified using a DNA purification column (TIANGEN, Beijing, China) and the scintillation counting of tritiated DNA was quantified by Beckman LS6500 for 10 min. The background radioactivity was determined by omitting the enzyme from the reaction solution. All the reactions were performed in triplicate.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>35753</offset><text>SUPPLEMENTARY FIGURES AND TABLES</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>35786</offset><text>CONFLICTS OF INTEREST</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>35808</offset><text>The authors declare that they have no conflicts of interest.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>35869</offset><text>ACCESSION CODES</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>35885</offset><text>Atomic coordinates and structure factors for apo-M1.HpyAVI and cofactor-bound M1.HpyAVI have been deposited in the PDB, with accession codes 5HEK and 5HFJ 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+<collection><source>PMC</source><date>20201222</date><key>pmc.key</key><document><id>5603727</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/ncomms11032</infon><infon key="article-id_pii">ncomms11032</infon><infon key="article-id_pmc">5603727</infon><infon key="article-id_pmid">27010430</infon><infon key="elocation-id">11032</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Janowski;given-names:Robert</infon><infon key="name_1">surname:Heinz;given-names:Gitta A.</infon><infon key="name_10">surname:Niessing;given-names:Dierk</infon><infon key="name_11">surname:Heissmeyer;given-names:Vigo</infon><infon key="name_12">surname:Sattler;given-names:Michael</infon><infon key="name_2">surname:Schlundt;given-names:Andreas</infon><infon key="name_3">surname:Wommelsdorf;given-names:Nina</infon><infon key="name_4">surname:Brenner;given-names:Sven</infon><infon key="name_5">surname:Gruber;given-names:Andreas R.</infon><infon key="name_6">surname:Blank;given-names:Michael</infon><infon key="name_7">surname:Buch;given-names:Thorsten</infon><infon key="name_8">surname:Buhmann;given-names:Raymund</infon><infon key="name_9">surname:Zavolan;given-names:Mihaela</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">7</infon><infon key="year">2016</infon><offset>0</offset><text>Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>77</offset><text>The RNA-binding protein Roquin is required to prevent autoimmunity. Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40). A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin. Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE). Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin. Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1004</offset><text> Roquin is an RNA-binding protein that prevents autoimmunity by limiting expression of receptors such as Ox40. Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1306</offset><text>Posttranscriptional gene regulation is involved in a wide range of cellular functions and its critical importance has been described for many developmental and differentiation processes. Consistently, mutations of factors involved in posttranscriptional gene regulation pathways were found associated with a number of genetically inherited diseases. The Roquin protein is essential in T cells for the prevention of autoimmune disease. This is evident from the so-called sanroque mutation in Roquin-1, a single amino acid exchange from Met199 to Arg that causes the development of systemic lupus erythematosus-like symptoms in homozygous mice. The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells. Consistently, CD4+ and CD8+ T cells with the combined deletion of Roquin-encoding genes are spontaneously activated and CD4+ T-helper cells preferentially differentiate into the Th1, Tfh or Th17 subsets. Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2652</offset><text>We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA. The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin. At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain. The structural basis for CDE recognition by the Roquin-2 ROQ domain has also been recently reported.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3652</offset><text>We found that the posttranscriptional activity of Roquin-1 and Roquin-2 is regulated through cleavage by the paracaspase MALT1 (refs). Enhanced MALT1-dependent cleavage and inactivation of Roquin, and thus less effective repression of target genes, result from increased strength of antigen recognition in T cells. These findings suggest that dependent on the strength of cognate antigen recognition differential gene expression and cell fate decisions can be established in naive T cells by a graded cleavage and inactivation of Roquin. In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4622</offset><text>Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain. We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments. Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>5275</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>5283</offset><text>SELEX identifies novel RNA ligands of Roquin-1</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5330</offset><text>We set out to identify Roquin-bound RNA motifs in an unbiased manner by performing SELEX experiments. A biotinylated amino-terminal protein fragment of Roquin-1 (residues 2–440) was used to enrich RNAs from a library containing 47 random nucleotides over three sequential selection rounds. Next-generation sequencing (NGS) of the RNA before and after each selection round revealed that the starting pool represented about 99.6% unique reads in ∼4.2 × 106 sequences. Bioinformatic analysis of NGS data sets derived from the starting pool and enriched selection rounds revealed that the complexity was reduced to 78.6% unique reads in 3.7 × 106 sequences that were analysed after 3 rounds of selection and enrichment. For NGS data analysis, the COMPAS software (AptaIT, Munich, Germany) was applied. Enriched sequences were clustered into so-called patterns with highly homologous sequences. Hereby, the algorithm at first identified frequent motifs of five to eight nucleotides length and subsequently used iterative cycles of proto-pattern formation to cluster sequences bearing two of such frequent motifs. A final aptamer pattern was built up by sequences bearing two frequent motifs and, at the same time, having high similarities also in other sequence parts. Based on this so-called co-occurrence approach, patterns on the basis of frequent motifs were generated and were searched for prominent hexamer sequences (Supplementary Fig. 1a). We identified 5′-CGTTTT-3′, 5′-GCGTTT-3′, 5′-TGCGTT-3′ and 5′-GTTTTA-3′ motifs that were also reconfirmed in an independent experiment (Supplementary Fig. 1a) and are located within highly similar sequences (Fig. 1a and Supplementary Fig. 1b). Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b). Notably, our SELEX approach did not reveal the previously identified CDE sequence. We assume that the region of sequence identity in the CDE is too short for our sequence clustering algorithm. Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5–8 nt stem, with one base in the stem not being paired (Supplementary Fig. 1c). Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b). Importantly, this motif is present across species in the 3′-UTRs of respective mRNAs and showed highest conservation in the loop and the upper stem sequences with a drop of conservation towards the boundaries of the motif (Fig. 1c,d). The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8579</offset><text>NMR analysis of Roquin-bound SL RNAs</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8616</offset><text>We used NMR to analyse the secondary structure of Roquin-1-binding motifs derived from SELEX. Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs. Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson–Crick G–G base pair at this position (Fig. 2a). Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red). No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. In contrast, all expected base pairs are observed for the Ox40 CDE-like SL RNA (Fig. 2c; see also Supplementary Notes).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10018</offset><text>Structures of ROQ bound to ADE SL RNAs</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10057</offset><text>To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements. The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively. In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b). The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e). As may be expected, the recognition of the hexaloop is significantly different from the triloop in the CDE RNA (Fig. 3b,c and Supplementary Fig. 2b). Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e). The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11492</offset><text>The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop. Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts. For example, the main chain atoms of Phe255 form two hydrogen bonds with the Watson–Crick face of the U11 base (Fig. 3d). Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). In addition, the Tyr250 main-chain carbonyl interacts with U13 imino proton (Fig. 3d,e). Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop. The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f). The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA). Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b). The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex. This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e). The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f). This is possible by exposing the base C12 of the Ox-40 ADE-like SL towards the solvent, which accordingly does not show any contacts to the protein. In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts. However, these involve an extended binding surface on the ROQ domain with a number of additional residues compared with the triloop RNA.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13852</offset><text>NMR analysis of ROQ interactions with ADE SLs</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13898</offset><text>We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution. CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). This is fully consistent with the interactions observed in the crystal structure (Supplementary Fig. 2c–e) and indicates a similar binding surface. However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d). On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs. This is consistent with the very similar structural features and mode of RNA recognition of the ROQ domain with these RNAs (Supplementary Fig. 2a,d,e).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>15076</offset><text>Mutational analysis of the ROQ-ADE interaction</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15123</offset><text>To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4). Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2). We next tested a set of mutants (Supplementary Fig. 4), which were designed based on contacts observed in the crystal structure (Fig. 3) and the NMR CSPs (Fig. 4a,b). In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine. We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a). This underlines the central role of Tyr250 for stabilization of the hexaloop structure and recognition by stacking interactions (Fig. 3b,e). Mutation of Ser253, which shows large CSPs in the NMR titrations (Fig. 4a,b), does not significantly impair complex formation (Supplementary Fig. 4). The large chemical shift change is probably caused by ring current effects induced by the close proximity of the U11 and U13 bases. Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4). This indicates that replacement by Tyr does not strongly affect the RNA interaction, and that some conformational variations are tolerated. Thus, the mutational analysis is fully consistent with the recognition of the hexaloop observed in our crystal structures.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16950</offset><text>To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells. Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion. The cells were then transduced with doxycycline-inducible retroviral vectors to reconstitute Roquin-1 expression (Fig. 5b). Depletion of Roquin proteins on tamoxifen treatment (Supplementary Fig. 5a) strongly increased surface expression of Ox40 and Icos (Fig. 5c). This increase in surface expression of both costimulatory receptors was partially corrected by the doxycycline-induced reconstitution with Roquin-1 WT protein (Fig. 5c left panels). Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels). The observed partial rescue may relate to the low, close to endogenous expression of these constructs (Supplementary Fig. 5b). However, it is also possible that continuous overexpression of targets following Roquin deletion induces a hyperactivated state in the T cells. This hyperactivation, compared with the actual posttranscriptional derepression, may contribute even stronger to the increased Icos and Ox40 expression levels. Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18696</offset><text>We also investigated the role of individual nucleotides in the Ox40 ADE-like SL for complex formation with the ROQ domain. We designed four mutants (Mut1–4, see Supplementary Fig. 6) that were expected to disrupt key interactions with the protein according to our co-crystal structure (Fig. 3d–f and Supplementary Fig. 2). NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6). We next used surface plasmon resonance experiments to determine dissociation constants for the ROQ-RNA interaction (Table 2 and Supplementary Fig. 7). Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. As intended, the mutation Mut 1 allows the formation of an additional base pair and thus leads to the formation of a tetraloop with a new G-C closing base pair (Supplementary Fig. 6a). Consistent with the structural analysis, we assume that this variant alters the hexaloop conformation and thus reduces the interaction with ROQ. Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7). No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d). Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ. Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>20620</offset><text>Roquin binding to different SLs in the Ox40 3′-UTR</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20673</offset><text>We have recently shown that Roquin-1 binds to a CDE-like motif in the 3′-UTR of Ox40 mRNA (Figs 1d and 4c). We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR. The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2). The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2). This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR. We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a). These RNAs were then tested in EMSAs with the Roquin-1 N terminus (residues 2–440) (Fig. 6b). Gel shift assays show that binding to the wild-type 3′-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively. Consistent with this, both bands are strongly reduced when mutations are introduced that interfere with the formation of both SLs. Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL. We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>22420</offset><text>Regulation of Ox40 expression via two motifs in its 3′-UTR</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22481</offset><text>To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells. Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts. Doxycycline treatment of cells from this cell line enabled ectopic Roquin-1 and co-translational mCherry expression due to the stable integration of an inducible lentiviral vector (Supplementary Fig. 8c). The expression of Ox40 in cells with and without doxycycline treatment was then quantified by flow cytometry (Supplementary Fig. 8c). Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c). In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40. In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24230</offset><text>To further analyse the functional consequences of Roquin binding to the 3′-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d). Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min). A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min). Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively. These findings underscore the importance of both structural motifs and reveal that they have an additive effect on the regulation of Ox40 mRNA expression in cells.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>25165</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>25176</offset><text>Recent structural and functional studies have provided first insight into the RNA binding of Roquin. Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. Considering that the CDE RNA recognition is mostly structure specific and not sequence dependent, a wide spectrum of target mRNA might be recognized by Roquin. Some evidence for this is provided by a recent study by Landthaler and colleagues.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>25783</offset><text>Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes. Our experiments show that this SELEX-derived ADE shows functional activity comparable to the previously established CDE motif. The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop. Notably, the recognition of the respective RNA-helical stem regions by the ROQ domain is identical for the triloop and hexaloop motifs. However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). Of particular importance for the hexaloop recognition is Tyr250, which acts as a stabilizing element for the integrity of a defined loop conformation. It stacks with nucleotides in the hexaloop but not the CDE triloop (Fig. 3b,c). The functional role of Tyr250 for ADE-mediated mRNA regulation by Roquin-1 is thus explained by our experiments (Fig. 5b,c). The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL. Consistent with this, loss of ROQ binding is observed on replacement of U11 and U13 by other bases (Table 2). In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively. A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c). Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>27734</offset><text>Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work. Most probably, the experimental setup of Murakawa et al. revealed both high- and low-affinity target motifs for Roquin, whereas our structural study reports on a high-affinity binding motif. Notably, Murakawa et al. neither found the Roquin-regulated Ox40 nor the Tnf 3′-UTRs, as both genes are not expressed in HEK 293 cells. However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement. It remains to be seen which exact features govern the recognition of the A20 SL by Roquin.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28712</offset><text>The regulatory cis RNA elements in 3′-UTRs may also be targeted by additional trans-acting factors. We have recently identified the endonuclease Regnase-1 as a cofactor of Roquin function that shares an overlapping set of target mRNAs. In another study, the overlap in targets was confirmed, but a mutually exclusive regulation was proposed based on studies in lipopolysaccharide (LPS)-stimulated myeloid cells. In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target. In CD4+ T cells, Ox40 does not show derepression in individual knockouts of Roquin-1 or Roquin-2 encoding genes, but is strongly induced upon combined deficiency of both genes. In addition, conditional deletion of the Regnase-1-encoding gene induced Ox40 expression in these cells. Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes. However, in cultures of CD4+ T cells, Ox40 is translated on day 4–5 and is expressed much higher in T cells with combined deficiency of Roquin-1 and Roquin-2. At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30235</offset><text>Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays. Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3′-UTR by both proteins. In contrast, structural details on how Regnase-1 can interact with these SL RNAs are currently missing. Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene. Both were required for Regnase-1-mediated repression. These findings therefore raise the possibility that Regnase-1 interacts with ADE-like hexaloop structures either in a direct or indirect manner.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>31199</offset><text>Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation. The novel 3′-UTR loop motif that we have identified as a bona fide target of Roquin now expands this multilayer mode of co-regulation. We suggest that differential regulation of mRNA expression is not only achieved through multiple regulators with individual preferences for a given motif or variants thereof, but that regulators may also identify and use distinct motifs, as long as they exhibit some basic features regarding shape, size and sequence.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>31958</offset><text>The presence of distinct motifs in 3′-UTRs offers a broader variability for gene regulation by RNA cis elements. Their accessibility can be modulated by trans-acting factors that may bind regulatory motifs, unfold higher-order structures in the RNA or maintain a preference for duplex structures as was shown recently for mRNAs that are recognized by Staufen-1 (ref.). In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain. The exact stoichiometry of Roquin bound to the Ox40 3′-UTR is unknown. The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs. In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site. This opens the possibility that one Roquin molecule may cluster two motifs in a given 3′-UTR and/or cluster motifs from distinct 3′-UTRs to enhance downstream processing. Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA. We therefore hypothesize that the combination of multiple binding sites may be more commonly used to enhance the functional activity of Roquin. At the same time, the combination of cis elements may be important for differential gene regulation, as composite cis elements with lower affinity may be less sensitive to Roquin. This will lead to less effective repression in T cells when antigen recognition is of moderate signal strength and only incomplete cleavage of Roquin by MALT1 occurs. For understanding the intricate complexity of 3′-UTR regulation, future work will be necessary by combining large-scale approaches, such as cross-linking and immunoprecipitation experiments to identify RNA-binding sites, and structural biology to dissect the underlying molecular mechanisms.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>34078</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>34086</offset><text>SELEX experiments</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34104</offset><text>Selection of Roquin-1-bound RNAs from a random RNA library was performed in three rounds of selection with increased stringency of washing (3 × 100 μl, 4 × 100 μl and 5 × 100 μl washing steps) and with decreased protein concentrations (250, 150 and 50 nM). Before selection, 100 μg recombinant Roquin-1 and Roquin-1 M199R N-terminal protein (residues 2–440) were biotinylated: proteins were incubated for 30 min on ice with 10 × molar excess of EZ-link PEG4-NHS-Biotin (Pierce) in PBS (0.1 mg ml−1). Subsequently, the biotinylated protein was purified via gel filtration (MicroSpin column P6, BioRad) and the loss of protein during the biotinylation procedure was estimated by SDS–PAGE and Coomassie staining. The efficiency of the biotinylation reaction was evaluated after spotting of unlabelled and labelled proteins onto a nitrocellulose membrane. After blocking the membrane with 1% BSA in PBS, it was incubated in streptavidin–PE (R-Phycoerythrin) diluted 1:1,000 in PBS for 30 min at room temperature (RT). Subsequently, the membrane was washed three times with PBS and fluorescence intensity of PE bound to biotinylated protein was determined by fluoroimaging (Raytest, FLA5000, 473 nm, Y510 filter).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35349</offset><text>The RNA startpool containing the 47-nt random sequence as well as the RNA pools for the second and third selection rounds were transcribed in vitro from double-stranded PCR DNA, and protein-bound RNA was isolated and reverse transcribed before PCR amplification, as previously described. Following transcription, the samples were separated on an 8% PAGE, the bands excised and RNA purified. Every round of selection started by combining the RNA pool (400 pmol) with biotinylated protein and incubating the mix for 30 min at 37 °C. Subsequently, binding buffer-equilibrated streptavidin-magnetic beads were added and incubated (10 min, 37 °C) to bind the protein–RNA complexes, followed by washes. By boiling the beads in 0.2 mM EDTA in water for 3 min, protein and RNA molecules were released. After removal of beads, the solution served as template for reverse transcription (One-Step RT-PCR Kit, Qiagen) and from the obtained complementary DNA the RNA pool of the next round of selection was transcribed. The cDNAs from every selection round (startpool, round 1, round 2 and round 3) were used for Index-PCRs to analyse the pool composition at every stage during selection. Comparable amounts of the PCR products were combined to one cDNA library and analysed by Solexa Illumina sequencing.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>36660</offset><text>Sequence motif and structural analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36699</offset><text>To identify sequence motifs to which Roquin specifically binds, we counted the number of occurrences of each hexamer (46=4,096 motifs) in the sequences obtained by SELEX. We then generated a data set of randomized sequences of the same nucleotide composition as the SELEX-derived sequences, by permuting the SELEX-derived sequences with a custom script. Finally, we counted the number of occurrences of each hexamer in the set of randomized sequences and computed the log2 ratio of the number of occurrences of each motif in the real and randomized sequence sets. To identify a shared sequence motif in the SELEX patterns that showed the strongest enrichment in our selection experiments, the top 100 patterns were analysed with the Motif-based sequence analysis tool MEME (http://meme-suite.org) using the default settings. This analysis revealed three sequence motifs of which the first is shown in Fig. 1b. For the construction of sequence logos, we screened the obtained nucleotide sequences from SELEX replicate 1 and extracted the nucleotide sequences including the 7-nt flanking regions. Sequence logos were constructed with WebLogo 2.8.2 (http://weblogo.berkeley.edu/).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>37877</offset><text>For the Ox40 3′-UTR sequence alignment, we extracted Multiz alignments of 60 Vertebrates from the UCSC mouse GRCm38/mm10 assembly for the genomic region chr4:156,016,498–156,016,520. For each species contained in the alignment, we extracted genomic coordinates of the aligned sequence, extended the coordinates by 10 nt upstream and downstream, and retrieved the extended sequences from the corresponding genome assemblies. Finally, sequences were aligned with ClustalW 2.1 with standard settings and the alignment was visualized using Jalview.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38428</offset><text>To evaluate the structural context the inferred motif is located in, we first appended to the nucleotide sequences obtained from the SELEX experiment the SELEX primers 5′-GGAGAGATGTGAACTT-3′ and 5′-AGTTTCGTGGATGCCAC-3′ to the 5′- and 3′-end, respectively. Next, we screened for sequences that contained the inferred motif and performed secondary structure prediction on those sequences with RNAfold from the ViennaRNA package version 1.8 with parameters '-p -d2'. Next, we used a custom Perl script to parse the base-pairing probability file generated by RNAfold and to calculate an average base-pair probability over all sequences that contained the inferred motif.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>39107</offset><text>Production of proteins</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>39130</offset><text>Cloning of expression vectors for Roquin-1 ROQ (residues 147–326), ROQ (residues 171–326) and Roquin-1 N-term (residues 2–440) was carried out by standard procedures as described previously. Briefly, PCR-amplified fragments were put into pETM11 and pETTrx1a vectors based on pET24d as provided by the Protein Expression and Purification Facility at Helmholtz Zentrum München. All vectors contained tobacco etch virus (TEV) protease recognition sites for subsequent proteolytic removal of the tags. All length-variable Roquin-1 expression constructs were designed and cloned via restriction sites NcoI (5′) and XhoI (3′). ROQ domain RNA-binding mutants were cloned by Quick change PCR with high-fidelity Phusion DNA polymerase and subsequent treatment with DnpI. Alternatively, we used conventional cloning with a two-step PCR protocol and enzymatic restriction.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40003</offset><text>The Roquin-1 fragments (147–326) and (171–326) were expressed as N-terminal His6-thioredoxin fusion proteins as recently described. Isotope-labelled protein for NMR studies was expressed in M9 minimal medium supplemented with 0.5 g l−1 15N ammonium chloride and 2 g l−1unlabelled or [U-13C] glucose. For the preparation of deuterated proteins, cells were adapted and grown as described previously. Briefly, we used a protocol with stepwise adaptation of cells to deuterium changing buffer from no D2O, low glucose to 50% D2O, low glucose and finally 99.5% D2O with deuterated glucose. The Roquin-1 N-terminal domain (residues 2–440) was expressed and purified essentially as described above for the ROQ domain, but no thioredoxin tag was used. For Roquin-1 N-terminal domain, all expression media and the final buffer contained 100 or 25 μM of zinc chloride, respectively.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>40897</offset><text>RNA preparation</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40913</offset><text>RNAs were synthesized and purchased from IBA GmbH (Göttingen, Germany), purified via PAGE followed by two steps of desalting. No major impurities were seen in NMR spectra. Complex formation for crystallography and NMR experiments was achieved by dissolving the lyophilized RNA in water or NMR buffer. This stock solution was snap-cooled by boiling at 95 °C for 5 min and transferred to an ice-cold bath for 10 min before aliquoting. All RNAs were stored at −80 °C, to avoid degradation and thermodynamically favoured duplex formation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>41461</offset><text>Full length and fragments of Ox40 3′-UTR mRNA were produced by in vitro transcription (IVT) from DNA templates harbouring a T7 promoter site either with direct incorporation of α-32P-labelled UTP or subsequent 3′-labelling of purified RNA with γ-32P-labelled ATP. DNA templates were cloned by primer extension PCR. For IVT, 50–150 nM of DNA were incubated with 11 mM magnesium chloride, 8% (w/v) PEG8000, 1.25 mM of each NTP and 0.05 mg ml−1 of T7 polymerase in 1 × standard reaction buffer for 3–5 h at 37 °C. Labelled RNAs were produced in 50 μl reactions and purified via spin columns and directly subjected to EMSA assays. Unlabelled RNAs were produced in reactions of 500–5,000 μl. After IVT, the reactions were separated on 8% denaturing SDS–PAGEs, RNA of interest excised and eluted from the gel using the Elutrap kit (GE Healthcare). After elution, RNAs were dialysed against water and lyophilized. Subsequently, RNAs were dissolved in water and stocks generated by boiling them at 95 °C for 5 min and transferred to an ice-cold bath for 10 min before aliquoting. Labelling for EMSA assays was carried out as for short motifs and described recently. As a modification, dephosphorylation was performed for 30 min and 3′-phosphorylation with γ-32P-labelled ATP and T4 polynucleotide kinase for 90 min for higher efficiency, respectively.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>42857</offset><text>NMR spectroscopy</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>42874</offset><text>NMR measurements of Roquin-1 ROQ (147–326) and ROQ (171–326) were performed in buffers as described, mixed with 10% D2O. Backbone chemical shift assignments of ROQ (171–326) with 1.1- to 1.2-fold excess of the Ox40 ADE-like SL motif or consensus ADE SL RNAs were recorded with protein concentrations of 350–400 μM. HNCA, HNCACB, HNCO, HNcaCO and 3D 15N-edited NOESY spectra were acquired at 298K on Bruker Avance III spectrometers at field strengths corresponding to 600 and 800 MHz proton Larmor frequency, equipped with TCI cryogenic probe heads. Spectra of ROQ in complex with Ox40 CDE-like SL RNA and the RNA alone have been reported before. Spectra were processed with Topspin3.2 and analysed with CCPNMR Analysis and Sparky. For RNA motifs, one- and two-dimensional imino NOESY spectra with water-flip-back WATERGATE were recorded at 600–900 MHz, at 278 and 298 K at 150–350 μM RNA concentrations. Sequential assignments were guided by secondary structure predictions with mfold and supported by 15N chemical shifts from natural abundance SOFAST-HMQC experiments.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>43968</offset><text>Electrophoretic mobility shift assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>44006</offset><text>The EMSAs with ROQ domain and individual motifs were performed as described previously. In short, for the binding reaction a mastermix containing transfer RNA, 32P-labelled SL RNA and reaction buffer was prepared and then mixed with dilutions of the recombinant proteins to achieve the indicated protein concentrations. The binding was performed for 10 min at RT or 20 min on ice in 20 μl reaction volume in the presence of 2.5 μg μl−1 tRNA from baker’s yeast (Sigma), 500 pM 32P-labelled RNA, 20 mM HEPES (pH 7.4), 50 mM NaCl, 1 mM MgCl2, 1 mM dithiothreitol and 1 μg μl−1 BSA. For the binding reaction of Roquin-1 N-terminal with full-length Ox40 3′-UTRs or fragments thereof, ∼1 pmol of RNA was incubated with protein concentrations between 0 and 1,000 μM in a volume of 20 μl. RNP complexes were resolved by PAGE (6% polyacrylamide, 5% glycerol, 0.5 × TBE) at 120 V for 40 min at RT. Gels were then fixed, dried and exposed to a phosphor imager screen.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>45015</offset><text>X-ray crystallography</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>45037</offset><text>The crystallization experiments for ROQ–RNA complexes were performed at the X-ray Crystallography Platform at Helmholtz Zentrum München. The crystals of both, Roquin-1 ROQ (171–326) with Ox40 ADE-like SL motif (22mer, 5′-UCCACACCGUUCUAGGUGCUGG-3′) and with the consensus SELEX-derived ADE SL motif (20mer, 5′-UGACUGCGUUUUAGGAGUUA-3′) were obtained from the same condition: 100 mM Bis-Tris buffer pH 5.5, 200 mM sodium chloride and 25% (v/w) PEG 3350. Crystallization was performed using the sitting-drop vapour-diffusion method at 292 K in 24-well plates and a protein concentration of 12 mg ml−1. The crystals appeared after 1 day. For the X-ray diffraction experiments, the crystals of both co-complexes were mounted in a nylon fibre loop and flash cooled to 100 K in liquid nitrogen. The cryoprotection was performed for 2 s in reservoir solution complemented with 20% (v/v) ethylene glycol. Diffraction data for ROQ Ox40 ADE-like motif was collected on the ID29 beamline (ESRF, Grenoble, France) using a Pilatus 6M at a wavelength of 1.25363 Å. Diffraction data for the ROQ-ADE complex were collected using Pilatus 2M detector at 1.00003 Å wavelength at PXIII beamline at SLS (Villigen, Switzerland). All data sets were indexed and integrated using XDS and scaled using SCALA. Intensities were converted to structure–factor amplitudes using the programme TRUNCATE. Table 1 summarizes data collection and processing statistics for both data sets.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>46524</offset><text>Structure determination and refinement</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>46563</offset><text>The structure of both ROQ-Ox40 ADE-like SL and ROQ-ADE SL were solved by molecular replacement using the native Roquin-1 ROQ (147–326) structure as a search model (PDB: 4QI0 (ref.)). Model building was performed in COOT. RNA molecules were modelled manually. The refinement of both structures was done in REFMAC5 (ref.) using the maximum-likelihood target function including translation, libration and screw-rotation displacements of a pseudo-rigid body (TLS). For the ROQ-ADE SL structure, non-crystallographic symmetry (NCS) averaging was implemented. The final models are characterized by R and Rfree factors of 21.8 and 25.7% for ROQ-Ox40 ADE-like SL, and 18.6 and 23.4% for ROQ-ADE SL (Table 1), respecively. The stereochemical analysis of both final models was done in PROCHECK and MolProbity. It indicates that there are no residues with generously allowed or unfavourable backbone dihedral angles, and that 99.4% (for ROQ-Ox40 ADE-like SL structure) and 92.3% (for ROQ-ADE SL structure) of all residues are in the core region of the Ramachandran plot.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>47625</offset><text>Functional assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>47643</offset><text>Functional assays determining the Roquin-mediated regulation of Ox40 with different 3′-UTR variants were performed as described previously. In brief, Rc3h1/2−/− mouse embryonic fibroblast (MEF) cells, stably transduced with a doxycycline-inducible Roquin-1-p2A-mCherry construct, were retrovirally infected with Ox40 constructs of different 3′-UTR length or mutation, which led to the expression of Ox40 on the cell surface (CDE-like mutation changing nt 14–16 GCA to CGT, ADE-like mutation changing nt 15–17 from GGT to CCA). Forty-eight hours after infection, the cells were split and one half of the cells was treated with doxycycline (1 μg ml−1), to induce expression of Roquin-1 and mCherry, connected via the self-cleaving peptide p2A. Thus, Roquin-expressing cells were marked by mCherry expression. Sixteen to 20 h after induction, the cells were harvested, stained with allophycocyanin (APC)-conjugated anti-Ox40 and analysed by flow cytometry. To compare the Ox40 expression levels achieved by different constructs, the relative Ox40 mean fluorescence intensity (MFI) was determined by dividing the MFI of treated (mCherry+) cells by the MFI of untreated (mCherry−) cells.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>48849</offset><text>Mouse experiments</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>48867</offset><text>Compound mutant mice with the Rc3h1fl/fl (ref.) and Rc3h2fl/fl (ref.) (denoted Rc3h1/2fl/fl), as well as Cd4-Cre-ERT2 (ref.) and Gt(ROSA)26Sortm1(rtTA*M2)Jae alleles were maintained on a C57BL/6 genetic background. All animals were housed in a pathogen-free barrier facility in accordance with the Ludwig-Maximilians-University München institutional, state and federal guidelines.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>49249</offset><text>Generation of overexpression vectors</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>49286</offset><text>Expression constructs of Roquin-1 and Ox40 were cloned into a modified pRetroX-Tight vector (Clontech). The puromycine-resistance cassette was removed and a cassette containing attR1-ccdB-attR2 was inserted, to generate a Gateway destination vector. Roquin-1 and Ox40 constructs were inserted by LR reaction (Invitrogen). Any mutants thereof were generated by site-directed mutagenesis.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>49673</offset><text>Virus production</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>49690</offset><text>Replication-deficient retrovirus production and T-cell transduction was performed as previously described. Briefly, retroviral and packaging plasmids were introduced into HEK293T cells by calcium-phosphate transfection. Forty-eight hours after transfection, cell culture supernatants containing the retrovirus particles were harvested, passed through 0.45-μm filters and stored at −80 °C.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>50085</offset><text>Cell isolation and culture</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>50112</offset><text>Splenocytes were isolated from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice. CD4+ T cells were isolated by negative selection with magnetic beads according to the manufacturer’s instructions (Stem Cell Technologies). CD4+ T cells were cultured in DMEM medium supplemented with 10% (vol/vol) fetal bovine serum, 1 × nonessential amino acids (Lonza), 10 mM HEPES pH 7.4 (Invitrogen), 50 μM β-mercaptoethanol (Invitrogen) and 100 U ml−1 penicillin–streptomycin (Invitrogen). Rc3h1/2fl/fl deletion was induced by addition of 4′OH-Tamoxifen (0.3 μM) for 24 h. For TH1 differentiation, CD4+ T cells were cultured in six-well plates pre-coated with goat anti-hamster IgG (MP Biochemicals) and DMEM medium further supplemented with anti-CD3 (0,25 μg ml−1), anti-CD28 (2,5 μg ml−1), IL-12 (10 ng ml−1) and anti-IL-4 (10 μg ml−1) for 40 h. Cells were then infected with retroviral constructs, allowing reconstitution with either Roquin-1, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A, and cultured in IL-2 containing media (20 U ml−1). Forty-eight hours after transduction, the cells were split and one half of cells was treated with doxycycline (1 μg ml−1), to induce expression of Roquin-1 WT and Roquin-1 mutants. Twenty-four hours after induction, the cells were harvested for analysis by immunoblot and flow cytometry with the indicated antibodies (1:200 anti-mouse Icos-biotin clone 7E–17G9 (eBioscience); 1:200 Streptavidin-PerCP (Becton Dickinson); 1:200 anti-mouse Ox40-PE clone OX-86 (eBioscience)).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>51678</offset><text>Immunoblot analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>51698</offset><text>CD4+ T cells were incubated for 15 min on ice with lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.25% (vol/vol) Nonidet-P40, 1.5 mM MgCl2 and protease inhibitor mix without EDTA (Roche) and 1 mM dithiothreitol). Lysate was cleared by centrifugation (10 min, 10 000 g, 4 °C). Immunoblotting was performed by standard protocols with hybridoma supernatants containing monoclonal antibody recognizing Roquin-1 and Roquin-2 (anti-roquin, clone 3F12).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>52168</offset><text>mRNA decay experiments</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>52191</offset><text>Hela Tet-Off Advanced Cells (Clontech 631156) were stably transduced with retroviruses expressing different Ox40 constructs. FACS analysis 41 h post transduction revealed similar Ox40 surface expression levels on all five cell samples. After transduction, the cell lines were initially cultured for at least 48 h without doxycycline, to ensure high Ox40-expression levels. For each time point, 400 000 cells were spread on one well in a six-well plate. To switch off Ox40-transcription, doxycycline was supplied with the medium at time point 0. After one washing step with PBS, cells were directly harvested from each well with Trizol before Dox application (0 h), as well as 2, 3 and 4 h after Dox application. RNA was isolated using standard Trizol protocols. Reverse transcription was performed with the Qiagen Quantitect Reverse Transcription Kit following the manufacturer’s protocols. Quantitative PCR was carried out on a Roche Light Cycler 480 using the Light Cycler 480 Probes Master Mix and primer-/probe-combinations from Roches Universal Probe Library. Relative mRNA expression levels were calculated by normalization to the housekeeper gene ywhaz.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>53364</offset><text>Surface plasmon resonance</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>53390</offset><text>ROQ–RNA binding experiments were performed on a BIACORE 3000 instrument (Biacore Inc.). ROQ domain was diluted to a final concentration of 35 μg ml−1 in 10 mM HEPES pH 7.0 and chemically immobilized (amine coupling) onto CM5 sensor chips (Biacore Inc.). The RNA samples were diluted in the running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 2 mM MgCl2 and 0.005% Tween 20) to the final concentration of 31.25, 62.5, 125, 250 and 500 nM, and 1 and 2 μM, and injected over the sensor chip surface at 30 μl min−1 at 10 °C. The RNA samples were injected onto the sensor chip from the lowest to the highest concentration. Each RNA-type sample was tested three times with the exception of Mut1–3 two times. Injection of 250 nM RNA was always performed in duplicate within each experiment. To subtract any background noise from each data set, all samples were also run over an unmodified sensor chip surface. Data were analysed using BIAevaluation programme (Biacore Inc.) (Supplementary Fig. 7). For each measurement, the equilibrium dissociation constant was calculated (KD) from steady state binding. The KD from three independent experiments were used to calculate the mean values of these variables and the s.e.m. The results for all tested RNA samples are compared in Table 2.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>54700</offset><text>Additional information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>54723</offset><text>Accession codes: Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5F5H and 5F5F for the ROQ-Ox40 ADE-like SL and ROQ-ADE SL, respectively. Chemical shifts of the ROQ-Ox40 ADE-like SL and ROQ-ADE SL have been deposited in the Biological Magnetic Resonance Data Bank under accession codes 26587 and 26588, respectively.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>55100</offset><text>How to cite this article: Janowski, R. et al. Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40. Nat. Commun. 7:11032 doi: 10.1038/ncomms11032 (2016).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>55278</offset><text>Supplementary Material</text></passage><passage><infon key="fpage">160</infon><infon key="lpage">165</infon><infon key="name_0">surname:Turner;given-names:M.</infon><infon key="name_1">surname:Hodson;given-names:D.</infon><infon key="pub-id_pmid">22326859</infon><infon key="section_type">REF</infon><infon key="source">Curr. Opin. 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Crystallogr.</infon><infon key="type">ref</infon><infon key="volume">66</infon><infon key="year">2010</infon><offset>58667</offset><text>MolProbity: all-atom structure validation for macromolecular crystallography</text></passage><passage><infon key="fpage">e1001674</infon><infon key="name_0">surname:Sledzinska;given-names:A.</infon><infon key="pub-id_pmid">24115907</infon><infon key="section_type">REF</infon><infon key="source">PLoS Biol.</infon><infon key="type">ref</infon><infon key="volume">11</infon><infon key="year">2013</infon><offset>58744</offset><text>TGF-beta signalling is required for CD4(+) T cell homeostasis but dispensable for regulatory T cell function</text></passage><passage><infon key="fpage">725</infon><infon key="lpage">733</infon><infon key="name_0">surname:Glasmacher;given-names:E.</infon><infon key="pub-id_pmid">20639877</infon><infon key="section_type">REF</infon><infon key="source">Nat. Immunol.</infon><infon key="type">ref</infon><infon key="volume">11</infon><infon key="year">2010</infon><offset>58853</offset><text>Roquin binds inducible costimulator mRNA and effectors of mRNA decay to induce microRNA-independent post-transcriptional repression</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>58985</offset><text>The authors declare no competing financial interests.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>59039</offset><text>Author contributions A.S. carried out cloning, protein expression and purification, and NMR experiments. R.J. performed crystallization and structure determination. G.A.H. carried out EMSA assays and SELEX experiments, and functional studies were performed by G.A.H, N.W. and S.B. M.B. and R.B. helped setting up the SELEX experiments and identified patterns from NGS data, on the basis of which A.G. and M.Z. analysed the motifs and secondary structures. T.B. contributed unpublished reagents and advice. A.S., G.A.H., R.J., V.H., D.N. and M.S. designed the project and wrote the paper. All authors discussed the results and commented on the manuscript.</text></passage><passage><infon key="file">ncomms11032-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>59694</offset><text>SELEX identifies a novel SL RNA ligand of Roquin-1.</text></passage><passage><infon key="file">ncomms11032-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>59746</offset><text>(a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences. (c) Conservation of the motif found in Ox40 3′-UTRs for various species as indicated. The labels correspond to the versions of the genome assemblies in the UCSC server (see Method section). rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL. The broken line between the G–G base pair in the ADE SL indicates a putative non-Watson–Crick pairing. The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison. See also Supplementary Fig. 1.</text></passage><passage><infon key="file">ncomms11032-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>60600</offset><text>NMR analysis of the SL RNAs used in this study.</text></passage><passage><infon key="file">ncomms11032-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>60648</offset><text>Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). The respective SL RNAs and their base pairs are indicated. Red asterisks indicate NMR signals of the protein. Black asterisks in a indicate a second conformation (see Supplementary Notes). Green lines in the secondary structure schemes on the left refer to visible imino NMR signals and thus experimental confirmation of the base pairs indicated. Red nucleotides indicate significant chemical shift changes observed. The dotted green line between G6 and G15 in a highlights a G–G base pair.</text></passage><passage><infon key="file">ncomms11032-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>61352</offset><text>Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA.</text></passage><passage><infon key="file">ncomms11032-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>61417</offset><text>(a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta). Selected RNA bases and protein secondary structure elements are labelled. (b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green). Only RNA-interacting residues that are different in both structures are shown. Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA. The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257). The protein chain is shown in turquoise and the RNA is shown in grey. Atoms are colour coded according to charge. (e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop. U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247. The side chain of Tyr250 makes hydrophobic interactions with the pyrimidine side chain of U10 on one side and U11 on the other side. Lys259 interacts with the phosphate groups of U10 and U11. (f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11. In d – f, amino acids are shown in turquoise and blue, nucleotides in grey colour. See also Supplementary Notes and Supplementary Fig. 2.</text></passage><passage><infon key="file">ncomms11032-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>63093</offset><text>NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA.</text></passage><passage><infon key="file">ncomms11032-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>63170</offset><text>(a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red). Selected shifts of amide resonances are indicated. (b) Plot of chemical shift change versus residue number in the ROQ domain (residues 171–326) from a. Grey negative bars indicate missing assignments in one of the spectra. Gaps indicate prolines. (c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green). See also Supplementary Notes and Supplementary Fig. 3.</text></passage><passage><infon key="file">ncomms11032-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>63754</offset><text>Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR.</text></passage><passage><infon key="file">ncomms11032-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>63840</offset><text>(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).</text></passage><passage><infon key="file">ncomms11032-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>64624</offset><text>Functional importance of Roquin-1 target motifs in cells.</text></passage><passage><infon key="file">ncomms11032-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>64682</offset><text>(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). Arrows indicate the individual binding events to either motif. It is noteworthy that the higher bands observed at large protein concentrations are probably additional nonspecific, lower-affinity interactions of Roquin-1 with the 3′-UTR or protein aggregates. (c) Relative Ox40 MFI normalized to expression levels from the Ox40 CDS construct. Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments. Statistical significance was calculated by one-way analysis of variance (ANOVA) Kruskal–Wallis test followed by Dunn’s multiple comparison test (**P&lt;0.01). (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). Error bars represent the mean of technical duplicates in one experiment. mRNA half-life times were calculated with Graph Pad Prism. Data are representative of two experiments with similar results.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>66346</offset><text>Data collection and refinement statistics.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml">&lt;?xml version=&quot;1.0&quot; encoding=&quot;UTF-8&quot;?&gt;
+&lt;table frame=&quot;hsides&quot; rules=&quot;groups&quot; border=&quot;1&quot;&gt;&lt;colgroup&gt;&lt;col align=&quot;left&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;col align=&quot;center&quot;/&gt;&lt;/colgroup&gt;&lt;thead valign=&quot;bottom&quot;&gt;&lt;tr&gt;&lt;th align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;ROQ-&lt;/bold&gt;&lt;italic&gt;&lt;bold&gt;Ox40&lt;/bold&gt;&lt;/italic&gt;
+&lt;bold&gt;ADE-like SL&lt;/bold&gt;&lt;/th&gt;&lt;th align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;bold&gt;ROQ-ADE SL&lt;/bold&gt;&lt;/th&gt;&lt;/tr&gt;&lt;/thead&gt;&lt;tbody valign=&quot;top&quot;&gt;&lt;tr&gt;&lt;td colspan=&quot;3&quot; align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;Data collection&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; space group&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;P&lt;/italic&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;P&lt;/italic&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;2&lt;sub&gt;1&lt;/sub&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;3&quot; align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Cell dimensions&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;italic&gt;a&lt;/italic&gt;, &lt;italic&gt;b&lt;/italic&gt;, &lt;italic&gt;c&lt;/italic&gt; (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;89.66, 115.79, 42.61&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;72.90, 89.30, 144.70&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;italic&gt;α, β, γ&lt;/italic&gt; (°)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90, 90, 90&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;90, 90, 90&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Resolution (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;50–2.23 (2.29–2.23)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;50–3.0 (3.08–3.00)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;merge&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;5.9 (68.3)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;14.8 (93.8)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;italic&gt;I&lt;/italic&gt;/σ&lt;italic&gt;I&lt;/italic&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;14.9 (2.1)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;16.7 (3.1)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Completeness (%)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;98.7 (97.7)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.9 (99.9)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Redundancy&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3.9 (3.7)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;13.2 (12.7)&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;3&quot; align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;Refinement&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Resolution (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2.23&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;3.00&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; No. reflections&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;21,018&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;18,598&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;work&lt;/sub&gt;/&lt;italic&gt;R&lt;/italic&gt;&lt;sub&gt;free&lt;/sub&gt;&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;21.8/25.7&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;18.6/23.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;3&quot; align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; No. atoms&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Protein&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;2,404&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;4,820&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Ligand/ion&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;894&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1,708&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Water&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;49&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt; B&lt;/italic&gt;-factor overall&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;47.2&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;60.4&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;3&quot; align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;&lt;italic&gt;Root mean squared deviations&lt;/italic&gt;&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Bond lengths (Å)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.006&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.014&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Bond angles (°)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.07&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.77&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; &lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td colspan=&quot;3&quot; align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;Ramachandran plot&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Most favoured (%)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;98.6&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;99.8&lt;/td&gt;&lt;/tr&gt;&lt;tr&gt;&lt;td align=&quot;left&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt; Additional allowed (%)&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;1.4&lt;/td&gt;&lt;td align=&quot;center&quot; valign=&quot;top&quot; charoff=&quot;50&quot;&gt;0.2&lt;/td&gt;&lt;/tr&gt;&lt;/tbody&gt;&lt;/table&gt;
+</infon><offset>66389</offset><text> 	ROQ-Ox40ADE-like SL	ROQ-ADE SL	 	Data collection	 	 space group	P21212	P212121	 	 	 	 	 	 Cell dimensions	 	 a, b, c (Å)	89.66, 115.79, 42.61	72.90, 89.30, 144.70	 	 α, β, γ (°)	90, 90, 90	90, 90, 90	 	 Resolution (Å)	50–2.23 (2.29–2.23)	50–3.0 (3.08–3.00)	 	 Rmerge	5.9 (68.3)	14.8 (93.8)	 	 I/σI	14.9 (2.1)	16.7 (3.1)	 	 Completeness (%)	98.7 (97.7)	99.9 (99.9)	 	 Redundancy	3.9 (3.7)	13.2 (12.7)	 	 	 	 	 	Refinement	 	 Resolution (Å)	2.23	3.00	 	 No. reflections	21,018	18,598	 	 Rwork/Rfree	21.8/25.7	18.6/23.4	 	 	 	 	 	 No. atoms	 	 Protein	2,404	4,820	 	 Ligand/ion	894	1,708	 	 Water	99	49	 	 B-factor overall	47.2	60.4	 	 	 	 	 	Root mean squared deviations	 	 Bond lengths (Å)	0.006	0.014	 	 Bond angles (°)	1.07	1.77	 	 	 	 	 	Ramachandran plot	 	 Most favoured (%)	98.6	99.8	 	 Additional allowed (%)	1.4	0.2	 	</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>67293</offset><text>ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>67373</offset><text>For each data set, only one crystal has been used.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>67424</offset><text>*Values in parentheses are for highest-resolution shell.</text></passage><passage><infon key="file"></infon><infon key="id">t2</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>67481</offset><text>KD for selected RNAs obtained from SPR measurements with immobilized ROQ domain of Roquin-1.</text></passage></document></collection>